JANUS - FACED IMMUNITY TO AN ENTERIC PATHOGEN: HOW CAMPYLOBACTER CAUSES COLITIS AND AUTOIMMUNITY By A nkit M alik A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Microbiology and Molecular G enetics Doctor of Philosophy 2014 ABSTRACT JANUS - FACED IMMUNITY TO AN ENTERIC PATHOGEN: HOW CAMPYLOBACTER CAUSES COLITIS AND AUTOIMMUNITY By Ankit Malik Campylobacter jejuni is an enteric bacterium present ubiquitously in food animals and is one of the top two etiologic agents of gastroenteritis worldwide . While many healthy individuals can be colonized asymptomatically or experience self - limiting gastroenteritis, serious autoimmune disease sequelae can also follow infections with C. jejuni . Inflammatory Bowel Disease, Reactive A rthritis and acute neuropathies Guillain Barré Syndrome and Miller Fisher Syndrome , have also been demonstrated to be strongly associated with antecedent C . jejuni infection. GBS annual ly in the USA. Yet very little i s known about how Campylobacter jejuni interacts with the immune system to esta blish asymptomatic colonization, induce gas troenteritis and/or lead to autoimmune sequelae . Lack of functional IL - 10 is a predispos ing factor to IBD, and thus the IL - 10 deficient mice reiterate human disease by develop ing colitis after C. jejuni infection, making it a useful model for studying colo n inflammation. In these studies, w e have demonstrate d that C. jejuni mediated colitis in specific pathogen free C57BL/6IL - 10 - / - mice was IFN - and IL - 17 cytokine dependent. We also show ed that both Innate Lymphoid Cells and T cells participate in IFN - and IL - 17 elicitation, albeit with different kinetics. We also show ed that T cells are essential for C. jejuni induced colitis. The s e results yield new cytokine and cellular targets for immunomodulatory therapy against IBD. This work also provides the fir st demonstration of a time - dependent role of I nnate L ymphoid C ells and T cells in mediating Type1 ( IFN - ) and Type17 ( IL - 17 ) cytokine responses following infection with a human pathogen. Furthermore, w e demonstrate d that depleting IFN - and/or IL - 17 pivots the immune response away from sustaining inflammation in the colon to inducing a contrasting Type2 (IL - 4) cytokine response . Following infection with C. jejuni isolate s from GBS patient s , th is T helper cell dependent IL - 4 cytokine response lead to elicitation of circulating autoantibodies. The se autoantibodies cause d histological and phenotypic changes in IL - 10 deficient mice that we re consistent with this d that Siglec - 1 is a central antigen presenting cell - expressed receptor that mediates uptake of C. jejuni and subsequent T H 2 maturation in a sialylation dependent manner. Therefore, IL - 4 and Siglec - 1 are novel and rational therapeutic targets against this peripheral autoimmunity, against which only invasive, non - specific and inefficient therapies like plasmapheresis exist. iv ACKNOWLEDGEMENTS I would like to express my profound gratitude towards my advisor Dr. Linda Mansfield . Sh e has been exceptionally patient and encouraging in giving me sound advice and support. Sh e has encouraged me to be independent, motivated and grounded in science and life in general . I fee l extremely fortunate to have her as my mentor and will strive my best to imbibe these qualities as I take the next step in my scientific career. I also feel immense gratitude towards my committee members for providing me constant guidance through my PhD. Their support, constructive discussions and continual encouragement have helped me take the research in a better direction. I am particularly grateful to Dr. Hoag for helpful advice on my projects and review o f my manuscripts . I am also thankful to Dr. Louis King for managing the flow cytometry facilit y and Dr. Narayanan Parameswaran for letting me use his laboratory hardware and software, and letting me attend journal clubs organized by him . I am thankful to the Microbiology and Molecular genetics department, Dr. Esselman and Dr. Hausinger for accepting me into their programs and giving me the opportunity to enhance my scientific career. I sincerely thank, Angie Zell, Betty Miller and Becky Mansel for their he lp in administrative work through the academic years. I would also like to thank College of Natural science for continuation and completion fellowships that providing great support and encouragement. I would al so like to extend my s pecial gratitude towards the endowers of Graduate Student Scholarships at Michigan State University for recognizing and encouraging scientific endeavors of graduate students. v I am deeply obliged to Dr. Barbie J. Gadsden , Leslie A. Dybas , Dr. Julia Bell, Deepika Sharma and Alexander Ethridge for their help in my research work. My sincere thanks to all current and former lab members of Dr. laboratory, especially Jessica St. Charles , Andy Flies, Philip Brooks , J.P. Jerome , Jean Brudvig, Amanda Staunton , Hahyung Kim , Vijay Rathinam, Dr. Anne P. Jones , Jamie Kopper and Eric Smith for their kind help, cooperation and sustaining a friendly atmosphere. I also want to thank all my friends and colleagues in Molecular Biology and Molecular Genetics and Physiolog y department. I am greatly indebted to my friends here at MSU especially Disha, Rajasi, Rewatee, Nandha, Renga, Shipra, Vishal, Ashwini, Gaurav, Aparajita, Sunetra, S atyaki, Kishore , Arjun, Haritha, Sarguru and Dr. Taehyung Lee for their companionship and support through these years. I greatly appreciate ULAR and URCF staff and histopathology core lab, especially Jeff Daman, Amy and Kathy Porter for their invaluable help in my research. I feel immense gratitude towards my fam ily and relatives for their continual love, support and encouragement. Finally special thanks to Deepika who has provided me immense help, support and motivation through these years. vi TABLE OF CONTENTS LIST OF TABLES....................................................................................................... ............ .......i x LIST OF FIGURES.......................................................................................................... ............ ..x KEY TO ABBREVIATIONS............................................................... . ... .............. ..........................x i CHAPTER 1..................................................................................................... .................. .......... .1 LITERATURE REVIEW.............................................................................................. ............. 1 ABSTRACT ...................................................................... ....... ....... ................................... ...... .2 ............................................................ ....... .4 Inflammatory Bowel Disease.... .. ..................................................... ............... ..... .................... 5 Importance of IL - ...............................................................5 Campylobacter jejuni as a causative organism of inflammatory ..................................... .................... ................. ............ ............... 6 Murine models of C. jejuni .......................................... . .............. 7 Immune basis of C. jejuni induced colitis.. .. .............................................................. . ... ........... 9 Guillain Barré Syndrome: Epidemiology and Clinical Manifestations ............................................................................................ ............. ....... ........ 13 Molecular mimicry and autoimmune basis of GBS .......... ........................................................................................ .....................................1 5 Immune recognition in the context of auto - reactive oligosaccharides ............................................................. ...................................... ......... ........1 9 ............................................................. ....... ...... .................... ......... ....... .23 REFERENCES. ......... ..................... ......................................... . ..... ............................. .............2 6 CHAPTER 2 37 ABSTRACT 38 INTRODUCTION................................................................................................... ............ . . . .. 39 RESULTS ............................................................................... .................................. ........ . ... . . 42 C. jejuni induced a mixed Type1 and Type17 cytokine and cellular response in IL - 10 - / - mice ...... ........ .......................................................................... ..... .. . ....................... 42 Both Innate Lymphocytes and T cells contribute to Type1 (IFN - ) and Type17 (IL - 17 and IL - 22) cytokine production in an organ, time and cell type specific manner 44 Thy - 1 + lymphocytes are necessary for C. jejuni mediated colitis .. .................... .... . ........ . .. 4 5 Innate immunity is insufficient to mediate severe C. jejuni induced colitis ..... . ..... . .. 46 Both IFN - and IL - 17 participate in C. jejuni induced colitis ....................................... 47 Infection with GBS associated C. jejuni strains induces Type2 immunity that is protective for colitis but leads to ................................................... 48 DISCUSSION............................................................................ ............. . .................. .. .. ........ . 50 .................................................... .. ................................... .. 54 Mice 54 Antibodies for in - vivo 54 Immnuohistochemical analysis of mononuclear cells Preparation of colonic lamina propria leukocytes Flow cytometry Enzyme - linked Immunosorbent Assay vii Quantitative and reverse transcriptase PCR Splenocyte challenge by gentamicin killing assay Epithelia l challenge by gentamicin killing assay Statistical analysis . REFERENCES... .......................................................................... ....... .. ................................. 74 CHAPTER 3..................................................................................... ......................................... .. 79 ABSTRACT................................................................................................... ............ ........... 80 INTRODUCTION....................................................................................... ..... ......... ............. 8 1 RESULTS .............................................................................. ...............................................8 4 C. jejuni induced autoimmunity is IL - 4 Siglec - ........................................... 8 5 IL - 10 is a negative regulator for autoantibody production .... . ........................................... 8 7 C jejuni GBS isolate infection leads to abnor mal hind limb movements in a subset of IL - 10 - / - mice .................. ..................................................... ................................ ....... .. .......... 88 DISCUSSION ....................................................................................... .......... ......................... 9 0 MATERIALS AND METHODS ........................... ............ ................................ . ....................... . . 9 2 Mice , inoculation and antibodies for in vivo neutralization 9 2 Tissue samples . .9 2 Immunohistochemical analysis of mononuclear cells 3 Evaluation of cell densities 3 Preparation of Lamina Propria lymphocytes 3 Flow cytometry 4 Enzyme - linked Immunosorbent Assay 5 Splenocyte challenge by gentamicin killing assay . 5 APPEND REFERENCES ......... ........................... .. .......................................................... . .........1 0 6 CHAPTER 4......................................................................................................... .......... ........... 11 2 Conclusions, Significance and Future Directions . ................................. . .......................................................1 1 3 Future Direction 1 ........................... ........... ................... . ......................................................1 1 5 Future Direction 2 ............................... ........................... . ...... ...............................................1 1 7 Future Direction 3 .... ..... ...................... ... ......................... . ....................................................1 19 ... ..... ...................... ... ......................... ....................................................1 21 REFERENCES .................................................. ..................... ...... . ........................12 3 viii LIST OF TABLES Table 1 : Contrasting C. jejuni induced colitis and autoimmune neuropathy ......... ........... 1 22 ix LIST OF FIGURES Figure 1 .1 : Schematic representation of lymphocyte population s involved in C. jejuni infections ... ......................................................................... ................. 24 Figure 1. 2 : Schematic representation of immune basis of C. jejuni 25 Figure 2. 1: ... . ................. 64 Figure 2 .2 : Temporal analysis of colon leukocyte and C. jejuni specific antibody plasma a ntibody 65 Figure 2. 3 : ........... ................................... 66 Figure 2. 4 : Colon cytokine - . Figure 2. 5 : Mesenteric Lymph Node cytokine - lymphocyte .............. ..... ... . ...... 68 Figure 2. 6 : Role of Thy - 1+ lymphocytes in C. jejuni ....... . ....... 69 Figure 2. 7 : Innate immunity is insufficient to induce colitis after C. jejuni infection ........ ....... .... .. 70 Figure 2. 8 : Both IFN - and IL - 17 are involved in C. jejuni mediated colitis and humoral . 71 Figure 2. 9 : ....................................................... 72 Figure 2. 10 : Whole splenocytes and epithelial cell challenge by gentamycin killing assay .........73 Figure 3. 1 : IL - ............... . ............. .99 Figure 3. 2 : IL - 4 depletion experiment sciatic nerve and DRG macrophage ............. 10 0 Figure 3 .3 : IL - 4 depletion experiment c o lon flow cytometry, histology and colonization.. ... ...... 10 1 Figure 3. 4 : Siglec - ............................................1 0 2 Figure 3. 5 : Siglec - 1 blocking in IL - 10 - / - mice, along with auto antibody analysis in IL - 10 +/+ mice . ....... ... 1 0 3 Figure 3. 6 : Long term phenotyping experiment with GBS isolate infected IL - 10 - / - mice ... ........ .10 4 Figure 3. 7 : Long term phenotyping experiment: colonization, colon flow cytometry and plasma antibody analysis .... ..... .......... ...............................................................1 0 5 x KEY TO ABBREVIATIONS ATP: Adenosine triphosphate AIDP: Acute Inflammatory Demyelinating Polyneuropathy AMAN: Acute Motor Axonal Neuropathy AMSAN: Acute Motor Axonal and Sensory Neuropathy BM D M: Bone marrow derived macrophages BM DC : Bone marrow derived dendritic cells CIA: Collagen induced arthritis cAMP: cyclic adenosine monophosphate CD: Cluster of differentiation C - terminus: carboxy terminus DSS: Dextran sodium sulfate EAE: Experimental autoimmune encephalomyelitis GBS: Guillain Barré Syndrome IFN: Interferon Ig: Immunoglobulin : nuclear factor of kappa light polypeptide gene enhancer in B - cells inhibitor, alpha IL: Interleukin ILC: Innate Lymphoid Cell JAK: Janus kinase JNK: c - Jun N - terminal kinases LPS: Lipopolysaccharide LOS: Lipoligosaccharide MAPK: Mitogen activated protein kinase MFS: Miller Fisher Syndro me xi MMP: matrix metalloproteases MS: multiple sclerosis MyD88: Myeloid differentiation primary response gene 88 NK: Natural Killer NO: Nitric oxide NOS: Nitric oxide synthase PMA: phorbol myristate acetate PRR: Pa ttern recognition receptor ROS: reactive oxygen species SIRS: Systemic inflammatory response syndrome STAT: Signal Transducers and Activators of Transcription TGF: T ransforming growth factor TCR: T cell receptor TNF: Tumor necrosis factor TRIF: TIR - domain - conta ining adapter - inducing interferon - Treg: regulatory T TLR: Toll like receptor UC: Ulcerative colitis 1 CHAPTER 1 LITERATURE REVIEW 2 ABSTRACT Campylobacter jejuni is a spiral, G ram - negative, microaerophilic bacterium that is the most common bacterial cause of gastroenteritis . Previous infections with C. jejuni has been linked with several chronic autoimmune disorders including development and flare - u p of Inflammatory Bowel Disease, Irritable Bowel Syndrome , and peripheral neuropathy Guillain Barré Syndrome (GBS). C. jejuni affects about 1.4 million individuals in the USA every year, leading to about 10,000 deaths. The occurrence of GBS is temporally associated with development of autoantibodies that target gangliosides on peripheral nerves. The C. jejuni strains isolated fr om GBS patients have modifications of the outer core of the lipooligosaccharide that mimic several peripheral nerve ganglioside s. P lasmapheresis and intravenous immunoglobulin (IVIg) treatment are the only known treatments with beneficial effect, but only 60% of GBS patients improve. Thus, new therapeutics are critically needed, but drug development pipelines are limited by lack of appropriate animal models for efficacy testing. In an effort to develop a model to study enteric and systemic manifestations of this bacterium , we s howed that a number of isolates of C. jejuni from human enteritis patients induce colitis in the IL - 10 - / - mouse model whereas isolates from human GBS patients colonize these mice without inducing colitis. Our overarching hypothesis was that fundamentally different immune responses mediate C. jejuni induced enteritis, asymptomatic colonization and GBS. The thesis presented here addresses the C. jejuni strain dependent immunological mechanisms behind induction of colitis by gastroenteritis patient - derived isolates versus induction of an asymptomatic colonization by the GBS patient - derived isolate s in murine models . Further, we show that infection of mic e with C. jejuni strains from GBS patients, but not from colitis patients, elicits autoantibodies that react with nerve gangliosides consistent with those seen in human GBS cases. Production of these autoantibodies correlate s temporally with a moderate per ipheral neuropathic phenotype and causes the histo pathological changes that are This review discusses the 3 considerable recent advances in our understanding of the immunological underpinnings of C. jejuni associated enteritis and autoimmunity , along with future challenges . 4 Basic mechanism of inflammatory reactions. A pathogen typically displays a set of D anger A ssociated M olecular P atterns (DAMPs) that are recognized by P attern R ecognition R eceptor s ( PRRs, expressed by antigen presenting cells and non - hematopoietic cells like epithelial cells and fibroblasts) leading to production of one or more primary/innate cytokines and chemokines (such as interleukin ( IL ) - 8, IL - 12, TN F - - 18, IL - 15, IL - - 6, IL - 23 and IL - 10) 1 . These primary cy tokines perform multiple roles su ch as mediating the infiltration and activation of immune effector cells, inducing production of anti - bacterial compounds, activating the coagulation cascade to localize the infection and priming a regulated secondary/adaptive immune response. Inflammatory adaptive immune responses can be broadly classified into 4 types Type1, Type2, Type17 and T regulatory responses. IL - 12, IL - 15 and IL - 18 direct the maturation of naïve T cells and innate (Rag independent) lymphocytes into Type1 cells that are characteri zed by production of IFN - 2 . IL - 4 is the most powerful trigger for Type2 cells that are themselves characterized by production of IL - 4, IL - 5 and IL - 13 3 . Therefore, IL - 4 both induces and maintains its own expression, although the initial source of IL - 4 is not known 3 . Type 17 cells require IL - 6, TGF - - 23 for induction and maintenance and are characterized by production of one or more of IL - - - 17F, IL - 21 and IL - 22. 4 , 5 Type1, 2 and 17 cytokines have both microbi o cidal and tissue protective effects. T regulatory (T reg) responses typically involve cytokine IL - 10 . Innate and adaptive immune cells and also epithelial cells and fibroblasts can produce IL - 10. It - inflammatory cytokine production and suppress proliferation in mononuclear cells. The nature of the ensuing adaptive immune response depends on the stimulatory innate signals. The first three types enhance infiltration and activation of effector cells and potentiate a humoral response that is in accordance with the nature of the innate response, while Tregs hel p resolve the inflammation and repair the affected tissue. An immune response may encompass one or more types of innate and adaptive responses in a tissue - and time - dependent manner. A 5 repairs the affected tissue, while a dysregulated immune response can cause severe morbidity and even mortality. Inflammatory Bowel Disease. Inflammatory Bowel Disease (IBD) refers to a group of auto inflammatory conditions of the small intestine and colo n. Based on pathological manifestations in humans, Inflammatory Bowel Disease (IBD) is often divided into two subsets metaplasia, glandular atrophy and architectural di stortion, the presence of fissuring ulcers, deep granulomas, transmural inflammation and skip lesions classify it as CD 6 . CD can affect any part of the gastrointestinal tract including the colon. In case of UC, lesions are present throughout the colon and while backwash ileitis may be present, other parts of the gut are unaffected and fissuring ulcers or deep granulomas are not present 6 . Both forms can be relapsing and remitting in some individuals and progressive in others. Both forms of the disease are highly prevalent in developed countries (21 246 per 100,00 for UC and 8 214 per 100,00 for UC) and represent major burdens for morbidity and the economy 7 . Importance of IL - 10 in IBD. Interleukin - 10 (IL - 10) is a regulatory cytokine that prevents overt self damaging inflammatory response to pathogens or autoimmune stimulators. IL - 10 is produced by many leukocytes (including T regulatory cells, B cells and myeloid cells) after ligation of pathogen recognition receptors and/or co stimulatory molecules and acts on hematopoietic and non hematopoietic cells to regulate diverse immune response s 8 . IL - 10 signals via its cognate receptor, IL - 10R, which is composed of two chains, IL - 10RA and IL - 10RB, which activates Janus kinase family members and STAT transcri ption factors to mediate its downstream effects 9 . In Antigen Presenting Cells (APCs), it suppresses the release of pro - inflammatory mediators, decreases antigen presentation and decreases expression of MHCII, co - stimulatory and adhesion molecules while enhancing phagocytosis and scavenging 6 functions 10 . It also acts on CD4 + T cells directly to reduce their proliferation and cytokine synthesis 10 . As the intestine is continuously exposed to dietary antigens and commensal microflora, sophisticated immune regulatory pathways must exist to prev ent chronic intestinal inflammation, and IL - 10 has been implicated in them 11 . A genome wide association analysis discovered Single Nucleotide Polymorphisms (SNPs) flanking the IL10 gene as the most significant locus associated with UC, outside the MHC locus 12 . Association of CD with SNPs in IL10 was also significant. Furthermore, a recently identified monogenic form of CD was shown to be associated with mutations in cognate rece ptor chains IL - 10RA or IL - 10 RB 12 . Therefore, impairment of IL - 10 associated regulatory pathway is critical for both forms of IBD. Campylobacter jejuni as a causative organism of inflammatory diarrhea. Campylobacter jejuni is a spiral , G ram - negative, bipolar and microaerophilic bacterium and is the most common cause of gastroenteritis worldwide 13 , 14 . C. jejuni is most commonly associated with poultry and farm animals, and consequently raw/undercooked meat and unpasteurized milk are the most common sources of infection in humans 13 . Enteritis caused by C. jejuni is debilitating but usually self - limiting, but can be life threatening in immune - compromised individuals 15 . Histopathological manifestations include colonic cry pt distortion, crypt abscesses, mucin depletion of epithelium, edema in the colonic lamina propria (cLP) along with infiltration of granulocytes, plasma cells and lymphocytes 16 . In spite of the medical and economic importance of the pathogen, little wa s known about the immunological mechanisms initiated and susta ined during the course of the d isease. Most of the data regarding C. jejuni virulence factors comes from challenge infections in epithelial cell culture or chicken colonization models . In these models it has been shown that CadF (Campylobacter adhesion to Fibronectin) and FlpA (Fibronectin - like protein A) are to the epithelial extracellular matrix , while injection of 7 flagella mediated Cia (Campylobacter invasion a ntigens) proteins induced phagocytosis and IL - 8 release 17 . Most strains of C. jejuni produce CDT (Cytolethal Distending Toxin) that has DNase activity that trigger s apoptosis and IL - 8 release 18 . PorA (porin), cj0091 (putative lipoprotein), JlpA ( a surface - exposed lipoprotein), pldA (phospholipase A), CapA (putative autotransporter), DNA J (transcriptional regulatory) and FlaA (flagellin subunit A) are some other proteins shown to be involved in epithelial adherence/ invasion 19 , 20 , 21 , 22 , 23 . The capsule and LOS also play a role in modulating epithelial invasion and cytokine secretion 24 , 25 . On the s ide of the epithelial cells, PI - 3 kinase, c - src and Focal Adhesion Kinase (FAK) and Pho GTPases cdc42 and Rac1 are necessary for C. jejuni uptake 17 , 26 . Recently, we and others have confirmed the positive role of CiaD and HtrA (High temperature requirement A) in mediating enteritis in IL - 10 - / - mice 27 , 28 . However, confirmation of the role of other virulence factors in an in vivo model of enteritis has been lacking. Murine model s of C. jejuni induced colitis . Natural and mutant isolates of C. jejuni have been shown to colonize a large number of in - bred mouse lines, including BALB/c, CBA, C3H and Swiss Webster mice 29 , 30 , 31 , 32 . However colonization in these models was not associated with clinical or histopathological changes typical of campylobacter i osis in humans . Fox et al., showed that mice deficient in NF - B subunits (p50 - / - p65 - / - ) in a C57BL/129 background developed gastritis and proximal duodenitis but large bowel inflammation was mild 33 . Due to the anti - inflammatory and inflammation resolving roles of IL - 10, i t was hypothesized that IL - 10 deficient mice will experience exacerbated Type1, 2 or 17 cytokines in the colon after C. jejuni challenge. Consistent with this hypothesis, w ork in the Mansfield laboratory has showed that wild type (IL - 10 +/+ ) and IL - 10 - / - mice of various genetic backgrounds coloniz e C. jejuni while only the IL - 10 - / - mice develop enteritis 11 . While the IL - 10 +/+ mice of C57BL/6, C3H/HeJ and NOD background stably colonized C. jejuni ( isolate NCTC11168) for 35 days post oral inoculation without any adverse clinical or histopathological effects, the IL - 10 - / - mice of all three genetic backgrounds 8 developed typhlocolitis (inflammation of cecum and colon) 11 . The histopatho logical features of colitis in IL - 10 - / - mice also replicated the histopathological features of C. jejuni associated colitis in humans 16 , 34 . These features include infiltration of mononuclear and polymorphonuclear cells in the colonic lamina propria and occasionally within the muscularis (smooth muscle tissue), necrosis/ulceration of colonic epithelium and edema. A d ose range of 10 2 10 10 CFU/mouse caused similar level of pathology in these mice 31 , 34 . The immune parameters that sustain inflammation in IL - 10 - / - mice were also largely unkn own before our work . Several key findings suggest that diet and gastrointestinal microbiome play a role in C. jejuni pathogenesis and immunity. In a serendipitous discovery, it was observed that BL/6 IL - 10 - / - 35 . The reason for diet dependence of phenotype remains unknown. Multiple reports in the last few years have demonstrated that mice with reduced/altered microflora are more readily colonized with C. jejuni and experience more severe clinical and histological signs of colitis. Chang et al have shown that C3H SCID mice with Limited Flora (LF) experience significant colitis after infection with 81 - 176, an isolate that does not stably colonize mice with conventional flora 36 . Bereswill et al showed that mice in which conventional microbiota is eradicated by a quintuple antibiotic treatment and then reconstituted with a complete human flora (in contrast with mice reconstituted with mouse flora) dev elop colitis after C. jejuni infection 37 . Furthermore, the colitis respo nse depended upon sensing of LOS and DNA by TLR 4 and TLR 9 37 . Requirement of TLR4 for full - blown C. jejuni induced colitis was also confirmed in a more severe model of colitis - the quintuple antibiotic treated IL - 10 - / - mice 38 . Direct eviden ce of microbiota components affecting C. jejuni colonization and virulence was provided by Haag et al. by demonstrating that E. coli feeding alone could facilitate C. jejuni colonization and colitis induction 39 . Stahl et al., have recently developed a new model of C. 9 jejuni colitis that requires vancomycin pretreatment in SIGIRR - / - (Single IgG IL - 1 Re lated Receptor a negative regulator of MyD88 signaling ) mice. This , along with the IL - 10 - / - mouse model , provides evidence for the requirement of dysregulation of the host immune system and /or perturbation of the host microbiota as pre - requirements for C. jejuni to cause colitis in mice , and by extension probably also humans . The vancomycin treated SIGIRR model also confirmed the pro - inflammatory role of TLR4, but demonstrated an anti - inflammatory role of host TLR2 and capsule in colitis indu ction 40 . Recent studies in germ free or antibiotic pre treated IL - 10 - / - mice from Sun et al., has demonstrated the pro - inflammatory actions of mTOR (mammalian target of rapamycin) 41 and phosphatidylinositol 3 kinase - (PI3 - ) 42 by upregulaing pro - inflammatory cytokine production and neutrophil recruitment 43 . Conversely , an anti - inflammatory role for NOD2 in C. jejuni induced colitis has been shown that is mediated by enhancement of Nitrous Oxide (NO) mediated bacterial killing 43 . Thus, these observations tie the microbiome and innate PAMP recognition to C. jejuni mediated disease . However, the direct role of inflammatory mediators particularly lymphocytes and their secreted cytokines had not been established in vivo before our work. Immune basis of C. jejuni induced colitis : Infecting IL - 10 - / - mice with multiple isolates of C. jejuni produces a range of phenotypes, from failure of coloniza tion to colonization without enteritis and colonization with high rate of enteritis 35 , 44 . In chapter 2 , w e show that during infection with multiple human enteritis patient isolates, there is a significant increase in the levels of IFN - , IL - 17, IL - 22, IL - 6, TNF - , IL - 1 and IL - 12, but not IL - 4 or IL - 13, in the colon and/or mesenteric lymph nodes of IL - 10 - / - mice 45 . These findings are consistent with elicitation of a mixed Type1 and Type17 cytokine response, with the absence of a Type2 response. Similar findings were made by Edwards et al., that showed the elicitation of Type1 and 17 cytokine responses after challe nge of human colon explants and peripheral blood - derived cells with a 10 hypervirulent isolate of C. jejuni 11168 46 . We directly confirmed the pathogenic role of IFN - and IL - 17 in C. jejuni induced colitis by depleting them with neutralizing antibody injections during infection. Depleting I F N - and/or IL - 17 le d to a significant decrease in overt histopatholog ical changes including the extent of neutrophil , macrophage and T cell infiltration in the colon 45 . Strikingly, not all C. jejuni isolates are associated with colitis in humans or produce colitis in animal models. But exposure/ colonization alone often leads to detectable anti - C. jejuni IgG antibodies in circulation 35 , 47 . IgG subtype class switching in activated B cells is known to be modulated by Type1, Type2 and Type17 cytokines , and can therefore be used to proxy the specific type of immune response elicited by C. jejuni in vivo . Using IFN - and IL - 4 reporter mice, Reinhardt et. al. showed that IFN - induces isotype class switching to IgG2a, while IL - 4 primarily induces IgG1 class switch 48 . In vitro , B cells stimulated with LPS in the presence of IFN - preferentially switch to IgG2a and IgG3, while in the presence of IL - 4 they preferentially switch to IgG1 49 , 50 . IL - 17 was shown to be required for induction of autoreactive IgG2b (but not IgG1 or IgG2a) antibodies in an experimental autoimmune myasthenia gravis model by knock out and supplementation strategies 51 . IL - 21 is produced by a s ubset of Type17 cells and has been shown to induce IgG1 and IgG3 switching in CD40L stimulated B cells 52 . Furthermore, TGF - a cytokine involved in differentiation of Type17 cells and Tregs, can also induce IgG2b class switching in LPS stimu lated B cells 49 . Therefore, Type1, Type17 and Type2 responses seem to have overlapping but distinct class switching ability Type1 (IgG2a and IgG3) v/s Type17 (IgG2b, IgG3 and possibly IgG1) v/s Type2 (IgG1). Consistent with our finding of upre gulation of a Type1 and Type17 but not Type2 response by colon and plasma cytokine analysis, levels of C. jejuni specific IgG2b (IL - 17 dependent), IgG2c (allelic isoform of IgG2a, IFN - dependent) and IgG3 but not IgG1 (IL - 4 dependent) w ere significantly e nhanced in infected mice 45 . D epleting IFN - (with or without IL - 17) le d to an increase in the level of colonic 11 IL - 4 and plasma anti - C. jejuni IgG1 antibodies . This, along with decrease in IgG2b and IgG2c isotypes, demonstrat ed a compe nsatory shift towards a Type2 response . Elicitation of pro - inflammatory cytokines in response to C. jejuni related with progressive infiltration of neutrophils, macrophages and lymphocytes (T cells, B cells and innate lymphocytes) into the three layers of the colon the epithelium, lamina propria and the muscularis. Innate lymphocytes are functionally heteroge neous and can be broadly divided into NK cells (CD3 - NKp46 + ) and Innate - Lymphoid Cells (Thy1 hi CD3 - NKp46 - ). T cells and innate lymphocytes are known to be major producers of secondary/adaptive cytokines in an inflammatory lesion. T cells can further be divid ed into two subsets based on the nature of T cell receptor to two subsets on the basis of their co - receptor CD4 + T helpers (T H ) and CD8 + T cytotoxic cells (T C ). NK cells can be further divided into conventional NK cells (that depend on transcription factor NLIF3 for development and are potent producers of IFN - ) and NKR cells (that express transcription factor - 22) 53 , 54 , 55 . ILCs are further categorized as Lymphoid hi CD4 + ) and LTi - like cells (Thy1 hi CD4 - Sca - 1 + ) and nuocytes (Thy1 hi CD4 - CD117 + Sca - 1 + ) 56 , 57 are necessary for development of l ymphoid follicles and have also been shown to produce IL - 17 and IL - 22 in dextran sodium sulfate and Citrobacter rodentium model of innate colitis 58 . LTi - like cells were discovered as principal IL - 17 and IFN - producers in Helicobacter hepaticus and CD40 ligand induced models of innate colitis respectively 59 . Nuoc ytes are akin to T H 2 cells as they produce IL - 5 , IL - 9 and IL - 13 in response to helminth infection 60 (summarized in Fig 1.1) . 12 Remarkably, we observed an opposite regulation of ILCs and T cells in the colon and draining lymph nodes as the ILCs expand earlier before the expansion of T cells and then contracted with the expansion of T cells. This the inflammatory response that seems to be initiated by ILCs and is eventually taken over by adaptive T cells. Intra cellular cytokine staining demonstrated upregulated expression of IFN - , IL - 17 and/or I L - 22 from these cell types in a cell type, time and organ - specific manner 45 . We confirmed that non - B cell lymphocytes are necessary for development of colitis as depleting Thy - 1 + cells during infection completely abrogated inflammation and production of IFN - , IL - 17 and IL - 22 in the gut. Innate immunity alone was found to be insufficient for colitis as T and B cell deficient Rag1 - / - nor IL - 10R blocked Rag1 - / - mice developed colitis after infection. Therefore, T cells are necessary for C. jejuni to cause colitis in SPF BL/6 IL - 10 - / - mice. However, in Rag2 / IL - 10 double KO germ free mice of 129/SvEV background, it has been shown that 11168 (a human colitis isolate) d id induce colitis 42 . This point ed to wards a p rotective role of the interaction between the microbiota and host genetic makeup in determining susceptibility to a pathogen. Furthermore, it has been shown that CD4 + T cell depletion in infected gnotobiotic IL - 10 - / - mice did not decrease the colitis severity 42 . This finding does suggest a role for non CD4 + T cell and ILC subsets in driving colitis, but is complicated by the fact that macrophages can also express this marker. Therefore , further target ed studies are needed to resolve the role of each of the cellular subs e ts in C. jejuni induced colitis, and for the development of rational therapeutic interventions that can target particular pathogenic cell subsets. We also observed that the extent of C. jejuni colonization in IL - 10 - / - mice correlated with the extent of inflammation in the colon. In the kinetic studies, we observed that colonization extent increased with increasing time, and that correlated with incre ase in the histopathological score in the colon. Furthermore, treatments that decreased the extent of inflammation in the colon, like 13 depletion of Thy - 1 + cells or depletion of IFN - and IL - 17, decreased the colonization extent to a basal level observed at a time point that was prior to the onset of inflammation . Therefore, in this aspect C. jejuni res embles Salmonella enteric sero var T yph i murium wherein inflammation facilitates enhancement of the pathogen burden 61 , 62 . For salmonella, i nflammation has been shown to create a better niche for the pathogen by eliminat ing competing microbes or by utilizing substrates that are provided by the dying inflammatory cells 52 , 53 . P re - existing enteritis also overcomes colonization resistance of certain C. j ejuni isolates , but the mechanisms are unknown 39 . Gu illai n Barr é Syndrome: Epidemiology and C linical M anifestations. With the near eradication of polio, GBS is now the leading cause of acute neuromuscular paralysis with 6,000 9,100 cases annually in the US 63 . GBS typically involves an immune attack on peripheral nerves that may lead to irrever sible nerve damage and muscle wasting. Unlike most autoimmune diseases, i ncidence rates do not increase with age, and GBS is 1.5 times more prevalent in men than women 62 . About 1/3 rd of the patients require ventilation support and 3 - 10% die due to diaphragm paralysis 64 . Furthermore, while most patients recover from this syndrome, about 20% are unable to walk after 6 months, and up to 60% suffer pain and fatigu e that lasts for years 65 . Incidence rates and mortality is likely higher in develo ping countries due to inadequate diagnosis, treatment and reporting. Based on the type of the peripheral nerve fibers affected and the nature of the immune damage, GBS can be divide d into multiple subtypes. Acute Inflammatory Demyelinating Poly neuropathy (AIDP) refers to a multifocal immune attack on the myelin sheath that surrounds the axons . Macrophages, neutrophils and T cells invade the myelin and denude the axons 66 . As the myelin sheath acts as an insulator for nerve conductance, its damage leads to decrease d nerve signals reaching the neuromuscular junction. As appropriate nerve conduction signals are required f or 14 the integrity of both the axon and muscle, demyelination eventually leads to axon die back and muscle wasting 67 . Acute Motor Axonal Neuropathy ( AMAN ) and Acute Motor and Sensory Neuropathy ( AMSAN ) subtypes primar ily involve an antibody mediated attack on the ax o ns as macrophages invade the nodes of Ranvier and nerve root ganglia, blocking the nerve conductance but leaving the myelin sheath intact 68 . In severe cases, the whole axon may be permanently damaged 64 . The idea that this pathology is antibody based comes from the observation that only macrophages, and almost no lymphocytes, are p resent in the infiltrate 69 . Both AIDP and AMAN only affect motor fibers, while AMSAN also has sensory involvement. AIDP is the predominant subtype in North America and Europe accoun ting for about 90% of the GBS cases 70 while AMAN and AMSAN are more common in South and Central America and Asia accounting for about 50% of the GBS cases 71 , 72 . Miller - Fisher Syndrome (MFS) is a variant of GBS that primarily affects oculomotor nerves leading to paralysis of eye muscles 73 . It can also involve ataxia and paralysis of the diaphragm 74 . 5% of the GBS inc idences in western countries belongs to this variant, but it is more common in Asia 75 . GBS is frequently preceded by gastrointestinal or lung infections, usually 1 - 4 weeks before onset of clinical signs 76 . Based on culture and/or serologi cal data, more than 50% of the GBS patients were positive for C. jejuni infections 76 . However, many patients that present clinical symptoms of GBS do not present preceding signs of gastroenteritis 75 , 76 . The refore, the correlation, if any, between the extent of gastroenteritis or pneumonia and subsequent GBS is un clear 77 , 78 . Other pathogens associated with GBS incidence include Heamophilus influenzea, Mycoplasma pneumonia, Epstein - Barr Virus and cytomegalovirus 79 . A slight increase in risk for GBS after swine flu and influenza vaccination ha ve also been suggested 80 . However, for many GBS cases, especially the AIDP variant, no prior infections have been detected. This may be attributable to a different underlying etiology of AIDP and/or transient n ature of the preceding infection. 15 Molecular mimicry and autoimmune basis of GBS. Certain strains of C. jejuni (and other GBS associated pathogens) express particular epitopes that are structurally similar to self - molecules present on the nerves 81 . Sialic acids are 9 chain carbon monosaccharides found in terminal position in glycan st ructures in mammals and some microbes. Gangliosides are sia lic - acid containing glycan motifs that are highly presented on the surface of the myelin sheath and axons, including the nodes of Ranvier. Gangliosides are anchored on the outer leaflet of the plas ma membrane by ceramide 82 . They can differ based on the position of sialic acid linkage on the galactose residue, and can be mono - or di - sialylated. Gangliosides, namely GM1, GD1a, GM1b and GalNac - GD1a are monosial ylated with 2 3 linkage and commonly found on myelin and axonal membranes of peripheral nerves , while GQ1b is a common disialylated ganglioside with both , 2 - 3 and , 2 - 8 linkage and found commonly on cranial oculomotor nerves 79 . These sialylated oligosaccharide motifs are structurally similar to those found on the outer surface of the lipooligosaccharide (LOS) of certain C. jejuni strains, thereby providing molecular mimicry for the onset of autoimmunity 81 , 83 . C. jejuni LOS is a glycolipid entity consisting of a hydrophobic membrane anchor called lipid A, and surface exposed oligosaccharide motif that contains the sialic acid. T hus , it is speculated that after infection, antibodies are elicited against multiple epitopes presented by C. jejuni , including the LOS outer surface gangliosides. The se antibodies can cross - react with distal limb nerve gangliosides (in case of GBS) or oculomotor nerve gangliosides (in case of MFS), leading to antibody mediated immune attack at the se sites 82 . Antibody deposition onto the myelin or axon at the nodes of Ranvier can concievably mount an immune attack by two mechanisms : 1. Classical activation of the complement cascade, wherein C1 co mplex is activated when it binds to the Fc portion of the antibody, leading to activation of C3 convertase and deposition of C3b and C5b on to the tissue. The released fragments, C3a and C5a, act as chemoattractants for macrophages, while the bound C3b and C5b can lead to 16 further deposition of complement factors C6, 7, 8 and 9, that together form the membrane attack complex (MAC). MAC causes loss of ionic balance in the cells leading to their apoptosis. IgG and C3b deposition on axolemma in the case o f AMAN , 84 and the sc hwann cell membrane in the case of AIDP patients has been demonstrated 8 5 . MAC formation on GD1a over - expressing phrenic nerves has been demonstrated ex vivo after deposition of anti - ganglioside antibodies and heterologous complement 86 , However its formation in vivo lacks evidence . Antibody deposition at Nodes of Ranvier can cause a block in nerve conductance, that can eventually cause the axon and surrounding schwann cells to under apoptosis. Apoptotic cells can secrete multiple macrophage chemoattractants 87 , 88 . Infiltrating macrophages can then be activated further by binding to Fc port ion of the deposited antibodies by their Fc receptors . These mechanisms further unde rscore the idea of using IL - 10 - / - mice as a model for GBS, because in the absence of IL - 10 there is reduced deactivation of inflammatory cells and auto antibody production , that would be expected to intensify the disease . I nteresting ly, in humans and our mouse model of GBS , autoantibodies associated with C. jejuni asso ciated GBS are predominantly of the IgG1 isotype, while patients with C. jejuni associated MFS or H. influenzea induced GBS have IgG3 as the predominant autoreactive IgG isotype 89 , 90 . Further, in human GBS patients, titers of autoreactive IgG1 correlates with enhanced severity and poor long - term prognosis 89 , 91 . After IgM, IgG1 (and to a lesser extent , IgG3 ) is th e isotype most adept at complement fixation and binding to FcR for activation induced degranulation 92 . But because IgM is too big for extravasation from circulation into tissue structures, IgG1 is well suited for the task of carrying out an immune attack in peripheral tissue by complement fixation and inducing activation and degranulation of immune cells. Because isotype class switching to IgG1 or IgG3 isotype requires T helper cells, it is safe to conclude that T cells are n ecessary for GBS and MFS induction, even if they are not observed as a nerve - infiltrating cell type during AMAN and MFS. Indeed, in chapter 2 we have directly demonstrated the role of T cells in 17 antibody elicitation after C. jejuni challenge. It is also po ssible that antibody production is incidental to T cell activation, and that the primary role of T cells is to directly activate the macrophages and other phagocytic cells , and direct th eir infiltration into the nerve bundle . It is important to note that w hile IgG3 is predominantly IFN - dependent, IgG1 is IL - 4 dependent. IL - 4 itself is required for T H 2 induction, and is a strong stimulator for T H 2 cells 93 . And because the mucosal organs have a concentration of IL - 4 in homeostatic conditions , it is likely that the T H 2 response against C. jejuni GBS isolates is initiated there. Indeed, in chapter 2 we have sh own that infection of mice with GBS isolate s lead to a small but significant increase in IL - 4 transcripts and T H 2 cells in the colon. This suggests that C. jejuni induced GBS is IL - 4 and T H 2 cell dependent while C. jejuni induced MFS is IFN - and T H 1 cell dependent. However, it should be noted that IL - 21, a cytokine predominantly produced by T follicular helper cells, has also been shown to be independently capable of inducing IgG3 and IgG1 class switching 48 , 52 . Therefore, further work is necessary to work out exact T cell, B cell, APC and phagocytic cell interactio ns that orchestrate GBS and MFS syndromes, and provide therapeutic interventions that can target the central cell types/ cytokines to ameliorate these syndromes. It is also interesting to note that nerves that innervat e the colon, including the myen t e ric plexus, are often found inflamed (infiltrated with macrophages and T cells) in the C. jejuni induced IL - 10 - / - colitis model 34 . Thus, it is likely that the nervous tissue in colon, the primary site for infection, may provide fodder for further epitope spreading onto the antigens of the limbs and /or craniofacial nerves. However, the exact nature , distribution and density of gangliosides in the enteric and peripheral nervous system, in mice or humans is lacking, and will be important for the development of a robust animal model. 18 The underlyin g genetic makeup of LOS biosynthesis locus in a C. jejuni isolate determines the sialylation pattern on the LOS 94 . Consequently , the LOS biosynthesis locus de termines if the LOS will have molecular mimicry with the limb and/or craniofacial nerve gangliosides and by extension whether the infection may lead to the AMAN/AMSAN or MFS variant s of GBS. Atleast 19 different subclasses of LOS biosynthesis loci have been identified and classified in classes A through S 94 . Class A is most commonly associated with GBS, while class B confers structural similarity to cr aniofacial nerve gangliosides and therefore MFS 95 , 96 . Sialytransferase - II (cst - II) is an enzyme central to determining the sialylation pattern of the oligosaccharide in the outer core of the LOS, and is consequently commonly encoded in the genomes of C. jejuni strains isolated from GBS patients 97 . The genomes of two GBS isolates that we have utilized experimentally , HB93 - 13 and 260.94, both contain this gene, while the colitogenic isolates 11168 and CG8421 do not. However, g enome of 11168 does contain cst - III, which leads to the presentation of a low level of GM1 - , but not GD1a - like sialylation pattern on its LOS 98 , 99 . Enhanced fitness can be conferred by surface - structure modifying genes in microbes. H aving a sia l ylated outer core of LOS in this case provides a n evolutionary advantage by enhanc bacteriophage resistance when compared to asialylated isolates 100 . Antibody based pathophysiology for C. jejuni associated AMAN is supported by the fact that i ntravenous Ig, alongside plasmapheresis, have been the only succ essful treatments for GBS. However, these therapies are untargeted and only beneficial in about 60% of the cases 101 . The exact mechanism of action for IVIg is not known, but it can be speculated that i t may bind to Fc receptors on phago c ytic cells and prevent their activation/infiltration at the site of autoreactive IgG binding, and/or form immune complexes with autoreactiv e IgG a nd boost its clearance. A robust mouse model of C. jejuni induced GBS will therefore not only be the first demonstration of a bacterial pathogen causing autoimmunity, it will also serve as a model to screen new and targeted therapeutics for this syndrome . 19 Immune recognition in the context of auto - reactive oligosaccharides. While innate immune stimulation and recognition is well established for protein based antigens, antigen recognition and stimulation by carbohydrate based antigens, especially fo r sialic acid containing gangliosides , is less well studied. Sialic acid - binding immunoglobulin - like (SIGLEC) family of receptors are expressed mainly by immune cells that bind to sialic acid containing oligosaccharides 102 . They contain a single N - terminus variable domain that binds to the exposed sialic acid residue, and a numb er of constant domains that connects the sialic acid binding domain to the plasma membrane anchor, and a variable length cytoplasmic domain 103 . There are 14 different Siglec receptors that have been discovered in humans, and they have different binding specificity towards sialic acid containing oligosaccharides. Based on structural similarity, Siglecs can be divided into two groups: 1. Siglec - 1, 2, 4 and 15, that are conserved throughout mammals, and 2. CD33 - related Siglecs. Most siglecs contain cytoplasmic domain that contains an immunoreceptor tyrosine - based inhibitor motif (ITIM). ITIM leads to dephosphorylation of activating kinases, thereby provid ing negative immune stimul atory role to these receptors during proliferation, activation, cytokine production and apoptosis 104 . Humans have nine CD33 - related Siglecs while mice have five , and they share 50 - 90% sequence identity 102 . Siglec - 1/Sialoadhesin/Sn (CD169) has a short cytoplasmic chain that is devoid of any known signaling motifs. H owever , it has - - like domains, placing its sialic acid - bably prevents cis - interactions and allows Siglec - 1 to make the initial contact with sialylated pathogens or other cells during cell - cell interactions 105 . Siglec - 1 is exp ressed by macrophages located in prime positions for macrophages in the spleen, the site of afferent lymphatics for incoming blood. In the lymph nodes, Siglec - 1 is expr essed by the subcapsular sinus macrophages at the site of incoming lymph, and medullary cord macrophages exposed to the efferent lymphatics. In the colon, 20 Siglec - 1 expressing phagocytes are found in the lamina propria surrounding the crypts, a common site for bacterial invasion 106 . Therefore, anatomical location s of Siglec - 1 expressing cells sug gest its role as a primary sent inel for pathogens or apoptotic and cancer cells. Consistent with its role as a sentinel, expression of Siglec - 1 is usually upregulated under inflammatory conditions, particularly upon exposure to primary interferons. In pri mary cell cultures, Siglec - 1 has recently been shown as a primary receptor for HIV uptake by activated human DCs and m acrophages, and its trans - infection int o T cells 107 , 108 . It has also been shown to act as a specific receptor for phagocytosis of heat - killed C. jejuni isolates that carry GBS , 2 - 3 sialyla tion motifs by human blood - derived macrophages , and also upregulates IL - 6 production 99 . Activated m urine BMDMs and BMDCs f rom recently generated Siglec - 1 - / - mice have also been shown to have decreased uptake of heat - killed and 2 - 3 sialylated C. jejuni , and decreased expression of Type1 interferons and MyD88 dependent cytokines in vitro 109 , 110 . Siglec - 1 requirement for the rapid IFN - production was also confirmed by i.v. injection of inactivated sialylated C. jejuni in the knockout mice 109 , 110 . Recently, a role for Si glec - 1 in clearance of intravenou sly initiated group B streptococcus infection and resulting pathology has also been demonstrated 111 . However, these reports did not examine the role of Siglec - 1 in maturation of T cell subsets or IgG isotypes, and its consequence in related diseases. Multiple recent reports have also suggested a critical role for Siglec - 1 in T cell activation during au toimmunity and cancer. Siglec - 1 is highly expressed by circulating monocytes during chronic MS and rheumatoid arthritis 112 . It has been shown to play a pro - inflammatory role in t he EAE model of MS, by binding to Tregs and preventing their expansion 113 . It has also been shown to play a role in T cell activation and proliferation in coronary artery disease 114 . And the prese nce of Siglec - 1 + macrophages in local lymph nodes correlates with enhanced survival from colorectal cancer as well . Siglec - 1 + macrophages have also been associated with presentation of lipid antigens for activation of iNKT cells 115 . Further, targeting delivery of microbial/tumor 21 anti gens through Siglecs has shown promise for inducing strong T cell activation 116 . A r ecent publication has also shown that Siglec - 1 + macrophages are required to generate and maintain an anti - tumor T C popu lation. These reports support a view that deregulation of phagocytosis and T cell presentation by Siglec - 1 + APCs can lead to activation of auto - reactive T cells or failure to clear tumor cells. In case of pathogenic infections, it can lead to a failure in clearing systemic infection that result in morbidity and mortality. However, the role of Siglec - 1 in influencing T H 1/T H 2 balance, and subsequent B cell maturation in the context of a pathogenic infection or an autoimmune disease, has also not been explored before our work. As described in chapter 3 , w e found that Sigec - 1 blocking significantly decreased uptake of GBS isolates by adherent splenocytes and IL - 6 production from whole or adherent splenocytes. However, Siglec - 1 blocking did not affect elicitation of Type1 cytokines (IFN - , TNF - - 22). Siglec - 1 blocking also did not affect the uptake or any cytokine elicitation by colitogenic isolates. In vivo, blocking Siglec - 1 by neutralizing antibody injections in GBS isolate infected IL - 10 - / - mice lead to a decrease in T H 2 cells in the colon, and decrease in C. jejuni specific and auto - reactive IgG1 isotype antibodies in circulation. However, the effect of Siglec - 1 blocking in vivo does not appear to be T H 2 specific, as C. jejuni reactive I gG2b subtype also trended towards a decrease by Siglec - 1 blocking. Furthermore, blocking Siglec - 1 mirrored IL - 4 neutralization as both lead to a decrease in C. jejuni specific as well as auto - reactive antibodies in circulation. Decreased autoantibodies in circulation correlated with decreased macrophage infiltration in the sciatic nerve s and its roots. Thus, sialylated oligosaccharide motifs on the LOS of GBS associated C. jejuni is unique in the sense that it acts as the ligand for phagocytosis by antigen presenting cells, and also as a cross - reactive epitope that leads to autoimmunity. The r ole of IL - 10 in negatively affecting autoimmunity was also confirmed for GBS as IL - 10 +/+ mice, unlike the IL - 10 - / - mice, did not develop a significant level of autoanti bodies after infection with GBS isolates. IL - 22 10 +/+ mice did develop a significant anti - C. jejuni IgG2b response, suggesting an IL - 10 independent mechanism for its elicitation. Similar to Siglec - 1 bind ing to , 2 - 3 mono - sialylated gangliosides that are common in LOS of GBS associated C. jejuni , Siglec - 7 binds to , 2 - 3, , 2 - 8 di - sialylated gangliosides that are common in LOS of MFS associated C. jejuni 117 . Siglec - 7 is a CD33 - related Siglec that contains ITIMs in the cytoplasmic tail and is known to be expressed by human NK cells and myeloid cells 102 . The refor e , ganglioside sialylation pattern 1) forms the basis of autoi mmunity , by being conserved between GBS LOS and peripheral nerve gangliosides (mono - sialylation), and MFS LOS and craniofacial nerves (disialylation) ; 2) it also determines the Siglec receptor interaction and likely subsequent T and B cell maturation (summ arized in Fig. 1.2) . However, f urther work is required to pinpoint the role of Siglec - 7 in C. jejuni uptake, T cell activation and subsequent disease. These Siglecs that are not expressed by the mouse genus , but it is possible that other Siglecs in mice have similar sialic acid binding specificities and perform the same functions . Therefore, other Siglec receptors appear to play a role in GBS and MFS pathogenesis. 23 APPENDIX 24 Figure 1. 1. Schematic representation of Lymphocyte populations involved in C. jejuni infections Figure 1. 1 . On the basis on Rag dependence, lymphocytes can be divided as Adaptive ( Rag dependent) or Innate ( Rag Independent). Adaptive lymphocytes can be divided as B cells (CD19+B220+CD3 - ) and T cells (CD19 - on T cell - receptor, or CD4+T H and CD8+T C based on co - receptor expression. Innate lymphocytes can be div ided as NK cells (NK1.1+) and innate - like lymphocytes (Thy1 hi CD3 - ). NK NKp46+NK1.1 lo ) and NKT cells (CD3+NK1.1+). Innate - (Thy1 hi CD4+), LTi - like cells (Thy1 hi CD4 - Sca - 1+ROR t+) and Nuocytes (Thy1 hi CD4 - Sca - 1+IL - 13+) 25 Figure 1. 2. Schematic representation of immune basis of C. jejuni induced autoimmu nity. Figure 1. 2 . The structure of the sialylation of LOS (endotoxin) determines the biological site of it autoreactive potential and specificity towards a Siglec receptor. Almost all sialylation is sialyltransferase (cst) - II dependent. - 2,3 monosialylation mimics gangliosides on nerves of distal limbs and enables binding to Siglec - 1, which in turns facilitates C. jejuni uptake and T H 2 maturation. - 2,8 disialylation , on the other hand , mimics gangliosides on oculomotor nerves and facilitates binding to Siglec - 7. Role of Siglec - 7 in bacterial uptake and T ce ll maturation is not known. 26 REFERENCES 1. Murphy K. Janeway's immunobiology . Garland Science, 2011. 2. Farrar MA, Schreiber RD. The Molecular Cell Biology of Interferon - gamma and its Receptor. Annual Review of Immunology 1993, 11 (1) : 571 - 611. 3. Paul WE, Zhu J. How are TH2 - type immune responses initiated and amplified? Nat Rev Immunol 2010, 10 (4) : 225 - 235. 4. Mangan PR, Harrington LE, O'Quinn DB, Helms WS, Bullard DC, Elson CO , et al. Transforming growth factor - [beta] induces development of the TH17 lineage. Nature 2006, 441 (7090) : 231 - 234. 5. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M , et al. Reciprocal developmental pathways for the generation of pathogenic effector TH1 7 and regulatory T cells. Nature 2006, 441 (7090) : 235 - 238. 6. Clayburgh DR, Shen L, Turner JR. A porous defense: the leaky epithelial barrier in intestinal disease. Lab Invest 2004, 84 (3) : 282 - 291. 7. Loftus EV, Jr. Clinical epidemiology of inflammatory bowel disease: Incidence, prevalence, and environmental influences. Gastroenterology 2004, 126 (6) : 1504 - 1517. 8. Sabat R, Grütz G, Warszawska K, Kirsch S, Witte E, Wolk K , et al. Biology of interleukin - 10. Cytokine & growth factor reviews 2010, 21 (5) : 331 - 344. 9. Rossi D, Zlotnik A. The biology of chemokines and their receptors. Annual review of immunology 2000, 18 (1) : 217 - 242. 10. Saraiva M, O'Garra A. The regulation of IL - 10 production by immune cells. Nat Rev Immunol 2010, 10 (3) : 170 - 181. 11. Mansfie ld L, Patterson J, Fierro B, Murphy A, Rathinam V, Kopper J , et al. Genetic background of IL - pathogen interactions with Campylobacter jejuni and influences disease phenotype. Microbial Pathogenesis 2008, 45 (4) : 241 - 257. 27 12. Glocker E - O, Kotlarz D, Boztug K, Gertz EM, Schäffer AA, Noyan F , et al. Inflammatory Bowel Disease and Mutations Affecting the Interleukin - 10 Receptor. New England Journal of Medicine 2009, 361 (21) : 2033 - 2045. 13. Dasti JI, Tareen AM, Lugert R, Zautner AE, Groß U. Campylobacter jejuni: A brief overview on pathogenicity - associated factors and disease - mediating mechanisms. International Journal of Medical Microbiology 2010, 300 (4) : 205 - 211. 14. Butzler JP. Campylobacter, from obscurity to celebrity. Clinical Micro biology and Infection 2004, 10 (10) : 868 - 876. 15. Tee W, Mijch A. Campylobacter jejuni bacteremia in human immunodeficiency virus (HIV) - infected and non - HIV - infected patients: comparison of clinical features and review. Clinical infectious diseases 1998, 2 6 (1) : 91 - 96. 16. van Spreeuwel JP, Duursma GC, Meijer CJ, Bax R, Rosekrans PC, Lindeman J. Campylobacter colitis: histological immunohistochemical and ultrastructural findings. Gut 1985, 26 (9) : 945 - 951. 17. secreted proteins promote maximal Campylobacter jejuni invasion of host cells by stimulating membrane ruffling. Cellular microbiology 2012, 14 (2) : 226 - 238. 18. Hick ey TE, McVeigh AL, Scott DA, Michielutti RE, Bixby A, Carroll SA , et al. Campylobacter jejuni cytolethal distending toxin mediates release of interleukin - 8 from intestinal epithelial cells. Infection and immunity 2000, 68 (12) : 6535 - 6541. 19. Ziprin RL, Yo ung CR, Byrd JA, Stanker LH, Hume ME, Gray SA , et al. Role of Campylobacter jejuni potential virulence genes in cecal colonization. Avian diseases 2001 : 549 - 557. 20. Brás AM, Chatterjee S, Wren BW, Newell DG, Ketley JM. A novel Campylobacter jejunitwo - com ponent regulatory system important for temperature - dependent growth and colonization. J Bacteriol 1999, 181 (10) : 3298 - 3302. 21. Nuijten PJ, van den Berg AJ, Formentini I, van der Zeijst BA, Jacobs AA. DNA Rearrangements in the Flagellin Locus of an flaAMu tant of Campylobacter jejuni during Colonization of Chicken Ceca. Infection and immunity 2000, 68 (12) : 7137 - 7140. 22. Oakland M, Jeon B, Sahin O, Shen Z, Zhang Q. Functional characterization of a lipoprotein - encoding operon in Campylobacter jejuni. PloS o ne 2011, 6 (5) : e20084. 28 23. Ashgar SS, Oldfield NJ, Wooldridge KG, Jones MA, Irving GJ, Turner DP , et al. CapA, an autotransporter protein of Campylobacter jejuni, mediates association with human epithelial cells and colonization of the chicken gut. J Bact eriol 2007, 189 (5) : 1856 - 1865. 24. Rose A, Kay E, Wren BW, Dallman MJ. The Campylobacter jejuni NCTC11168 capsule prevents excessive cytokine production by dendritic cells. Medical microbiology and immunology 2012, 201 (2) : 137 - 144. 25. Louwen R, Heikema A, van Belkum A, Ott A, Gilbert M, Ang W , et al. The sialylated lipooligosaccharide outer core in Campylobacter jejuni is an important determinant for epithelial cell invasion. Infection and immunity 2008, 76 (10) : 4431 - 4438. 26. de M, Hartig R, Genth H, Schmidt G, Keo T , et al. Role of the small Rho GTPases Rac1 and Cdc42 in host cell invasion of Campylobacter jejuni. Cellular microbiology 2007, 9 (10) : 2431 - 2444. 27. Samuelson DR, Eucker TP, Bell JA, Dybas L, Mansfield LS, Konkel ME. The Campylobacter jejuni CiaD effector protein activates MAP kinase signaling pathways and is required for the development of disease. Cell Commun Signal 2013, 11: 79. 28. Heimesaat MM, Alutis M, Grundmann U, Fischer A, Tegtmeyer N, Böhm M , et al. Th e role of serine protease HtrA in acute ulcerative enterocolitis and extra - intestinal immune responses during Campylobacter jejuni infection of gnotobiotic IL - 10 deficient mice. Frontiers in Cellular and Infection Microbiology 2014, 4: 77. 29. Jesudason M V, Hentges D, Pongpech P. Colonization of mice by Campylobacter jejuni. Infection and immunity 1989, 57 (8) : 2279 - 2282. 30. Baqar S, Pacheco ND, Rollwagen FM. Modulation of mucosal immunity against Campylobacter jejuni by orally administered cytokines. Antimicrobial agents and chemotherapy 1993, 37 (12) : 2688 - 2692. 31. Mansfield L, Schauer DB, Fox JG, Nachamkin I, Szymanski C, Blaser M. Animal models of Campylobacter jejuni infections. Campylobacter, 3rd ed ASM Press, Washington, DC 2008 : 367 - 379. 32. Hodgson A, McBride B, Hudson M, Hall G, Leach S. Experimental campylobacter infection and diarrhoea in immunodeficient mice. Journal of medical microbiology 1998, 47 (9) : 799 - 809. 29 33. Fox JG, Rogers AB, Whary MT, Ge Z, Taylor NS, Xu S , et al. Gastroenterit is in NF - B - Deficient Mice Is Produced with Wild - Type Camplyobacter jejuni but Not with C. jejuni Lacking Cytolethal Distending Toxin despite Persistent Colonization with Both Strains. Infection and Immunity 2004, 72 (2) : 1116 - 1125. 34. Mansfield LS, Bell JA, Wilson DL, Murphy AJ, Elsheikha HM, Rathinam VAK , et al. C57BL/6 and Congenic Interleukin - 10 - Deficient Mice Can Serve as Models of Campylobacter jejuni Colonization and Enteritis. Infection and Immunity 2006, 75 (3) : 1099 - 1115. 35. Bell JA, St Charles JL, Murphy AJ, Rathinam VAK, Plovanich - Jones AE, Stanley EL , et al. Multiple factors interact to produce responses resembling spectrum of human disease in Campylobacter jejuni infected C57BL/6 IL - 10 - / - mice. BMC Microbiology 2009, 9 (1) : 57. 36. Chang C, M iller JF. Campylobacter jejuni Colonization of Mice with Limited Enteric Flora. Infection and Immunity 2006, 74 (9) : 5261 - 5271. 37. Bereswill S, Fischer A, Plickert R, Haag L - M, Otto B, Kühl AA , et al. Novel murine infection models provide deep insights in innate immunity. PLoS One 2011, 6 (6) : e20953. 38. Haag L - M, Fischer A, Otto B, Plickert R, Kühl AA, Göbel UB , et al. Campylobacter jejuni Induces Acute Enterocolitis in Gnotobiotic IL - - Like - Receptor - 2 and - 4 Signaling. PLoS ONE 2012, 7 (7) : e40761. 39. Haag L - M, Fischer A, Otto B, Plickert R, Kühl AA, Göbel UB , et al. Intestinal microbiota shifts towards elevated commensal Escherichia coli lo ads abrogate colonization resistance against Campylobacter jejuni in mice. PloS one 2012, 7 (5) : e35988. 40. Stahl M, Ries J, Vermeulen J, Yang H, Sham HP, Crowley SM , et al. A Novel Mouse Model of Campylobacter jejuni Gastroenteritis Reveals Key Pro - infla mmatory and Tissue Protective Roles for Toll - like Receptor Signaling during Infection. PLoS pathogens 2014, 10 (7) : e1004264. 41. Sun X, Threadgill D, Jobin C. Campylobacter jejuni Induces Colitis Through Activation of Mammalian Target of Rapamycin Signali ng. Gastroenterology 2012(0). 42. Sun X, Liu B, Sartor RB, Jobin C. Phosphatidylinositol 3 - Kinase - Campylobacter jejuni Induced Colitis through Neutrophil Recruitment in Mice. The Journal of Immunology 2013, 190 (1) : 357 - 365. 30 43. Sun X, Jobin C. Nucleotide - Binding Oligomerization Domain Containing Protein 2 Controls Host Journal of Infectious Diseases 2014 : jiu148. 44. Bell JA, Jerome JP, Plovanich - Jones AE, Smith EJ, Gettings JR, Kim HY , et al. Outcome of infection of C57BL/6 IL - 10( - / - ) mice with Campylobacter jejuni strains is correlated with genome content of open reading frames up - and down - regulated in vivo. Microbial Pathogenesis 2013, 54: 1 - 19. 45. Malik A, Sharma D, St Charles J, Dybas L, Mansfield L. Contrasting immune responses mediate Campylobacter jejuni - induced colitis and autoimmunity. Mucosal immunology 2013. 46. Edwards LA, Nistala K, Mills DC, Stephenson HN, Zilbauer M, Wren BW , et al. Del ineation of the Innate and Adaptive T - Cell Immune Outcome in the Human Host in Response to Campylobacter jejuni Infection. PLoS ONE 2010, 5 (11) : e15398. 47. Black RE, Levine MM, Clements ML, Hughes TP, Blaser MJ. Experimental Campylobacte r jejuni infection in humans. Journal of infectious diseases 1988, 157 (3) : 472 - 479. 48. Reinhardt RL, Liang H - E, Locksley RM. Cytokine - secreting follicular T cells shape the antibody repertoire. Nat Immunol 2009, 10 (4) : 385 - 393. 49. Stavnezer J. Immunogl obulin class switching. Current Opinion in Immunology 1996, 8 (2) : 199 - 205. 50. Deenick EK, Hasbold J, Hodgkin PD. Decision criteria for resolving isotype switching conflicts by B cells. European Journal of Immunology 2005, 35 (10) : 2949 - 2955. 51. Bai Y, L iu R, Huang D, La Cava A, Tang Y - y, Iwakura Y , et al. CCL2 recruitment of IL - 6 - producing CD11b+ monocytes to the draining lymph nodes during the initiation of Th17 - dependent B cell - mediated autoimmunity. European Journal of Immunology 2008, 38 (7) : 1877 - 188 8. 52. Pène J, Gauchat J - F, Lécart S, Drouet E, Guglielmi P, Boulay V , et al. Cutting Edge: IL - 21 Is a Switch Factor for the Production of IgG1 and IgG3 by Human B Cells. The Journal of Immunology 2004, 172 (9) : 5154 - 5157. 53. Satoh - Takayama N, Dumoutier L, Lesjean - Pottier S, Ribeiro VSG, Mandelboim O, Renauld JC , et al. The Natural Cytotoxicity Receptor NKp46 Is Dispensable for IL - 22 - Mediated Innate Intestinal Immune Defense against Citrobacter rodentium. The Journal of Immunology 2009, 183 (10) : 6579 - 6587 . 31 54. Vonarbourg C, Mortha A, Bui VL, Hernandez PP, Kiss EA, Hoyler T , et al. Regulated Expression of Nuclear Receptor ROR³t Confers Distinct Functional Fates to NK Cell Receptor - Expressing ROR³t+ Innate Lymphocytes. Immunity 2010, 33 (5) : 736 - 751. 55. Sa toh - Takayama N, Lesjean - Pottier S, Vieira P, Sawa S, Eberl G, Vosshenrich CAJ , et al. IL - 7 and IL - - dependent precursors. The Journal of Experimental Medicine 2010, 2 07 (2) : 273 - 280. 56. Diefenbach A, Vonarbourg C. Innate lymphocytes induce inflammatory bowel disease. Immunology and Cell Biology 2010, 88 (7) : 694 - 696. 57. Spits H, Di Santo JP. The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodeling. Nature Immunology 2010, 12 (1) : 21 - 27. 58. Sonnenberg GF, Monticelli LA, Elloso MM, Fouser LA, Artis D. CD4+ Lymphoid Tissue - In ducer Cells Promote Innate Immunity in the Gut. Immunity 2011, 34 (1) : 122 - 134. 59. Buonocore S, Ahern PP, Uhlig HH, Ivanov II, Littman DR, Maloy KJ , et al. Innate lymphoid cells drive interleukin - 23 - dependent innate intestinal pathology. Nature 2010, 464 (7293) : 1371 - 1375. 60. Moro K, Yamada T, Tanabe M, Takeuchi T, Ikawa T, Kawamoto H , et al. Innate production of TH2 cytokines by adipose tissue - associated c - Kit+Sca - 1+ lymphoid cells. Nature 2010, 463 (7280) : 540 - 544. 61. Thiennimitr P, Winter SE, Winter MG, Xavier MN, Tolstikov V, Huseby DL , et al. Intestinal inflammation allows Salmonella to use ethanolamine to compete with the microbiota. Proceedings of the National Academy of Sciences 2011, 108 (42) : 17480 - 17485. 62. Stecher B, Robbiani R, Walker AW, W estendorf AM, Barthel M, Kremer M , et al. Salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota. PLoS biology 2007, 5 (10) : e244. 63. CDC. Gullian Barre Syndrome. http://wwwcdcgov/h1n1flu/vaccination/factsheet_gbshtm 2013. 64. Anand BABP, Nimisha NK. Guillain - Barré syndrome (GBS). Pharmacol Rep 2010, 62 (2) : 220 - 232. 65. Garssen M, Van Koningsveld R, Van Doorn P. Residual fatigue is indep endent of antecedent events and disease severity in Guillain - Barré syndrome. Journal of neurology 2006, 253 (9) : 1143 - 1146. 32 66. Hughes RA, Cornblath DR. Guillain - Barre syndrome. Lancet 2005, 366 (9497) : 1653 - 1666. 67. Vucic S, Kiernan MC, Cornblath DR. Gui llain - Barré syndrome: An update. Journal of Clinical Neuroscience 2009, 16 (6) : 733 - 741. 68. Griffin J, Li C, Macko C, Ho T, Hsieh S - T, Xue P , et al. Early nodal changes in the acute motor axonal neuropathy pattern of the Guillain - Barré syndrome. Journal o f neurocytology 1996, 25 (1) : 33 - 51. 69. Griffin JW, Li CY, Ho TW, Tian M, Gao CY, Xue P , et al. Pathology of the motor - sensory axonal Guillain - Barré syndrome. Annals of Neurology 1996, 39 (1) : 17 - 28. 70. Hadden R, Cornblath D, Hughes R, Zielasek J, Hartung HP, Toyka K , et al. Electrophysiological Annals of neurology 1998, 44 (5) : 780 - 788. 71. McKhann G, Cornblath D, Griffin J, Ho T, Li C, Jiang Z , et al. Acute motor axonal neuropathy: a frequent cause of acute flaccid paralysis in China. Annals of neurology 1993, 33 (4) : 333 - 342. 72. Paradiso G, Tripoli J, Galicchio S, Fejerman N. Epidemiological, clinical, and electrodiagnostic findings in childhood Guil Annals of neurology 1999, 46 (5) : 701 - 707. 73. Fisher M. An unusual variant of acute idiopathic polyneuritis (syndrome of ophthalmoplegia, ataxia and areflexia). New England Journal of Medicine 1956, 255 (2) : 57 - 65. 74. Overell JR, Willison HJ. Recent developments in Miller Fisher syndrome and related disorders. Current opinion in neurology 2005, 18 (5) : 562 - 566. 75. Mori M, Kuwabara S, Fukutake T, Yuki N, Hattori T. Clinical features and prognosis of Miller Fisher syndro me. Neurology 2001, 56 (8) : 1104 - 1106. 76. Hadden R, Karch H, Hartung H - P, Zielasek J, Weissbrich B, Schubert J , et al. Preceding infections, immune factors, and outcome in Guillain Barré syndrome. Neurology 2001, 56 (6) : 758 - 765. 77. Zhang M, Li Q, He L, Meng F, Gu Y, Zheng M , et al. Association study between an outbreak of Guillain - Barre syndrome in Jilin, China, and preceding Campylobacter jejuni infection. Foodborne Pathogens and Disease 2010, 7 (8) : 913 - 919. 33 78. Kalra V, Chaudhry R, Dua T, Dhawan B, Sa hu JK, Mridula B. Association of Campylobacter jejuni Infection with Childhood Guillain - Barré Syndrome: A Case - Control Study. Journal of Child Neurology 2009, 24 (6) : 664 - 668. 79. Yu R, Ariga T, Usuki S, Kaida K - i. Pathological Roles of Ganglioside Mimicry in Guillain Barré Syndrome and Related Neuropathies. In: Wu AM (ed). The Molecular Immunology of Complex Carbohydrates - 3 , vol. 705. Springer US, 2011, pp 349 - 365. 80. Salmon DA, Halsey NA. Editorial Commentary: Guillain - Barré Syndrome and Vaccinations. C linical Infectious Diseases 2013, 57 (2) : 205 - 207. 81. Aspinall GO, Fujimoto S, McDonald AG, Pang H, Kurjanczyk LA, Penner JL. Lipopolysaccharides from Campylobacter jejuni associated with Guillain - Barré syndrome patients mimic human gangliosides in struct ure. Infection and Immunity 1994, 62 (5) : 2122 - 2125. 82. Chang Y - C, Nizet V. The interplay between Siglecs and sialylated pathogens. Glycobiology 2014, 24 (9) : 818 - 825. 83. Godschalk PC, Kuijf ML, Li J, Michael FS, Ang CW, Jacobs BC , et al. Structural char acterization of Campylobacter jejuni lipooligosaccharide outer cores associated with Guillain - Barre and Miller Fisher syndromes. Infection and immunity 2007, 75 (3) : 1245 - 1254. 84. , et al. Acute motor axonal Annals of neurology 1996, 40 (4) : 635 - 644. 85. , et al. Immune attack on the Schwann cell surface in acute inflammatory demy elinating polyneuropathy. Annals of neurology 1996, 39 (5) : 625 - 635. 86. Goodfellow JA, Bowes T, Sheikh K, Odaka M, Halstead SK, Humphreys PD , et al. Overexpression of GD1a ganglioside sensitizes motor nerve terminals to anti - GD1a antibody - mediated injury in a model of acute motor axonal neuropathy. The Journal of neuroscience 2005, 25 (7) : 1620 - 1628. 87. Gude DR, Alvarez SE, Paugh SW, Mitra P, Yu J, G riffiths R , et al. Apoptosis induces expression of sphingosine kinase 1 to release sphingosine - 1 - - and - get - The FASEB Journal 2008, 22 (8) : 2629 - 2638. 34 88. Lauber K, Bohn E, Kröber SM, Xiao Y - j, Blumenthal SG, Lindemann RK , et al. Apoptotic cells induce migration of phagocytes via caspase - 3 - mediated release of a lipid attraction signal. Cell 2003, 113 (6) : 717 - 730. 89. Koga M, Yuki N, Hirata K, Morimatsu M, Mori M, Kuwabara S. Anti - GM1 antibody IgG subclass A clinical recovery p redictor in Guillain Barré syndrome. Neurology 2003, 60 (9) : 1514 - 1518. 90. l and immunohistochemical studies. Neurology 1993, 43 (10) : 1911 - 1911. 91. Kaida K, Ariga T, Yu RK. Antiganglioside antibodies and their pathophysiological effects on Guillain Barré syndrome and related disorders A review. Glycobiology 2009, 19 (7) : 676 - 692 . 92. Jefferis R, Kumararatne DS. Selective IgG subclass deficiency: quantification and clinical relevance. Clinical & Experimental Immunology 1990, 81 (3) : 357 - 367. 93. Rengarajan J, Szabo SJ, Glimcher LH. Transcriptional regulation of Th1/Th2 polarizati on. Immunology today 2000, 21 (10) : 479 - 483. 94. Parker CT, Gilbert M, Yuki N, Endtz HP, Mandrell RE. Characterization of lipooligosaccharide - biosynthetic loci of Campylobacter jejuni reveals new lipooligosaccharide classes: evidence of mosaic organization s. J Bacteriol 2008, 190 (16) : 5681 - 5689. 95. Godschalk PC, Heikema AP, Gilbert M, Komagamine T, Ang CW, Glerum J , et al. The crucial role of Campylobacter jejuni genes in anti - ganglioside antibody induction in Guillain - Barré syndrome. Journal of Clinical Investigation 2004, 114 (11) : 1659 - 1665. 96. Islam Z, van Belkum A, Wagenaar JA, Cody AJ, de Boer AG, Tabor H , et al. Comparative genotyping of Campylobacter jejuni strains from patients with Guillain - Barré syndrome in Bangladesh. PloS one 2009, 4 (9) : e725 7. 97. Nachamkin I, Liu J, Li M, Ung H, Moran AP, Prendergast MM , et al. Campylobacter jejuni from patients with Guillain - Barré syndrome preferentially expresses a GD1a - like epitope. Infection and Immunity 2002, 70 (9) : 5299 - 5303. 98. Gilbert M, Karwaski M - F, Bernatchez S, Young NM, Taboada E, Michniewicz J , et al. The genetic bases for the variation in the lipo - oligosaccharide of the mucosal pathogen, Campylobacter jejuni Biosynthesis of sialylated ganglioside mimics in the core oligosaccharide. Journal of Biological Chemistry 2002, 277 (1) : 327 - 337. 35 99. Heikema AP, Koning RI, Duarte dos Santos Rico S, Rempel H, Jacobs BC, Endtz HP , et al. Enhanced, Sialoadhesin - Dependent Uptake of Guillain - Barré Syndrome - Associated Campylobacter jejuni Strains by Human M acrophages. Infection and Immunity 2013, 81 (6) : 2095 - 2103. 100. Louwen R, Horst - Kreft D, de Boer AG, van der Graaf L, de Knegt G, Hamersma M , et al. A novel link between Campylobacter jejuni bacteriophage defence, virulence and Guillain Barré syndrome. Eu r J Clin Microbiol Infect Dis 2013, 32 (2) : 207 - 226. 101. Hughes R, Raphaël JC, Swan AV, van Doorn PA. Intravenous immunoglobulin for Guillain - Barré syndrome. Cochrane Database Syst Rev 2006, 1 . 102. Crocker PR, Paulson JC, Varki A. Siglecs and their role s in the immune system. Nature Reviews Immunology 2007, 7 (4) : 255 - 266. 103. Crocker PR. Siglecs in innate immunity. Current opinion in pharmacology 2005, 5 (4) : 431 - 437. 104. Avril T, Floyd H, Lopez F, Vivier E, Crocker PR. The Membrane - Proximal Immunorec eptor Tyrosine - Based Inhibitory Motif Is Critical for the Inhibitory Signaling Mediated by Siglecs - 7 and - 9, CD33 - Related Siglecs Expressed on Human Monocytes and NK Cells. The Journal of Immunology 2004, 173 (11) : 6841 - 6849. 105. Klaas M, Crocker PR. Sial oadhesin in recognition of self and non - self. Seminars in immunopathology; 2012: Springer; 2012. p. 353 - 364. 106. Hiemstra IH, Beijer MR, Veninga H, Vrijland K, Borg EG, Olivier BJ , et al. The identification and developmental requirements of colonic CD16 9(+) macrophages. Immunology 2014, 142 (2) : 269 - 278. 107. Zou Z, Chastain A, Moir S, Ford J, Trandem K, Martinelli E , et al. Siglecs Facilitate HIV - 1 Infection of Macrophages through Adhesion with Viral Sialic Acids. PLoS ONE 2011, 6 (9) : e24559. 108. Izqu ierdo - Useros N, Lorizate M, Puertas MC, Rodriguez - Plata MT, Zangger N, Erikson E , et al. Siglec - 1 Is a Novel Dendritic Cell Receptor That Mediates HIV - 1 Trans - Infection Through Recognition of Viral Membrane Gangliosides. PLoS Biol 2012, 10 (12) : e1001448. 109. Huizinga R, Easton AS, Donachie AM, Guthrie J, van Rijs W, Heikema A , et al. Sialylation of Campylobacter jejuni Lipo - Oligosaccharides: Impact on Phagocytosis and Cytokine Production in Mice. PLoS ONE 2012, 7 (3) : e344 16. 36 110. Klaas M, Oetke C, Lewis LE, Erwig LP, Heikema AP, Easton A , et al. Sialoadhesin Promotes Rapid Proinflammatory and Type I IFN Responses to a Sialylated Pathogen, Campylobacter jejuni. The Journal of Immunology 2012, 189 (5) : 2414 - 2422. 111. Chang Y - C, Olson J, Louie A, Crocker P, Varki A, Nizet V. Role of macrophage sialoadhesin in host defense against the sialylated pathogen group B Streptococcus. J Mol Med 2014, 92 (9) : 951 - 959. 112. Xiong Y - S, Cheng Y, Lin Q - S, Wu A - L, Yu J, Li C , et al. I ncreased expression of Siglec - 1 on peripheral blood monocytes and its role in mononuclear cell reactivity to autoantigen in rheumatoid arthritis. Rheumatology 2014, 53 (2) : 250 - 259. 113. Wu C, Rauch U, Korpos E, Song J, Loser K, Crocker PR , et al. Sialoadhesin - positive macrophages bind regulatory T cells, negatively controlling their expansion and autoimmune disease progression. The Journal of Immunology 2009, 182 (10) : 6508 - 6516. 114. Xiong Y - s, Wu A - l, Lin Q - s, Yu J, Li C, Zhu L , et al. Contribut ion of monocytes Siglec - 1 in stimulating T cells proliferation and activation in atherosclerosis. Atherosclerosis 2012, 224 (1) : 58 - 65. 115. Kawasaki N, Vela JL, Nycholat CM, Rademacher C, Khurana A, van Rooijen N , et al. Targeted delivery of lipid antigen to macrophages via the CD169/sialoadhesin endocytic pathway induces robust invariant natural killer T cell activation. Proceedings of the National Academy of Sciences 2013, 110 (19) : 7826 - 7831. 116. Kawasaki N, Rillahan CD, Cheng T - Y, Van Rhijn I, Macaule y MS, Moody DB , et al. Targeted Delivery of Mycobacterial Antigens to Human Dendritic Cells via Siglec - 7 Induces Robust T Cell Activation. The Journal of Immunology 2014, 193 (4) : 1560 - 1566. 117. Heikema AP, Jacobs BC, Horst - Kreft D, Huizinga R, Kuijf ML, Endtz HP , et al. Siglec - 7 specifically recognizes Campylobacter jejuni strains associated with oculomotor weakness in Guillain Barré syndrome and Miller Fisher syndrome. Clinical Microbiology and Infection 2012 : n/a - n/a. 37 CHAPTER 2 Contrasting immune responses mediate Campylobacter jejuni induced colitis and autoimmunity publishing group, (2014) 7, 802 817; doi:10.1038/mi.2013.97 Contributing authors: Ankit Malik, Deepika Sharma, Jessica St. Charles, Leslie A. Dybas and Linda S. Mansfield . 38 ABSTRACT Campylobacter jejuni is a leading cause of foodborne enteritis that has been linked to the autoimmune neuropathy, Guillain Barré Syndrome(GBS) . C57BL/6 IL - 10 +/+ and congenic IL - 10 mice serve as C. jejuni colonization and colitis models, respectively, but a mouse model for GBS is lacking. We demonstrate that IL - 10 - / - m ice infected with a C. jejuni colitogenic human isolate had significantly upregulated Type1 and 17 but not Type2 cytokines in the colon coincident with infiltration of phagoc and T cells participated in IFN - , IL - 17 and IL - 22 upregulation but in a time - and organ - specific manner. T cells were however necessary for colitis as mice depleted of Thy - 1 + cells were protected w hile neither Rag1 - / - nor IL - 10R blocked Rag1 - / - mice developed colitis after infection. Depleting IFN - , IL - 17 or both significantly ameliorated colitis and drove colonic responses towards Type2 cytokine and antibody induction. In contrast, C. jejuni GBS p atient strains induced mild colitis associated with blunted Type1/17 but enhanced Type2 responses. Moreover, the Type2 but not Type1/17 antibodies cross - reacted with peripheral nerve gangliosides demonstrating autoimmunity. 39 INTRODUCTION Campylobacter jejuni is a spiral, gram - negative microaerophilic bacterium that is the second most common cause of gastroenteritis in the United States with over 2.4 million campylobacteriosis cases reported annually and many sporadic cases unreported 1 . The majority of patients ingesting C. jejuni in raw/undercooked meat and unpasteurized milk develop mild to severe gastroenteritis targeting the colon, which is debilitating but self - limiting within 7 to 10 days 2 , 3 . Histopathological manifestations include colonic crypt distortion, crypt abscesses, mucin depletion, edema of the colonic lamina propria (c LP) and significant infiltration of granulocytes and mononuclear cells 4 . Lesions resolve in most patients, but cam pylobacteriosis can be life threatening in immune - compromised individuals with systemic spread and multi - organ damage 5 , 6 . Furthermore, infection with C. jejuni has been linked with serious autoimmune sequelae such as development or flare - up of Inflammatory Bowel Diseases 7 , Irritable Bowel Syndrome 8 9 and Guillain Barré Syndrome (GBS) 10 . Campylobacter jejuni infection is the most common predisposing factor for developing the peripheral neuropathy GBS with 40% of US cases triggered by this bacterium 11 , 12 . Recently, the GBS disease burden was estimated at 3000 to 6000 cases per year 13 . GBS syndrome consists of at least three different subtypes including acute inflammatory demyelinating polyradiculoneuropthy (AID P), acute motor axonal neuropathy (AMAN) and acute motor and sensory axonal neuropathy (AMSAN). AMAN and AMSAN are axonal subtypes associated with development of autoantibodies that target gangliosides on peripheral nerves; these autoantibodies are thought to result from molecular mimicry 10 . Indeed, the lipooligosaccharide (LOS) o f C. jejuni isolates from GBS patients with antecedent infections have been shown to mimic gangliosides on peripheral nerves including GM1, GD1a and others 10 , 14 , 15 . When bound to peripheral nerv es, these antibodies are expected to block nerve conduction by activation of complement and/or by cellular mechanisms 16 . At present, plasmapheresis and Intravenous 40 Immunoglobulin (IVIg) treatment are the only known treatments with beneficial effect, but are effective in only 60% of GBS patients 17 . Little is known about host immunological mechanisms that lead to self - limiting gastrointestinal (GI) disease versus severe enteritis or neurological sequelae. Our rationale was to utilize inbred mice deficient in IL - 10 to study factors mediating the development of C. jejuni - induced enteritis and autoimmune manifestations. In nate and adaptive immune cells, epithelial cells and fibroblasts can produce IL - 10. It functions to desensitize - inflammatory cytokine production and suppress proliferation of mononuclear cells 18 , 19 . Genome wide association analysis studies discovered Single N ucleotide Polymorphisms (SNPs) flanking the il10 gene as the most significant locus outside the MHC locus to associate with Ulcerative Colitis, a form of IBD affecting 8 - 24/10,000 individuals in the US and Europe. SNPs in IL10 also show a significant form of IBD with a similar incidence 20 . We have previously establis hed wild type (IL - 10 +/+ ) and IL - 10 - / - mice of various genetic backgrounds as models of C. jejuni colonization and colitis respectively 21 , 22 . While the IL - 10 +/+ mice of C57BL/6, C3H/HeJ and NOD background were stably colonized with C. jejuni (strain NCTC11168) for 35 days post oral inoculation without any adverse clinical or histopathological effects, the IL - 10 - / - mice of these three genetic backgrounds developed typhlocolitis (inflammation of cecum and colon) 22 . Thus, the enteritis model of oral inoculation of IL - 10 - / - mice with C. jejuni essentially involves combining the most strongly associated pathway for susceptibility to IBD ( IL 10) with the most common causative bacterium for coli tis ( Campylobacter jejuni ) through the natural route of infection. The histopathological features of colitis in IL - 10 - / - mice also replicate the histopathological features of C. jejuni associated colitis in humans 4 , 21 , including C. jejuni invasion of the colonic epithelium followed by ulceration, necrosis and neutrophilic exudates, infiltration of mononuclear and polymorphonuclear cells into the 41 colonic lamina propria and occasionally the muscularis, and crypt distension with abscesses and edema most prominent in the submucosa. These effects were dose independent as the dose range of 10 2 10 10 CFU/mouse produced similar levels of pathology 21 , 23 . Furthermore, C57BL/6 IL - 10 - / - mice inoculated with C. jejuni strains obtained from human GBS patients were colonized, but developed little or no colitis 24 . Recent studies have revealed the importance of diet 25 , Pattern Recognition Receptors (TLR 2, 4 and 9) 26 and particular signaling molecules (NF B, mTOR, PI3K - ) 27 , 28 in C. jejuni colonization and induced pathology in wild type or gnotobiotic IL - 10 - / - mouse models. However, the role of inflammatory medi ators - particularly lymphocytes and their secreted cytokines - has not been established in vivo . We hypothesized that differential cytokine responses mediated by lymphocytes in the colon are responsible for C. jejuni induced colitis, protection from colitis a nd initiation of autoimmune sequelae in the IL - 10 - / - murine host. In humans, autoreactive IgG1 is the commonly associated antibody subtype after C. jejuni infection and enhanced IgG1 titers also associate with enhanced severity and a poor long term prognos is for GBS cases 29 . Because IgG1 isotype classically req uires T H 2 mediated class switching, we further hypothesized that a C. jejuni specific T H 2 response generated by the GBS but not the colitogenic strains will lead to induction of autoreactive IgG1. 42 RESULTS C. jejuni induced a mixed Type1 and Type17 cytokine and cellular response in IL - 10 - / - mice All mice infected with C. jejuni strain 11168 were colonized, with 40 - 90% of them exhibiting premature mortality 24 , 25 . Furthermore, colon histopathology scores were significantly enhanced compared to t he T SB inoculated controls 24 , 25 . To assess the type of inflammatory reaction(s) C. jejuni induces to cause colitis, C57BL/6 IL - 10 - / - (IL - 10 - / - ) mice were orally infected with 10 9 CFU of C. jejuni 11168 or Trypticase Soy Broth (TSB sham) and observed for clinical signs of enteritis. Mice were euthanized upon showing a severe enteric disease endpoint or at day 35 - post inoculation. Tissues were collected during necropsy and analyzed simultaneously. When colon protein extracts were analyzed, prim ary/innate cytokines (IL - 1 and IL - 6), Type1 (IFN - ) and Type17 (IL - 17A and IL - 22), but not type 2 (IL - 4) cytokines were significantly increased in the infected group ( Figure 2. 1A ). Similar to the colon, plasma IFN - , IL - 17A and IL - 6 were increased significantly in the infected group ( Figure 2. 1C ), while other cytokine were not detectable. The numbers of macrophages (F4/80 + ) and T cells (CD3 + ) infiltrating the lamina propria as quantified by IHC were significantly increased in the infected group ( Fig ure 2. 1B ). These data suggest that C. jejuni infection induces a mixed Type1 and Type17 response in infected IL - 10 - / - mice. Furthermore, there were consistently high correlations between histopathology score and colonic IFN - , IL - 1 , IL - 6, IL - 17A and IL - 22 levels (Spearman rank correlation factor, r s =0.7 - 0.9, Suppl. Table 1 ). IgG subtype class switching in activated B cells is modulated by Type1, Type2 and Type17 cytokines. It has been long established that IFN - induces isotype clas s switching to IgG2a (or IgG2c, an isoform of IgG2a in C57BL/6 background) and IgG3, while IL - 4 primarily induces IgG1 class switching 30 - 32 . IL - 17 was shown to be necessary for autoreactive IgG2b (but not IgG1 or IgG2a) antibodies in an experimental autoimmune myasthenia gravis model 33 . Therefore, 43 antibody responses can inform the nature of C. jejuni specific cytokine responses. Consistent with the upregulation of a Type1 and Type17 but not Type2 response by colon and plasma cytokine analysis, plasma levels of C. jejuni specific IgG2b (published previously 24 ), IgG2c and IgG3 but not IgG1 were significantly enhanced in i nfected mice ( Figure 2. 1D ). Therefore, plasma IgG analysis reinforced our findings of a mixed Type1 and Type17 response, and also demonstrated the C. jejuni specificity of the response. We further analyzed the kinetics of cellular and cytokine changes in a kinetic study wherein mice were sacrificed at days 4, 7 and 11 post C. jejuni inoculation. Day 11 was chosen as the end point to avoid the need for premature euthanasia in the infected mice. Histopathology scores evaluated on hematoxylin and eosin stained sections of the ileocecocolic junctions increased with time, reaching significance at day 7 and 11 ( Figure 2. 2A) . On day 7, histological changes were moderate exhibiting slight epithelial hyperplasia and diffuse or patchy infiltration of mononuclear and polymorphonuclear cells in the colonic and cecal lamina propria that sometimes extended into the muscularis. On day 11 histological changes were more drastic with marked increase in mononuclear and polymorphonuclear cells in the lamina propria and the musc ularis. The epithelium was ulcerated along with crypt dysplasia and abscess formation. Neutrophilic exudates were also apparent in the lumen. The numbers of infiltrating neutrophils (CD11b hi Gr - 1 + ) and inflammatory myeloid cells (CD11b + MHCII + Gr - 1 - ) were significantly increased at the earliest time point examined (day 4) and continued to increase until the end of experiment (day 11), as assessed by flow cytometry (Figure 2. 2B) . C. jejuni specific IgG2b levels were increased significantly at days 7 and 11 p ost inoculation, while levels of IgG2c and IgG3 were found to be significantly enhanced on day 11. IgG1 levels (Type2 dependent) were not significantly different at any time point after inoculation ( Figure 2. 2C ) . Therefore, C. jejuni specific IgG responses corroborate Type1 and Type17 cytokine responses. Also, as ascertained by Q - PCR for C. jejuni specific gyrA in fecal DNA ( Figure 2. 2D ) the degree of colonization did 44 increase with time. However, it cannot be determined at this stage if increase in colonization is a cause of and/or a consequence of inflammation. Remarkably, levels of IFN - , IL - 17A and IL - 22 were increased in draining lymph nodes as early as day 4, and c ontinued to rise until day 11 ( Figure 2. 3A ). However, in the colon, an increase in IFN - day 7 and continued to rise at day 11. IL - 17, IL - 22, TNF - and MCP - 1 were also highest at day 11 in both organs ( Figure 2. 3B ). Thi s continuous increase in the levels of pro - inflammatory cytokines from colon and lymph nodes reflects the continuous increase in the number of colon infiltrating neutrophils and inflammatory myeloid cells. Both Innate Lymphocytes and T cells contribute t o Type1 (IFN - ) and Type17 (IL - 17 and IL - 22) cytokine production in an organ, time and cell type specific manner C. jejuni induced colitis is elicited upon infiltration of mononuclear cells and neutrophils in the colon, both in humans and IL - 10 - / - mice 4 , 21 . These mononuclear cells can belong to myeloid or lymphoid lineages. Lymphoid cells can further be categorized into adaptive lymphocytes B cells (CD3 - CD19 + ) and T cells (CD 3 + CD19 - ), and Rag - independent innate lymphocytes. Mucosal innate lymphocytes can further be divided into NK cells (CD19 - NKp46 + ) and relatively novel Innate Lymphoid Cells (ILCs) (Thy1 hi Lin - ) where Lin has the following markers (CD3 - CD19 - NKp46 - Gr - 1 - MHC - II - CD11b - producers of IFN - , IL - 17 and IL - 22 in a number of inflammatory diseases 34 - 36 . Therefore in a increase in production of IL - 17, IL - 22 and IFN - in the colo n and mesenteric lymph nodes (MLN). In both organs, there were significant increases in the absolute number of T cells (CD3 + CD19 - decreased to the level of controls by day 11 ( F igure 2. 4 A - C and 2. 5 A - C ). The maintenance of elevated numbers of T cells and NK cells and basal levels of ILCs was also confirmed in a 45 separate experiment at a later time point of day 21 (not shown). Within the T cell compartment, the proportion of CD4 + T H cells doubled in the colon by day 11, while the proportion of + T cells was unchanged during the course of the experiment ( Figure 2. 4A ). The proportion of the IFN - + cells was increased, in T H , T C , T and NK cell compartments on day 11 post infection in both organs ( Figure 2. 4A - B and 2. 5A - B ). The proportion of IL - 17 + (ILC17) increased as early as day 4 in the MLN ( Figure 2. 5C ), but decreased to control levels at day 7 while the proportion of IL - 17 + T H cells increased on days 7 and 11 ( Figure 2. 5A ). This pattern of IL - 17 production initially from ILCs and subsequently by T cells correlated with the kinetics of infiltration of these cells in the colon and MLN. In contrast, the proportion of ILC17 in the colon did not increase until day 11 (Figure 2. 4C) , at which time the total number of these cells had decreased to basal level. This demonstrates a selective organ specific activation of this cell type. Furthermore, increases in IL - 22 + T H cells and N K cells were observed at day 7 and/or 11 in both the colon and MLN ( Figure 2. 4A - B and 2. 5A - B ). Therefore, a time and cell type specific Type1 and Type17 response was observed in the colon and MLNs of C. jejuni challenged IL - 10 - / - mice. Thy - 1 + lymphocytes are necessary for C. jejuni mediated colitis Thy - 1 (CD90) is a cell surface marker expressed by both innate lymphocytes and T cells and consequently Thy - 1 depleting antibody was utilized to deplete these cells in the mouse model. Infected and Thy - 1 depleted IL - 10 - / - mice had significantly lower histopathology scores ( Figure 2. 6A ) as well as decreased numbers of neutrophils, inflammatory myeloid cells, T cells and DC infiltration of colonic tissues ( Figure 2. 6B ) as compared to mice that were inf ected but given control antibody injections (11168+CIgG). In fact, the infected and depleted group (11168+ - Thy - 1) had numbers similar to the uninfected group (TSB+ - Thy - 1). Out of ten mice in the positive control group (11168+CIgG group), two mice had to be euthanized early (day 9 and 21 46 p.i.) because they reached the threshold of clinical signs. Their tissues were collected during necropsy and appropriately fixed or frozen and analyzed alongside the other samples after the end of the experiment (Day 25 p.i.). Because T cells are necessary for IgG isotype class switching in B cells, Thy - 1 depleted and C. jejuni 11168 infected mice had lower levels of C. jejuni specifi c IgG2b, IgG2c and IgG3 in the plasma as compared to the 11168+CIgG group ( Figure 2. 6C ). Furthermore, colonic levels of IFN - , IL - 17 and IL - 22 were decreased to basal levels in Thy1 - depleted infected mice reinforcing that Thy1 + cells are the major producer s of these cytokines ( Figure 2. 6D ). The success of depleting Thy - 1 positive cells with antibody treatments was confirmed in the blood and colon by flow cytometry ( Figure S1B ). Innate immunity is insufficient to mediate severe C. jejuni induced colitis To assess if innate immunity is sufficient for C. jejuni mediated colitis, Rag1 - / - mice on the C57BL/6 background were challenged with C. jejuni alongside C57BL/6 wt mice that serve as a known positive colonization control. While all infected mice were colonized, neither group developed any clinical signs of colitis, and did not have differences in the histopathology scores ( Figure 2. 7A ) or the nu mbers of colonic neutrophils, inflammatory myeloid cells or dendritic cells (not shown) at day 32 - 35 p.i. as assessed by flow cytometry. To determine if IL - 10 was responsible for lack of inflammation in Rag1 - / - mice, they were given IL - 10R blocking antibod y after infection. Even after IL - 10R blocking, Rag1 - / - mice did not develop any clinical signs of colitis, and had no increase in histopathology scores ( Figure 2. 7B ) or numbers of phagocytes in the colon (not shown) as compared to TSB+ - IL - 10R or 11168+CIg G treated groups. C57BL/6 wt mice did develop C. jejuni specific antibody responses similar in nature to that of IL - 10 - / - mice with significant increases in the IgG2b, IgG2c and IgG3 isotypes and with no difference in IgG1, IgM or IgA ( Figure 2. 7C ), demons trating that the nature of C. jejuni antibody responses are independent of IL - 10. Taken together, these data, along with the requirement of Thy - 1 + cells to 47 produce colitis, show that T cells are necessary for severe C. jejuni induced colitis. C. jejuni col onization correlated with colitis induction in as much as the mouse genetic background that supports inflammation (IL - 10 - / - ) had higher colonization extent by the end of the experiment than the mouse genotypes/treatments that did not (BL/6wt or Rag1 - / - or Rag1 - / - IL - 10R blocked mice) ( Figure 2. 7D ). Both IFN - and IL - 17 participate in C. jejuni induced colitis To account for the relative contribution of IFN - and IL - 17 in C. jejuni mediated pathology, one or both were depleted during the course of the disease by intra - peritoneal injection of neutralizing antibodies. Results showed that both of these cytokines have a positive role in mediating colitis because depleting either IFN - o r IL - 17 or both led to significant decreases in histopathology scores ( Figure 2. 8A ) . Furthermore, d epleting IFN - or IL - 17 significantly decreased or trended towards decreasing the extent of colonic infiltration of neutrophils, inflammatory myeloid cells and T cells, while depleting both had a significant effect ( Figure 2. 8B nd the proportion of CD4 + T H and + T cells were unchanged between all groups (not shown). The number of NK cells, however decreased in the IFN - depleted groups, suggesting a positive feedback mechanism ( Figure 2. 8B ). Notably, depleting IFN - and IL - 17 p ushed the response towards Type2 cytokine and antibody induction. IFN - depleted groups had decreased levels of C. jejuni specific plasma IgG2c but increased plasma IgG1 ( Figure 2. 8C ) along with modestly increased levels of IL - 4 and IL - 13 in the proximal c olon ( Figure 2. 8D and not shown). IL - 17 depleted groups had decreased IgG2b and increased IgM ( Figure 2. 8C ). The double depleted group also had increased levels of C. jejuni - specific IgA ( Figure 2. 8C ). Thus, depleting IFN - and IL - 17 after infection with t he colitogenic 48 11168 prevented colitis and shifted the immune response towards Type2 cytokines and antibodies. Infection with GBS associated C. jejuni strains induces Type2 immunity that is protective for colitis but leads to autoimmunity . Having observed a Type2 response in 11168 infected IL - 10 - / - mice after IL - 17 and IFN - neutralization, we hypothesized that the non - colitogenic GBS strains, 260.94 and HB93 - 13, also induce a blunted Type1/17 but enhanced Type2 response in the IL - 10 - / - mouse. Inde ed, the GBS strains induced significantly blunted Type1/17 mediators (IFN - , IL - 17, IL - 22, IL - 6, T - bet) ( Figure 2. 9A and not shown ) locally in the colon and systemically in the plasma (not shown) when compared to the colitogenic strains 11168, CF93 - 6 (show n) and CG8421 (not shown). C. jejuni specific IgGb (published previously 24 ), IgG2c and IgG3 responses by the GBS strains were also trending towards a decrease as compared to the colitogenic strains (Figure 2. 9B ). In contrast, the GBS strains significantly enhanced the C. jejuni specif ic Type2 mediators in the colon (IL - 4, IL - 13, Gata - 3) (Figure 2. 9A) and antibody (IgG1) responses (Figure 2. 9B) in the plasma. Remarkably, Type2 but not Type1/17 autoantibodies were detected in plasma of C. jejuni GBS strain challenged mice. These IgG1 subclass autoantibodies reacted against the peripheral nerve gangliosides, GM1 and GD1a, in an ELISA format (Figure 2. 9C and Figure S2) . Molecular mimicry along with cytokine milieu was essential for autoimmunity bec ause only the GBS strains induced autoantibodies. Challenge infections with C. jejuni 11168 even in Type2 inducing conditions, i.e., after depletion of IFN - and IL - 17 (from Figure 8C), did lead to Type2 antibody induction against the C. jejuni antigen, bu t these antibodies failed to cross - react with the nerve ganglioside autoantigens (not shown). The GBS strains colonized stably to the same extent as the colitogenic 11168 strain at the early time point of day 4 (from Figure 2D), but had lower fecal coloniz ation at the later time points of day 11 (from Figure 2D) or day 16 ( Figure 49 2. 9D ). Therefore, 11168 has enhanced colonization prowess that may be related to its enhanced colitogenic ability. For the GBS strain HB93 - 13, all stably colonized mice (8 out of 1 0), but none of the uncolonized mice (2 out of 10) tested positive for autoantibodies against GD1a, suggesting stable colonization as essential for inducing significant autoantibody responses. The blunted Type1 responses elicited by the GBS strain HB93 - 13 were also confirmed ex vivo by measuring secreted cytokines from wild type mouse splenocytes challenged with colitogenic 11168 and CF93 - 6, or GBS associated HB93 - 13 and 260.94 strains using a gentamicin killing assay. 72 hours post challenge; GBS strains produced significantly less IFN - , TNF - , IL - 22 and IL - 10 than either of the two colitogenic strains, consistent with the in vivo data ( Figure 10A ). However, they induced similar IL - 12p40 and more IL - 6 which points towards differential innate immune activa tion and/or T cell maturation downstream of T H 1/T H 17/T H 2 pathways. To evaluate IL - 12p70 induction by the colitogenic/GBS strains, adherent splenocytes alone were challenged individually ( Figure 2. 10B ). Consistent with enhanced IFN - production by colitogenic strains, higher IL - 12p70 was produced by them when compared to the GBS strains. In contrast the GBS strains produced more IL - 6 and IL - 10. As i nvasion is likely an important feature of Campylobacter - mediated enteritis, epithelial invasion by gentamicin killing assay was performed using the young adult mouse epithelial cell line (YAMC) ( Figure 2. 10C ). While the non colitogenic GBS strain 260.94 invaded to a slightly lower extent as the colitogenic 11168, the other GBS strain HB93 - 13 invaded significantly more. Therefore, different outcomes to infection by colitogenic versus GBS strains cannot be explained by differences in epithelial cell invasion but rather by the Type1/17 or Type2 induction by individual strains. 50 DISCUSSION In thi s study, we show that C. jejuni 11168 a known colitogenic strain induced a mixed Type1 and 17 cytokine response in the colon and the draining mesenteric lymph nodes of the C57BL/6 IL - 10 - / - mouse colitis disease model. This is consistent with the observatio ns made by Edwards et al, that demonstrated the induction of Type1 and 17 responses from C. jejuni 11168H challenged human colon explants and peripheral blood derived cells ex vivo , along with a novel role for IL - 17 in reducing intracellular survival in in testinal epithelial cells 37 . In our study, these cytokines orchestrated infiltration of neutrophils, macrophages, T cells, NK c colonic mucosa and sub - mucosa that closely resembled lesions seen in patients with Campylobacter - 4, peaked at day 7 and decreased to background level b y day 11. Increases in T cells and NK cells did not reach significance until day 7 or 11 respectively. By day 4 post infection, the amount of secreted IFN - , IL - 17 and IL - - 3 fold, which indicated innate sources for these cytokines since this response is too early for adaptive immunity to arise. We also found that innate lymphocytes as well as T cells participated in the upregulation of IFN - , IL - 17 and IL - 22, but in a time and organ - specific manner. Mirroring the kin - 17 as early as day 4 in the lymph nodes that switched to an IFN - response by day 7 at which time IFN - + and IL - 17 + cells were also increased in the T H and cell compartments. Because only ad aptive immunity can be downregulation is a mechanism of ensuring an acute response to infection whilst preventing non - specific inflammatory responses that pred ispose to autoimmunity. However, in the colon, studies are needed to determine their functional discrepancy in the two organs. Neutrophil and inflammatory m yeloid cell numbers in the colon were enhanced significantly at day 4 and 51 continued to increase until the later time points (e.g. day 11) post infection. This was another reflection of the continuous significant increases in pro - inflammatory factors from t he colon and - responses. It should be recognized that although ILCs participated in C. jejuni colitis, the innate immune system was unable to induce full blown c olitis. Neither Rag1 - / - nor IL - 10R blocked Rag1 - / - mice developed colitis after C. jejuni inoculation. However, Thy - 1 + lymphocytes were necessary for the colitogenic response as significantly decreased pathology was observed in adaptive immune competent IL - 10 - / - mice that were depleted for Thy - 1 + cells. These findings demonstrated that T cells were necessary for C. jejuni to elicit colitis. In contrast, Jobin et al., have very recently shown that germ - free Rag2 IL - 10 double KO mice of the 129/SvEv backgroun d develop colitis after C. jejuni 11168 inoculation 28 . The difference in outcomes may be attributable to differences in the host genetics (C57BL/6 v/s 129/SvEv) and/or protective effect of host gut microbiota on degree of colonization and immune stimulation. Jobin et al have also recently shown that d epletion of CD4 + cells does not affect the pathology in C. jejuni infected gnotobiotic C57BL/6 IL - 10 - / - mice 27 . This observation is complicated by the fact that in addition to immature thymocytes and mature T H cells, CD4 is also expressed by macrophages and therefore CD4 depletion cannot be used as a specific tool for T H cell depletion. Nevertheless it does suggest a pro - inflammatory role of other lymphocytic populations in C. jejuni colitis and, in our model, we indeed observed the increase in numbers of IFN - + and IL - 17 + cells in non - T H cell compartments, including and CD4 - (CD8 + Lymphoid Tissue Inducer cells (Lin - Thy1 hi CD4 + ) and LTi - like cells (Lin - Thy - 1 hi Sca - 1 + ) that have been shown to perform both pro - and anti - inflammatory functions 38 - 40 . Early IL - 22 production Citrobacter rodentium infected BL/6 IL - 10 +/+ mice was shown to be protective 41 while the production of IFN - and IL - 17 fro m LTi - like cells in the colons of 52 Helicobacter hepaticus infected Rag.129 mice was shown to be pro - inflammatory 42 . Further studies will be necessary to determine the exact role of these subsets in C. jejuni mediated colon pathology. We have shown that enhanced IFN - and I L - 17 cytokine secretion induces lesions in C. jejuni induced colitis. Neutralizing either or both of these cytokines prevented the overt histopathological changes associated with the disease, including the infiltration of neutrophils, inflammatory myeloid cells and T cells into the colon. It was notable that neutralizing these cytokines shifted the immune response towards Type2 cytokines (IL - 4 and IL - 13) and antibody responses (IgG1), alongside increases in C. jejuni specific IgM. The non - colitogenic GBS st rains also induced similarly enhanced Type2 and blunted Type1/17 responses, suggesting that C. jejuni mediated colitis can be explained by the balance between Type1/17 and Type2 responses. This may also suggest the mechanism underlying the development of a utoantibodies and autoimmune diseases after treatment with TNF - blockers in humans 43 - 45 . It remains to be seen if blockin g the Type2 cytokines in the mouse model could lead to induction of colitis thus diverting the expected autoimmune outcomes after challenge with the non - colitogenic GBS patient strains. GBS, especially the AMAN form, has most commonly been associated with antecedent C. jejuni infection 17 . It has also been published that antecedent gastrointestinal symptoms are only observed in 50% of GBS patients that are serologically positive for C. jejuni 46 , 47 . This suggests that there is no clear cut association between extent of colitis and GBS in humans and at least half of the human GBS cases mirror our mouse model in that infection with GB S strains leads to autoantibodies without antecedent diarrhea. Furthermore, identical to what has been observed in human GBS patients, autoreactive antibodies exclusively of the IgG1 isotype were found in mice infected with the GBS strains but not the coli togenic strains. Molecular mimicry and not just the cytokine milieu is essential for autoantibody development because the colitogenic non - GBS 53 strain C. jejuni 11168 failed to induce autoantibodies even when the immune response was biased towards Type2 afte r depleting IFN - and IL - 17. T he non - colitogenic GBS strains were colonized to a similar extent as the colitogenic 11168 strain, but only at the early time point (day 4). Colonization with C. jejuni 11168 was higher at later time points (day 11/16). Therefore 11168 has enhanced colonization prowess that may be related to its enhanced colitogenic ability and Type1/17 over Type2 maturation. However there is still proof of strain - intrinsic but colonizat ion - independent bias of Type1/17 maturation from ex vivo experiments. Stable colonization was nevertheless necessary to develop autoantibodies because 2 of mice infected with the GBS strain HB93 - 13 strain that were not colonized failed to develop a signifi cant autoantibody level when compared to their 8 colonized group mates. Consistent with our in vivo findings, Bax et al have shown in vitro that LOS purified from GBS strains induced blunted T H 1 and enhanced T H 2 response when compared to LOS from non - GBS s trains 48 . The T H 2 response depended on the nature of sialylation of the LOS and correlated with bin ding to Sialoadhesin, a DC and macrophage cell surface receptor. The role of Sialoadhesin in C. jejuni phagocytosis and primary interferon induction has also been established 49 , 50 . These results suggest that surface modification of the C. jejuni GBS strains play an important role in development of autoimmu nity, but more work is needed to determine the relevant ligand - receptor and downstream interactions in this model. Future studies are underway to determine the pathological and histological consequences of developing these autoantibodies, and the innate im mune mechanism of differential T cell responses by colitogenic versus GBS strains. It is imperative to understand the host factors and bacterial ligands that are responsible for GBS and IBD in order to develop the needed novel therapeutic interventions for these conditions. 54 MATERIALS AND METHODS Mice. C57BL/6J wild type (WT), BL/6.129P2 - IL - 10 tm1Cgn / J (IL - 10 - / - ) and B6.129S7 - Rag1 tm1Mom /J (Rag1 - / - ) mice were purchased from The Jackson Laboratory (Bar Harbor, MA) and bred in a specific pathogen free breeding colony. Mice at 8 12 weeks of age were orally inoculated with Tryptone Soy Broth (TSB vehicle control) or 10 9 CFU of C. jejuni 11168 in 0.2ml TSB, as described previously 21 . All animal protocols were approved by the Michigan State University Institutional Animal Care & Use Committee. Colonization by C. jejuni was confirmed and Hematoxylin and eosin stained sections of the ileocecocolic junction were scored as described previously 21 . Antibodies for in - vivo neutralization . Anti - IFN - (XMG1.2), - IL - 10R (1B1.3A) and - Thy - 1 (30H12) was purchased from Bio - X - Cell, and - IL - 17 (ebioMM17F3) was purchased from eBiosciences. XMG1.2 and ebioMM17F3 injected with 500µg and 200µg i.p. respectively per mouse twice weekly, starting on the day of inoculation. - IL - 10R (1B1.3A) was injected with 1mg/mouse i.p. at the time of inocul ation, and weekly thereafter. - Thy - 1.2 (30H12) was injected with 250µg/mouse i.p. twice a week, starting 3 days before inoculation. Rat or mouse IgG (Jackson ImmunoResearch) were used as controls where appropriate. Immunohistochemical analysis of mononuc lear cells. 5 m serial sections were cut, deparaffinized in two changes of xylene and rehydrated in graded alcohol series. Slides were incubated in 1% H 2 0 2 in TBS and boiled for 15 minutes in citrate buffer (Vector laboratories) for CD3 or incubated at 37 C in Proteinase K (Genemed biotechnologies) for F4/80. Thereafter, sections were blocked for 1 3 hours with 1% BSA + 1.5% Goat serum in TBS and then incubated with respective primary antibody (1:500 for CD3 , 1:50 for F4/80) at 4 C overnight. 55 Slides wer e then stained with the Vectastain ABC kit (Vector laboratories) for CD3 or rat on instructions. Non - specific IgG (Vector laboratories) for CD3 or irrelevant isotype control (RTK4530, Biolegend) for F4/80 were used as negative controls. From each section, ten non overlapping 400x magnification fields were chosen randomly, photographed and positively staining cells were counted using the ImageJ cell counter (N.I.H., Bethesda, M D). Preparation of colonic lamina propria leukocytes. Lamina propria leukocytes were isolated as previously described 51 , 52 . Briefly, fo r removal of epithelial cells, the colon was washed; cut into small pieces, and then the pieces were incubated with calcium - and magnesium - free HBSS supplemented with 5% FBS and 5 mM EDTA (Sigma - Aldrich) at 100 rpm at 37°C for 30 min. The tissues were then incubated with RPMI 1640 containing 10% FBS and 0.5 mg/ml collagenase type IV and 0.5mg/ml DNaseI (Sigma - Aldrich) for 1 hour at 37°C with shaking at 150 rpm. The liberated cells were collected by passage through a 70 m nylon mesh. The isolated cells were separated on a 40/80% discontinuous Percoll gradient (GE Bioscience). Flow cytometry. The following monoclonal antibodies (eBiosciences or Biolegend) were used: anti - CD3 (clone 145 2C11), anti - CD4 (clone RM 4 - 5), CD8 (clone 53 - 67); anti - TCR (clone GL3), CD19 (clone 1D3); anti NKp46 (clone 29A1.4); anti - CD11b (clone M1/70), anti - Gr1 (clone - RB6 - 8C5), anti - CD90.2 (clone53 - 2.1) and anti - CD16/CD32 (clone 2.4G2). The cells were preincubated for 20 minutes with anti - CD16/CD32 to block Fc receptors then was hed and labeled with appropriate mixture of antibodies or isotype matched controls for 30 minutes, centrifuged at 650 g , and resuspended in FACS buffer. To exclude dead/dying leukocytes were gated according to forward and side scatter. For intracellular cyt okine staining, cells were restimulated for 4 hours with cell stimulation cocktail (eBioscience) and fixed and permeabilized 56 using fixation and permeabilization solution (eBioscience). All cells were analyzed on a LSRII flow cytometer (BD Biosciences) usin g FlowJo software (Tree Star). Enzyme - linked Immunosorbent Assay. All cytokines were measured according to the manufacturer's protocol (Ready - Set - Go ELISA kits, eBioscience). To prepare protein extracts from colon, flash frozen tissue was homogenized with 0.5 ml HBSS 1% Triton X - 100 with the mini protease inhibitor cocktail (Roche). The homogenates were centrifuged at 12,000 g for 30 min at 4 ° C, and the supernatants were collected for ELISA. For ex vivo culture, RBC - depleted MLN cells at 5X10 6 cells/ml or 0 .5cm of proximal colon in 0.5ml of RPMI 1640 supplemented with 10% FBS and 100µg/ml penicillin G, 10µg/ml of streptomycin and gentamicin and 5µg/ml amphotericin B were incubated at 37°C for 48 hours. Supernatants were clarified and stored at - 70 o C. C. jeju ni specific antibody ELISA has been described previously 21 . The following plasma dilutions were used: 1:10 for IgA, 1:50 for IgG1, IgG2c and IgG3, 1:100 for IgM and 1:400 for IgG2b. Only absorbance values more than 2 SD away from mean of negative control were considered positive. GM1 (Sigma) and GD1a (USBio) were u and handled similarly. Quantitative and reverse transcriptase PCR. RNA was extracted from flash frozen proximal colon samples using RNaeasy plus extraction kit (Qiagen). cDNA was subsequently synthesized by GoScript Rever se Transcriptase kit (Promega). Real - time PCR was performed for the target and hprt genes using Quantifast primers and probe assay (Qiagen) in ABI 7500 PCR machine (Applied Biosystems) or iQ5 iCycler (Bio - Rad). For C. jejuni DNA estimation in feces, DNA wa s extracted as described previously 21 . 50ng of fecal DNA was used in Q - PCR reaction with C. jejuni specific gyr A primers 53 and iQ SYBR green supermix. C. jejuni DNA in fecal DNA was estimated by running a parallel standard curve of pure C. jejuni DNA. 57 Splenocyte challenge by gentamicin killing assay. RBC deplet ed splenocytes from naïve C57BL/6 wt mouse (10 6 cells/ml) were plated in antibiotic free R10 medium and challenged with indicated C. jejuni strains at multiplicity of infection (M.O.I) of 0.1, 1 or 10. One hour after infection, gentamicin (250µg/ml) was added to all the wells to kill extracellular bacteria. Supernatants were collected after 72 hours for cytokine measurement by ELISA. To obta in adherent cells, splenocytes were plated at 10 7 cells/ml for 90 minutes upon which the non - adherent cells were washed off. Epithelial challenge by gentamicin killing assay. 1.5X10 6 Young Adult Mouse Colon (YAMC) cells were grown in permissive media (RP MI 1640 with 5% FBS, ITS and 5 IU/ml IFN - 2 . At 80% confluence media is changed to ITS and IFN - C. jejuni is then added at M.O.I of 100 followed by two ho ur incubation and three washed in PBS. For measuring invasion 0.1% Triton X - 100 and released bacteria is enumerated by serial dilution. For cytokine measurement cel ls are incubated for further 24 hours in media containing gentamicin and supernatant is clarified. Sensitivity of all strains to this concentration of gentamicin was also confirmed. Statistical analysis. All statistical tests were performed in Prism 6.0 (GraphPad Software) and described in figure legends. *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant. 58 APPENDIX 59 FIGURE LEGENDS Figure 2. 1 . IL - 10 - / - mice were inoculated with C. jejuni 11168 or TSB and euthanized at humane end point or 35 days post inoculation. A) Colon homogenate ELISA. IL - 12p70, TNF - and IL - 17F were only detected in colons of 20% of the mice and are not shown. B) Colon CD3 and F4/80 IHC, at the time of necropsy, the ileocecocolic junction (junction of ileum, cecum and colon) was fixed in formalin and embedded in paraffin. Its sections were stained for CD3 or F4/80 and the number of positively staining cells in the colonic lamina propria were quantified in 10 random hi gh - powered fields. C) Plasma cytokine ELISA. Other cytokines from Figure 1a were not detectable in majority of mice from infected/control group, and are not shown. D) Plasma C. jejuni specific antibody analysis. Anti C. jejuni IgM and IgA was detectable bu t did not change significantly and is not shown. Data represented is one of three independent experiments with 8 10 mice per group. Bar indicates the median, Mann Whitney U test. Figure 2. 2. IL - 10 - / - mice were inoculated with C. jejuni or TSB, and sacrif iced at indicated days post inoculation. At the time of necropsy, the ileocecocolic junction (junction of ileum, cecum and colon) was fixed in formalin and embedded in paraffin. A) H&E sections were scored in blinded fashion. B) Colon leukocytes were prepa red and analyzed for indicated populations by flow cytometry . Dead/dying cells were excluded on the basis of forward and side scatter. All cells were gated on CD19 - gate. C) Mice were bled at time of necropsy, and serum was analyzed for indicated antibody subtypes reactive against C. jejuni antigen. Anti - C. jejuni IgM and IgA was detectable but did not change significantly for any time point and is not shown. D) C. jejuni colonization was measured in the feces at the time of necropsy by Q - PCR. E) Representa tive photomicrographs from H&E stained proximal colon sections. Data is represented as mean + s.e.m; two independent experiments; 5 - 8 mice per group per time point. 60 Infected group for each time point was compared with control group pooled for each time point s by Kruskal - Figure 2. 3 . IL - 10 - / - mice were inoculated with C. jejuni (open circles) or TSB (filled circles) and euthanized at indicated day post inoculation. A) 5 mm of proximal colon or B) 2.5X10 6 RBC - depleted cells from MLN were incubated in 0.5ml tissue culture media for 48 hours. Supernatant media was clarified and analyzed for indicated cytokines, as well as IL - - 4, IL - 23 and IL - 13, which were not detectable. IL - 12p70 was detectable from M LN but not the colon. Data is represented as mean + s.e.m; two independent experiments; 5 - 8 mice per group per time point. Infected group for each time point was compared with control group pooled for each time points by Kruskal - est. Figure 2. 4 . IL - 10 - / - mice were inoculated with TSB or C. jejuni and sacrificed at indicated day post inoculation. Single cell suspension was prepared from colon. T cells, ILCs and NK cells were analyzed for IFN - and IL - 17 or IFN - and IL - 22 productio n by intracellular cytokine staining and flow cytometry following brief restimulation with PMA and ionomycin in presence of brefeldinA. Double positives were relatively rare and data presented here represents total positive. Dead/dying cells were excluded on the basis of forward and side scatter. Data is represented as mean + s.e.m; two independent experiments with 5 - 8 mice per group per time point. Infected group for each time point was compared with control group pooled for each time points by Kruskal - Wal - 17+ or IL - 22+ cells from CD3+CD4 - cells (which are likely CD8+) was not significantly different at any time point, and is not shown. Figure 2. 5 . IL - 10 - / - mice were inoculated with TSB or C. jejuni and sacrificed at indicated day post inoculation. Single cell suspension was prepared from MLN. T cells, ILCs and NK cells were analyzed for IFN - , IL - 17 or IL - 22 production by intracellular cytokine staining and flow 61 cytometry following brief restimulati on with PMA and ionomycin in presence of brefeldinA. Data is represented as mean + s.e.m; two independent experiments with 5 - 8 mice per group per time point. Infected group for each time point was compared with control group pooled for each time points by Mann Whitney U test. Proportion of IL - 17+ or IL - 22+ cells from CD3+CD4 - cells (which are likely CD8+) was not significantly different at any time point, and is not shown. Figure 2. 6. IL - 10 - / - mice were orally inoculated with C. jejuni or TSB and injected with - Thy - 1 or CIgG twice weekly starting 3 days before inoculation, and sacrificed at Day 23 - 24 post inoculation. At the time of necropsy, the ileocecocolic junction (junction of ileum, cecum and colon) was fixed in formalin and embed ded in paraffin. A) H&E sections were scored in blinded fashion. B) Colon leukocytes were prepared and analyzed for indicated populations by flow cytometry. C) Mice were bled at time of necropsy, and plasma was analyzed for indicated antibody subtypes reac tive against C. jejuni antigen. D) 5mm of proximal colon was washed and incubated in 0.5ml tissue culture media for 48 hours. Supernatant media was clarified and analyzed for indicated cytokines, as well as other cytokines from Figure.1, which were not sig nificantly different. Bar indicates the median, n=10 mice per group. Kruskal - Wallis test Figure 2. 7. A) C57 BL/6 wt or Rag1 - / - mice were orally inoculated with C. jejuni or TSB and sacrificed at Day 32 - 34 post inoculation. B) Rag1 - / - - IL - 10R or CIgG after infection. At the time of necropsy, the ileocecocolic junction (junction of ileum, cecum and colon) was fixed in formalin and embedded in paraffin. H&E sections were scored in blinded fashion. C) Mice were bled at time of necropsy, and plasma was analyzed for indicated antibody subtypes reactive against C. jejuni antigen. D) C. jejuni colonization was measured in the feces at the time of necropsy by Q - PCR. Data is represented as median with 9 - 10 mice pe r group. Kruskal - 62 Figure 2. 8. IL - 10 - / - mice were orally inoculated with C. jejuni and sacrificed at Day 21 - 22 post inoculation. IFN - , IL - 17 or both were neutralized by i.p. injection of neutralizing antibodies twice a week for three weeks, starting on the day of inoculation. A) H&E sections were scored in blinded fashion. B) Colon leukocytes were prepared and analyzed for indicated pop ulations by flow cytometry. C) Mice were bled at time of necropsy, and plasma was analyzed for indicated antibody subtypes reactive against C. jejuni antigen. D) Level of IL - 4 was measured in the proximal colon homogenate by ELISA. Data is represented as m edians or mean + s.e.m., with 10 mice per group. Kruskal - group are shown. Figure 2. 9. IL - 10 - / - mice were orally inoculated with given C. jejuni strains and sacrificed at Day 35 post inocul ation, or earlier if requiring euthanasia. A) Expression of indicated mediators in the colon by real time PCR. B) Antibody subtypes reactive against C. jejuni antigen. C) Peripheral nerve gangliosides autoantigens by ELISA. D) C. jejuni colonization was measured in the feces at the indicated days post inoculation for the GBS strains and the time of necropsy for all strains by Q - PCR. Data is represented as median or mean + s.e.m., with 10 mice per group. Kruskal - Wallis test followed by Dunn Figure 2. 10. Whole s plenocytes A) or adherent splenocytes B) from naïve C57BL/6 wt mouse added after one hour, and cultures were further incubated for 72 hours for whole or 24 hours for adherent splenocytes, upon which supernatants were clarified and analyzed for indicated cytokines by ELISA. IL - 17, IL - 4 and IL - 23 were not detectable with whole splenocytes wh ile IL - 12p70 was only detectable with adherent cell culture. C) Invasion and IL - 6 elicitation by the indicated strains was assessed on YAMC cells at M.O.I of 100. Data represents mean + s.e.m. of three wells and analyzed by two - way ANOVA for splenocytes or o ne way ANOVA for epithelial 63 or HB93 - 13 for epithelial cells are shown. One of three independent replicates is shown. 64 Figure 2. 1. Endpoint cytokine, antibody and colonic cellular infiltration analysis 65 Figure 2. 2. Temporal analysis of colon leukocyte and C. jejuni specific plasma antibody. 66 Figure 2. 3. Temporal cytokine analysis of colon and MLN. 67 Figure 2. 4. Colon cytokine - lymphocyte subset analysis 68 Figure 2. 5. Mesenteric Lymph Node cytokine - lymphocyte subset analysis s 69 Fi gure 2. 6. Role of Thy - 1 + lymphocytes in C. jejuni mediated colitis 70 Figure 2. 7. Innate immunity is insufficient to induce colitis after C. jejuni infection 71 Figure 2. 8. Both IFN - and IL - 17 are involved in C. jejuni mediated colitis and humoral responses . 72 Figure 2. 9. Strain dependent colitis and autoimmunity . 73 Figure 2. 10. Whole splenocyte and epithelial cell challenge by gentamicin killing assay. 74 REFERENCES 75 REFERENCES 1 CDC. Incidence of laboratory - confirmed bacterial and parasitic infections, and postdiarrheal hemolytic uremic syndrome (HUS), by year and pathogen,. http://www.cdc.g ov/foodnet/data/trends/tables/table2a - b.html#table - 2b . 1996 - 2011. (2012). 2 Dasti, J. I., Tareen, A. M., Lugert, R., Zautner, A. E. & Groß, U. Campylobacter jejuni: A brief overview on pathogenicity - associated factors and disease - mediating mechanisms. In ternational Journal of Medical Microbiology 300 , 205 - 211, doi:10.1016/j.ijmm.2009.07.002 (2010). 3 Butzler, J. P. Campylobacter, from obscurity to celebrity. Clinical Microbiology and Infection 10 , 868 - 876, doi:10.1111/j.1469 - 0691.2004.00983.x (2004). 4 van Spreeuwel, J. P. et al. Campylobacter colitis: histological immunohistochemical and ultrastructural findings. Gut 26 , 945 - 951, doi:10.1136/gut.26.9.945 (1985). 5 Blaser, M. J. Epidemiologic and clinical features of Campylobacter jejuni infections. Jou rnal of Infectious Diseases 176 , S103 - S105 (1997). 6 Tee, W. & Mijch, A. Campylobacter jejuni bacteremia in human immunodeficiency virus (HIV) - infected and non - HIV - infected patients: comparison of clinical features and review. Clinical infectious diseases 26 , 91 - 96 (1998). 7 Gradel, K. O. et al. Increased Short - and Long - Term Risk of Inflammatory Bowel Disease After Salmonella or Campylobacter Gastroenteritis. Gastroenterology 137 , 495 - 501, doi: http://dx.doi.org/10.1053/j.gastro.2009.04.001 (2009). 8 Qin, H. - Y. et al. Systematic review of animal models of post - infectious/post - inflammatory irritable bowel syndrome. J Gastroenterol 46 , 1 64 - 174, doi:10.1007/s00535 - 010 - 0321 - 6 (2011). 9 Garg, A. X., Pope, J. E., Thiessen - Philbrook, H., Clark, W. F. & Ouimet, J. Arthritis risk after acute bacterial gastroenteritis. Rheumatology 47 , 200 - 204 (2008). 10 Yuki, N. et al. Association of IgG anti - GD1a antibody with severe Guillain Barré syndrome. Muscle & Nerve 16 , 642 - 647, doi:10.1002/mus.880160610 (1993). 11 Vucic, S., Kiernan, M. C. & Cornblath, D. R. Guillain - Barré syndrome: An update. Journal of Clinical Neuroscience 16 , 733 - 741, doi:10.1016/ j.jocn.2008.08.033 (2009). 12 Hughes, R. A. & Cornblath, D. R. Guillain - Barre syndrome. Lancet 366 , 1653 - 1666, doi:10.1016/S0140 - 6736(05)67665 - 9 (2005). 13 CDC. Gullian Barre Syndrome. http://www.cdc.gov/flu/protect/vaccine/guillainbarre.htm (2012). 76 14 Jacobs, B. C. et al. Campylobacter jejuni infections and anti - GM1 antibodies in guillain - barré syndrome. Annals of Neurology 40 , 181 - 187, doi:10.1002/ana.410400209 (1996). 15 Islam, Z. et al. Guillain - Barré Syndrome - Related Campylobacter jejuni in Bangladesh: Ganglioside Mimicry and Cross - Reactive Antibodies. PLoS ONE 7 , e43976, doi:10.1371/journal.pone.0043976 (2012). 16 Goodfellow, J. A. et al. Overexpression of GD 1a ganglioside sensitizes motor nerve terminals to anti - GD1a antibody - mediated injury in a model of acute motor axonal neuropathy. The Journal of neuroscience 25 , 1620 - 1628 (2005). 17 Kieseier, B. C. et al. Advances in understanding and treatment of immun e mediated disorders of the peripheral nervous system. Muscle & Nerve 30 , 131 - 156 (2004). 18 Ouyang, W., Rutz, S., Crellin, N. K., Valdez, P. A. & Hymowitz, S. G. Regulation and Functions of the IL - 10 Family of Cytokines in Inflammation and Disease. Annua l Review of Immunology 29 , 71 - 109, doi:10.1146/annurev - immunol - 031210 - 101312 (2011). 19 Saraiva, M. & O'Garra, A. The regulation of IL - 10 production by immune cells. Nat Rev Immunol 10 , 170 - 181 (2010). 20 Franke, A. et al. Sequence variants in IL10, ARPC2 and multiple other loci contribute to ulcerative colitis susceptibility. Nat Genet 40 , 1319 - 1323, doi: http://www.nature.com/ng/journal/v40/n1 1/suppinfo/ng.221_S1.html (2008). 21 Mansfield, L. S. et al. C57BL/6 and Congenic Interleukin - 10 - Deficient Mice Can Serve as Models of Campylobacter jejuni Colonization and Enteritis. Infection and Immunity 75 , 1099 - 1115, doi:10.1128/iai.00833 - 06 (2006). 22 Mansfield, L. et al. Genetic background of IL - pathogen interactions with Campylobacter jejuni and influences disease phenotype. Microbial Pathogenesis 45 , 241 - 257, doi:10.1016/j.micpath.2008.05.010 (2008). 23 Mansfield, L. et al . Animal models of Campylobacter jejuni infections. Campylobacter, 3rd ed. ASM Press, Washington, DC , 367 - 379 (2008). 24 Bell, J. A. et al. Outcome of infection of C57BL/6 IL - 10( - / - ) mice with Campylobacter jejuni strains is correlated with genome content of open reading frames up - and down - regulated in vivo. Microbial Pathogenesis 54 , 1 - 19, doi:10.1016/j.micpath.2012.08.001 (2013). 25 Bell, J. A. et al. Multiple factors interact to produce responses resembling spectrum of human disease in Campylobacter j ejuni infected C57BL/6 IL - 10 - / - mice. BMC Microbiology 9 , 57, doi:10.1186/1471 - 2180 - 9 - 57 (2009). 26 Bereswill, S. et al. Gnotobiotic IL - - Colitis Following Campylobacter jejuni Infection. PLoS ONE 4 , e7413, doi:10.1371/journal.pone.0007413 (2009). 77 27 Sun, X., Threadgill, D. & Jobin, C. Campylobacter jejuni Induces Colitis Through Activation of Mammalian Target of Rapamycin Signaling. Gastroenterology , doi:10.1053/j.gastro.2011.09.042 (2012). 28 Sun, X., Liu, B., Sartor, R. B. & Jobin, C. Phosphatidylinositol 3 - Kinase - Promotes Campylobacter jejuni Induced Colitis through Neutrophil Recruitment in Mice. The Journal of Immunology 190 , 357 - 365 (2013). 29 Koga, M. et al. Anti - GM1 antibody IgG subclass A clinical recovery predictor in Guillain Barré syndrome. Neurology 60 , 1514 - 1518 (2003). 30 Reinhardt, R. L., Liang, H. - E. & Locksley, R. M. Cytokine - secreting follicular T cells shape the antibody repertoire. Nat Immunol 10 , 385 - 393, doi: http://www.nature.com/ni/journal/v10/n4/suppinfo/ni.1715_S1.html (2009). 31 Stavnezer, J. Immunoglobulin class switching. Current Opinion in Immunology 8 , 199 - 205, doi:10.1016/s0952 - 7915(96 )80058 - 6 (1996). 32 Deenick, E. K., Hasbold, J. & Hodgkin, P. D. Decision criteria for resolving isotype switching conflicts by B cells. European Journal of Immunology 35 , 2949 - 2955, doi:10.1002/eji.200425719 (2005). 33 Bai, Y. et al. CCL2 recruitment of IL - 6 - producing CD11b+ monocytes to the draining lymph nodes during the initiation of Th17 - dependent B cell - mediated autoimmunity. European Journal of Immunology 38 , 1877 - 1888, doi:10.1002/eji.200737973 (2008). 34 Bernink, J. H. et al. Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues. Nature Immunology (2013). 35 Sonnenberg, G. F. & Artis, D. Innate lymphoid cell interactions with microbiota: implications for intestinal health and disease. Immunity 37 , 601 - 610 (2012). 36 Diefenbach, A. & Vonarbourg, C. Innate lymphocytes induce inflammatory bowel disease. Immunology and Cell Biology 88 , 694 - 696, doi:10.1038/icb.2010.82 (2010). 37 Cua, D. J. & Tato, C. M. Innate IL - 17 - producing cells: the sentinels of the immun e system. Nature Reviews Immunology 10 , 479 - 489, doi:10.1038/nri2800 (2010). 38 Spits, H. & Di Santo, J. P. The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodeling. Nature Immunology 12 , 21 - 27, doi:10.1038 /ni.1962 (2010). 39 Cherrier, M., Ohnmacht, C., Cording, S. & Eberl, G. Development and function of intestinal innate lymphoid cells. Current Opinion in Immunology , doi:10.1016/j.coi.2012.03.011 (2010). 40 Sonnenberg, G. F., Monticelli, L. A., Elloso, M. M., Fouser, L. A. & Artis, D. CD4+ Lymphoid Tissue - Inducer Cells Promote Innate Immunity in the Gut. Immunity 34 , 122 - 134, doi:10.1016/j.immuni.2010.12.009 (2011). 41 Buonocore, S. et al. Innate lymphoid cells drive interleukin - 23 - dependent innate intest inal pathology. Nature 464 , 1371 - 1375 (2010). 78 42 Caramaschi, P. et al. Anti - TNFalpha blockers, autoantibodies and autoimmune diseases. Joint Bone Spine 76 , 333 - 342, doi: http://dx.doi.org/10.1016/j.jbspin.2008.12.008 (2009). 43 Lozeron P, D. C. L. C. A. D. Long - term course of demyelinating neuropathies occurring during tumor necrosis factor - blocker therapy. Arch Neurol 66 , 490 - 497, doi:10.1001/archneurol.2009.11 (2009). 44 Tristano, A. G. Neurological adverse events associated with anti - tumor necrosis factor alpha treatment. Journal of neurology 257 , 1421 - 1431 (2010). 45 Zhang, M. et al. Association study between an outbreak of Guillain - Barre syndrome in Jilin, China, and preceding Campylobacter jejuni infection. Foodborne Pathogens and Disease 7 , 913 - 919 (2010). 46 Kalra, V. et al. Association of Campylobacter jejuni Infection with Childhood Guillain - Barré Syndrome: A Case - Control Study. Journal of Child Neurology 24 , 664 - 668, doi:10.1177/0883073808325649 (2009). 47 Bax, M. et al. Campylobacter jejuni lipooligosaccharides modulate dendritic cell - mediated T cell polarization in a sialic acid linkage - dependent manner. Infection and Immunity 79 , 2681 - 2689 (2011). 48 Hui zinga, R. et al. Sialylation of Campylobacter jejuni Lipo - Oligosaccharides: Impact on Phagocytosis and Cytokine Production in Mice. PLoS ONE 7 , e34416, doi:10.1371/journal.pone.0034416 (2012). 49 Klaas, M. et al. Sialoadhesin Promotes Rapid Proinflammatory and Type I IFN Responses to a Sialylated Pathogen, Campylobacter jejuni. The Journal of Immunology 189 , 2414 - 2422, doi:10.4049/jimmunol.1200776 (2012). 50 Weigmann, B. et al. Isolation and subsequent analysis o f murine lamina propria mononuclear cells from colonic tissue. Nature Protocols 2 , 2307 - 2311, doi:10.1038/nprot.2007.315 (2007). 51 Sanos, S. L. et al. of mucosal interleukin 22 producing NKp46+ cells. Nature Immunology 10 , 83 - 91, doi:10.1038/ni.1684 (2008). 52 Wilson, D. L., Abner, S. R., Newman, T. C., Mansfield, L. S. & Linz, J. E. Identification of Ciprofloxacin - ResistantCampylobacter jejuni by Use of a Fluorogenic PCR Assay. Journal of clinical microbiology 38 , 3971 - 3978 (2000). 79 CHAPTER 3 This chapter represents a manuscript title d as Campylobacter jejuni induces autoimmune peripheral neurop athy via Siglec - 1 and IL - that is under preparation for publication. Contributing authors: Ankit Malik, Barbie J. Gadsden, Alexander Ethridge, Leslie A. Dybas and Linda S. Mansfield . 80 ABSTRACT Campylobacter jejuni is a spiral, gram - negative, microaerophilic bacterium that is the most common bacterial cause of gastroenteritis worldwide . C. jejuni infection has also b een causally linked with development of the peripheral neuropathy called Guillain Barré Syndrome (GBS). We have previously shown that C. jejuni isolates from human enteritis patients induce a Type1/17 cytokine dependent colitis response in IL - 10 - / - mice . I n contrast, isolates from human GBS patients colonize the IL - 10 - / - mice without inducing colitis but instead induce autoantibod y elicitation targeted against peripheral nerve antigens . We show here that the autoantibody response is dependent upon blunted T ype 1 / 17 but enhanced Type2 cytokine production by T helper cells . Autoantibody elicitation also correlated with enhanced macrophage infiltration in the sciatic nerve and its dorsal root ganglia. Autoantibodies and t hese histological changes were significan tly decreased in mice depleted of IL - 4 , without leading to colitis induction . Histological damage in the sciatic nerve was associated with abnormal gait and hind limb movements in the IL - 10 - / - mice . Furthermore, we show here that Siglec1 is a central a ntigen p resenting c ell receptor that mediates GBS but no t colitogenic isolate uptake, T cell differenti ati on and autoantibody elicitation . Therefore, t his is the first mouse model of an autoimmune diseas e induced directly by a bacterium and it is dependent upon Siglec1 and IL - 4 axes . 81 INTRODUCTION Campylobacter jejuni is a gram negative enter ic bacterium that is a leading cause of food - borne illness worldwide and affects 1 .4 million individuals an nually in the United States 1 . It is found ubiquitously in the gastrointestinal tract s of chicken and food animals 2 . Th us, consumpt ion of raw or undercooked poultry, other meats and unpasteurized milk are the most common sources of infection 3 . Campylobacteriosis is an inflammatory diarrhea with polymorphonuclear exudates affecting mainly the colon when the organism invades and induces inflammation. While the majority of healthy adults experience d isease for 7 - 10 days followed by resolution, it has been a cause of mortality in high risk individuals 4 . Histopathological manifestations include colonic crypt distortion, crypt abscesses, mucin depletion of epithelium, edema in the colonic lamina propria (cLP) as well as infiltration of granulocytes, pl asma cells and lymphocytes 5 . Infection with C. jejuni has also been linked to development a nd flare - ups of other chronic diseases of the gut like Irritable Bowel Syndrome and Inflammatory Bowel Disease 6 , 7 . Besides these e ffects o n the gut, C. jejuni infection has also been strongly linked with development of distal autoimmune diseases Gullian Barr é Syndrome (GBS) and Reactive Arthritis 8 , 9 . GBS is a term used for a number of autoimmune peripheral neuropathy conditions including Acute Inflammatory Demye linating Polyradiculoneuropathy ( AIDP ) , Acute Motor Axonal Neuropa thy ( AMAN ) and Acu te Motor and Sensory Neuropathy ( AMSAN ) . AIDP primarily involves demyelination of motor axons by inflammatory cell infiltration while AMAN and AMSAN involve axon death without marked inflammatory infiltrates 10 . Early symptoms of GBS appear as tingling or numbness in the extremities that rapidly progresses to ascending paralysis and can cause death du e to paralysis of the diaphragm 11 , 12 . GBS affects about 9,100 individuals annually in the US and has a mortality rate of 10% 13 . At present, plasmapheresis and int ravenous immunoglobulin (IVIg) are the only known treatments with beneficial effect, but only 60% of GBS patients improve 11 , 14 . Furthermore, up to 90% of patients face long term disability 82 after recovery from acute stage of the disease 11 . C. jejuni infection is primarily linked with the AMAN form of GBS which has been associated with development of autoantibodies that target gangliosides on peripheral nerves 9 , 15 . Gangliosides are sialic acid containing gylco lipids moieties in the outer leaflet of the plasma membrane of the myelin sheath and neurons. Oligosaccharide motifs on the outer surface of C. jejuni e ndotoxin (lipooligosaccharide) isolated from AMAN patients ha s been shown to mimic the peripheral nerve gangliosides, namely GM1, GD1a and others 16 , 17 . The development of effective treatments has been limited by lack of appropriate animal model s 18 . We have pre viously shown that a number of isolates of C. jejuni from human enteritis patients induce colitis in IL - 10 - / - mice whereas isolates from human GBS patients colonize the IL - 10 - / - mice but do not induce colitis 19 . We have also shown C. jejuni induced colitis respo nse depends on an upregulated Type1 and Type 17 but not Type2 cytokine response in the colon and draining lymph nodes. Both I nnate L ymphoid C ells (ILCs) and T cell subsets participated in this IFN - , IL - 17 and IL - 22 upregulation but in a time and organ spec ific manner. In contrast, the two C. jejuni strains isolated from human GBS patients did not induce colitis and elicited blunted Ty pe1/Type17 cytokine and antibodies, but enhanced Type2 responses. Moreover, the se GBS isolate s induced Type2 , but not Type1/1 7 antibodies cross - reacted with peripheral nerve gangliosides GM1 and GD1a 20 . However , little i s known about the histological or phenotypic consequences of elicitation of these autoantibodies, and what factors control the contrasting T and B cell differentiation following C. jejuni colitogenic versus GBS isolate infection in vivo . Siglec - 1 or Sialo adhesin is a type1 transmembrane protein, member of the Sialic acid binding Ig - like lectin (Siglec) family. It is expressed by metallophilic macrophages and IFN - activated dendritic cells and monocytes. It binds to N - acetylneuraminyl alpha 2 - 3 - galactose ( - 2 - 3 Nan - gal) containing glycolipids and glycoproteins, and has recently been shown to be a major 83 mediator for uptake of HIV by activated dendritic cells and macrophages in vitro 21 , 22 . Bax et al. have shown that C. jejuni LOS has - 2,3 siaylation , which depends on siayltran sfer a s e - II and mediates binding to Siglec - 1 (CD169) - 2,3 siaylation is required to induce a significant T ype 2 response in a human DC - T cell coculture system 23 . This ligand is also present on lipooligosaccharides of GBS associated but not colitogenic isolates of C. jejuni 16 . The role of s ialoadhesin in C. jejuni phagocytosis and primary IFN i nduction ha ve also been established 24 , 25 , 26 . Yet , there is no direct evidence of the role for Siglec - 1 in regulating adaptive immune Type1/Type2 balance in any inflammatory process . Further, it is we ll established that IL - 10 is a principal anti - inflammatory mediator for many auto - inflammatory diseases including IBD and MS/EAE 27 . It has also been shown that naive IL - 10 - / - mice have a higher number of T cells with autoreactive TCR in their lymphoid organs 28 . T herefore , w e hypothesized that C. jejuni induced autoimmune response in vivo will depend on IL - 4 and Siglec - 1 axes , and also that the absence of IL - 10 will amplify this autoimmune response . 84 RESULTS C. jejuni induced autoimmunity is IL - 4 and T helper cell dependent. We have previously shown that C. jejuni isolates from human GBS patients colonize the IL - 10 - / - mice without inducing colitis but do induce autoantibody elicitation. These responses were associated with decreased Type1/17 cytokine response but an enhanced Type2 cytokine response 20 . We therefore hypothesized that autoimmunity in GBS isolate infected IL - 10 - / - mice will be blocked by administration of IL - 4 neutralizing antibodies . Consistent with our previously published results, GBS isolate HB93 - 13 infected and co ntrol antibody injected mice developed a significant Type2 associated IgG1 response ( but not Type1/17 associated IgG2b, IgG2c or IgG3 response) against the C ampylobacter and peripheral nerve gangliosides GM1 and GD1a , 4 weeks after infection ( Fig . 3. 1A and B ) . The extent of both C ampylobacter specific and autoantibodies were decreased by administering IL - 4 neutralizing antibody injections . We have also previously shown that colitogenic isolate (11168) infection followed by IFN - and/or IL - 17 depletion leads to reciprocal upregulation of C ampylobacter specific (but not autoreactive) Type 2/ Ig G 1 response 20 . However, IL - 4 depletion did not significantly increase the Type1/17 dependent IgG2c, IgG2b or IgG3 isotypes ( Fig. 3. 1 A ) . Because the AMAN form of GBS is associated with damage in the sciatic nerve s and their roots 29 , 30 , we ascertain ed the histological manifest at ions of development of these autoantibodies in the sciatic nerve, dorsal root and the phrenic nerve . While no differences were observed upon evaluation o f the formalin fixed and H&E stained sections, greater number s of F4/80 + macrophages were found infiltrated into the sciatic nerve and the dorsal root in the infected mice when compared to sham inoculated mice ( Fig . 3. 2 A and B ) . This macrophage infiltrat ion was ascertained by immuohistochemistry and found to be particul arly marked in the 85 dorsal roots . Furthermore , macrophage infiltration was s ignificantly decreased in mice given IL - 4 neutralizing antibodies and correlated with decreased autoantibody titer s in circulation. Previously we have also shown that both Innate and adaptive lymphocytes had contributed towards upregulation of IFN - - 17 and IL - 22 after colitogenic isolate infection. In contrast, after GBS isolate infection, upregulation of IL - 4 wa s exclusively observed in CD4 + T cells and not in other T cell subsets or ILCs, as judged by intra - cellular cytokine staining and flow cytometry from the single cell suspension of the colon ( Fig . 3. 3 A ). Therefore , C. jejuni induced autoimmunity is IL - 4 and T helper cell dependent. However, IL - 4 depletion did not lead to an increase in T H 1 response n or inflammation in the gastrointestinal tract nor were any clinical signs of colitis exhibited by these or mice injected with control antibody ( Fig. 3. 3B ) . We ha ve also previously shown that colitis induction by colitogenic isolates correlates with an increase in their colonization extent. However, IL - 4 depletion did not lead to any difference in colonization extent n mice infected with GBS isolate ( Fig. 3 .3 C ) . Th us, lack of colitis induction by the GBS isolates is independent of th eir IL - 4 inducing capability . Siglec - 1 is essential for GBS isolate invasion into APCs and cytokine and autoantibody elicitation. It has been demonstrated that LOS - 2,3 siaylation, that is structurally similar to mono - or di - sialyated peripheral nerve gangliosides, acts as a ligand for Siglec - 1 (CD169) 23 . Also, the ganglioside presentation on the LOS surface is required to induce a significant T H 2 response towards the purified LOS in a human DC - T cell coculture system 23 . However, the role of Siglec - 1 , if any, in modulating an adaptive Type1/Type2 cytokine res ponse is not known . We used naïve splenocyte (adherent fraction and whole cell preparation) challenge by gentamycin killing assay as an ex vivo model to determine the role of Siglec - 1 in uptake and immune stimulation by GBS and colitogenic C. jejuni isolat es. Consistent with our previous observations, 86 GBS strains induced more IL - 6 while the colitogenic strains induced more TNF - and IFN - after challenge to whole splenocytes ( Fig. 3. 4A and B ) . We found th at a 20 min pre - treatment with anti - Siglec - 1 antibody, but not isotype control antibody , significantly decreased IL - 6 elicitation from GBS isolates but did not affect IL - 6 elici tation from colitogenic isolates. This effect was dependent on the antibody dose, a 5 g/ml dose was found to be sufficient for maximal effect to decrease IL - 6 elicitation by GBS isolates ( Fig. 3. 4A ) . Furthermore, elicitation of Type 1 cytokines like TNF - and I F N - were not affected by Siglec - 1 blocking at any dose from GBS or colitogenic isolates ( Fig. 3. 4A and B ) . As Siglec - 1 is a cell surface receptor known to be involved in uptake of HIV by macrophages and DCs 21 , 22 , we also hypothesized that Siglec - 1 blocking will lead to similarly decreased invasion of GBS but not colitogenic isolates into the adherent fraction of the splenocytes (that are enriched for phagocytes) . Consistent with this hypothesis, Siglec - 1 blocking significantly reduced invasion of GBS but not colitogenic isolates into the adherent splenocytes in the gentamycin killing assay ( Fig 3. 4C ). To corroborate the specificity of Siglec - 1 receptor towards ganglioside presenting C. jejuni isolates, we also included a n isolate from an enteritis patient that lacks all ganglioside mimics, C. jejuni C G8421 , in the cytokine elicitation and invasion assa y. We have previously shown that CG8421 causes a high degree of colitis in IL - 10 - / - mice that i s consistent with its high Type 1 and 17 cytokin e elicitation characteristics 20 . This isolate has been shown to lack any ganglioside presentation on its surface 31 , and consistent with that Siglec - 1 blocking had no affect on its cytokine elicitation ( Fig. 3. 4B ) or invasion ( Fig. 3. 4C ) properties in whole and adherent splenocytes respectively. Due to decreased bacterial uptake and IL - 6 produc tion a fter Siglec - 1 blocking ex vivo , we further hypothesize d that Siglec - 1 blocking in vivo will lead to decreased T cell activation that also lead s to decreased C. jejuni - and auto - reactive antibody elicitation by GBS isolates . Consistent with the tissue culture data , administering IL - 10 - / - mice anti - Siglec - 1 antibody for 6 87 weeks significantly decreased T H 2 differentiation in the colon but without affecting T H 1 differentiation , which was similar to sham - inoculated mice ( Fig 3. 5A ). Further , both C ampylobac ter specific ( Fig. 3. 5 B ) and also autoreactive anti - GM1 and anti - GD1a IgG1 antibodies ( Fig . 3. 5 C ) in circulation were significantly decreased after Siglec - 1 blocking in vivo . However, the affect of Siglec - 1 blocking does not seem to be T H 2 specific as anti - C. jejuni IgGb antibody levels also trended towards a decrease after blocking ( Fig. 3. 5B ) . Consistent with our previous data, 2 /10 mice in the infected + CIgG group that failed to be colonized at the end of the experiment were low/negative for C. jejuni s pecific or autoreactive antibodies. Only one mouse in the i nfected and S iglec - 1 blocked group failed to be colonized at the end of the experiment. IL - 10 is a negative regulator for antibody production . We have also previously shown that C. jejuni induced colitis in mice depends upon absence of IL - 10 because IL - 10 +/+ mice become colonize d by the colitogenic isolate but do not experience any clinical signs of disease 32 . But colitogenic isolate infected IL - 10 +/+ mice do develop anti - C ampylobacter antibodies that are of the same classes (IgG2b, IgG2c and IgG3) as that elicited in the IL - 10 - / - mice 20 . Therefore we asked if infection of IL - 10 +/+ mice with the GBS isolate lead s to autoantibody elicitation like that observed in the IL - 10 - / - mice. To this end, BL/6 IL - 10 +/+ mice were either sham inoculated or inoculated with HB93 - 13 alongside the IL - 10 - / - mice from the Siglec - 1 blocking experim ent, and analyzed for C. jejuni - and auto - reactive antibody elicitation 6 weeks post inoculation. We found that the infected IL - 10 +/+ mice did develop a significant anti - C ampylobacter IgG2b response when compared to sham inoculated mice , and this was not s ignificantly different from the IgG2b response in the IL - 10 - / - mice ( Fig. 3. 5 B ) . Intriguingly , the T H 2 dependent IgG1 response, both C ampylobacter specific and rea c tive to peripheral nerve gangliosides , was not induced to a significant extent in the IL - 10 + /+ mice as opposed to the IL - 10 - / - mice ( Fig 3. 5 B and C ) . Therefore, IL - 10 functions as a negative regulator of C. jejuni 88 induced T H 2 response and consequent autoantibody production , but does not affect C. jejuni reactive IgG2b elicitation . C. jejuni GBS isolate infection leads to abnormal hind limb movements in a subset of IL - 10 - / - mice. We have shown that BL/6 IL - 10 - / - mice infected with GBS isolates of C ampylobacter jejuni develop T H 2 associated antibodies that bind to peripheral nerve gangliosides GM1 and GD1a , and lead to significant macrophage infiltration in sciatic nerves and its dorsal root ganglia . We wanted to evaluate the phenotypic consequences associated with development of these autoantibodies , and to this end sham and GBS isolate HB93 - 13 in fected IL - 10 - / - mice were subjected to reaching reflex and open field test s weekly , for up to 17 weeks post inoculation. During the reaching reflex, mice were videotaped while being hung from the tail for 5 - 10 seconds and scored blinded for abnormal leg sp lay ( m ouse will spread legs out to the side and raise them up towards the tail; they will no longer remain in line with the forelimbs) and leg flexing ( m During open f ield testing, mice were videotaped for one minute after being put in a rat cage that wa s divided into 4 quadrants. Videotapes of each mice were scored for wide gait stance, foot drag and knuckling . They were considered affected if they were scored positive ly for at least three of the above features. The incidence of neurologically affected mice varied from 0 to 6 (out of 18 for infected ) and 0 (out of 11 for control) , with the highest incidence between weeks 4 - 7 post inoculation ( Fig. 3. 6 ) . Infected female mice trended towards a higher incidence rate than males but the maximum incidence rate for either sex did not reach above 50%. The number of rears and quadrants crossed in the open field test were quantified separately during the open field test. While th e infected mice trended toward s less quadrants crossed and lower number of rear s , it did not reach statistical significance (Fig 6B) . Digigait©® analysis on 89 the mice was also attempted but not found useful as these mice gradually stopped cooperating toward s running on the treadmill at any speed. After the fourth tri a l (that includes one tri a l before infection and 3 after infection) only 36% of the infected group and 45% of the controls ran on the treadmill, consistent with a previous finding of recalcitranc e of different strains of mice towards treadmill running 33 . Therefore Digigait testing was discontinued and only the reach ing reflex and open field test applied. We have also previously shown that the exten t of colonization in BL/6 IL - 10 - / - mice after infection with colitogenic isolates increases with time, and that this increase correlates with increasing extent of inflammat ion in the colon. Colonization with this GBS strain was in contrast found to be unstable with time , as the extent of camplylobacter DNA in feces decreased significantly at 17 weeks post inoculation when compared to 1, 4 or 8 weeks post inoculation ( Fig. 3. 7 A ) . This decrease in colonization probably explains the absence of elevated numbers of T H 2 (or T H 1) cells in the colon ( Fig. 3. 7 B ) , the absence of campylobacter specific ( Fig. 3. 7C ) or autoreactive antibodies ( Fig. 3. 7D ) in circulation and a decrease in the clinical signs of the disease at the time of necropsy ( Fig. 3. 6 ) . We also determined the histological manifestati ons of long term infection in the sciatic nerve and dorsal roots. While no gross differences were observed upon evaluation of the formalin fixed and H&E stained sections, slightly enhanced number of macrophages were found infiltrated into the sciatic nerve , but not the dorsal root in the infected mice that had demonstrated clinical signs ( Fig. 3. 7 E ) . This macrophage infiltration was ascertained by F4/80 immuohistochemistry . Therefore, C. jejuni induced peripheral neuropathy in IL - 10 - / - mice is transient and infrequent. 90 DISCUSSION We show here that C. jejuni induced autoimmune response in SPF IL - 10 - / - mice is dependent upon blunted Type1 / 17 but enhanced Type2 cytokine production. Moreover, these IL - 4 and CD4 + T cell dependent Type2 antibodies , but not the Type1 / 17 antibodies, cross - reacted with peripheral nerve antigens. Presence of autoantibodies in circulation co r related with enhanced macropha ge infiltration in the sciatic nerve and its dorsal root ganglia, while the phrenic nerve was largely unaffected (not shown) . These findings are consistent with human clinical reports that IgG1 isotype is the most commonly associated autoreactive isotype i n C. jejuni infection, and that its titer directly correlates with severity of clinical signs and worsening prognosis of C. jejuni associated GBS 34 , 35 . Macrophage infiltration wa s most marked in DRGs consistent with its weaker nerve blood barrier 36 , 37 . This contrasts with our previous findings that C. jejun i mediated colitis in specific pathogen free mice is T cell, IFN - and IL - 17 dependent 20 and de monstrates how differential T cell maturation by different C. jejuni strains leads to different disease outcomes in a susceptible host . Anatomical location of Siglec - 1 expressing macrophages suggests it s role as a sentinel for primary contact with pathog ens, apoptotic and cancer cells. Siglec - 1 is expressed by macrophages at the site of afferent lymphatics in the spleen and the lymph nodes , and at the base of the crypts in the colon, which are sites of frequent invasion by pathogens 38 , 39 . Consistent with our find ings, Heikema et al., have recently also shown that Siglec - 1 blocking decreases heat killed - GBS isolate uptake and IL - 6 (but not TNF - ) elicitation from human b lood monocyte - derived and LPS primed macrophages in vitro 26 . Recently it has also been shown that Siglce - 1 plays a critical role in phagocytosis and primary IFN and early cytokine production after challenge with sialylated pathogens 24 , 25 , 40 . We extend these findings to demonstrate that 91 colonic T H 2 differentiation and a utoimmune sequelae after C. jejuni GBS isolate infection is also Siglec - 1 dependent. Sigl e c - 1 has also been shown to be highly expressed by circulating myeloid and local lymph node cells during MS and other autoinflammatory diseases in humans 41 , 42 and plays a critical pro - inflammatory role by binding to Tregs and preventing their expansion, as shown in the EAE model 43 . Siglec - 1 + macrophages were also associated with presentation of lipid antigens for activation of iNKT cells 44 . Further, targeting delivery of microbial/tumor antigens throu gh Siglecs has shown promise for inducing strong T cell activation 45 . Therefore, Siglec - 1 is a unique recept or involved in both pha gocytosis and cell - cell interaction during antigen presentation to T cell s . While we have demonstrated a direct role for Siglec - 1 in sialylated C. jejuni uptake, it remains to be determined if it has an independent role in blocking APC - T cell or T cell - B cell interactions. Nevertheless , sialylated oligosaccharide motifs on the L OS of GBS associated C. jejuni is unique in the sense that it acts as the ligand for phagocytosis by antigen presenting cells, and also as epitopes that lead to autoimmunity. Histological damage in the sciatic nerve manifested with abnormal gait and hind limb movements in a subset IL - 10 - / - But the reasons for mild or no disease in some infected mice are not clear. Nevertheless, the extent of macrophage infiltration w as significantly decreased in mice given IL - 4 neutralizing or Siglec - 1 blocking antibodies, without a side effect of colitis. This demonstrates that down modulation of the T H 2 response alone is not sufficient to upregulate a colitogenic Type1/17 response after C. jejuni infection. This work is the first demonstration of a role for Siglec - 1 in T H 1/T H 2 balance regulation. We also confirmed the negative regulatory role of IL - 10 in C. jejuni induced autoimmunity. Therefore, SPF BL/6 IL - 10 - / - mice serve as excellent models of C. jejuni i nduced colitis, autoantibody elicitation and sub - clinical inflammation in the peripheral nervous system, but an insufficient model for studying sever clinical changes associated with C. jejuni induced GBS such as paralysis and respiratory insufficiency . As IL - 10 is a strong locus of 92 susceptibility for IBD and other inflammatory diseases 28 , this model essentially combines the most frequent genetic perturbation underlying inflammatory disorders with the most common causative organism of colitis and GBS through its natural route of infection. MATERIALS AND METHODS Mice , i noculati on and a ntibodies for in vivo neutralization . C57BL/6J IL - 10 +/+ (IL - 10 +/+ ) and BL/6.129P2 - IL - 10 tm1Cgn / J (IL - 10 - deficient) mice were purchased from Jackson lab oratories and maintained in specific pathogen free conditions and transported to University Resear ch Containment Facility at 8 12 weeks of age for experimental infection . Mice were housed individually and inoculated with tyrptone soy broth (TSB vehicle control) or 10 9 CFU of C. jejuni in 0.2ml TSB as described previously 32 . - Siglec - 1 (3D6.112) and isotype control (RTK2758) were purch retro - orbital injection, weekly for 6 weeks starting at 2 days before inoculation. - IL - 4 (11B11) was purchased from Bio - X - Cell (West Lebanon, NH) and injected intraperitoneally at use biweekly, starting at the day of inoculation . All animal protocols were approved by Michigan State University Institutional Animal Care & Use Committee and conformed to National Institutes of Health guidelines. Tissue samples. C olon and spleen was ta ken from infected and non - infected mice at the time of necropsy and rinsed in PBS. For ELISA or RNA extraction , tissue was frozen immediately using an ethanol - dry ice bath. For immunohistochemistry, sciatic nerves were exposed on the mouse carcass and fixe d in 10% buffered formalin for 48 hours, then stored in 60% ethanol followed by paraffin embedment. lleocecocolic junction was fixed for 20 - 24 hours. H&E sections were evaluated by a board certified pathologist . 93 Immunohistochemical analysis of mononuclear cells. 10% formalin buffer pH 7.0 and paraffin - embedded. 3 - 5 m thick serial sections were cut and attached to silanized glass slides and stained for F4/80 (BM8, eBiosciences). Sections were deparaffinized in two changes o f xylene and rehydrated in graded alcohol series. Endogenous peroxidase was blocked in the sections by incubating in 1% hydrogen peroxide in TBS (50mM tris, 150mM NaCl, pH 7.4) . Subsequently , the sections were boiled for 15 minutes in citrate buffer (Vecto r laboratories). The sections were blocked for 1 hour at room temperature with 1% BSA + 1.5% Goat s erum in TBS 0.025% Triton X - 100, the incubated with the respective primary antibody at 4 C overnight (Dilution 1:100 , in TBS 0.025% Triton X - 100). Sections were then washed twice with TBS 0.025% Triton X - 100 and stained with the Vectastain ABC kit (Vector oxylin, differentiated with 1% acetic acid, dehydrated and mounted with permount (sigma) . Negative controls were stained as above, except that primary antibodies were replaced with irrelevant isotype control (RTK4530, Biolegend) for F4/80. Evaluation of c ell densities. Sections were stained and analyzed in a blinded fashion. From each section at 20 x magnifica tion contiguous fields of view were photographed so as to include the entire area of the nerve and the root by the micrographs . T he area of the nerve/ root was calculated with the help of free hand tool of Image J, (N.I.H) and positively staining cells were counted manually with the help of cell counter tool of ImageJ. Preparation of Lamina Propria lymphocytes. LP lymphocytes were isolated as previousl y described 46 , 47 . Briefly, for removal of epithelial cells, the colon was washed, cut into small pieces, and then the pieces were incubated with calcium - and magnesium - free HBSS 94 supplemented with 5 % FBS and 5 mM EDTA (Sigma - Aldrich) at 1 4 0 rpm at 25 °C for 30 min. The tissues were then incubated with RPMI 1640 containing 10% FBS and 0.5 mg/ml collagenase type IV for 1 hour at 37°C with shaking at 150 rpm. The liberated cells were collected by passage through a 70 m nylon mesh. The isolated cells were pooled together and separated on a 40/80% discontinuous Percoll gradient (GE Bioscience). The cell yield was typic ally 1 2 X 10 6 cells per mouse with 90% cell viability, as ascertained by Propidium Iodide staining. Flow cytometry. The following monoclonal antibodies (eBiosciences) were used in appropriate combinations: anti - CD3 (clone 145 2C11), ant i - CD4 (clone RM 4 - 5), anti - TCR (clone GL3), anti CD19(clone 1D3), anti - CD11b (clone M1/70), anti Gr1 (clone - RB6 - 8C5), eFlour 780 anti - CD90 (clone53 - 2.1) and anti - CD16/CD32 (clone 2.4G2). The cells were preincubated for 20 minutes with anti - CD16/CD32 to block Fc rec eptors, thus avoiding nonspecific binding. Cells were then washed and labeled with appropriate mixture of antibodies or isotype matched controls for 30 minutes, centrifuged at 650 g , and resuspended in FACS buffer. To exclude dead/dying cells and therefore nonspecific antibody - binding cells, leukocytes were gated according to forward and side scatter. The percentages of CD4 + , CD8 + , CD4 + CD8 + and T cells subsets were calculated on CD19 - CD3 + gate. For intracellular cyto kine staining, cells were restimulated for 4 h with 50ng/ml PMA and 1 µg/ml Ionomycin (Sigma) and Golgi Stop and block (BD biosciences) were added for the last two hours . The cells were fixed and permeabilized using fixation and permeabilization solution ( eBioscience). Staining was performed for IL - 4 (clone 11B11 ) and IFN - (clone XMG1.2) antibodies, and the cells were analyzed on a LSRII flow cytometer (BD Biosciences) using FlowJo software (Tree Star). 95 Enzyme - linked Immunosorbent Assay. IFN - , IL - 6 and T NF - , were measured in tissue culture supernatants according to the manufacturer's protocol (Ready - Set - Go ELISA kits, eBioscience). C. jejuni antibody ELISA was used as described earlier 20 . Briefly, t he protein concentration was - Immuno Maxisorp plates were coated with the an tigen overnight at 4°C, blocked overnight at 4°C in blocking buffer (3% BSA in PBS with 0.05% Tween 20). Next day, plates were washed 4 times with wash buffer (PBS with 0.05% Tween 20) plasma samples diluted in blocking buffer (or blocking buffer alone as negative control) were applied to the plate and incubated overnight. Next day, plates were washed 4 times and i ncubated with biotinylated anti - mouse IgG1, IgG2b, IgG2c, IgG3 or IgM (Jackson Imumunoresearch) at 1:20000 dilution in blocking buffer, incubated for 1 hour at room temperature, washed 4 times with wash buffer and incubated for 1 hour at room temperature with Extravidin Peroxidase reagent (Sigma) diluted 1:2,000 in PBS with 1%BSA and 0.05% Tween 20. Plates were washed four times, developed with TMB (Sigma), stopped with 2N sulphuric acid and absorbance was read at 450 nm with 562 nm as reference wavelength. Serum concentrations were 1:25 for IgG1, IgG2c and IgG3; 1:100 for IgG2b. GM1 (Sigma) and GD1a (US bio) were used at 2 and 20µg/ml respectively and handled similarly. Splenocyte challenge by gentamicin killing assay. Red blood cell depleted splenocytes from naive C57BL/6 wt mouse ( 3X 10 6 cells / ml) were plated in antibiotic - free R10 medium and challenged with the indicated C. jejuni strains at mul tiplicity of infection of 1 . One hour af ter g / ml) was added to all the wells to kill extracellular bacteria. Supernatants were collected after 72 h for cytokine measurement by ELISA. To obtain adherent cells, splenocytes were pla ted at 10 7 cells / ml for 90 min upon which the non - adherent cells were washed off. For measuring invasion, cells are further incubated for 1 h with 250 mg / ml gentamicin, washed in PBS, lysed in 0.1% Triton X - 100, and released bacteria wa s enumerated by limi ting serial dilution assay . 96 APPENDIX 97 FIGURE LEGENDS Figure 3. 1. IL - 10 - / - mice were orally gavaged with TSB or C. jejuni GBS isolate HB93 - 13 or colitogenic 11168 . Sham and HB93 - 13 infected mice were injected with CIgG or IL - 4 neutr alizing antibody biweekly for 4 weeks starting at the time of inoculation. Plasma IgG isotypes reactive to C. jejuni antigen (A) or peripheral nerve ganglioside autoantigens (B) were analyzed by ELISA at the time of necropsy. N=8 - 10 mice per group. Data i s represented as mean + s.e.m and . Figure 3. 2. IL - 10 - / - mice were orally gavaged with TSB or indicated isolates of C. jejuni and injected with CIgG or IL - 4 neutralizing antibody . Formalin fixed sciat ic nerve and root sections were stained for F4/80 (A) . Positively staining cells and tissue area were quantified with the help of ImageJ cell counter and area tools respectively. N=8 - 10 mice per group. Data is represented as mean + s.e.m and was analyzed by Figure 3 .3 . IL - 10 - / - mice were orally gavaged with TSB or indicated isolates of C. jejuni and injected with CIgG or IL - 4 neutralizing antibody. Single cell suspension of the colon leukocytes was prepared and analyzed for indicated cell populations by ICCS and flow cytometry (A) . Formalin fixed illeocecococlic junctions were stained for H&E (B) . Colonization load was determined by Q - PCR on fecal DNA with C. jejuni specific primers (C) . N=8 - 10 mice per group . Data is represented as mean + s.e.m and was analyzed by Kruskal Wallis test followed by Figure 3. 4. Single cell suspension of whole splenocytes from naïve wt mouse was challenged with the indicated C. jejuni isola tes at M.O.I of 1 by gen tamycin killing assay. Cells were pre treated for 20 minutes by anti - Siglec1 or control antibody (1 , 5 or 10 for A ; 5 for B) . Gentamycin was added 1 hour after challenge and 72 hours later, indicated cytokine levels were determined by ELISA in c larified supernatant media (A and B ) . For invasion assay cells 98 were lyzed 1 hour after washing off gentamycin ( C ) . Gentamycin sensitivity to all the strains was also confirmed. Data represents mean + s.e.m of three wells, and was repeated atleast twice indep endently. Analyzed by two way A Figure 3. 5. Wt or IL - 10 - / - mice were orally gavaged with TSB or C. jejuni HB93 - 13 and injected with CIgG or anti - Siglec - 1 antibody weekly for 6 weeks starting at two days before i noculation. Plasma IgG isotypes reactive to C. jejuni antigen (A) or peripheral nerve ganglioside autoantigens (B) were analyzed by ELISA at the time of necropsy . N=8 - 15 mice per group. , horizontal bar represents the median . Figure 3. 6. IL - 10 - / - mice were orally gavaged with TSB or of C. jejuni HB93 - 13 and phenotyped weekly for 17 weeks. N=10 - 18 mice per group. Error bar represents mean + s.e.m. Data was an alyzed by two way A NOVA test fo Figure 3. 7. IL - 10 - / - mice were orally gavaged with TSB or of C. jejuni HB93 - 13 and phenotyped weekly for 17 weeks. Colonization load was determined by Q - PCR on fecal DNA with C. jejuni specific primers. Single cell suspens ion of colon leukocytes was prepared and analyzed for indicated cell populations by ICCS and flow cytometry. Plasma IgG isotypes specific to given antigens were quantified by ELISA at the time of necropsy. Formalin fixed sciatic nerve and root sections we re stained for F4/80. Positively staining cells and tissue area was quantified with the help of ImageJ cell counter and area tools respectively. N=10 - 18 mice per group. Colonization data was analyzed by one way A , and the rest by Mann Whitney test. 99 FIGURE 3. 1. IL - 4 depletion experiment serum antibodies 100 FIGURE 3. 2. IL - 4 depletion experiment sciatic nerve and DRG macrophage IHC A F4/80 B CIgG - IL - 4 11168 101 F IGURE 3. 3. IL - 4 depletion experiment colon f low cytometry, histology and colonization 102 FIGURE 3. 4. Siglec - 1 blocking in splenocytes ex vivo 103 FIGURE 3. 5. SIglec - 1 blocking in IL - 10 - / - mice, along w ith autoantibody analysis in IL - 10 +/ + mice. 104 Figure 3. 6. Long term phenotyping experiment with GBS isolate infected IL - 10 - / - mice. 105 Figure 3. 7. Long term phenotyping experiment: colonization, c olon flow cytometry and plasma antibody analysis. 1 06 REFERENCES 107 REFERENCES 1. CDC. Campylobacter: general information. wwwcdcgov/nczved/divisions/dfbmd/diseases/campylobacter/ 2012. 2. Manser P, Dalziel R. A survey of Campylobacter in animals. Journal of hygiene 1985, 95 (01) : 15 - 21. 3. Dasti JI, Tareen AM, Lugert R, Zautner AE, Groß U. Campylobacter jejuni: A brief overview on pathogenicity - associated factors and disease - mediating m echanisms. International Journal of Medical Microbiology 2010, 300 (4) : 205 - 211. 4. Tee W, Mijch A. Campylobacter jejuni bacteremia in human immunodeficiency virus (HIV) - infected and non - HIV - infected patients: comparison of clinical features and review. Cl inical infectious diseases 1998, 26 (1) : 91 - 96. 5. van Spreeuwel JP, Duursma GC, Meijer CJ, Bax R, Rosekrans PC, Lindeman J. Campylobacter colitis: histological immunohistochemical and ultrastructural findings. Gut 1985, 26 (9) : 945 - 951. 6. Gradel KO, Niel sen HL, Schønheyder HC, Ejlertsen T, Kristensen B, Nielsen H. Increased Short - and Long - Term Risk of Inflammatory Bowel Disease After Salmonella or Campylobacter Gastroenteritis. Gastroenterology 2009, 137 (2) : 495 - 501. 7. Qin H - Y, Wu JC, Tong X - D, Sung JJ , Xu H - X, Bian Z - X. Systematic review of animal models of post - infectious/post - inflammatory irritable bowel syndrome. J Gastroenterol 2011, 46 (2) : 164 - 174. 8. Garg AX, Pope JE, Thiessen - Philbrook H, Clark WF, Ouimet J. Arthritis risk after acute bacterial gastroenteritis. Rheumatology 2008, 47 (2) : 200 - 204. 9. Yuki N, Yamada M, Sato S, Ohama E, Kawase Y, Ikuta F , et al. Association of IgG anti - GD1a antibody with severe Guillain Barré syndrome. Muscle & Nerve 1993, 16 (6) : 642 - 647. 10. Hughes RA, Cornblath DR. Guillain - Barre syndrome. Lancet 2005, 366 (9497) : 1653 - 1666. 11. Anand BABP, Nimisha NK. Guillain - Barré syndrome (GBS). Pharmacol Rep 2010, 62 (2) : 220 - 232. 12. Vucic S, Kiernan MC, Cornblath DR. Guillain - Barré syndrome: An update. Journal of Clinical Neuroscience 2009, 16 (6) : 733 - 741. 108 13. CDC. Gullian Barre Syndrome. http://wwwcdcgov/h1n1flu/vaccination/factsheet_gbshtm 2013. 14. Kieseier BC, Kiefer R, Gold R, Hemmer B, Willison HJ , Hartung H - P. Advances in understanding and treatment of immune - mediated disorders of the peripheral nervous system. Muscle & Nerve 2004, 30 (2) : 131. 15. Jacobs BC, van Doorn PA, Tio - Gillen AP, Visser LH, van der Meché FGA, Schmitz PIM , et al. Campylobac ter jejuni infections and anti - GM1 antibodies in guillain - barré syndrome. Annals of Neurology 1996, 40 (2) : 181 - 187. 16. Aspinall GO, Fujimoto S, McDonald AG, Pang H, Kurjanczyk LA, Penner JL. Lipopolysaccharides from Campylobacter jejuni associated with G uillain - Barré syndrome patients mimic human gangliosides in structure. Infection and Immunity 1994, 62 (5) : 2122 - 2125. 17. Nachamkin I, Liu J, Li M, Ung H, Moran AP, Prendergast MM , et al. Campylobacter jejuni from patients with Guillain - Barré syndrome pre ferentially expresses a GD1a - like epitope. Infection and Immunity 2002, 70 (9) : 5299 - 5303. 18. Mansfield L, Schauer DB, Fox JG, Nachamkin I, Szymanski C, Blaser M. Animal models of Campylobacter jejuni infections. Campylobacter, 3rd ed ASM Press, Washingto n, DC 2008 : 367 - 379. 19. Bell JA, Jerome JP, Plovanich - Jones AE, Smith EJ, Gettings JR, Kim HY , et al. Outcome of infection of C57BL/6 IL - 10( - / - ) mice with Campylobacter jejuni strains is correlated with genome content of open reading frames up - and down - regulated in vivo. Microbial Pathogenesis 2013, 54: 1 - 19. 20. Malik A, Sharma D, St Charles J, Dybas L, Mansfield L. Contrasting immune responses mediate Campylobacter jejuni - induced colitis and autoimmunity. Mucosal immunology 2013. 21. Zou Z, Chastain A, Moir S, Ford J, Trandem K, Martinelli E , et al. Siglecs Facilitate HIV - 1 Infection of Macrophages through Adhesion with Viral Sialic Acids. PLoS ONE 2011, 6 (9) : e24559. 22. Izquierdo - Useros N, Lorizate M, Puertas MC, Rodriguez - Plata MT, Zangger N, Erikson E , et al. Siglec - 1 Is a Novel Dendritic Cell Receptor That Mediates HIV - 1 Trans - Infection Through Recognition of Viral Membrane Gangliosides. PLoS Biol 2012, 10 (12) : e1001448. 23. Bax M, Kuijf ML, Heikema AP, van Rijs W, Bruijns SCM, García - Vallejo JJ , et al. Campylobacter jejuni lipooligosaccharides modulate dendritic cell - mediated T cell polarization in a sialic acid linkage - dependent manner. Infection and Immunity 2011, 79 (7) : 2681 - 2689. 109 24. Huizinga R, Easton AS, Donachie AM, Guthrie J, van Rijs W, Heikema A , et al. Sialylation of Campylobacter jejuni Lipo - Oligosaccharides: Impact on Phagocytosis and Cytokine Production in Mice. PLoS ONE 2012, 7 (3) : e34416. 25. Klaas M, Oetke C, Lewis LE, Erwig LP, Heikema AP, Easton A , et al. Sialoadhesin Promotes Rapid Proinflammatory and Type I IFN Responses to a Sialylated Pathogen, Campylobacter jejuni. The Journal of Immunology 2012, 189 (5) : 24 14 - 2422. 26. Heikema AP, Koning RI, Duarte dos Santos Rico S, Rempel H, Jacobs BC, Endtz HP , et al. Enhanced, Sialoadhesin - Dependent Uptake of Guillain - Barré Syndrome - Associated Campylobacter jejuni Strains by Human Macrophages. Infection and Immunity 201 3, 81 (6) : 2095 - 2103. 27. Tian G, Li J - L, Wang D - G, Zhou D. Targeting IL - 10 in Auto - immune Diseases. Cell biochemistry and biophysics 2014 : 1 - 13. 28. Anderson AC, Reddy J, Nazareno R, Sobel RA, Nicholson LB, Kuchroo VK. IL - 10 Plays an Important Role in th e Homeostatic Regulation of the Autoreactive Repertoire in Naive Mice. The Journal of Immunology 2004, 173 (2) : 828 - 834. 29. Griffin J, Li C, Macko C, Ho T, Hsieh S - T, Xue P , et al. Early nodal changes in the acute motor axonal neuropathy pattern of the Gu illain - Barré syndrome. Journal of neurocytology 1996, 25 (1) : 33 - 51. 30. , et al. Acute motor axonal Annals of neurology 1996, 40 (4) : 635 - 644. 31. Platts - Mills JA, Kosek M. CURRENT OPINION Update on the burden of Campylobacter in developing countries. Curr Opin Infect Dis 2014, 27: 000 - 000. 32. Mansfield LS, Bell JA, Wilson DL, Murphy AJ, Elsheikha HM, Rathinam VAK , et al. C57BL/6 and Congenic Interleukin - 10 - Deficient Mice Can Serve as Models of Campylobacter j ejuni Colonization and Enteritis. Infection and Immunity 2006, 75 (3) : 1099 - 1115. 33. Wooley CM, Xing S, Burgess RW, Cox GA, Seburn KL. Age, experience and genetic background influence treadmill walking in mice. Physiology & Behavior 2009, 96 (2) : 350 - 361. 110 34. Koga M, Yuki N, Hirata K, Morimatsu M, Mori M, Kuwabara S. Anti - GM1 antibody IgG subclass A clinical recovery predictor in Guillain Barré syndrome. Neurology 2003, 60 (9) : 1514 - 1518. 35. Kaida K, Ariga T, Yu RK. Antiganglioside antibodies and their pa thophysiological effects on Guillain Barré syndrome and related disorders A review. Glycobiology 2009, 19 (7) : 676 - 692. 36. Abram SE, Yi J, Fuchs A, Hogan QH. Permeability of Injured and Intact Peripheral Nerves and Dorsal Root Ganglia. Anesthesiology 2006 , 105 (1) : 146 - 153. 37. Griffin JW, Li CY, Ho TW, Tian M, Gao CY, Xue P , et al. Pathology of the motor - sensory axonal Guillain - Barré syndrome. Annals of Neurology 1996, 39 (1) : 17 - 28. 38. Klaas M, Crocker PR. Sialoadhesin in recognition of self and non - sel f. Seminars in immunopathology; 2012: Springer; 2012. p. 353 - 364. 39. Hiemstra IH, Beijer MR, Veninga H, Vrijland K, Borg EG, Olivier BJ , et al. The identification and developmental requirements of colonic CD169(+) macrophages. Immunology 2014, 142 (2) : 2 69 - 278. 40. Chang Y - C, Nizet V. The interplay between Siglecs and sialylated pathogens. Glycobiology 2014, 24 (9) : 818 - 825. 41. Xiong Y - S, Cheng Y, Lin Q - S, Wu A - L, Yu J, Li C , et al. Increased expression of Siglec - 1 on peripheral blood monocytes and its role in mononuclear cell reactivity to autoantigen in rheumatoid arthritis. Rheumatology 2014, 53 (2) : 250 - 259. 42. Xiong Y - s, Wu A - l, Lin Q - s, Yu J, Li C, Zhu L , et al. Contribution of monocytes Siglec - 1 in stimulating T cells proliferation and activation in atherosclerosis. Atherosclerosis 2012, 224 (1) : 58 - 65. 43. Wu C, Rauch U, Korpos E, Song J, Loser K, Crocker PR , et al. Sialoadhesin - positive macrophages bind regulatory T cells, negatively controlling their expansion and autoimmune disease progression . The Journal of Immunology 2009, 182 (10) : 6508 - 6516. 44. Kawasaki N, Vela JL, Nycholat CM, Rademacher C, Khurana A, van Rooijen N , et al. Targeted delivery of lipid antigen to macrophages via the CD169/sialoadhesin endocytic pathway induces robust invari ant natural killer T cell activation. Proceedings of the National Academy of Sciences 2013, 110 (19) : 7826 - 7831. 111 45. Kawasaki N, Rillahan CD, Cheng T - Y, Van Rhijn I, Macauley MS, Moody DB , et al. Targeted Delivery of Mycobacterial Antigens to Human Dendrit ic Cells via Siglec - 7 Induces Robust T Cell Activation. The Journal of Immunology 2014, 193 (4) : 1560 - 1566. 46. Weigmann B, Tubbe I, Seidel D, Nicolaev A, Becker C, Neurath MF. Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue. Nature Protocols 2007, 2 (10) : 2307 - 2311. 47. Sanos SL, Bui VL, Mortha A, Oberle K, Hener s C, Johner C , et al. are required for the differentiation of mucosal interleukin 22 producing NKp46+ cells. Nature Immunology 2008, 10 (1) : 83 - 91. 112 CHAPTER 4 Conclusions, Sig nificance and Future Directions 113 Conclusions and Significance Campylobacter jejuni is an ubiquitous enteric bacterium that is the most common bacterial cause of gastroenteritis and has also been linked with development or flare - up of Inflammatory Bowel Disease and Irritable Bowel Syndrome. C. jejuni infection has also been causally linked with development of the peripheral neuropathy , Guillain Barré Syndrome (GBS). The occurrence of GBS is associated with development of autoantibodies that target gangliosides on peripheral nerves. The lipooligosaccharide of C. jejuni isolated from GBS patients with antecedent inf ections have been shown to mimic the peripheral nerve gangliosides GM1, GD1a and others. At present, therapies against GBS are untargeted and highly invasive or toxic, like plasmapheresis and intravenous immunoglobulin (IVIg) treatmen t. They too are not he l pful or stop working in almost 4 0% of the patients 1 . The development of effective treatments is limited by lack of appropriate animal models. We have previously shown that a number of isolates of C. jejuni from human enteritis patients induce colitis in the IL - 10 - / - mouse 2 whereas the isolates from human GBS patients colonize the IL - 10 - / - mice without inducing colitis 3 . The thesis presented here tested our overarching hypothesis that differential strain dependent immunological mechanisms are behind colitis induction by a colitogenic strain and asymptomatic colonization by the GBS strains. Further, we show that infection of mice with C. jejuni strains from GBS patients elicits autoantibody production that is consistent with that seen in human GBS cases 4 . We found that the colitogenic C. jejuni isolate elicits a Type1 and Type17 cellular, cytokine and antibody response as measured in the colon, mes enteric lymph node and plasma. We also determined that Innate Lymphoid Cells as well as adaptive T cells participated in producing a mixed Type1 and Type17 response in colon and draining lymph nodes in a time dependent manner by intracellular cytokine staining and flow cytometry. T cells were found to be neces sary for severe colitis as 114 mice depleted of Thy - 1 + cells (a common T cell and innate lymphocyte marker) were protected while the T cell deficient mice (Rag1 - / - and IL - 10R blocked Rag1 - / - ) failed to develop colitis after infection with the colitogenic isola te. The pro - inflammatory role of IFN - and IL - 17 in C. jejuni colitis was demonstrated by neutralizing these proteins in vivo which led to colon - protective Type2 cytokine and antibody responses. On the other hand, GBS C. jejuni isolates induced blunted Typ e1 and Type17 but enhanced Type2 colon cytokine and C. jejuni specific antibody responses. Furthermore, Type2 but not Type1/17 systemic autoantibodies were produced following infection with C. jejuni GBS isolates that also reacted with peripheral nerve gangliosides. These autoantibodies also cause peripheral neuropathic phenotype and Therefore, in support of our overarching hypothes is, we have demonstrated that C. jejuni mediated colitis in specific pathogen free C57BL/6IL - 10 - / - mice is T cell, IFN - and IL - 17 dependent. This work is also the first demonstration of the time - dependent role of Innate Lymphoid Cells and T cells in media ting Type1 and Type17 responses following infection with a human pathogen. We also show that a contrasting CD4 + T cell and IL - 4 dependent Type2 immune response pivots the disease away from inflammation in the colon to generation of circulating autoantibodi es. This work yields new cytokine and cellular targets for immunomodulatory therapy against IBD. It also suggests a potential mechanism for the situation where patients with rheumatoid arthritis or IBD receive TNF - to deve lop different autoimmune diseases that manifest with autoantibodies. Viral myocarditis is sequelae. This is the first mouse model of an autoimmune disease induced dir ectly by a bacterium. We further provide IL - 4 and Siglec - 1 as novel and rational therapeutic targets against this peripheral autoimmunity. 115 Future direction 1. To determine if early ILC activation facilitates T cell activation in C. jejuni mediated colitis . Thy - 1 is a marker common to both T cells and ILCs, and we have shown that depletion of these cells ameliorates C. jejuni colitis. Furthermore, since neither Rag1 - / - nor IL - 10R blocked Rag1 - / - mice developed colitis after infection, we can conclude that T cells are necessary for C jejuni to induce colitis. This aim is designed to directly determine if T cell activation is dependent on early activation of ILCs. However, as there are no known gene(s) that are exclusively expressed by, or required for the development of innate but not adapti ve lymphocytes, de novo models that contain adaptive lymphocytes but are devoid of innate lymphocytes do not exist. However, to study the effect of adaptive lymphocytes independent of innate lymphocytes, we can engineer a mouse model that contains adaptive that it will be devoid of all innate lymphocytes. Rag1 - / - c - / - (double KO) mice are devoid of all lymphocytes, adaptive and innate, and by injecting positively sorted splenic B and T cells into these mice, one can generate a model of transiently isolated adaptive immunity that is of devoid of all innate lymphocytes. Similar strategy was used by Sonnenberg et al., required for protective immunity against Citrobacter rodentium inf ection 5 . These mice can then be inoculated with Campylobacter jejuni to evaluate the inflammatory versus protective role of adaptive lymphocytes in C. jejuni mediated colitis . T - cell dependent antibody response specific to C. jejuni in the adaptive lymphocyte replete and innate lymphocyte deficient host can be used as an internal positive control to check the ability of introduced B and T cells in mounting an immune response similar to that of an unmanipulated host. Primary Hypothesis . Innate lymphocytes are dispensable for colitis in adaptive lymphocyte - replete host. 116 Experimental Design. 6 week old Rag1 - / - c - / - C57BL/6 mice will be injected i.p. with 6X10 7 positively sorted splenic B cells (B220 + ) and T cells (CD3 + ) from donor wild type mice. 3 weeks post injection, mice will be orally infected with 10 10 CFU of C. jejuni 11168 and monitored for clinical signs of diarrhea or colitis for up to 5 weeks. Age and sex matched mice inoculated with TSB will serve as controls. Parameters to be analyzed: Mice will be analyzed for weight loss and clinical signs of diarrhea and colitis, and also for histopathological changes in the colon. Alternative theory: Adaptive lymphocytes replete but innate lymphocyte deficient host will not d evelop colitis after C. jejuni infection. This could be the case due to two reasons: 1. IL - 10 produced in the host was sufficient to prevent colitis and/or 2. ILCs are necessary for T cells to induce colitis. If the alternative scenario turns out to be tru e, we will distinguish between the two possible reasons by depleting IL - 10 in the host during the infection. This will be achieved by injecting 400 g of IL - 10 neutralizing antibody every 3 days and starting 6 days before the infection. If IL - 10 depleted mi ce develop colitis, it will suggest that adaptive lymphocytes in the absence of IL - 10 and innate lymphocytes are sufficient to induce colitis. If even after IL - 10 neutralization mice do not develop colitis, then it will suggest that innate lymphocytes are indeed necessary to cause colitis. Potential Pitfall: A major cause of concern is that the splenic adaptive lymphocyte population injected into the Rag1 - / - c - / - host will not be able to self - renew efficiently without continuous reconstitution from the bone marrow. Therefore this is model of transiently isolated adaptive immunity only. 117 Future Direction 2. To determine the role of IL - 22 in C. jejuni induced colitis and a utoimmunity. In this study, we show that all C. jejuni isolates, colitogenic, non - sialylated and GBS associated, induce a n IL - 22 cytokine response in the colon and mesenteric lymph nodes ( in vivo ) and/or in the splenoc t yes ( ex vivo ). IL - 22 is a dichotomous cytokine that has been shown to be both pro - inflammatory as well as anti - inflammatory. It has bee n demonstrated as a major factor in maintain ing epithelial barrier integrity in the lung and gut and preventing systemic dissemination of colonized mircobiota 6 . I L - 22 is typically upregulated under inflammatory conditions and v ia STAT3 signaling, it plays a significant role in mucosal wound healing and goblet cell hyperplasia and enhanced mucus production 3 . Especially in conjunction with IL - 17, IL - 22 leads to elicitation of multiple antimicrobial peptides like - defensins, S100, lipocalin - 2 and Regenerating islet - from the epithelial cells 7 , 8 , 9 . Consequently, a bsence of IL - 2 2 results in increased systemic spread and subsequent morbidity and mortality following gut and lung infections like C. rodentium and K. pneumonia 5 , 10 . IL - 22 has also been shown to be p rotective in ulcerative colitis, hepatitis and lung fibrosis models 11 , 12 , 13 . We and others have shown that NK cells, ILCs and CD4 + T cells participate in the production of IL - 22, with NK cells and ILCs as the dominant source at early time points (within first week post infection), and CD4 + T cells as a dominant source later in the infection 14 , 15 . On the other hand, IL - 22 has been shown to be pro - inflammatory in experimental psoriasis/atopic dermatitis and collagen - induced arthritis models by upr egulating expression of IL - 23 and IL - 1 family of cytokines by keratinocytes and promoting autoreactive IgG elicitation 16 , 17 , 18 . It will therefore be interesting to reveal the pro - versus anti - inflammatory role of IL - 22 in C. jejuni mediated colitis and autoimmunity. Its role can be tested by either depleting IL - 22 in IL - 10 - / - mice by neutralizin g antibody injections or generating IL - 22 and IL - 10 double knock - out mice. It will also be interesting to infect single IL - 22 - / - mice to determine if it is responsible for preventing systemic dissemination and inflammatory response in IL - 10 sufficient host s. 118 Primary Hypothesis: IL - 22 is primarily protective in C. jejuni induced colitis and autoimmunity. Consequently, its absence during colitogenic or GBS isolate infection will lead to systemic dissemination of C. jejuni and subsequent systemic inflammation, enhanc ing morbidity and mortality. Experimental Design: 8 - 12 week old IL - 10, IL - 22 double KO mice will be orally i noculated with C. jejuni 11168 or HB93 - 13. Mice will be monitored for weight loss and clinical signs o f colitis and GBS for up to 5 weeks post inoculation. After sacrifice, histopathological changes in the colon, sciatic nerve and its roots, and plasma autoantibody titers will also be evaluated. Alternative Theory: It is possible that IL - 22 is redundant in preventing systemic dissemination of C. jejuni . Instead , akin to arthritis and skin inflammation models, its primary role after C. jejuni infection is to upregulate pro - inflammatory gene expression from epithelial cells and antigen presenting cells. Conse quently , its absence may ameliorate colitis after colitogenic isolate infection and/or decrease autoreactive IgG production after GBS isolate infection. 119 Future Direction 3. To evaluate the role of complement in C. jejuni induced autoimmunity. It is well established that human GBS patients with antecedent infections usually have antibodies in circulation that cross - react with peripheral nerve gangliosides and the outer core of the - of - concept in IL - 10 - / - mice infected with C. jejuni GBS isolates. Autoantibodies against cell surface antigens can lead to cellular damage by complement activation via classical pathway and/or lead to ADCC by NK cells. Using explanted mouse diaphragm nerves, Plomp et al., examined the effect of anti - ganglioside antibodies on neuromuscular transm ission. While autoantibody deposition alone did not affect transmission, addition of complement lead to a 300 fold decrease in neuromuscular transmission 19 . Therefore, complement can serve to enhance the effect of antibody deposition in blocking nerve conductance that can eventually lead to axonal death. Furthermore, antibody mediated classical complement activation leads to formation of membrane attack complex and release of C3a and C 5a . C3a and C5a are strong macrophage chemoattractants, consequently complement ma y also be involved in recruitment of macrophages to the peripheral nerves . Deposition of complement factors on the nerves can be evaluated by IHC. Role of complement in C. je juni induced GBS can be tested with C3 - / - mice. C3 is a central component of complement, essential for C5 cleavage and membrane attack complex formation. Primary Hypothesis: Complement activation is necessary for anti - ganglioside antibody mediated nerve damage and macrophage infiltration. Therefore complement deposition will be found in the sciatic nerve of GBS isolate infected IL - 10 - / - mice, and it will be co - localized with macrophage marker F4/80. Furthermore, infected C3 and IL - 10 double KO mice will h ave reduced infiltration of macrophages in sciatic nerves and its roots when compared to single IL - 10 - / - mice. Potential Pitfall. C3 - / - mice are hypersusceptible to certain bacterial infections , like intra - venous Group B Streptococcus infection results in increased mortality as compared to 120 immunocompetent controls (LD 50 dose: control 6.3 x 10 4 vs. 1.3 x 10 3 in C3 - deficient mice) 20 . Therefore, it may be necessary to choose a lower dose of C. jejuni for this experiment. We have shown that a dose of 10 2 cf u/mouse is sufficient to obtain stable colonization , and this low dose should be sub - lethal in these mice . Alternative theory: Another approach to test the importance of complement towards C. jejuni induced GBS can be to use a mouse model that lacks complement regulatory checkpoint(s) . CD59, also known as MAC - inhibitory protein, is a cell surface expressed protein that prevents formation of MAC complex by inhibiting C9 polymerization with the deposit ed C5b678 complex 21 . So if MAC formation aids in development of GBS lesions, CD59 IL - 10 double KO mice will have a greater extent of phenotypic and histological changes associated with GBS when compared to single IL - 10 - / - mice. As CD59 deficie ncy will not affect the elicitation of released products C3a and C5a, this model alongside the C3 - / - mice , will also help isolate the role of MAC formation versus chemoattractant generation as the primary function of complement in GBS. 121 APPENDIX 122 Table 1. Contrasting C. jejuni induced colitis and autoimmune neuropathy C. jejuni induced colitis C. jejuni induced autoimmunity Pathogenic Cytokine IFN - , IL - 17, IL - 22? IL - 4, IL - 21? IL - 6? T cell dependent? Yes, including CD4+, CD8+ and + Yes Role for ILCs? Yes, ILC1 and ILC17 No Siglec dependent? No Yes B cell dependent? No Yes Exacerbated by IL - 10 deficiency? Yes Yes Major IgG isotype IgG2c, IgG3, IgG2b IgG1, IgG2b 123 R EFERENCES 124 REFERENCES 1. Bril V, Ilse W, Pearce R, Dhanani A, Sutton D, Kong K. Pilot trial of immunoglobulin versus plasma exchange in patients with Guillain - Barré syndrome. Neurology 1996, 46 (1) : 100 - 103. 2. Mansfield LS, Bell JA, Wilson DL, Murphy AJ, Elsh eikha HM, Rathinam VAK , et al. C57BL/6 and Congenic Interleukin - 10 - Deficient Mice Can Serve as Models of Campylobacter jejuni Colonization and Enteritis. Infection and Immunity 2006, 75 (3) : 1099 - 1115. 3. Bell JA, Jerome JP, Plovanich - Jones AE, Smith EJ, Gettings JR, Kim HY , et al. Outcome of infection of C57BL/6 IL - 10( - / - ) mice with Campylobacter jejuni strains is correlated with genome content of open reading frames up - and down - regulated in vivo. Microbial Pathogenesis 2013, 54: 1 - 19. 4. Koga M, Yuki N, Hirata K, Morimatsu M, Mori M, Kuwabara S. Anti - GM1 antibody IgG subclass A clinical recovery predictor in Guillain Barré syndrome. Neurology 2003, 60 (9) : 1514 - 1518. 5. Sonnenberg GF, Monticelli LA, Elloso MM, Fouser LA, Artis D. CD4+ Lymp hoid Tissue - Inducer Cells Promote Innate Immunity in the Gut. Immunity 2011, 34 (1) : 122 - 134. 6. Shih VF - S, Cox J, Kljavin NM, Dengler HS, Reichelt M, Kumar P , et al. Homeostatic IL - 23 receptor signaling limits Th17 response through IL - 22 mediated containm ent of commensal microbiota. Proceedings of the National Academy of Sciences 2014, 111 (38) : 13942 - 13947. 7. Pickert G, Neufert C, Leppkes M, Zheng Y, Wittkopf N, Warntjen M , et al. STAT3 links IL - 22 signaling in intestinal epithelial cells to mucosal woun d healing. The Journal of experimental medicine 2009, 206 (7) : 1465 - 1472. 8. Eyerich S, Eyerich K, Cavani A, Schmidt - Weber C. IL - 17 and IL - 22: siblings, not twins. Trends in Immunology 2010, 31 (9) : 354 - 361. 9. Wolk K, Kunz S, Witte E, Friedrich M, Asadull ah K, Sabat R. IL - 22 increases the innate immunity of tissues. Immunity 2004, 21 (2) : 241 - 254. 10. Aujla SJ, Chan YR, Zheng M, Fei M, Askew DJ, Pociask DA , et al. IL - 22 mediates mucosal host defense against Gram - negative bacterial pneumonia. Nature medicin e 2008, 14 (3) : 275 - 281. 125 11. Sugimoto K, Ogawa A, Mizoguchi E, Shimomura Y, Andoh A, Bhan AK , et al. IL - 22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis. The Journal of clinical investigation 2008, 118 (2) : 534 - 544. 12. protect against lung fibrosis via IL - 22. The Journal of Experimental Medicine 2010, 207 (10) : 2239 - 2253. 13. Zenewicz LA, Yancopoulos GD, Valenzuela DM, Murphy AJ, Karow M, Fla vell RA. Interleukin - 22 but not interleukin - 17 provides protection to hepatocytes during acute liver inflammation. Immunity 2007, 27 (4) : 647 - 659. 14. , et al. Th22 cells are an important sourc e of IL - 22 for host protection against enteropathogenic bacteria. Immunity 2012, 37 (6) : 1061 - 1075. 15. Takatori H, Kanno Y, Watford WT, Tato CM, Weiss G, Ivanov II , et al. Lymphoid tissue inducer like cells are an innate source of IL - 17 and IL - 22. The Jou rnal of experimental medicine 2009, 206 (1) : 35 - 41. 16. Geboes L, Dumoutier L, Kelchtermans H, Schurgers E, Mitera T, Renauld JC , et al. Proinflammatory role of the Th17 cytokine interleukin 22 in collagen induced arthritis in C57BL/6 mice. Arthritis & Rhe umatism 2009, 60 (2) : 390 - 395. 17. Ma H - L, Liang S, Li J, Napierata L, Brown T, Benoit S , et al. IL - 22 is required for Th17 cell mediated pathology in a mouse model of psoriasis - like skin inflammation. The Journal of clinical investigation 2008, 118 (2) : 597 - 607. 18. Zheng Y, Danilenko DM, Valdez P, Kasman I, Eastham - Anderson J, Wu J , et al. Interleukin - 22, a TH17 cytokine, mediates IL - 23 - induced dermal inflammation and acanthosis. Nature 2006, 445 (7128) : 648 - 651. 19. Plomp JJ, Molenaar PC, O'Hanlon GM, Jacobs BC, Veitch J, Daha MR , et al. Miller Fisher anti - GQ1b antibodies: alpha - latrotoxin - like effects on motor end plates. Ann Neurol 1999, 45 (2) : 189 - 199. 20. Levy O, Jean - Jacques RM, Cywes C, Sisson RB, Zarember KA, Godowski PJ , et al. Critical role o f the complement system in group B streptococcus - induced tumor necrosis factor alpha release. Infect Immun 2003, 71 (11) : 6344 - 6353. 126 21. Meri S, Morgan B, Davies A, Daniels R, Olavesen M, Waldmann H , et al. Human protectin (CD59), an 18,000 - 20,000 MW compl ement lysis restricting factor, inhibits C5b - 8 catalysed insertion of C9 into lipid bilayers. Immunology 1990, 71 (1) : 1.