MRGPRX2 MEDIATED MAST CELL RESPONSES ARE SUPPRESSED BY LACTIC ACID By Meesum H. Syed A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Physiology—Master of Science 2021 ABSTRACT MRGPRX2 MEDIATED MAST CELL RESPONSES ARE SUPPRESSED BY LACTIC ACID By Meesum H. Syed Mas-related G-protein coupled receptor X2 (MRGPRX2) is a G-protein coupled receptor (GPCR) expressed in human mast cells that plays an important role in facilitating pseudo-allergic reactions as well exacerbating inflammation during asthmatic and other allergic diseases. Lactic acid, a byproduct of anaerobic glycolysis, is abundantly present in inflamed tissues and has been shown to regulate functions of several immune cells. Because the endogenous ligands for MRGPRX2 (substance P and LL-37) are upregulated during pathologic conditions such as cancer and asthma, the role of lactic acid in regulating mast cells response via MRGPRX2 and MrgprB2, the murine orthologue of the human receptor, is important to define. In this study, lactic acid suppressed both the early (Ca2+ mobilization and degranulation) and late (chemokine/cytokine release) phases of mast cell activation; this data was confirmed in LAD2, human skin and mouse peritoneal mast cells. In LAD2 cells, the reduction in degranulation and chemokine/cytokine production mediated by lactic acid was partially dependent on the pH. In agreement with the in vitro studies, lactic acid also reduced passive systemic anaphylaxis induced by compound 48/80 (a known MRGPRX2/MrgprB2 ligand) and inflammation in an LL-37 induced murine model of rosacea that is dependent on MRGPRB2 expression in skin mast cells. This data suggests that lactic acid may serve to inhibit mast cell-mediated inflammation during asthma and reduce immune response during cancer by affecting mast cell activation through MRGPRX2. Copyright by MEESUM H. SYED 2021 I dedicate this thesis to my peers and fellow students. iv ACKNOWLEDGEMENTS Several schools offer a master’s degree in physiology, my inclination towards attending this program at Michigan State University was based mostly on its research focused objective. Learning how to conduct research and developing investigative techniques are constantly high priority items in my education and I must give a sound thanks to the University for developing this program and admitting me as a student. The biggest thanks, however, must go to Dr. Hariharan Subramanian. Dr. Subramanian took a chance and decided to work with me, a post baccalaureate, self-described “undergraduate failure” of a student at the time. His mentorship was the most influential and critical force in developing my skills as a researcher. Dr. Subramanian not only taught me how to think as a scientist but also how to live as one. I don’t think you fully realize the confidence your mentorship instilled in me. My scientific acumen is a result of your guidance in basic cell culture technique to expressing my own conclusions and ideas. We’ve been through almost too much: freezer meltdowns, cannibalistic mice, malfunctioning plate readers, and a worldwide pandemic—to name a few. Thank you for getting me through it and for pushing me to realize my capabilities. A large thank you must also be given to my committee members, Dr. Julia Busik and Dr. Susanne Mohr, both of whom have taught me in undergraduate and graduate courses. Thank you, Dr. Busik, for encouraging me to pursue graduate school and research, Nermin’s lab minion has come a bit of a ways thanks to your support. A big thank you to Dr. Mohr, who was also instrumental in pushing me to continue my pursuit of research and lab experience. PSL 829, a class impossible without your coordination and instruction, is my standing favorite for all-time best academic course at MSU and will always remain as such in my eyes. Your prompting and drilling questions required in depth investigation and fostered skills I desperately needed to address my new curiosities in biology. v I cannot go further without thanking Dr. Rupali Das. I have never met anyone with more infectious passion for science nor anyone who could fully understand the complexities of optimizing a chocolate chip cookie recipe. You’ve taken me aside many times not only to advise me in improving my research methods but also just to make sure I was well and joke around with me. There are many instances where you made sure I was motivated to continue making progress despite my unexpected, out of left field setbacks, namely a ravaging skin infection that apparently isn’t even common in adults. Thank you for your commendation and praise, I will never forget your confidence in me. I am deeply indebted to the Molecular, Cellular and Integrative Physiology (MCIP) department at MSU for funding my studies. I cannot thank Dr. Narayanan Parameswaran enough for securing me positions as an instructor assistant. Because of your efforts I found a new love in teaching. My gratitude extends to Dr. Katherine Krueger and Dr. Kyle Miller with whom I taught introductory biology, and a special thanks must be given to the director of the Biological Sciences program, Dr. Jon Stoltzfus. I am extremely grateful for getting to work with Dr. Erica Wehrwein who is ever devoted to her students and to her teaching team. Thank you for teaching me how to teach and for showing me how to navigate the surprisingly rough waters an instructor faces. In contrast to those waters, Dr. Wehrwein also showed me how much I enjoy kayaking calm rivers—I am still planning on buying a boat. I wish to express my enormous appreciation for my instructors in my graduate education in and outside of the MCIP department at MSU. There are too many professors to list here but without all of you, my pursuit of scientific education would be fruitless. At every turn in this program, there has been an MSU professor supporting my education. A special thanks to Dr. L. Karl Olson and Dr. Marty Spranger for their advice and personal support. Thank you, you both have taken time to make sure I didn’t fall to the wayside. In that same line, I must thank Dr. Andrea Doseff for her constant and unwavering support of me and my fellow graduate students. vi There are many people in the Subramanian lab that I cannot even find the right words to thank. Dr. Ananth Kammala, thank you for so many things. For being the first to answer any of my questions, for teaching me how to handle mice and experiment timelines, for celebrating my small victories with me and of course, for being my older brother in lab. Thank you to the undergraduate students who each took time to teach me different experimental methods I needed for all my research on mast cells. Thank you, Christopher Occhiuto for introducing me to Dr. Subramanian and for your expert advice on preparing histology samples. Thank you to Canchai Yang and his magic hands. I never would have gotten clean western blots without your help nor would I have survived the last 2 years without your friendship. Brianna Callahan, thank you for all your help in my experiments and for discussing research and mast cells with me. I miss having all of you in lab with me and again, I can’t even articulate how much I appreciate your friendships. Devika Bahal and Tanwir Hashem, thank you for your friendship and helpful advice and for walking to class with me. We were the coolest clique, undoubtedly. Tanwir, thank you for helping me focus my energies on my studies and for showing me that fiery passion for my work is nothing to be ashamed about it. Devika, I will forever be thanking you for so much. You brought insight to my own scientific inquiries and thought process, our discussions have always brought clarity and I strive to be as stringent as you in my technique and work. I owe much of my resilience and ambition to you. You have such a bright outlook on the future, I have looked to you many times when I needed hope, and you have never turned me away. The kinship I shared with my fellow graduate students is of no small consequence. One of the best parts of this program is the students with whom I attended class, practiced presentations, and debated. My education relied on you and your solidarity. Thank you, Mama and Abu, my parents, who have encouraged my affinity for research and science since I was a young child. And a big thanks to my brothers and my sister-in-law who have debated with me about many biological and medical topics as well as other incredibly important items such as whether my apple vii pie was too salty or too sour. The biggest thank you goes to the smallest man—my nephew, Ghazanfur, who is the center of the brightest moments of my life. I’m going to make a researcher out of you, no matter what your dad says. viii PREFACE My study of mast cells began in the winter of 2019 when Dr. Hariharan Subramanian invited me to tour his lab. Mast cells have proven to have expansive and more diverse roles than the first mention I encountered of them in undergraduate textbooks. This thesis presents one context of mast cell biology that I researched and was fortunate enough to publish but not all contexts that I investigated with Dr. Subramanian. Although I can finally close out of the windows and tabs with articles and journal pieces about mast cells, my future research will likely acknowledge their impact in physiology. ix TABLE OF CONTENTS LIST OF FIGURES .................................................................................................................................... xii KEY TO ABBREVIATIONS .................................................................................................................... xiii CHAPTER 1 INTRODUCTION .................................................................................................................. 1 Mast cells ........................................................................................................................................ 2 Mast cells develop from myeloid progenitors released from bone marrow..................................... 2 Discovery of mast cells and initial investigations ............................................................................ 3 Mast cell types ................................................................................................................................. 4 Role in immunity ............................................................................................................................. 5 Psuedo-allergy ................................................................................................................................. 6 Psuedo-allergic mast cell response is mediated by Mas-related G protein-coupled receptor X ...... 7 Lactic acid and inflammatory conditions ......................................................................................... 8 Mast cell activation and lactic acid .................................................................................................. 9 MRGPRX2 and lactic acid............................................................................................................. 10 APPENDIX .................................................................................................................................... 12 CHAPTER 2 LACTIC ACID SUPPRESSED MAST CELL RESPONSE TO MRGPRX2 LIGANDS ... 18 Introduction .................................................................................................................................... 19 Materials and Methods ................................................................................................................... 22 Cells .................................................................................................................................. 22 Calcium assays .................................................................................................................. 23 Degranulation assays ........................................................................................................ 24 Cytokine assays................................................................................................................. 25 Results and Discussion .................................................................................................................. 27 Lactic acid reduces MRPGRX2 mediated calcium mobilization ..................................... 17 Lactic acid treatment reduced MRGPRX2 mediated early phase activation in mast cells ...................................................................................................... 28 Degranulation of LAD2 cells is reduced with lactic acid treatment ................................. 28 MRGPRX2-mediated degranulation of primary skin-derived human mast cells is reduced by lactic acid ....................................................................... 29 Lactic acid suppresses late phase response in MRGPRX2 stimulated mast cells............. 30 APPENDIX .................................................................................................................................... 31 CHAPTER 3 MRGPRB2/MAST CELL MEDIATED INFLAMMATION IS REDUCED WITH LACTIC ACID TREATMENT IN VIVO ............................................................... 41 Introduction .................................................................................................................................... 42 Materials and Methods ................................................................................................................... 44 Mice .................................................................................................................................. 44 Passive systemic anaphylaxis (PSA) ................................................................................ 44 Ex vivo culture of peritoneal mast cells ............................................................................ 44 Cathelicidin LL-37 induced rosacea model ...................................................................... 44 Epidermal thickness .......................................................................................................... 45 Degranulated mast cell count in dorsal skin ..................................................................... 45 x mRNA analysis of dermal site of inflammation ............................................................... 45 Results and Discussion .................................................................................................................. 46 Lactic acid administration reduced MrgprB2/MC dependent anaphylaxis in vivo and ex vivo......................................................................................... 46 LL-37 induced rosacea is diminished with lactic acid treatment in a chronic, murine model of inflammation..................................................................... 47 APPENDIX .................................................................................................................................... 49 CHAPTER 4 SUPPRESSION OF MRGPRX2 MEDIATED MAST CELL RESPONSES BY CHEMICAL MOEITES OF LACTIC ACID ....................................................................................... 58 Introduction .................................................................................................................................... 59 Results and Discussion .................................................................................................................. 61 Lactic acid exposure did not reduce phosphorylation of pathways downstream of MRGPRX2 ligation.................................................................................. 61 Lactic acid reduced of MRGPRX2 mediated responses largely independent of the lactate anion ....................................................................................... 61 Degranulation of mast cells exposed to compound 48/80 was only partially reduced by acidic buffers .................................................................... 62 APPENDIX .................................................................................................................................... 64 CHAPTER 5 CONCLUSION..................................................................................................................... 71 Conclusion ..................................................................................................................................... 72 Lactic acid as a possible therapeutic for LL-37 associated rosacea .................................. 72 MRGPRX2-substance P-cancer axis ................................................................................ 73 Possible mechanism of suppression by lactic acid............................................................ 73 REFERENCES ........................................................................................................................................... 76 xi LIST OF FIGURES Figure 1. Lineage and flow chart of mast cell development ....................................................................... 13 Figure 2. Metachromatic staining of primary and cultured murine mast cells ........................................... 14 Figure 3. MRGPRX2 ligands employed for experiments ........................................................................... 16 Figure 4. Viability of mast cells in lactic acid concentrations .................................................................... 17 Figure 5. Fluorescence measured in ligand exposed HEK 293T-MRGPRX2 cells is reduced by pretreatment with lactic acid .................................................................................................... 32 Figure 6. MRGPRX2 mediated Calcium mobilization is reduced in lactic acid treated mast cells ............ 34 Figure 7. Early phase, MRGPRX2 mediated degranulation in LAD2 mast cells was significantly attenuated in a dose dependent manner with lactic acid pretreatment ............................ 36 Figure 8. Lactic acid inhibits MRGPRX2 mediated degranulation in primary human skin mast cells ...... 38 Figure 9. Lactic acid suppresses cytokine/chemokine production LAD2 cells exposed to MRGPRX2 ligands .................................................................................................................. 40 Figure 10. Lactic acid reduces systemic anaphylaxis to compound 48/80 in vivo...................................... 50 Figure 11. Peritoneal derived mast cells showed significantly inhibited MrgprB2 mediated degranulation to MRGPRX2 ligands with lactic acid pretreatment ............................................ 51 Figure 12. Lactic acid prevents thickening of the epidermis in an LL-37 induced rosacea model ............. 53 Figure 13. Lactic acid treated mice in LL-37 induced rosacea model showed a significantly smaller number of degranulated mast cells than the vehicle treated cohort ........... 54 Figure 14. mRNA expression of mast cell derived products post LL-37 injection was reduced in mice treated with lactic acid ................................................................................ 56 Figure 15. Phosphorylation events downstream of MRGPRX2 ligation was not reduced in LAD2 mast cells pretreated with lactic acid ................................................................ 65 Figure 16. Lactic acid modulation of MRGPRX2 mediated degranulation of LAD2 mast cells is dependent on proton moiety ........................................................................................ 66 Figure 17. Sodium lactate has no effect on chemokine/cytokine production in LAD2 cells ...................... 68 Figure 18. Acidic buffer partially reduced MRGPRX2 mediated degranulation of LAD2 mast cells ....... 70 xii KEY TO ABBREVIATIONS AKT Protein kinase B AM Acetoxymethyl AMP Adenosine monophosphate ATP Adenosine triphosphate BSA Bovine serum albumin C3 Complement protein 3 C3a Fragment A of C3; anaphylatoxin C3aR C3a anaphylatoxin receptor C5 Complement protein 5 C5a Fragment A of C5; anaphylatoxin C5aR C5a anaphylatoxin receptor cAMP Cyclic adenosine monophosphate CCL2 Chemokine (C-C motif) ligand 2; monocyte chemoattractant protein 1 CCR2 Chemokine (C-C motif) receptor 2 CHTN Cooperative Human Tissue Network CLP Common lymphoid progenitor CMA1 Chymase 1 CMP Common myeloid progenitor CTMC Connective tissue mast cell DMEM Dulbecco's Modified Eagle Medium DNA Deoxyribose nucleic acid DRG Dorsal root ganglion ELISA Enzyme-linked immunosorbent assay ERK 1/2 Extracellular signal-regulated kinases 1 and 2 xiii FBS Fetal bovine serum FcεRI Fc epsilon receptor 1; immunoglobulin E receptor FDA Food and Drug Administration of the USA GAPDH Glyceraldehyde 3-phosphate dehydrogenase GMP Granulocyte monocyte progenitor GPCR G-protein-coupled receptor GPR G-protein-coupled receptor; gene or protein GPR65 G-protein-coupled receptor 65; known proton sensor GPR81 G-protein-coupled receptor 81; known lactate anion sensor HEK Human embryonic kidney; fibroblast cell line HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HMR Human embryonic kidney 293 with SV40-T cells transfected to express MRGPRX2 hSCF Recombinant human stem cell factor i.d. intradermal; injection/administration i.p. intraperitoneal; injection/administration IgE Immunoglobulin E IgG Immunoglobulin G IL Interleukin IL-33 Interleukin 33 IL-6 Interleukin 6 IL-8 Interleukin 8 IP3 inositol 1,4,5-trisphosphate LAD2 Laboratory of allergic diseases 2; mast cell line LPS Lipopolysaccharide MAPK Mitogen-activated protein kinase MAS1 Angiotensin-II and angiotensin-(1-7) receptor; G-protein-coupled receptor xiv MC Mast cell MCT Mast cell containing tryptase; mucosal type mast cell MCTC Mast cell containing tryptase and chymase; connective tissue type mast cell McP Mast cell progenitor MCT-1 Monocarboxylate cotransporter-1; cotransporter for proton and lactate anion MDP Monocyte-dendritic cell progenitor MEP Megakaryocyte-erythroid progenitor MMP9 Matrix metalloprotease 9 Mrg Mas-related gene; sequence homology to MAS1-GPCR MrgprB2 Mas-related G protein-coupled receptor B2; murine basic secretagogue receptor MRGPRX2 Mas-related G protein-coupled receptor X2; human basic secretagogue receptor mRNA Messenger ribose nucleic acid mSCF Recombinant murine stem cell factor NAD+ Nicotinamide adenine dinucleotide NADH Nicotinamide adenine dinucleotide + hydrogen NMBD Neuromuscular blocking agents/drugs PAMP Pathogen associated molecular patterns PBS Phosphate buffered saline PI3K Phosphoinositide 3- kinase PIP2 phosphatidylinositol (3,4)- biphosphate PIP3 phosphatidylinositol (3,4,5)- triphosphate PKB Protein kinase B PLC Phospholipase C PMC Peritoneal mast cell PNAG p-Nitrophenyl-N acetyl-β-D-glucosamine PSA Passive systemic anaphylaxis xv PSG Penicillin-streptomycin-glutamine RFU Relative fluorescent unit RNA Ribose nucleic acid RPMI Rosswell Park Memorial Institute; culturing medium SCF Stem cell factor also known as c-kit ligand or steel factor SFM Serum free media SIR Siraganian buffer ST2 Interleukin 1 receptor-like 1; Interleukin-33 receptor SV40-T Simian vacuolating virus 40 T antigen TLR Toll-like receptor TNF Tumor necrosis factor TPSAB1 Tryptase alpha/beta 1 xvi CHAPTER 1 INTRODUCTION 1 Mast cells Mast cells typically function in the innate immune system amongst leukocytes, phagocytes, granulocytes and even several T-cell types. Primarily, mast cells are known for the potent release of vasodilatory and inflammatory mediators such as histamine. The function of mast cells is far more complex, however, when considering the range of mast cell derived factors and cytokines released upon activation1,2. A granulocyte by nature, mast cells appear to be packed tightly with dense secretory vesicles that can mobilize and empty in a process of vesicular release known as degranulation. Degranulation of mast cells is a hallmark of early phase responses that accomplishes substantial release of granular content, namely histamine and heparin. Aside from fast acting inflammatory mediators like heparin, histamine, vasoactive eicosanoids, and proteases3, secretory granules contain cytokines and chemokines that are released during degranulation. Stimuli can also induce transcriptional changes in mast cells to produce and secrete signaling molecules anew; this long-lasting state of activation constitutes the late phase of mast cell response. Ultimately, late phase activity results in recruitment of monocytes, eosinophils, neutrophils, memory T-cells, and dendritic cells to the site of insult or exposure and presents as an enduring state of inflammation. Mast cells develop from myeloid progenitors released from bone marrow. Much of the research investigating mast cell maturation is derived from rodent models and involves analyzing surface receptor expression as well as identifying progenitor sources by observing phenotypes of resulting cultures4. Like many components of the immune system, mast cells arise from hematopoietic lineage, specifically from the myeloid branch (Figure 1). These myeloid progenitors are released from the bone marrow and the spleen to the blood stream from where recruitment to peripheral tissues can begin 5. The common myeloid progenitor gives rise to a granulocyte progenitor and further on to a committed but immature mast cell6, this path of development is not necessarily strict as studies have identified a committed mast cell progenitor originating in the bone marrow7. The immature mast cell expresses integrins and 2 chemokine receptors that allow it to settle at certain tissues and begin maturing into a functional, tissue resident mast cell8. (See Figure 1, a schematic summary of mast cell development.) Mast cell deficient models have been developed and used critically for studying cell function and immunity. Initially two genotypes could separately produce an anemic mouse that lacked mature mast cells, mice with a mutation of one allele in the Dominant Spotting locus (W/Wv) or mice carrying a heterozygous mutation at the locus for stem cell factor (Sl/Sld) 9. Phenotypic anemia in W/Wv resulted from inappropriate production of hematopoietic cells10 while Sl/Sld mice suffer from inadequate production of stem cell factor, a protein necessary for mast cell development at the site of tissue residence. Kitamura et al., has described both genotypes in the context of mast cells and further showed that W/Wv mast cell deficiency could be rescued with bone marrow transplants from W/W or wild type mice whereas Sl/Sld could not be similarly rescued11,12. This landmark study was the first of many to verify that mast cells originate from hematopoietic lineages and bone marrow. Conditions in which tissues, such as the lungs in an asthmatic patient, contain higher numbers of mast cells also seem to coincide with increased mast cell progenitors in the blood 13,14, further implying that these populations first rely on recruitment to tissues rather than just proliferation at the site of residence. Discovery of mast cells and initial investigations Cells that stained densely with alkaline dyes were first described as “mastzellen” by Paul Ehrlich in his 1878 doctoral thesis focused on identifying hematological cell types15. Ehrlich would eventually earn a Nobel Prize in 1908, though not simply for his discovery of these “well fed” cells. The true study of mast cell function began with anaphylaxis. Within decades of the first description of hypersensitivity in 190216, a correlation was made between anaphylaxis and the protein heparin 17. Later, various animal models showed increased blood levels of histamine as well as heparin after induction of anaphylactic shock. Decades later, from 1937 to 1953, mast cells were implicated in the process of 3 anaphylaxis by way of providing the anticoagulant heparin17–19 with later studies proving histamine release as an additional, major function of mast cells20. Studies analyzing mast cell contents proved them to contain large quantities of histamine and other potent mediators of inflammatory mechanisms21,22. These new understandings did not, however, elucidate the mechanism of mast cell activation. The diverse location of mast cell residences detailed multiple phenotypes capable of similar inflammatory responses but to seemingly different stimuli. Most research involved inducing allergic response with foreign proteins collected from other organisms23. Allergic induction aside, the list of substances that stimulated allergy-like or inflammatory responses began to grow and include a wide range of origins from molecules derived of parasites and bacteria to endogenous peptides to synthetic compounds and polymers. This class of compounds that did not require repeated exposures for inflammatory symptoms was initially described as histamine liberators, compound 48/80, a cationic polymer amongst this class, would become a prominent agent used to study mast cells24–26. Mast cell types Ehrlich’s “mastzellen” was described as a cell found in connective tissues, embedded in the network forming organs and structures—parotid glands from goats and perivascular tissues of animals, for example27. Initial studies on metachromatic staining further supported his observations in a variety of mammals with some researchers controversially identifying a circulating mature mast cell28. Further experiments and observations with other dyes elucidated this “blood mast cell” as basophils which can appear quite similar to mast cells when staining with early methods. The first real account of a second type of mast cell was in 1905, by Dr. Alexander Maximow—a Russian scientist and contemporary of Ehrlich. Maximow described a population of mast cells appearing in the mucosa of rat intestines, separate from the mast cells in the lining of the intestinal samples. He went on to assert the identical morphology and staining of mast cells in the lining with those in the mucosa while 4 noting the significant differences in size28. In 1966, a “mucosal” mast cell was confirmed in rats by more advanced staining techniques and with careful attention to the fragility of this population of mast cells29. Primarily based on rodent models, the first classification of mast cells identified two mature populations: mucosal type and connective tissue type. Mucosal mast cells are associated to mucosal epithelium including the respiratory tract and intestines; they are considered analogous to human mast cells located in the same tissues30. Serosal mast cells are collected from serous membranes of the rodent, namely the peritoneum by lavage. Serosal mast cells have become synonymous with connective tissue type due to similar morphology, staining, and response to histamine liberating agents31. Serosal and mucosal mast cells stain differentially with certain dyes as seen in Figure 2. Human mast cells are classified broadly by protease content. Trypsin was identified as a biomarker for all mast cells but by the latter half of the 20th century, a second protease was identified in mast cells from connective tissue32. Mast cells collected from mucosa contain mostly tryptase and have been dubbed MCT. Mast cells from connective tissues such as skin contain tryptase, chymase and other proteases and have thus been named MCTC33. Maturation from a committed mast cell progenitor to either phenotype is likely due to factors present at the tissue of recruitment4. Role in immunity Allergic response, early and late phase, is mediated through immunoglobulin E (IgE) and its surface receptor, FcεR1. This path of activation demonstrates the role mast cells play in adaptive immunity, requiring a sensitization to antigens and subsequent B cell production of IgE which then binds FcεR1. This priming equips mast cells with the machinery necessary for immediate, potent response when the antigen is reintroduced. Other immunoglobulin receptors expressed by mast cells facilitate similar response to respective antigens; though the expression profile of these receptors varies, the IgE receptor is known to be expressed by all mature mast cell phenotypes34. 5 Mast cells express a range of Toll-like receptors (TLRs) and can react to pathogen associated molecular patterns (PAMPs) as part of the innate and adaptive immune system. In mice, expression of TLRs on mast cells is substantial, with mRNA analysis confirming expression of 8 of the 13 murine TLRs35. Human mast cells have also been shown to express mRNA for multiple TLRs with confirmed protein expression of 9 human TLRs on human mast cells lines, mature mast cells from lung and skin tissue, as well as mast cells cultured from human blood36,37. TLR mediated response of mast cells is differential, with some ligands and their respective TLRs inducing degranulation and others inducing only cytokine production38. The complement cascade also initiates mast cell activity through complement protein receptors. The C3a and the C5a receptor, both G protein coupled receptors (GPCR), expressed on mast cells initiate degranulation and late phase production of cytokines39,40. In addition to receptor expression, tryptase, a mast cell protease, has been shown to cleave complement protein C3 and C5 in allergic responses. The end products, C3a and C5a, further induce anaphylactic responses through respective receptors expressed on mast cells41,42. The line separating innate and adaptive immune responses in mast cells is further blurred with their expression of interleukin (IL) receptors. Notably, mast cells express ST2, an interleukin 33 (IL-33) receptor of the IL-1 receptor family43,44. Response to cytokines can positively modulate mast cell function in allergic pathways and potentially others. While the ST2/IL-33 axis in mast cells induces cytokine production and mast cell maturation on its own45–47, it also has been shown to increase IgE- allergic responses48,49. Psuedo-allergy Symptoms of acute inflammation can occur in patients after administration of certain drugs and present very much like allergic reactions mediated by IgE/FcεR1 and mast cells. The incidence of drug hypersensitivity supported a patient specific allergy explanation, implying that such patients were previously exposed to and had raised antibodies against these or similar compounds 50. In clinic, a wide 6 range of Food and Drug Administration (FDA)-approved drugs have presented hypersensitive reactions including antibiotics such as vancomycin, fluoroquinolones, neuromuscular blocking agents (NMBDs) like atracurium, and the opioid morphine51–58. The notion that drug hypersensitivity operates through immunoglobulins and mast cell expressed immunoglobulin receptors is of controversy59,60. After all, mast cells express many surface receptors including GPCRs, TLRs, and interleukin receptors, some of which can produce the same symptoms observed in drug hypersensitive reactions. Psuedo-allergic mast cell response is mediated by Mas-related G protein-coupled receptor X2 Non-allergic activation was first reported in 1957 when mast cells were observed to potently release histamine with exposure to compound 48/8061. From there, other compounds and substances that acted similarly on mast cells were identified and named basic secretagogues—a reference to their likeness in chemical moieties. Basic secretagogues were largely grouped by their ability to activate connective tissue type mast cells and induce degranulation, this response was clearly IgE independent as no immune priming of the cells was required. Preliminary understanding of the mechanism of IgE independent response emerged when pertussis toxin incubation abolished compound 48/80 degranulation of rat peritoneal mast cells62. Further studies revealed basic secretagogues interact with and activate G proteins when eliciting secretory responses in mast cells63,64,65. The receptor responsible for these IgE independent or pseudo-allergic responses was first identified in nociceptive neurons. Screening of cDNA libraries uncovered 49 previously unknown genes in murine dorsal root ganglion (DRG) neurons66. Of these 49, 31 genes were intact and coded for GPCRs bearing sequence homology to MAS1 thus Dong et al. named this class “Mas-related”. Searches of human databases using the murine Mrgs further uncovered at least 4 human equivalents that the group labelled MRGXs66. 7 Multiple neuropeptides such as cortistatin 14 and substance P, a known basic secretagogue, were shown to ligate the receptor coded by MRGX2, Mas-related G protein-coupled receptor X2 (MRGPRX2)67. By 2006, investigation of MRGX2 showed selective expression on DRG neurons as expected but also in skin, colon, bladder, and adipose tissue—locations that contain many connective tissue type mast cells (Figure 1). More interestingly, mast cells cultured to the MCTC phenotype that degranulates with basic secretagogue exposure substantially expressed MRGPRX2 while those cultured to be the mucosal (MCT) phenotype did not68. MRGPRX2 was soon implicated in innate immune responses for antimicrobial defense when LL-37, a cathelicidin also associated to psoriasis and rosacea, was shown to be dependent on the receptor when inducing degranulation and cytokine production in mast cells69,70,71,72,73. The curiosity of pseudo-allergic responses in clinical settings was later resolved by McNeil et. al., when the group screened drugs correlated with hypersensitive reactions for MRGPRX2 activity. Furthermore, classic basic secretagogues compound 48/80 and substance P were proven to stimulate activity depending on MRGPRX2 with RNA silencing and transfected cell lines74. With elucidation of MRGPRX2’s role in drug hypersensitivity, it is now known as the pseudo-allergic receptor. This is despite the numerous physiological ligands that have been demonstrated for MRGPRX2 indicating an endogenous role in inflammatory mediation. Interestingly, MRGPRX2 expression on mast cells in the lung, where mucosal type mast cells normally reside, specifically implicates it in the chronic condition of asthma75. A similar trend of upregulated MRGPRX2 has been observed in connective tissue mast cells isolated from patients with chronic urticaria76. Lactic acid and inflammatory conditions Glycolysis is a major metabolic process that provides an initial and immediate source of adenosine triphosphate (ATP) with catabolism of glucose to 3 carbon sugars. Glucose is split to two glyceraldehyde 3-phosphates which require oxidation to proceed with glycolysis; to accomplish this, the complement substrate, a positive ion of nicotinamide adenine dinucleotide (NAD+), is reduced with a hydride (to make NADH). When oxygen is not available to complete the subsequent steps of aerobic respiration, cells may 8 rely on glycolysis for energy and in turn need a replenishing supply of NAD+. The regeneration of NAD+ is another oxidation-reduction reaction of the pyruvate produced at the end of glycolysis and is catalyzed by lactate dehydrogenases. This process of fermentation produces lactic acid. Chronic inflammatory and pathologic conditions can present with ramped up metabolic needs out matching the available oxygen and result in increased lactic acid production by anaerobic respiration. In cancer, cells shift to a glycolytic metabolism, fermenting lactic acid despite aerobic conditions, in a phenomenon known as the Warburg Effect77. Asthma, a condition linked to immune dysregulation, shows higher serum levels of lactate in patients with severe allergen- or exercise-induced asthma78,79. Lactic acid levels have also been linked to outcomes and/or severity in sepsis80, and pulmonary embolism81. The effect of lactic acid on different inflammatory mediators has uncovered its potential role in immune modulation 82,83,84 and it has been shown that lactic acid suppresses the inflammatory functions of macrophages, dendritic cells, and T cells. Mast cell activation and lactic acid As previously mentioned, mast cells act in multiple contexts of immunity coincident with elevated levels of lactic acid. With a more nuanced view of lactic acid acting as a signaling molecule and not just a metabolite, investigation of effects on immune cells has been of recent interest. Exogenous introduction of lactic acid inhibits cytokine production in mast cells stimulated with lipopolysaccharide and in models of sepsis, implying suppressed mast cell activity results in impaired immune function85. Cytokine production induced by IL-33 was also negatively modulated by lactic acid exposure as was phosphorylation of downstream proteins86. The potent, anaphylactic IgE/ FcεR1 mediated activation of mast cells was suppressed by lactic acid incubation. Lactic acid inhibited both early and late phase allergic responses of cultured mast cell and mice treated with lactic acid prior to induction of IgE mediated anaphylaxis showed reduced symptoms of hypothermia87. 9 MRGPRX2 and lactic acid Lactic acid effect on the MRGPRX2 pathway has not been studied despite the aggressive response induced by basic secretagogues. Abnormally high levels of lactic acid in the cancer microenvironment are of special relevance as certain peptides are upregulated in cancerous tissue. Substance P expression is elevated in breast and colorectal cancers88,89. Another MRGPRX2 ligand, LL-37, is associated with lung and ovarian cancers as well as asthma90,91. Given the concurrent expression of MRGPRX2 ligands with elevated lactate levels in inflammatory conditions, the current study was conducted to further explicate the effects of lactic acid on mast cell activity by way of MRGPRX2. MRGPRX2 is no longer truly an orphan receptor but its “promiscuous” nature can be appreciated. To round out this analysis, multiple MRGPRX2 ligands were employed. The range includes at least 2 endogenous and 2 exogenous ligands for the receptor with differing levels of affinity but all 4 have been shown to induce early and late phase responses in mast cells specifically through MRGPRX2. The highest affinity ligand is newly discovered and shown to be extremely specific for MRGPRX2. The structure for this compound, (R)-ZINC 3573, can be viewed in Figure 3 along with the amino acid sequences and structures for the other ligands used in this study. The (R) stereoisomer of ZINC 3573 was found in a drug screen of MRGPRX2 while the (S) stereoisomer was shown to have little affinity in comparison, mast cell responses followed this trend in ligation as expected92. Before continuing the investigation, it was important to determine if mast cells were viable when exposed to lactic acid. Mast cell lines were incubated with lactic acid containing media for 24 hours and viability was assessed with trypan blue counting (Figure 4). Cells remained viable within physiological ranges of lactic acid and so protocols for lactic acid incubation and activity assays were tested and optimized. 10 To investigate the effect of lactic acid on MRGPRX2 mediated responses, in vitro assays specific to MRGPRX2 and mast cells were conducted as well as in vivo experiments assessing inflammation through an analogous receptor. With previous studies demonstrating lactic acid strongly suppresses late phase and anaphylactic responses, it is expected that the pseudo-allergic pathway of mast cell activation will also be negatively regulated by exogenous lactic acid exposure. 11 APPENDIX 12 Figure 1. Lineage and flow chart of mast cell development. 13 Figure 2. Metachromatic staining of primary and cultured murine mast cells. Peritoneal cells were cytospun to glass slides before staining and mounting of cover slips. (A) Primary mast cells collected by peritoneal lavage stained with alcian blue (blue) and safranin O (red). Positive safranin stain indicates serosal type mast cells. (B) Mast cells collected by peritoneal lavage stained with toluidine blue showing violet coloring of granules. Left most panel is an unstimulated mast cell, packed tightly with granules, second panel from the left is likely a mast cell in the process of degranulation. Two rightmost panels are degranulated mast cells making violet staining more apparent. (D) Skin sections of mice were sectioned and fixed to slides before staining with toluidine blue. Left two panels are intact and inactive mast cells, right two panels show less dense staining and individual granules, or granular content, can be discerned indicating degranulation. Scale bar found below right hand side of each panel is 10 μm. 14 Figure 2. (cont’d) Peritoneal mast cells were collected by lavage then cultured with recombinant murine stem cell factor (SCF) and varying concentrations of recombinant murine IL-3. Cells from each culture were then cytospun to glass slides and sequentially stained with alcian blue and safranin O. before mounting of coverslip. (D) PMCs cultured only in the presence of SCF showing serosal phenotype, compare to panel (A). (E) PMCs cultured in SCF and only 1 ng of IL-3 per mL of media stain less safranin, indicating a phenotype closer to mucosal type mast cells. (F) PMCs cultured with SCF and 10 ng IL-3 per mL media showing mostly alcian blue staining indicating a mucosal phenotype. Scale bars found under right hand side of panel is 10 μm. 15 Figure 3. MRGPRX2 ligands employed for experiments. (A) Compound 48/80, a synthetic polymer first known as a histamine liberator and later a basic secretagogue. (B) Substance P, a neuropeptide in nociceptive signaling found to induce mast cell degranulation through the pseudo-allergic pathway. (C) (R)-ZINC 3573, an MRGPRX2, high affinity ligand found through screening drug databases with computer modeling of the receptor. (D) LL-37, a human cathelicidin implicated in multiple inflammatory conditions including mast cell mediated rosacea, shown to activate connective tissue MCs through MRGPRX2. 16 Figure 4. Viability of mast cells in lactic acid concentrations. Human mast cells (LAD2) were incubated in media containing varying concentrations of lactic acid for 24 hours. Living and dead cells were counted by trypan blue staining and percent viability is represented as an average of three trials. 17 CHAPTER 2 LACTIC ACID SUPPRESSES MAST CELL RESPONSE TO MRGPRX2 LIGANDS 18 Introduction Post stimulation, mast cell activity can be described in two phases: early and late. The effect of lactic acid was assessed on both phases through in vitro experiments. Ligation of MRGPRX2, a GPCR, to agonists results in degranulation and production of cytokines to facilitate inflammatory response93.94. Although the naming of these phases implies a time course, the hallmarks of each phase can be measured and observed starting with receptor activation. Early phase response of mast cells is defined by degranulation, a process of vesicular release95. Degranulation is a well-known function of mast cells and--at times--the only function considered in literature. Many processes coordinate the release of vesicular contents, from mobilization of the vesicles to membrane fusion. Histamine and other mediators of inflammation, previously packed in vesicles within the mast cell, enter the extracellular environment and lead to the initial inflammatory response, clinically manifested as anaphylaxis96. MRGPRX2 ligation leads to activation of phospholipase C, an enzyme that splits phosphatidylinositol 4,5- bisphophate (PIP2)97. One of the resulting products, inositol 1,4,5-triphosphate (IP3), triggers the release of calcium from the smooth endoplasmic reticulum to the cytosol. This initial phase of the secondary signal, at this point a calcium modality, itself leads to a further influx of calcium to the cytosol by other mechanisms 94 . The transient result of this cascade is mast cell degranulation. Calcium influx is a requirement for vesicular release and mast cell activation has been characterized calcium dependent even in pathways that do not result in significant degranulation98. As such, tracking the changes in calcium influx to the cytoplasm gives insight into the events of the early phase. Calcium imaging, a powerful technique involving chelating agents with fluorescent properties, allows measurement of influx, post receptor ligation. 19 The late phase of mast cell activation is the period in which inflammatory mediators and cytokines are newly synthesized and released. A variety of chemokines are secreted during the late phase of activation, many of which coordinate recruitment and response from other immune cell types 1. For example, CCL2, otherwise known as MCP-1 or monocyte chemoattractant protein 1, is produced and secreted during the late phase and functions to recruit monocytes as well as other cells expressing the receptor CCR2. This communication by means of cytokines functions to link the innate immune response to adaptive immune responses, highlighting a prominent role for mast cells in immunity. The late phase of mast cells can be observed by assessing RNA expression of protein-based mediators or by testing supernatants for presence of those cytokines. MRGPRX2 is a Mas-related GPCR and has been classified as an orphan receptor with no confirmed ligands. Since its discovery as a GPCR on certain neuron populations, many ligands have been found to activate MRGPRX2, FDA approved drugs and compounds used for mast cell research amongst them66,74. Endogenous ligands have been discovered and demonstrated, meaning MRGPRX2 is now an “adopted” receptor. Substance P, a neuropeptide produced from splice variants and post translational modification of preprotachykinin-1, has been demonstrated to induce degranulation in mast cells through MRGPRX299. LL-37 is a cathelicidin that has also been shown to activate mast cells through MRGPRX2 72. These two endogenous ligands were used for in vitro assays, due to their action of MRGPRX2 as well as their activity with the murine orthologue, MrgprB2. Compound 48/80 is a cationic polymer employed in mast cell research since 1951 due to its capacity to potently release histamine24. Studies of compound 48/80 have elucidated its inflammatory and histamine releasing action as MRGPRX2/MrgprB2 dependent, making it a useful ligand for this investigation and further in vivo studies74. More recently, a drug screening analysis revealed a high affinity compound for MRGPRX2, (R)-ZINC 357392. This compound has been shown to be selective for MRGPRX2 and was thus used for multiple experiments in human mast cells and cell lines. 20 Previous research has investigated the effects of lactic acid on mast cell function. Inhibitory effects of lactic acid have been demonstrated on mast cells stimulated by antigens post IgE sensitization, interleukin 33 (IL- 33), and lipopolysaccharide (LPS). Much of this previous research involved in vitro experiments with human mast cell lines and peritoneal mast cells cultured from mice and focused on cytokine production during the late phase of activity. The ligands for MRGPRX2, a receptor that potently activates human mast cells, reveal a different context for mast cell function and the possible roles lactic acid may play in modulating inflammation. This pseudo allergic pathway does not require sensitization nor exogenous compounds of bacterial origin; ligands for MRGPRX2 include multiple pharmaceuticals and endogenous neuropeptides/peptides. The presence of lactic acid in the body is simply from anaerobic respiration, however, excess lactic acid production is also coincidental with asthma, wound healing, sepsis, and tumor microenvironments77,80. Other conditions of hyperlactatemia and lactic acidosis curiously show aberrant inflammation as well. Many immune cells are involved in the inflammatory process including mast cells which potentially play a first responder role in the context of MRGPRX2. Studies on the effect of lactic acid during immune response (both innate and adaptive) is ongoing, but the MRGPRX2 pathway which is specific to mast cells has yet to be investigated. Regardless of the origin of lactic acid and in which physiological context its presence coincides with mast cells, we decided to investigate MRGPRX2 stimulation with lactic acid treatment. Presence of lactic acid could potentially effect calcium mobilization, vesicular release, or cytokine production. Previous studies show lactic acid having an inhibitory effect on mast cell cytokine production and degranulation. Lactic acid was expected to show a similar pattern of inhibition on early and late phase activation through the MRGPRX2 pathway. 21 Materials and Methods Cells Human embryonic kidney cells (HEK-293T) were transfected with plasmids encoding MRGPRX2 and puromycin resistance under a cellularly active (?) promoter and cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/mL) and streptomycin (100 ug/mL) and L-glutamine (2mM) (PSG) as well as puromycin (3 ug/mL). After multiple passages with strong puromycin selection, the transfected cells were further stained for MRGPRX2 with fluorescent antibodies and sorted for high expression. This high expression set of cells was cultured further with puromycin and saved. HEK- MRGPRX2 transfectants (HMR) were maintained in DMEM supplemented with FBS and PSG along with 3ug/mL puromycin at 40-80% confluency. Laboratory of Allergic Diseases 2 (LAD2) cells are human mast cells commercially available as a cell line and endogenously expresses MRGPRX2. LAD2 cells were cultured in complete Stem-Pro-34 SFM medium containing penicillin (100 IU/mL), streptomycin (100 µg/mL) and L-glutamine (2 mM) (PSG) supplemented with recombinant hSCF (100 ng/mL). Media was hemi-depleted once every week and cells were maintained at a density of 0.8 × 106 cells/mL. Human skin mast cells grown in culture with hSCF (100 ng/ml) for 8 weeks were graciously provided by Dr. Carole A. Oskeritzian from the University of South Carolina. As approved by the Internal Review Board at the University of South Carolina, mast cells were purified from skin biopsies of human breast tissues that were purchased from the Cooperative Human Tissue Network (CHTN) of the National Cancer Institute. Donors were all Caucasian females ranging in age from 40 to 71 years old. Purity of the cultures was assessed by flow cytometry staining for FcεRI expression with PE-labeled anti-human FcεRI antibody [clone AER-37 (CRA)] and mouse IgG2bk isotype control (BioLegend). 22 Calcium assays The number of calcium dyes available is numerous and the development of these agents is an interesting study on its own. For all the experiments investigating lactic acid effects on calcium mobilization post MRGPRX2 ligation, the calcium indicator Fluo-8AM was utilized. Fluo-8AM is a ratiometric dye, as opposed to single wavelength dyes, that emits wavelengths at 520nm when excited with 490nm wavelength light in its activated form—when calcium is present. The “AM” in Fluo-8AM’s name is referring to the acetoxymethyl esters that allow the chelating, fluorescent agent to pass through cell membranes, by way of lipophilicity. Once in the cytoplasm, cell esterases cleave the AM groups from the compound, priming it for calcium binding. When a signal cascade brings calcium ions into the cytosol, those ions bind to the dye and the fluorescent properties, as in the excitation and emission wavelengths, shift. This mechanism of function allows confirmation of when a cell is undergoing a cell process that involves a calcium signal. For the adherent HMR line, the culture was trypsinized and 50,000 cells were plated per well to a 96 well black walled plate in DMEM only. Before plating, the wells were treated with poly-l-lysine and cleaned with PBS twice. 16-18 hours later, media was cleaned off the adherent cells and 4uM Fluo-8AM solution in Siraganian buffer (SIR BSA) (118 mM NaCl, 5 mM KCl, 25 mM HEPES, 5.5 mM glucose, 0.4 mM MgCl2, 1 mM CaCl2, 0.1% w/v bovine serum albumin) was added to the wells. The dye was allowed one hour to enter the cells and for the esterases to cleave the AM groups. The dye was aspirated, and the cells were cleaned twice with SIR BSA before being incubated for 30 minutes with varying concentrations of lactic acid in SIR BSA. After treatment with lactic acid, cells were cleaned twice again and 100 uL of SIR BSA was gently added to each well. At this point the cells contain the calcium dye and have been treated with lactic acid, but the well walls and medium (SIR BSA) do not contain either. Control groups were treated with empty SIR BSA and subjected to the same cleaning steps as lactic acid treated groups. All groups were exposed to the same concentrations of MRGPRX2 ligands prepared in SIR BSA. For calcium mobilization assays with LAD2 cells, cells were cleaned with SIR BSA and incubated in 4uM Fluo-8AM for an hour. After this incubation, cells were spun down, cleaned, and resuspended in lactic acid 23 concentrations prepared in SIR BSA or empty SIR BSA (vehicle) for 30 minutes. Prior to plating, all cells were again centrifuged and cleaned with SIR BSA and resuspended to 0.3x106 cells/mL. 100 uL of the suspensions were plated to the black walled wells, the plate was spun down to place the mast cells at the bottom of the well prior to the FlexStation 3 ® protocol. The ligands were diluted in SIR BSA to a 5X of the desired concentration and loaded to the Flex Station 3 ®. The Flex Station 3 is set to pipette 25 uL of 5X concentrations of compound 48/80, substance P, LL-37 or (R)-ZINC 3573 to the cell plate. The cell plate is loaded, and measurements begin. Excitation wavelengths are sent through the bottom of the well at 490 nm and the desired range of 520 nm emitted wavelengths is measured. The machine can be set up to measure for a certain amount of time, in the case of HMR cells, 120 seconds at 2 second intervals. The length of this period of measurement was determined beforehand with the cells in a non-lactic acid treated control to see when the tracing of the emitted wavelength plateaued. Finally, the output data was represented as maximum relative fluorescent units (RFU) minus minimum RFU within the tracing. With this protocol each well would have its own tracing and max-min value. Control wells were treated with volume matched SIR-BSA in lieu of lactic acid solutions. The transfection of MRGPRX2 proved to be rather stable, control wells showed similar levels of calcium mobilization in each trial. Nonetheless, each trial was done with the same passage of cells to ensure that trend could validly be observed. Degranulation assays Degranulation is a hallmark event of the early phase of mast cell activation and results in anaphylaxis. Granular content is released in a process of vesicular release either by exocytosis or other mechanisms that fuse the cell membrane to vesicle membranes. Preformed mediators act locally to induce vasodilation and inflammation3. Histamine is released potently in this process as well as matrix proteases and many 24 hydrolases. In vitro, the extent of degranulation can be measured by analyzing supernatants of activated mast cells for enzymatic activity. Beta-hexosaminidase is one such hydrolase that mast cells release in degranulation to the extracellular environment100. Incubating supernatants with beta-hexosaminidase’s substrate, p-nitrophenyl-N acetyl-β-D-glucosamine (PNAG), allows for an enzymatic assay which correlates to the amount of enzyme released and therefore, reflects the degree of degranulation. The reaction results in a yellow-colored solution once stopped and is tested for absorbance of 405 nm light. LAD2 cells or primary human skin-derived mast cells or mouse peritoneal mast cell were cleaned with SIR- BSA and incubated with concentrations of lactic acid for 30 minutes. Cells were cleaned with SIR-BSA and resuspended to 20,000 cells per 45μL of SIR-BSA (~0.45 x 106 cells/mL). 20,000 cells were plated in per well in a 96-well plate. Cells were then stimulated with 5μL of 10X concentrations of compound 48/80, substance P, LL-37 or (R)-ZINC 3573 for 30 minutes. The plate was centrifuged to pellet cells and cell- free supernatants (20μL) were collected and incubated with equal volumes of 4mM p-nitrophenyl-Nacetyl- β-D-glucosamine (PNAG, purchased from Sigma-Aldrich) for 1 hour. The assay was halted by 5X volume of 0.1M NaHCO3/0.1M Na2Co3 buffer. Absorbance was measured using FlexStation® 3 multi-mode plate reader (Molecular Devices; San Jose, CA, United States) at 405 nm. The total β-hexosaminidase content was measured by lysing 45μL of cell suspension with 1μL of 0.1% Triton X-100 and then incubating the supernatant of the lysed cells with PNAG for 1 hour. Percent β-hexosaminidase release was calculated by first blanking each absorbance value with the absorbance of SIR-BSA buffer incubated with equal volume of PNAG and quenched with 5X volume of NaHCO3/0.1M Na2Co3 buffer. Blanked values were then divided by the total absorbance value measured from the lysed cell supernatant samples. Cytokine assays The late phase of mast cell activation includes production and release of cytokines that coordinate inflammatory processes and recruit other immune cells to the locality. Mast cells can produce a large variety of cytokines in response to different stimuli. That is to say, the profile of cytokines released can differ 25 depending on the path of activation. However, there are a few cytokines that are classically measured to indicate late phase response. In the case of MRGPRX2, measuring presence of interleukin 8 (IL-8) and monocyte chemoattract protein 1 (MCP-1/CCL2) provides a way to analyze the level of late phase activity of mast cells. LAD2 cells were cleaned twice in cytokine-deprived complete Stem-Pro-34 SFM media and resuspended to 1x106 cells/mL. 150,000 cells were plated per well of a 96 well plate and incubated with different concentrations of lactic acid for 30 minutes. Cells were then stimulated with MRGPRX2 agonists for 6 hours at 37⁰C and 5% CO2. The plate was centrifuged to pellet the cells, and supernatants were collected. Cytokines/chemokines (IL-8 and CCL2) in the supernatants were quantified by ELISA (ELISA kits purchased from Invitrogen). Late phase activity was only assessed for compound 48/80 and substance P, one exogenous ligand and one endogenous ligand. Concentrations of these agonists was kept the same as in the corresponding degranulation assays. 26 Results and Discussion Lactic acid reduces MRGPRX2 mediated calcium mobilization Previous studies show that lactic acid significantly reduced degranulation of mast cells stimulated with IgE/ antigen and cytokine production in mast cells stimulated with IL-3387,86. Degranulation of mast cells and cytokine production are both calcium dependent events, it was pertinent to see the effect of lactic acid on calcium influx initiated by MRGPRX2 stimulation. To begin an isolated assessment of lactic acid on the initial GPCR activity of MRGPRX2, HEK-293T cells were transfected to express the receptor. Confirming functional expression of MRGPRX2 can be revealed with receptor ligation and calcium imaging. As the results indicate, this transfected line—HEK-MRGPRX2 (HMR)—shows an intense increase in fluorescence, a proxy for calcium mobilization in this assay, upon ligation with multiple MRGPRX2 specific ligands. Before addition of MRGPRX2 ligands, cells were treated with concentrations of lactic acid for 30 minutes. Concentrations of lactic acid ranged from 3.125 mM to 12.5 mM to parallel physiologically relevant levels. Lactic acid was not found to be cytotoxic for this range of concentrations (Figure 1). Lactic acid was cleaned off the cells before the assay began and fluorescence was recorded for 120 seconds with data represented as max-min values. Agonist binding to MRGPRX2 induces a flux of calcium into the cytosol from intracellular stores and subsequent extracellular sources through a, mostly, phospholipase C dependent pathway. Calcium ions are chelated into Fluo-8 molecules, changing absorbance and fluorescent properties of the dye, allowing a proxy measurement of calcium presence. Pretreatment of HMR cells with lactic acid significantly reduced max-min values, indicating a suppression of calcium mobilization post MRGPRX2 activation (Figure 3). Controls were recorded for cells treated with empty SIR-BSA and agonists, varying values amongst vehicle controls reflect the differing potencies of these agonists on MRGPRX2. Interestingly, lactic acid suppression of calcium influx was also variable; max-min values were not similarly reduced from vehicle 27 controls for cells treated with specific lactic acid concentrations for all the ligands tested (see values for 8.3mM in Figure 3). Lactic acid treatment reduced MRGPRX2 mediated early phase activation in mast cells HEK cells are not a mast cell line, they do not degranulate or produce the classic cytokines, mediators, or products indicative of mast cell activation. The influx of calcium may result from additional or differing mechanisms in an actual mast cell. Calcium mobilization assays were conducted on LAD2 cells, a human mast cell line that endogenously expresses MRGPRX2101. Previous studies and characterization of LAD2 cells show that MRGPRX2 ligands potently activate degranulation and late phase activity, making the LAD2 line an adequate in vitro model93,94. Protocol was adjusted to accommodate LAD2 cells which are not adherent. In brief, cells were stained with Fluo-8AM and cleaned before and after treatment with lactic acid. Similarly, the assay was not conducted in the presence of lactic acid to not confound results with any effects lactic acid may have on extracellular ligation. As expected, pretreatment of LAD2 cells with lactic acid significantly reduced calcium mobilization as measured by a Fluo-8AM assay (Figure 6). The trend of reduction with increasing concentrations of lactic acid was more consistent in the LAD2 data as compared to HMR calcium mobilization data. Degranulation of LAD2 cells is reduced with lactic acid treatment Pretreatment of LAD2 cells with lactic acid concentrations ranging from 3.1 mM to 12.5 mM reduced fluorometrically measured calcium mobilization in a dose dependent manner (Figure 6). Following calcium influx, mast cells exocytose preformed, high density granules containing inflammatory mediators, various proteases, and lysosomal hydrolases in a process defined as degranulation. Substance P, compound 48/80, LL-37, and (R)-ZINC 3573, induce calcium mobilization and β- hexosaminidase release in LAD2 cells independent of the IgE-allergic pathway through MRGPRX2. 28 Degranulation of MRGPRX2 ligated LAD2 cells, evaluated by β-hexosaminidase enzymatic assay, was reduced in a dose dependent manner with lactic acid pretreatment (Figure 7). Profiles of the lactic acid mediated reduction in β-hexosaminidase release did not exactly match the profiles in lactic acid mediated reduction in calcium mobilization in LAD2 cells. For example, fluorescence measurements of LAD2 cells exposed to substance P were significantly reduced from the vehicle treated control at pretreatment concentrations of 10.4 mM and 12.5 mM lactic acid, yet degranulation response was significantly reduced for cells treated with 6.3 mM lactic acid (Figure 6B, Figure 7B). Though assessment of both early phase activation events in LAD2 cells show significant reduction with lactic acid incubation, the discrepancy in trends indicates lactic acid effects may be multipronged. MRGPRX2-mediated degranulation of primary skin-derived human mast cells is reduced by lactic acid Connective tissue mast cells (CTMCs), a subpopulation of mast cells in humans, express MRGPRX2 and reside in dermal tissue amongst others68. CTMCs are truly known as “sentinels” of the immune system and potentiate immune response. Skin biopsies from 4 female, Caucasian donors ranging from 40-71 years old were digested and sorted for mast cells. These primary cells were cultured with recombinant human stem cell factor to proliferate mature CTMCs. Cells were treated with or without lactic acid (8.3 mM), and exposed to MRGPRX2 ligands (compound 48/80, substance P, LL-37 and (R)-ZINC 3573) for degranulation assays. We observed differences in percent degranulation for each ligand between donor samples (Figure 8). Four different forms of MRGPRX2 have been reported in humans and the variation in degranulation between donors possibly reflects this finding102. Regardless, lactic acid treatment reduced the degranulation of skin mast cells obtained from all donors to the MRGPRX2 agonists (Figure 8). 29 Lactic acid suppresses late phase response in MRGPRX2 stimulated mast cells Mast cell activation not only comprises of early events that include intracellular Ca2+ mobilization and degranulation but also includes a delayed phase that ultimately results in inflammatory chemokine/cytokine production and their release2. The late phase of mast cell activation consists of chemokine/cytokine secretion and, like degranulation, is calcium dependent. In this phase, other immune cells are recruited to the site of inflammation by mast cell derived messenger molecules. To test whether lactic acid regulated the longer standing response of mast cell activation, we exposed LAD2 cells to varying concentrations of lactic acid and assessed for chemokine and cytokine generation by ELISA. We specifically chose to examine the production of CCL2 and IL-8 since LAD2 cells release these effectors upon MRGPRX2 stimulation. LAD2 cells produced both CCL2 and IL-8 on stimulation with compound 48/80 (Figure 9A) and substance P (Figure 9B). Interestingly, lactic acid treatment inhibited the release of these inflammatory mediators. 30 APPENDIX 31 Figure 5. Fluorescence measured in ligand exposed HEK 293T-MRGPRX2 cells is reduced by pretreatment with lactic acid. HEK-293T cells expressing MRGPRX2 were preincubated with calcium dye and then treated with vehicle (SIR buffer) or lactic acid (indicated concentrations) for 30 min. prior to being cleaned and exposed to MRGPRX2 ligands. (A-D) Ca2+ mobilization assays were performed following incubation with (A) compound 48/80 (300 ng/ml), (B) substance P (100 nM), (C) LL-37 (0.5 μM) or (D) (R)-ZINC 3573 (0.5 μM). 32 Figure 5. (cont’d) Data are plotted as the change in fluorescence intensity [minimum (Min) subtracted from maximum (Max) value] measurements. Results shown are mean ± S.E. of 3 independent experiments. Statistical significance was determined by unpaired Student’s t-test and values from the lactic acid-treated group was compared with the vehicle group. * p < 0.05, ** p < 0.01 and *** p < 0.001. 33 Figure 6. MRGPRX2 mediated Calcium mobilization is reduced in lactic acid treated mast cells. (A- D) Intracellular Ca2+ mobilization as measured by fluorescence in LAD2, human mast cells was determined following pre-incubation with vehicle (SIR BSA) or concentrations of lactic acid (indicated) for 30 min. Mast cells were exposed to MRGPRX2 agonists (A) compound 48/80 (300 ng/ml), (B) substance P (300 nM), (C) LL-37 (3 μM) or (D) (R)-ZINC 3573 (2 μM) and changes in fluorescence intensities were recorded for 90 sec. 34 Figure 6. (cont’d) Data are plotted as the change in fluorescence intensity values following ligand additions. Data shown are mean ± S.E. of 3 independent experiments. Statistical significance was determined by unpaired Student’s t-test with values compared between the lactic acid- and vehicle-treated groups. * p < 0.05, ** p < 0.01 and *** p < 0.001. 35 Figure 7. Early phase, MRGPRX2 mediated degranulation in LAD2 mast cells was significantly attenuated in a dose dependent manner with lactic acid pretreatment. Vehicle- or lactic acid-treated cells were exposed to (A) compound 48/80 (300 ng/ml), (B) substance P (300 nM), (C) LL-37 (3 μM) or (D) (R)-ZINC 3573 (2 μM). Degranulation was assessed by β-hexosaminidase release. Values are plotted as percentages of total cell lysate β-hexosaminidase content. 36 Figure 7. (cont’d) Data shown are mean ± S.E. of 3 independent experiments. Statistical significance was determined by unpaired Student’s t-test with values compared between the lactic acid- and vehicle-treated groups. * p < 0.05, ** p < 0.01 and *** p < 0.001. 37 Figure 8. Lactic acid inhibits MRGPRX2 mediated degranulation in primary human skin mast cells. Human skin-derived mast cells from 4 different donors were pre-treated with vehicle (SIR buffer) or lactic acid (8.3 mM and 10.4 mM) and exposed to compound 48/80 (1000 ng/ml), substance P (1 μM), LL-37 (5 μM) or (R)-ZINC 3573 (10 μM). 38 Figure 8. (cont’d) Line graphs show degranulation of mast cells from different donors as estimated by β-hexosaminidase release in supernatants. Mast cells were derived from breast-skin biopsies of Caucasian women obtained from augmentation procedures. (A) Donor 401, age: 40 yrs. old, (B) Donor 510, age: 52 yrs. old, (C) Donor 611, age: 71 yrs. old, (D) Donor 650, age: 55 yrs. old. Data is represented as means of two trials ± SD. 39 Figure 9. Lactic acid suppresses cytokine/chemokine production LAD2 cells exposed to MRGPRX2 ligands. (A-D) LAD2 cells (0.15 × 106 cells/well) were exposed to vehicle (SIR buffer) or different concentrations of lactic acid for 30 min, washed and exposed to, (A, B) compound 48/80 (300 ng/ml) or (C, D) substance P (300 nM) for 6 h. Supernatants were collected and analyzed for (A, C) MCP-1 and (B, D) IL-8 by ELISA. Data shown are mean ± S.E. of 3 independent experiments, each performed in triplicates. Statistical analysis was done using unpaired Student’s t-test by comparing the vehicle- and lactic acid- treated groups. * p < 0.05, ** p < 0.01 and *** p < 0.001. 40 CHAPTER 3 MRGPRB2/MAST CELL MEDIATED INFLAMMATION IS REDUCED WITH LACTIC ACID TREATMENT IN VIVO 41 Introduction The murine analog of MRGPRX2, MrgprB2, was discovered in tandem and is also a mas-related orphan GPCR66. CTMCs and serosal tissue mast cells in mice selectively express MrgprB2 and are activated by MRGPRX2 ligands but usually at higher EC50 concentrations. Mast cells from MrgprB2 knockout mice do not degranulate or activate with exposure to basic secretagogues nor do the mice exhibit inflammatory reactions with exposure74. Therefore, murine models focused on MrgprB2 in mast cells and inflammation serve as an adequate analog to MRGPRX2 mediated inflammation. Intraperitoneal injection of compound 48/80 induces substantial histamine release from mast cells and anaphylaxis in mice. With the strong vasodilatory response comes a drop in body temperature that can be measured with rectal thermometers. Passive systemic anaphylaxis in mice induced by MRGPRX2/MrgprB2 ligands presents an acute model of mast cell mediated responses. Previous experiments with compound 48/80 on wild type mice show a two-hour time course of temperature fluctuating with lowest measurements occurring within 30 minutes (data not shown). Rosacea is an atopic inflammation of the skin resulting in swelling, redness, and thickened dermal tissue. Recently, the mechanism of rosacea has been proven to be mast cell mediated and induced by presence of the cathelicidin, LL-37—a potent MRGPRX2 ligand72. Rosacea symptoms in human skin were accompanied by high levels of LL-37 and serine proteases involved in cleaving precursors to form the cathelicidin73. Mast cell deficient mice do not develop inflammation and rosacea when exposed to subdermal LL-37103. An LL-37 induced rosacea model in mice consists of multiple subdermal injections of LL-37 followed by histology and analysis of the inflamed tissue. It was important to use this model not only for its dependance on mast cells and MrgprB2/LL-37 but also because it represents a chronic condition and lasting state of inflammation. In essence, the acute passive systemic anaphylaxis model parallels early 42 phase activation as seen in our degranulation experiments while the LL-37 induced rosacea model aligns more with late phase activity resulting from cytokine production and action. 43 Materials and Methods Mice Balb/c and C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME, United States). All mice were kept under specific pathogen-free conditions. All experiments had the approval of the Institutional Animal Care and Use Committee at Michigan State University. Both male and female mice (6-8 weeks old) were used for experiments. Passive systemic anaphylaxis (PSA) C57BL/6J mice were first injected i.p with lactic acid (125 mg/kg, dissolved in PBS) or PBS alone. Sixteen hours later, mice received i.p. injections of compound 48/80 (3.75 mg/kg) to induce anaphylaxis. Core body temperature was measured using a rectal thermometer probe (Physitemp Instruments, Clifton, NJ). Ex vivo culture of peritoneal mast cells Mouse peritoneal mast cells were cultured separately from peritoneal lavages of C57BL/6 mice at 6 to 8 weeks of age. Lavages were centrifuged and cell pellets were washed with RPMI media; cells were collected and cultured in RPMI media supplemented with 10% FBS, PSG and murine stem cell factor (mSCF) at 10 ng/ml for 3 weeks. Expansion of serosal mast cells was confirmed by heterochromatic staining with Safranin O and Alcian Blue dyes. Cathelicidin LL-37 induced rosacea model Balb/c mice received intraperitoneal (i.p.) injections of vehicle (PBS) or lactic acid (4mg/kg dosed with 4% w/v concentration prepared in PBS) for 3 days. Intradermal injections (i.d.) of LL-37 (50 µL of 320 µM) was administered to the dorsal skin twice a day for 2 days while continuing the lactic acid treatment. On the third day, mice were euthanized and dorsal skin at the site of the injections was excised. Dorsal skin per sample was divided so that half could be snap-frozen in liquid N2 for mRNA analysis and half could be fixed in 10% formalin solution for H&E or Toluidine Blue staining. 44 Epidermal thickness 10% formalin fixed skin samples were cut, fixed to slides, and stained with hematoxylin and eosin (H&E) by the Investigative HistoPathology Laboratory at Michigan State University. Briefly, tissue samples were fixed in paraffin wax blocks then transversely sectioned and set on to glass slides. Slides were then cleared of wax before staining and mounting of coverslips. 10 random, but spread out, epidermal areas in H&E stained skin sections from each mouse were chosen and measured following the acquisition of images using a Nikon ® ECLIPSE 50i microscope equipped with a Lumenera ® Infinity 3 color camera. Measurements were made using Image J software calibrated to micrometer images using the same microscope at the same objective (10X). Degranulated mast cell count in dorsal skin For assessing in vivo mast cell degranulation, 10% formalin fixed skin tissues were set in paraffin wax and transversely sectioned as previously described. Sections on glass slides were de-paraffinized prior to staining with toluidine blue (0.1% in PBS, pH 2.3).. Degranulated mast cells (as determined by the staining intensity, appearance and/or location of the granules) were counted and expressed as percentage of total mast cells in the total tissue section under 40X view. Representative images were captured as described above at 40X objective, Image J software was used to add a 25 μm scale bar. mRNA analysis of dermal site of inflammation Dorsal skin samples taken from mice were frozen in liquid N2 and were mechanically disrupted using a mortar and pestle before further disruption with a rotor-stator homogenizer. RNA was extracted using TRIzolTM reagent according to the manufacturer’s protocol. RNA (2 μg) was transcribed to cDNA using the high capacity cDNA reverse transcription kit from Applied Biosystems (Foster City, CA, United States). RNA levels (Ccl2, Il6, Tnf, Mmp9, Tpsab1 and Cma1) were quantified using gene expression assays with TaqMan™ Fast Advanced Master Mix and validated TaqMan™ probes obtained from Applied Biosystems. 45 Results and Discussion Lactic acid administration reduced MrgprB2/MC dependent anaphylaxis in vivo and ex vivo MrgprB2 is the murine GPCR that is activated by basic secretagogues and is expressed on certain populations of mast cells corresponding to human CTMCs. Given the strikingly similar expression profile and agonists, MrgprB2 is considered the mouse counterpart of the human MRGPRX274. Compound 48/80, substance P, and LL-37 have been previously shown to induce pseudo-allergic phenotypes in animal models, specifically, murine models of inflammation. To observe the role of lactic acid in regulating MrgprB2 mediated responses in vivo, a previously described model of compound 48/80- induced systemic anaphylaxis was adopted. This model depends on peritoneal mast cell expression of MrgprB2. Lactic acid was dosed intraperitoneally at 125 mg/kg, 18 hours prior to compound 48/80 exposure. Lactic acid and compound 48/80 solutions were prepared in phosphate buffered saline (PBS). Control-vehicle (PBS) or lactic acid-treated mice were injected with compound 48/80 (i.p., 3.75mg/kg) and core body temperature was measured rectally every 10 minutes for 120 minutes. Mice dosed with the PBS vehicle 18 hours prior to induction of anaphylaxis showed a drastic decrease in body temperature with recovery beginning at 40 minutes (Figure 10; solid circles). However, mice dosed with lactic acid showed significantly higher body temperatures and began to recover within 20 minutes (Figure 10; open circles) of compound 48/80 exposure. Both groups showed a decrease in body temperature by 2⁰C for the 10-minute timepoint but a difference in recovery from anaphylactic insult suggesting lactic acid pretreatment modified peritoneal mast cell activation. To assess this contention directly, peritoneal mast cells were collected and cultured ex vivo for degranulation assays as previously described for LAD2 cells and primary human skin mast cells. Compound 48/80 was tested as well as the MRGPRX2/MrgprB2 ligands, substance P and LL-37 on PMCs pretreated 46 with lactic acid. As expected, peritoneal mast cells degranulated in the presence of these ligands, but this response was significantly reduced when the cells were pre-exposed to lactic acid (Figure 11). LL-37 induced rosacea is diminished with lactic acid treatment in a chronic, murine model of inflammation Human cathelicidin, LL-37, is an MRGPRX2 ligand strongly linked to the pathogenesis of rosacea, a chronic inflammatory condition of the skin73. Curiously, LL-37, while being endogenous to humans is also a potent ligand for MrgprB272, the murine orthologue of MRGPRX2 and as such has been used in murine models studying rosacea and mast cell mediated inflammation. Mice deficient in mast cells do not show rosacea or rosacea-like inflammation when exposed to LL-37 further implicating mast cell activation as a component103. LL-37 exposure induced degranulation and Ca+2 mobilization in human mast cell lines that were reduced with lactic acid pretreatment (Figure 6D, Figure 7D). Lactic acid also had a substantial suppressive effect on compound 48/80 induced PSA/hypothermia and MRGPRX2 ligand induced degranulation of PMCs (Figure 10, Figure 11). To test the effect of lactic acid on a more chronic model of inflammation analogous to a human condition, Balb/c mice were treated with vehicle (PBS) or lactic acid intraperitoneally during a 3-day period that also included multiple subdermal exposures to LL-37. H&E staining of skin sections from the site of inflammation show markedly reduced trans-epidermal thickness in the lactic acid treated cohort (Figure 12B). Mice treated with lactic acid also showed reduced erythema at the site of LL-37 injection with less notable lesions compared to vehicle treated mice. Lactic acid was dosed by intraperitoneal injection to reach systemic circulation before being cleared by physiological mechanisms involved in maintaining homeostatic levels. The data suggests that lactic acid presence significantly reduces rosacea symptoms induced by LL-37. 47 To assess whether lactic acid attenuation of rosacea was due to mast cell activity, numbers of degranulated and non-degranulated mast cells from dorsal skin samples were enumerated. Dorsal skin sections were stained with toluidine blue, a cationic dye that binds to heparin found in mast cell granules. Degranulated mast cells and total mast cells in the full sections were counted manually with microscopy. Degranulated mast cells show less intense staining and undefined borders while granulated mast cells are densely stained violet and show compact, clean borders. Representative images of degranulated and granulated mast cells from these sections are shown (Figure 13A-B). Results reflect lactic acid reduction of mast cell mediated rosacea; while there was no difference in the total numbers of mast cells between the vehicle and the lactic acid-treated cohorts of mice, the percentage of degranulated mast cells was significantly reduced in the lactic acid-exposed mice as compared to the control vehicle-treated group (Figure 13C). RNA analysis of skin tissues further suggests lactic acid modulation of mast cell activity as the primary component of reduced rosacea symptoms. Specifically, mRNA levels of mast cell inflammatory mediators such as CCL2, IL-6, TNF and MMP9 were significantly decreased in the skin tissues of lactic acid treated mice (Figure 14A-D). Furthermore, classical markers of mast cell activation, TPSAB1 (tryptase α-1 and tryptase β-1) and CMA1 (chymase) were also significantly reduced in the lactic acid treated group compared to the vehicle treated group (Figure 14E-F). 48 APPENDIX 49 Figure 10. Lactic acid reduces systemic anaphylaxis to compound 48/80 in vivo. Vehicle- (PBS, volume matched) or lactic acid- (125 mg/kg by 0.1388 M lactic acid in PBS) treated (intraperitoneal injection 18 hours prior) C57BL/6 mice were exposed to compound 48/80, (C48/80, 3.75 mg/kg) and rectal temperature was measured at different time points. Line graph shows change in rectal temperature for the different groups of mice. Data shown are mean ± S.E. from 3 experiments (a total of n = 10 mice/group). Statistical significance was determined by Student’s t-test. 50 Figure 11. Peritoneal derived mast cells showed significantly inhibited MrgprB2 mediated degranulation to MRGPRX2 ligands with lactic acid pretreatment. (A-C) Mouse peritoneal mast cells, cultured from peritoneal lavages, were exposed to vehicle (SIR BSA) or lactic acid (8.3 mM) and stimulated with the indicated concentrations of (A) compound 48/80, (B) substance P or (C) LL-37. 51 Figure 11. (cont’d) Graphs shows degranulation of mast cells as estimated by β-hexosaminidase release in supernatants. Data shown are mean ± S.E. from 3 experiments (a total of n = 3–5 mice/group). Statistical significance was determined by unpaired Student’s t-test. *** p < 0.001. 52 Figure 12. Lactic acid prevents thickening of the epidermis in an LL-37 induced rosacea model. Vehicle- (LL-37/Vehicle) or lactic acid (LL-37/Lactic acid) treated Balb/c mice were injected with LL-37 into the dorsal skin twice daily for 2 consecutive days. Control mice received PBS. (A) H&E stained skin sections of mice from different cohorts are shown. Scale bar = 100 μm. (B) Graph represents epidermal thickness of the H&E stained skin sections as measured by Image J. Data are mean ± S.E. from n = 3–9 mice/group. Statistical significance was determined by unpaired Student’s t-test comparing the vehicle- vs lactic acid-treated groups. * p < 0.05 and ** p < 0.01. 53 Figure 13. Lactic acid treated mice in LL-37 induced rosacea model showed a significantly smaller number of degranulated mast cells than the vehicle treated cohort. 54 Figure 13. (cont’d) The paraffin embedded skin sections from different cohorts of mice were stained with toluidine blue to detect mast cells. (A) Representative pictures of the skin sections are shown. The inset figure is an enlarged image of the mast cell(s) shown in the pictures. Scale bar = 25 μm. Bold closed arrowheads indicate intact mast cells whereas open arrowheads represent degranulated mast cells. (B) Enlarged representative images of degranulated and granulated mast cells in skin sections with toluidine blue staining. (C) Graph shows the percentage of degranulated mast cells in the skin tissue of different cohorts of mice. Data are mean ± S.E. from n = 3–9 mice/group. Statistical significance was determined by unpaired Student’s t-test comparing the vehicle- vs lactic acid-treated groups. * p < 0.05 and ** p < 0.01. 55 Figure 14. mRNA expression of mast cell derived products post LL-37 injection was reduced in mice treated with lactic acid. 56 Figure 14. (cont’d) (A-F) mRNA expression of selected gene targets from the excised skin was analyzed by real-time PCR. Values are plotted as fold change (2-ΔΔCt) normalized to GAPDH levels. Data are mean ± S.E. from n = 3–9 mice/group. Statistical significance was determined by unpaired Student’s t-test comparing the vehicle- vs lactic acid-treated groups. * p < 0.05 and ** p < 0.01. 57 CHAPTER 4 SUPPRESSION OF MRGPRX2 MEDIATED MAST CELL RESPONSES BY CHEMICAL MOEITES OF LACTIC ACID 58 Introduction The low pH solutions used in the in vitro experiments were suspected to simply kill mast cells. Viability assays of LAD2 cells show survival without significant cell death in lactic acid concentrations (2.5 mM to 12.5 mM) whether prepared in buffering culture mediums or in SIR BSA. In addition to this finding, lactic acid buffers were cleaned off the cells before exposure to MRGPRX2 ligands for in vitro assays yet resulted in dose dependent reduction of calcium mobilization, degranulation, and cytokine release (Figures 6-9). Exactly how lactic acid functions to downregulate these responses is unclear and the following experiments were designed to begin elucidating the mechanism behind this downregulation. The exact intracellular events occurring during mast cell activation with basic secretagogues are not entirely understood but certain defining points of protein activity have been explicated. Given the data collected, lactic acid should exert some effect on these events. MRGPRX2 activation leads to downstream phosphorylation of extracellular signal regulated kinases 1 and 2 (ERK 1/2), members of the mitogen activated protein kinase (MAPK) family. Other intracellular signaling events downstream of MRGPRX2 ligation includes phosphorylation of phosphatidylinositol (3,4)- biphosphate (PIP2) to phosphatidylinositol 104 (3,4,5)- triphosphate by phosphoinositide 3-kinase (PI3K) at the plasma membrane . Protein kinase B, also known as Akt, binds to PIP3 resulting in conformational changes that allow phosphorylation. In the MRGPRX2 pathway, MAPK and PI3K are thought to be activated by Gαi proteins, making downstream phosphorylation of Akt and ERK 1/2 hallmarks of MRGPRX2 mediated mast cell activation105. Furthermore, inhibitors for PI3K and ERK 1/2 demonstrate nearly abolished degranulation of LAD2 cells stimulated with compound 48/80 or substance P106. In solution, lactic acid presents two components that may greatly affect mast cells: an acidic proton and a lactate anion. A recent report by Abebayehu et al., implicated the acidic proton of lactic acid in inhibiting cytokine release of mast cells following allergic, IgE/antigen stimulation. Treating mast cells with sodium lactate solutions confirmed the inhibition as pH dependent—cytokine release of mast cells treated with 59 sodium lactate matched that of vehicle controls87. Similarly, to parse out the effect of the lactate anion on MRGPRX2 mediated responses, concentration matched solutions of sodium lactate in SIR BSA were prepared. To extrapolate the effects of acidic moiety without presence of the lactate anion, HCl was added to SIR BSA until a pH matching that of lactic acid solutions was reached. 60 Results and Discussion Lactic acid exposure did not reduce phosphorylation of pathways downstream of MRGPRX2 ligation LAD2 cells were treated with 8.33 mM lactic acid in SIR BSA, the same concentration that significantly reduced calcium mobilization and degranulation post MRGPRX2 ligation with compound 48/80 (Figure 6A, Figure 7A). As with the in vitro assays, lactic acid was cleaned off the cells before addition of compound 48/80 to a final concentration of 300 ng/mL. Stimulation was halted with ice cold PBS and cells were collected at time points 0, 10, and 30 minutes before lysing and preparation of Western Blot samples. Each lactic acid time point was accompanied by a vehicle control that was pretreated with empty SIR BSA. Probing for B-actin was performed as a loading control (not shown). Probing for p-Akt, p-ERK 1/2, Akt (total Akt, T-Akt) and ERK 1/2 (total ERK 1/2, T-ERK 1/2) was performed to assess levels of phosphorylation and to serve as further loading controls (Figure 15). Curiously, no difference was discernable between the time points for vehicle treated controls and the lactic acid treated groups (Figure 15). This is in stark contrast to previous studies showing that agents reducing MRGPRX2 mediated activation are coincident with downregulated phosphorylation of ERK 1/2 and Akt 93,94. Lactic acid reduced of MRGPRX2 mediated responses largely independent of the lactate anion Lactic acid presents two chemical moieties that possibly play a role in suppression of mast cell response: an acidic proton and the lactate anion. To assess the effects of the lactate anion, matching sodium lactate concentrations were prepared for in vitro assays. The protocol for degranulations used previously was maintained for these assays, except that lactic acid and sodium lactate concentrations ranged from 5 to 10 mM. Otherwise, as before, LAD2 cells were treated with buffers for 30 minutes and cleaned with empty SIR BSA before exposure to MRGPRX2 ligands. Sodium lactate did not inhibit LAD2 degranulation as robustly as the matched lactic acid conditions when stimulated with compound 48/80 (Figure 16A) or substance P (Figure 16B). Interestingly, sodium lactate did significantly inhibit degranulation at 5, 7.5 and 10 mM compared to the control (Figure 16; white bars), 61 indicating that the lactate anion itself also plays a role in suppressing the MRGPRX2 pathway. Clearly, lactic acid suppression of MRGPRX2 mediated responses requires the presence of acidic protons. To further support this, pH of sodium lactate concentrations in SIR BSA from 5 to 10 mM ranged from 6.95 to 6.96 while SIR BSA on its own averaged a pH of 6.94 (data not shown). In contrast to degranulation assays with compound 48/80 or substance P on LAD2 cells, release of IL-8 and CCL2 due to MRGPRX2 activation was mostly unaffected by treatment with sodium lactate (Figure 17). Levels of IL-8 were significantly reduced in LAD2 cells treated with sodium lactate and exposed to compound 48/80 in what seems like a dose dependent trend (Figure 17B; white bars). This finding is incongruous with the release of CCL2 from LAD2 cells exposed to compound 48/80 (Figure 17A), which is significantly reduced in a dose dependent manner with lactic acid treatment but not at all by treatment with sodium lactate. Degranulation of mast cells exposed to compound 48/80 was only partially reduced by acidic buffers To further test whether acidity of lactic acid concentrations suppresses early phase responses to MRGPRX2 ligation, SIR BSA was pH matched to lactic acid buffers with concentrated HCl. These buffers were used as a pretreatment condition as described for in vitro assays performed earlier. 7.5 mM lactic acid was prepared in SIR BSA and pH was recorded with a bench top pH meter, the average of 6 trials showed a pH of about 4.76 (data not shown). For each trial, a pH matching buffer of SIR BSA was prepared with HCl— the pH average of the matched buffers was 4.78 (data not shown). Degranulation of LAD2 cells pretreated with these buffers for 30 minutes was performed using compound 48/80 to activate the mast cells through MRGPRX2. 7.5 mM lactic acid strongly reduced β-hexosaminidase release as expected, reproducing the previously found trend (Figure 7, Figure 16A; black bars). Sodium lactate minimally reduced degranulation of mast cells activated with compound 48/80 presumably due to lack of acidity (Figure 16A; white bars). However, degranulation of cells treated with pH-matched SIR 62 BSA was not reduced to the same level as cells treated with 7.5 mM lactic acid (Figure 18). This data indicates that lactic acid suppression of MRGPRX2 early phase responses depends on the moieties presented by both the acidic proton and the lactate anion. 63 APPENDIX 64 Figure 15. Phosphorylation events downstream of MRGPRX2 ligation was not reduced in LAD2 mast cells pretreated with lactic acid. Western blot of p-ERK 1/2 and p-AKT lysates from LAD2 cells treated with vehicle (SIR buffer) or lactic acid (8.3 mM) and then exposed compound 48/80 (300 ng/ml) for different time intervals. Western blotting was performed to detect phosphorylated ERK1/2 (p-ERK1/2) or AKT (p-AKT) proteins. The blots probed with ERK1/2 (T-ERK1/2) and AKT (T-AKT) antibodies for loading controls. Images of representative blots from 5 independent experiments are shown. 65 Figure 16. Lactic acid modulation of MRGPRX2 mediated degranulation of LAD2 mast cells is dependent on proton moiety. LAD2 cells were exposed to vehicle (SIR BSA, control) or varying concentrations of lactic acid or sodium lactate for 30 mins. The cells were washed and stimulated with the MRGPRX2 agonist (A) compound 48/80 (300 ng/ml), (B) substance P (300 nM). Degranulation was quantified by β-hexosaminidase release. 66 Figure 16. (cont’d) Values are plotted as percentages of total cell lysate β-hexosaminidase content. Data shown are mean ± S.E. of 3 independent experiments. Statistical significance was determined by unpaired Student’s t-test with values. *** p < 0.001 indicates values compared between the lactic acid- and vehicle-treated groups. ††† p < 0.001 indicates values compared between the sodium lactate- and vehicle-treated groups. 67 Figure 17. Sodium lactate has no effect on chemokine/cytokine production in LAD2 cells. LAD2 cells were exposed to vehicle (SIR buffer, indicated by “0″) or different concentrations of lactic acid or sodium lactate, washed and exposed to, (A, B) compound 48/80 (300 ng/ml) or (C, D) substance P (300 nM) for 6 h. Supernatants were collected and analyzed for (A, C) MCP-1 and (B, D) IL-8 by ELISA. Data shown are mean ± S.E. of 4 independent experiments performed in triplicates. 68 Figure 17. (cont’d) Statistical analysis was done using unpaired Student’s t-test by comparing the control vehicle- and lactic acid-treated groups. *** p < 0.001 indicates values compared between the lactic acid- and vehicle-treated groups. † p < 0.05 and ††† p < 0.001 indicates values compared between the sodium lactate- and vehicle- treated groups. 69 Figure 18. Acidic buffer partially reduced MRGPRX2 mediated degranulation of LAD2 mast cells. SIR BSA was acidified with HCl to match the pH of 7.5 mM (pH 4.76-4.78) and incubated with LAD2 cells for 30 min. before exposure to compound 48/80 (300ng/mL). Degranulation was quantified by β- hexosaminidase release. Values are plotted as percentages of total cell lysate β-hexosaminidase content. Data shown are mean ± S.E. of 6 independent experiments. Statistical significance was determined by unpaired Student’s t-test with values. *** p < 0.001 indicates values compared between each condition. 70 CHAPTER 5 CONCLUSION 71 Conclusion The exact role of lactic acid in inflammatory response is complicated, as demonstrated by this research. Lactic acid effects on other immune cell types can be anti- or proinflammatory depending on the cell type and stage of immune response. For instance, studies of lactic acid in the asthmatic condition show a proinflammatory effect with upregulated T-cell proliferation and activation 79. Coincidence of lactic acid with pathophysiology in any case, should not be overlooked, as a metabolite resulting from anaerobic respiration it is unlikely to be a simple bystander in physiological processes. In this research, lactic acid has been exogenously applied to mast cells in effort to elucidate its potential role in innate immune response. However, the term exogenous is used in reference to mast cells themselves, the implications of this research extend to mast cells in high lactic acid tissue environments but also to possible pharmacological interventions. Lactic acid as a possible therapeutic for LL-37 associated rosacea The LL-37 induced rosacea model showed significant reduction in inflammatory response when mice were treated with lactic acid (Figure 12-13). Further investigation showed that mast cell activity was reduced as the expression of mast cell derived mediators (Figure 14). Rosacea is not just a pathology used in rodent models but in fact is a true skin condition afflicting humans. Lactic acid was injected intraperitoneally to the mice in order to flush through systemically and hopefully effect tissue that was being assaulted by exposure to LL-37. Repeating the rosacea induced model with topical application of lactic acid and examining the effect on MrgprB2 mediated mast cell responses could lead to a direct, real-world application for reducing LL-37/MRGPRX2 mediated rosacea. Lactic acid is currently already employed as a topical agent and has shown potentially positive cosmetic effects107. This study shows it could also, potentially, be employed as a therapeutic for inflammatory conditions, however, it is unclear if topical application will 72 penetrate dermal tissue enough to effect mast cells. Further investigations of local application of lactic acid are necessary, whether that is by topical administration or subdermal injection local to site of pathology. MRGPRX2-substance P-cancer axis The Warburg effect is a well-known physiological attribute of cancerous tissue, where glycolysis is preferred even in aerobic conditions and results in excessive lactic acid production77. The role mast cells play in cancerous tissue is not well characterized and is a point of debate. Mast cells may play a largely inflammatory role that helps cancerous tissue progress, there are even suggestions that circulating immature mast cells are recruited to cancerous sites by cancer derived Kit factor (SCF), a protein that plays a large role in mast cell development108. Mast cells have recently been implicated in pancreatic tumor suppression and shown to infiltrate and reside inside tumor masses109. The theory of a mast cell-cancer tissue axis is further complicated by this study, as certain cancer tissue and microenvironments involve production of substance P, an MRGPRX2 ligand88,89. Mast cells may be stimulated by tumor microenvironment substance P to release inflammatory mediators but also display suppressed production of cytokines and proteases by lactic acid effects. Clearly, lactic acid downregulates proinflammatory, MRGPRX2 mediated mast cell responses examined in this study. It would be interesting to see the effect of lactic acid on mast cell derived, proangiogenic or growth factors such as vascular endothelial factor, nerve growth factor, or platelet derived growth factor110. Furthermore, other immune cells called into action by mast cells that may aid in protecting the host from progression of cancer may also be inhibited by lactic acid found in the tumor microenvironment111. Further investigation is needed to understand the effects of cancer derived factors and lactic acid on MRGPRX2 mediated mast cell response. Possible mechanism of suppression by lactic acid The mechanism of lactic acid inhibition of MRGPRX2 stimulated responses, both late and early phase, is still elusive. This current study proves some contrasting elements of lactic acid’s role. Intracellular signaling 73 of ERK 1/2 and AKT is not downregulated with mast cell response yet early and late phase responses to MRGPRX2 ligands is definitively suppressed (Figures 6-9, Figure 15). These kinases may be activated as part of another pathway induced by lactic acid however this is yet to be demonstrated and analysis of LAD2 lysates not stimulated with MRGPRX2 ligands but pretreated with lactic acid concentrations show that phosphorylation is reduced as expected (Figure 15). It is possible that the intracellular signaling examined is not affected by lactic acid and that overall responses are reduced by interference downstream of phosphorylation of these kinases. In vitro assays repeated with sodium lactate matched to lactic acid show minimal yet significant reduction in degranulation of mast cells stimulated with compound 48/80 and substance P (Figure 16) yet no real effect on cytokine production. This indicates that the lactate anion may play a suppressive role in early phase responses to MRGPRX2 ligands. The corollary of this observation would imply that suppression of mast cell activity is in large part due to low pH produced by lactic acid presence. Yet, degranulation of mast cells treated with a buffer pH matched to lactic acid concentration was not suppressed to the same level (Figure 18). A synergy may exist between acidic effects and those effects produced by the lactate anion. The mechanism allowing such synergy can be theorized. LAD2 mast cells are known to express monocarboxylate cotransporter 1 (MCT-1), a membrane protein that requires binding of a positive proton and the lactate anion to facilitate transport of both across a membrane 86 . Lactic acid inhibition of MRGPRX2 mediated mast cell responses may require entry into the cell. Sodium lactate solutions were not acidic and therefore lacked the proton required, but still produced a significant suppression across 3 trials of degranulation. Another unconfirmed component playing a role in lactic acid effects on mast cells may be a lactate anion sensor, of which there are a couple. GPR81, a lactate sensor expressed on many immune cell types 112, may be responsible for the results seen with sodium lactate pretreatment. In contrast, the pH matched degranulation also showed a significant reduction of degranulation post exposure to compound 48/80 (Figure 18). As with all the early phase, in vitro assays, 74 buffer was cleaned off the cells so as to not interfere with receptor ligation. Assuming no effect on MRGPRX2 expression or conformation with acidic buffer incubation, pH effects observed in the matched degranulation could be due to pH sensors. Of the pH sensors known to be expressed by mast cells, GPR65 specifically is implicated in upregulation cyclic adenosine monophosphate (cAMP), a secondary messenger that is itself implicated in stabilizing or reducing mast cell activity111. As the results stand, lactic acid has an inhibitory effect on MRGPRX2 mediated mast cell responses. Whether this effect is independent of other GPCRs is yet to be determined as is its dependence on cell entry of the monocarboxylic acid. Elucidation of these mechanisms would require several in vitro experiments that have been formulated and pondered upon. 75 REFERENCES 76 REFERENCES 1. Blank, U. The mechanisms of exocytosis in mast cells. 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