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EFFECTS OF DOCOSAHEXAENOIC ACID CONSUMPTION
ON ENTERIC REOVIRUS INFECTION: IMMUNOGLOBULIN
RESPONSES AND VIRUS CLEARANCE

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ELENI BELI

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EFFECTS OF DOCOSAHEXAENOIC ACID CONSUMPTION ON
ENTERIC REOVIRUS INFECTION: IMMUNOGLOBULIN RESPONSES
AND VIRUS CLEARANCE

By

Eleni Beli

A THESIS
submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Food Science

2008

ABSTRACT

EFFECTS OF DOCOSAHEXAENOIC ACID CONSUIVIPTION ON
ENTERIC REOVIRUS INFECTION: IMMUNOGLOBULIN RESPONSES
AND VIRUS CLEARANCE

By

Eleni Beli

The possibility that dietary factors can modulate the immune system is of great
interest to health professionals and the public. The n-3 polyunsaturated fatty acids, PUFA,
found in fish oil, have well recognized anti-inflammatory effects. However, there is
concern that these might impair resistance to infectious microbes. Herein, we tested the
hypothesis that consumption of the n-3 PUFA, docosahexaenoic acid, DHA, interferes
with the in vivo immune response to enteric reovirus infection. Mice fed DHA-enriched
diets for 4 wks had increased incorporation of DHA and other n-3 PUFA in their
membrane phospholipids at the expense of arachidonic acid. Mice fed with DHA-
enriched and high-oleic safflower diets were orally infected with reovirus to access
antibody production and viral clearance. DHA consumption only transiently interfered
with reovirus clearance, but did not affect the humoral responses in the gastrointestinal
and systemic compartments or the overall resolution of the intestinal infection.
Accordingly, these data are not indicative of a profound impairment of the immune

response to reovirus infection.

ACKNOWLEDGMENTS

I would like to thank my advisor Dr Pestka for all his guidance, patience and
support for the completion of this project. Also I would like to give special thanks to my
committee members, Dr Amalfitano and Dr Hord for the useful comments, specifications
and suggestions on the project. Last but not least, I cannot ignore the complete support
from Dr Maoxing Li, an experienced virologist researcher in our lab and the technical

and moral support from my friend and lab mate Chidozie Amuzie.

iii

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. v
LIST OF FIGURES ................................................................................ vi
CHAPTER 1. INTRODUCTION ............................................................ 1
CHAPTER 2. LITERATURE REVIEW .................................................... 5
2.1. EFFECTS OF N-3 PUFA ON HOST RESISTANCE ............ 5
2.2. REOVIRUS ............................................................. 12

CHAPTER 3. DHA SUPPLEMENTATION MODULATES THE LIPID PROFILE
OF LYMPHOID ORGANS AND PERITONEAL MACROPHAGES 29

3.1. ABSTRACT ............................................................. 29
3.2. RATIONALE ............................................................ 30
3.3. INTRODUCTION ...................................................... 30
3.4. MATERIAL AND METHODS ....................................... 34
3.5. RESULTS ................................................................ 38
3.6. DISCUSSION ............................................................ 42

CHAPTER 4. EFFECTS OF DOCOSAHEXAENOIC ACID CONSUMPTION ON
ENTERIC REOVIRUS INFECTION: IMMUNOGLOBULIN

RESPONSES AND VIRUS CLEARANCE .............................. 47
4.1. ABSTRACT ............................................................. 47
4.2. RATIONALE ............................................................ 48
4.3. INTRODUCTION ...................................................... 48
4.4. MATERIAL AND METHODS ........................................ 52
4.5. RESULTS ................................................................ 57
4.6. DISCUSSION ............................................................ 67
APPENDIX .......................................................................................... 75
BIBLIOGRAPHY ................................................................................... 81

iv

LIST OF TABLES

Table 3.1 Lipid composition in experimental diets ...................................

36
Table 3.2 Fatty acid composition of spleen phospholipids in mice fed control-
or DHA- enriched diet ........................................................ 39
Table 3.3 Fatty acid composition PP phospholipids in mice fed control- or
DHA- enriched diet ........................................................... 41
Table 3.4 Fatty acid composition of peritoneal macrophage phospholipids in
mice fed control- or DHA- enriched diet .................................. 43

Figure 2.1

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6
Figure 4.7
Figure 4.8

Figure 4.9

LIST OF FIGURES

Attachment, endocytosis, replication, assembly and release of
reovirus ......................................................................... 17

Effects of DHA on fecal IgA levels in response to reovirus ............ 59

Effects of DHA on the production of virus-specific antibodies in ex
vivo culture of PP ........................................................... 60

Effects of DHA on the production of reovirus-specific IgA in lamina
propria cultures ............................................................... 61

Effects of DHA on generation of plasma virus-specific antibody
responses ....................................................................... 63

Effects of DHA on the production of reovirus-specific IgA and

IgG2a in ex vivo spleen cultures. ......................................... 64
Effect of DHA diet on reovirus L2 RNA in feces ........................ 65
Effect of DHA diet on reovirus L2 RNA in PP .......................... 66
Effect of DHA diet on infectious reovirus in intestinal tissues........ 68
Effect of DHA diet on IFN-y mRNA expression in PP .................. 69

vi

CHAPTER 1

INTRODUCTION

The possibility that food constituents can modify the immune system and
attenuate disease is of great interest. Among the nutrients that have become popular as
supplements are the n-3 polyunsaturated fatty acids (PUF A) that are found in high
concentrations in fish oil. Millions of people in United States take n-3 PUF A supplements
in the form of commercially available fish oil capsules and other fortified products.

Nutritionists have long endorsed fish as part of a heart-healthy diet, and the Food
and Drug Administration (FDA) in 2004 gave "qualified health claim" status to the n-3
PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) especially against
coronary heart diseases (1). Besides heart-related diseases (2-4), there is cumulative
experimental and clinical evidence that other inflammatory diseases are benefited by n-3
PUFA consumption. These diseases include rheumatoid arthritis (5), inflammatory bowel
disease (IBD) and colitis (6, 7). Additionally, n-3 PUFA are claimed to be a promising
preventive therapy for the progression of renal diseases (8-15). There is also some
indication that n-3 PUFA contribute to cancer prevention (16). Moreover, studies have
shown that DHA promotes the normal brain development of the fetus and the newborn
(17-19). The role of n-3 PUFA on neurological and psychological diseases has also been
another recent research focus (20-23).

Studies in our laboratory have used an experimental mouse model of human IgA

nephropathy (IgAN) to examine the mechanisms of the beneficial effects of n-3 PUFA in

retarding renal disease progression. In this model, a mycotoxin, deoxynivalenol (DON),
specifically modulates mucosal immune responses of the mouse, targeting the
development, differentiation, and homing of IgA-producing plasma cells in mucosal sites
(24). DON dramatically increases IgA in the serum and IgA deposition in kidney.
Overall, DON induces in the mouse clinical signs that resemble human IgA nephropathy
(25). Dietary supplementation with fish oil retards the progression of DON-induced
IgAN- like disease in the mouse by reducing IgA immune complexes in blood and
mesangial IgA deposition in kidney (14). The mechanisms by which n-3 PUFA mediate
these effects are likely to involve altered balance of n-6 and n-3 derived eicosanoids and
reduced inflammatory responses. Among the inflammatory responses generated with
DON, interleukin 6, (IL-6), a critical cytokine in promoting differentiation and
proliferation of plasma cell precursors, is reduced significantly in n-3 PUFA-fed mice
(26). However, it is not known whether fish oil reduces IgA production during normal
responses to mucosal pathogens.

Interest in the health benefits of fish oil began with epidemiological studies on
Greenland Eskimos (27). It was found that consumption of fats high in n-3 PUFA
correlated with low incidence of cardiovascular and inflammatory/autoimmune diseases
among the indigenous population. On the other hand, additional studies indicated a higher
than normal incidence of tuberculosis infection among this population (28). Many people
believed that Eskimo nutritional habits could have reduced their resistance to infections.
However, a link between the Eskimo diet and high tuberculosis rates has never been
proven epidemiologically, rather, the poor and crowded living conditions among the

endogenous population was considered main factor for the prevalence of the epidemic

(29). Human studies addressing exposure to n-3 PUFA and infection did not give clear
evidence that a diet rich in n-3 PUFA increases susceptibility to infections (30).
Nevertheless, these concerns raised questions about the net health benefits of n—3 PUFA
in the view of potentially reduced resistance to infections and a number of animal studies

with fish oil gave evidence for possible immunosuppressive effects (31).

Several studies measuring specific cell ftmctions ex vivo have shown
immunomodulatory effects of n-3 PUFA. However, it is not clear whether modifying the
immune function with n-3 PUFA supplementation would have any effect on host
resistance to infections. Furthermore, it is not yet determined whether the incidence and
severity of infections is increased in populations that are exposed to rich in n-3 PUFA
diets during naturally occurring episodes. Thus, measuring the in vivo immune responses
to an infectious challenge is a reliable method to address potential modulation of the
immune system with nutritional interventions. Since, host resistance to infectious
diseases is the cumulative result of cellular incorporation, production and activation of
various molecules, whole animal infectious models can give strong evidence on the effect
of dietary supplementations in infections. Typical endpoints used in similar studies are,
general well-being, duration and magnitude of symptoms, clearance of the infectious
agent, assessment of antigen-specific antibodies, and markers of innate immunity and

anti-inflammatory mediators (32).

To evaluate immunosuppressive effects of fish oil and potential interference with
the clearance of infectious microorganisms, numerous mouse models have been used
involving both bacterial and viral infectious agents (31). The type of n-3 PUFA, the dose

and time period of supplementation, the nature of the pathogen, the route of

administration, and genetic variation of the host can affect determining whether n-3
PUFA impair, enhance, or have no effect on host resistance. To our knowledge, there are
no n-3 PUFA studies that address these questions using enteric viral infection as a model
for common gastroenteritis (33).

Enteric infection with respiratory _e_nteric orphan virus (reovirus) that is a well-
characterized viral host resistance model, has been used with success to evaluate
intestinal immunotoxicity (34-37). Reovirus belongs to the Reoviridae family, which is
the largest of the six families of viruses including some human pathogens that cause
common enteric diseases. Enteric reovirus infection has recently been used to study the
antiviral effects of 3,3-diindolylmethane, a derivative from indole-3 carbinol found in
Brassica vegetables that is considered therapeutic for numerous forms of cancer (38).
The same model was employed in this study to assess possible suppression of the normal
activation of the gut immune system by long-term supplementation with n-3 PUFA.

The objective of this study was to test potential immunosuppressive effects of n-3
PUFA in a mouse model that mimics human gastrointestinal viral infections (39). We had
two hypotheses: (1) n-3 PUFA consumption would increase incorporation of these fatty
acids into major immune cell populations involved in mucosal and systemic immunity;
and (2) n—3 PUFA consumption will result to reduced immunoglobulin responses to

reovirus infection and will interfere with viral clearance.

CHAPTER 2

LITERATURE REVIEW

2.1 EFFECTS OF N-3 PUF A ON HOST RESISTANCE

Host resistance models represent the most reliable approach to study the influence
of different agents, (toxicants or nutrients) on the functional integrity of the immune
system and its ability to eliminate invading pathogens (40). An intact immune system
integrates complex signals at the local and systemic level with innate and adaptive
mechanisms that initiate both humoral and cell-mediated mechanisms. A complete,
functional immune system eliminates most pathogens, while an immunocompromised
one encounters difficulties (40). In all these models, a comparison between groups that
receive a treatment (such as a drug, a nutrient, or a toxicant) and a placebo is made to
estimate the ability of the subjects to deal with the infectious agent. Whether the
treatment enhances or suppresses an immune response can be determined by accessing
several functions of the immunocompetent cell populations, and the overall clearance of
the pathogen from the host. Finding a significant difference in one of the immunological
parameters between treatment and control groups will not always yield a definite answer
about potential immune stimulation or suppression. This is because it is not always
known what percent change in each of the immunological parameters correlates with
increased susceptibility and what alternative compensatory mechanisms might be evoked
(40). However, clearance of the infectious agent is the outcome of an integrated immune

response and, therefore, a strong indicator of possible immune modulation.

Most host resistance models used to test possible adverse immunomodulatory
properties of n-3 PUFA, to date, have employed bacteria as the infectious agents (31).
Although influenza, cytomegalovirus and murine retrovirus have also been used, there
are no studies to our knowledge addressing susceptibility to enteric viruses (31). Relative
to foodbome diseases, enteric viruses are among the leader causes of gastroenteritis cases
correlated with consumption of contaminated fresh produce or water. Enteric infections
induce complex immune responses in the gut, including both non-specific innate
responses and adaptive immunity mediated by cytotoxic T-cells and B-cells. Since the
majority of diarrhea] diseases are attributed to enteric viral infections, a model studying

susceptibility towards an enteric viral infection is of great importance.

Effecfl of 11-3 PUFA on the immune responses

There is an extensive literature about the effects of n-3 PUFA on different
immunological parameters that ostensible serve as indicators for immune modulation.
Since adaptive immunity is absolutely necessary for clearance of most pathogens, the
effect of n-3 PUFA on antibody production and cytotoxic T-cell expansion is of interest.
In the mucosal compartment, secretion of mucosal IgA is very important protective
mechanism and therefore could determine susceptibility of the host. n-3 PUFA down-
regulate DON-induced IgA production in serum of the mouse (l3, 14), indicating that
their consumption might also affect regulation of IgA production in the mucosa. In an
influenza host resistance model, it was shown that virus-specific serum IgG and lung IgA
production is significantly lower by 7 d post infection (PI) in fish oil-fed mice compared

to control-fed mice (41). However, human patients suffering from distal proctocolitis,

administered with n-3 PUF A had the same percentage of IgA and IgG containing cells in
the rectal mucosa but significantly reduced IgM cells (7). Finally, significant positive
correlations were found between fish oil supplementation and the concentration of IgA in
breast milk (42).

Production of antibodies and cytotoxic responses is regulated by T lymphocytes
and therefore, the effect of fish oil on this cell population has been studied extensively in
vitro and in vivo (13, 43-48). Some studies report that administration of n-3 PUFA
reduces T-cell proliferation and proliferation ex vivo in an agonist specific fashion (49-
51). Other studies report no effect of n-3 PUFA on antigen-specific CD4+ and CD8+ T
lymphocyte proliferation in vivo (45, 52). When Byleveld et al, (2000) examined the
effect of fish oil on the proliferative and cytotoxic capacities of viral-specific T-cells,
they found that spleen T-cell proliferation ex vivo was higher, whereas cytotoxicity of
lung virus-specific T-cells was significantly lower with fish oil supplementation (46).

Lymphocyte proliferation takes place after antigen-priming of antigen presenting
cells (APC). It has been suggested that fish oil might affect antigen presentation process
(53-55). APC express major histocompatibility complexes (MHC) on their surfaces that
display both self and non-self antigens. When non-self antigens are presented, an immune
response is mounted. The nature of the peptide-MHC complex determines which set of
lymphocytes will be activated with MHC class I activating CD8+ T cytotoxic cells and
MHC class II activating CD4+ T helper cells. In vitro studies showed that treatment with
n-3 PUFA reduced the expression of MHC-II on IFN-y activated monocytes and their
ability to present antigens to autologous lymphocytes (53, 54). Similarly, treatment in

vitro with PUFA resulted in B lymphoblasts with lowered susceptibility to lysis by

cytotoxic CD8+ T-cells, suggesting an effect of PUFA on the HLA class I pathway, as the
human MHC is named (55). n-3 PUFA have also been shown to interfere with LPS-
induced dendritic cell maturation evidenced by the expression of cell surface markers,
critical intracellular signaling pathways and cytokine production (56). Thus, n-3 PUFA
supplementation might reduce MHC molecule expression and subsequent antigen
presentation that drives the adaptive T-cell responses.

The most desirable outcome of the immune responses generated during infection
is clearance of the infectious agent. While several n-3 PUFA studies have shown a delay
in pathogen clearance (41, 52, 57), the specific part of the immune system that is targeted
by n-3 PUFA is unknown. Apart from the adaptive responses the role of innate immune
system in mounting a strong immune response is important. EPA, more than DHA, has
been shown to reduce innate cellular responses as evidenced by decreased natural killer
activity against target cells (58). EPA and DHA were shown to inhibit pathogen killing
by macrophages, which are also important in innate immunity (59). One suggested
mechanism is that n-3 PUFA interfere with actin nucleation in phagosomes and
subsequent with phagocytosis and fusion of the phagosomes with late endocytic
organelles, an essential step to kill pathogens. Thus, n-3 PUFA supplementation affects
not only adaptive but also innate responses. The magnitude of their impact to immune
responses is determined by the specific pathogen encountered, since different pathogens
provoke different immune responses.

There also has been extensive research on the effects of n-3 PUFA on the
production of cytokines, which are central mediators of immune responses. n-3 PUFA

consumption was shown to reduce expression of Th1 cytokines, such as IL-1, IL-2, TNF-

a, and IFN-y ex vivo (41, 44, 60-62). Mice fed with fish oil and infected with influenza
virus, have impaired IFN-y production, whereas the IF Nor/B response remains unchanged
(41). Mice fed with fish oil and infected with murine retrovirus, produce less TNF-a, IL-
113, IL-2 and IFN-y in spleen cultures (63). Reduced IFN-y production is also observed in
studies with mice fed fish oil and infected with Listeria monocytogenes (64, 65),
sometimes even prior to infection (65). However, DHA has also been shown to impact
Th-2 cytokines, like IL-10. A study with piglet neonates immunized with an inactivated
influenza vaccine and fed a PUFA-enriched formula, showed significant up-regulation of
IL-10 in peripheral blood monocytes accompanied by reduced ex vivo virus specific

CD4+ and CD8+ T-cells responses (66).

Mechanism of n-3 PUFA immunomodulatory effects

The most widely accepted mechanism of n-3 PUFA anti-inflammatory properties
is their ability to change the lipid mediators of inflammation. Supplementation with n-3
PUFA changes the fatty acid profile of cell membranes and therefore products that are
generated by their oxidation. Upon stimulation, PUFA are released from cell membranes
and they are converted enzymatically to eicosanoids. Eicosanoids are the most important
class of lipid mediators of inflammation and are mainly derived from free C20 fatty acids
(67). Since ARA is the dominant C20 fatty acid found in cell membranes of mammals fed
with a typical meat-based diet, eicosanoids derived from ARA are important lipid
mediators. Metabolism of ARA by cyclooxygenase enzymes (COX) gives rise to the 2-
series prostaglandins (PCs) and thromboxanes (TXs), while metabolism by the 5-

lipoxygenase (5-LOX) gives rise to hydroxyl- and hydroperoxy- derivatives (5-HETE

and 5-HPETE) and the 4-series leukotrienes (LTs), LTA4, B4, C4, D4 and E4 (68). Both
PGEz and LTB4 have strong proinflammatory properties such as the induction of fever,
increase of vascular permeability, induction of chemokinesis and release of lysosomal
enzymes, and enhancing generation of reactive oxygen species and inflammatory
cytokines like TNF-a, IL-1 and IL-6 (30, 68, 69).

Increased consumption of n-3 PUFA results in high proportions of those fatty
acids in cell membrane phospholipids partly at the expense of ARA. The anti-
inflammatory action of n-3 PUFA has mainly been attributed to reduction of the
proportion of ARA as a substrate for synthesis of the subsequent proinflarnmatory
eicosanoids (67, 68). Furthermore, n-3 PUFA alter the phospholipid composition of cell
membranes reducing the activity of phospholipases involving in the release of ARA fiom
cell membranes (70). Additionally, EPA, as another C20 fatty acid, when is incorporated
in membrane phospholipids competitively gains access to the same enzymes that produce
eicosanoids from ARA and gives rise to alternative classes of eicosanoids, the 3-series
P63 and TXs and the 5-series LTs (68) and other novels lipid mediators termed E-series
resolvins. Similarly, DHA gives rise to D-series resolvins, docosatrienes and
neuroprotectins (71). These alternative series of lipid mediators are less potent inducers
of inflammation and in certain cases exert anti-inflammatory properties counteracting the
proinflammatory potential ARA-derived eicosanoids exert (67, 71).

Incorporation of n-3 PUFA into plasma membrane not only alters the lipid
products derived from membrane lipids, but also the microenvironment in the membrane.
Thus, they could modify the conformation, lateral organization and vertical orientation of

molecules involved in interactions with other cells or other molecules (72). Additionally,

10

because n-3 PUF A also incorporate into internal membranes, uptake of n-3 PUFA can
affect molecule trafficking on cell surface (55). Lipid modulation of APC membranes and
MHC presentation on their surface possibly has biologically significant effects on viral
clearance (72, 73). Moreover, DHA has been shown to inhibit trafficking of RAS and
other lipidated proteins to plasma membrane through the secretory pathway (55, 75).

The degree to which the aforementioned mechanisms can explain the effect of n-3
PUF A on host responses is not known. Host resistance is the cumulative result of cellular
incorporation and different molecule production and activation. Thus, animal infectious
models can give the best indications of the role of n-3 PUF A upon infectious disease
resistance. Both bacterial and viral infectious agents have been used to give insight in
possible immunosuppressive effects (31). These studies include a wide variety of
different bacteria, from Grarn-negative, such as Klebsiella sp (76), Salmonella sp (77,
78), Bacteroides spp (79), Pseudomonas aeruginosa (77, 80) to Gram-positive
microorganisms, such as Staphylococcus aureus (79), Group B Streptococcus sp (81),
and Listeria monocytogenes (43, 57, 61, 65, 82-84). The usual endpoint in these studies is
host survival, and occasionally, clearance of the infectious agent and specific
immunological parameters are measured. In these studies, fish oil has been reported to
both enhance and suppress host survival. These differential results indicate that failure to
survive a specific infection may be related to the degree in which an overwhelming
immune response is the cause of death or the pathogen itself. Apparently, the type of
pathogen used and the type of disease it elicits is of particular importance to predict the

outcome.

11

Viral models that have been used to estimate immunomodulatory effects of n-3
PUFA on host resistance include cytomegalovirus (CMV), murine retrovirus (MAIDS),
and influenza virus. Mice infected with cytomegalovirus and supplemented with fish oil
did not show effects on susceptibility to the infection (80). Mice infected with murine
retrovirus and fed with fish oil for 8 wk had significant enhanced survival compared
control mice (85). However, using same virus Xi et al, (63) demonstrated that serum
levels of IgG and IgM were reduced in mice fed fish oil. Additionally, fish oil
consumption reduced virus induction of TNF-a, ILl-B, IL-2 and IFN-y during the
infection. In studies with influenza virus, Byleveld et al (41) reported that fish oil feeding
interfered with viral clearance from lungs and reduced IgA secretion in lungs.
Furthermore, IFN-y mRNA expression was found to be down regulated by fish oil
supplementation. Overall, those studies suggest that fish oil can enhance or impair

immune responses to virus depending on the type of virus used.

2.2 REOVIRUS

Enteric reovirus infection, a well-studied viral host resistance model, has been
used effectively to estimate immunotoxicity in the gastrointestinal tract (37). Li et al
successfully utilized this model to access modulation of the normal immune responses to
an enteric viral infection by natural occurring mycotoxins (34-36, 86). We chose to use
the same model to predict possible immunosuppression of the normal immune responses

in the gut by long-term supplementation with n-3 PUFA. The following section describes

12

the enteric reovirus model, and reviews both aspects of reovirus molecular biology and

immunity induced upon infection

Genome and life cycle

Reoviridae is the biggest of the six families of viruses with double stranded RNA
genome that includes many genera: Orthoreovirus, Aquareovirus, Cypovirus, F ijivirus,
Oryzavirus, Orbivirus, Phytoreovirus and the human pathogens Rotavirus and C oltivirus
(Colorado tick fever virus). Orthoreovirus genus is the prototype of the Reoviridae
family called also mammalian orthoreovirus or reovirus (respiratory enteric orphan

virus).

Reoviruses are grouped in three serotypes: 1, 2 or 3 based on hemagglutination-
inhibition tests (87). Prototypical viruses are the Type 1 Lang (TIL) which was isolated
from a healthy child, and Type 2 Jones (T21) and Type 3 Dearing (T3D) that were
isolated from children with diarrhea (87). From serological studies it has been shown that
the reovirus strains differ mostly in a capsid protein, the ol protein. TIL 81 gene is more
closely with the T21 than the T3D S] gene. Despite these differences 0] protein in all
strains share at least five conserved domains. The rest capsid or core proteins of all

strains are quite conserved, sharing a 90-98% homology (87).

The reovirus genome consists of 10 distinct segments of double stranded RNA
that encode all the proteins the virus needs and comprises a genome of around 23,600 kb
(88). The 10 RNA segments have different sizes and are categorized in three groups as
large (L1, L2, L3), medium (M1, M2, M3) or small (SI, 82, S3, S4) (87). During

infection with different viral strains, reassortment might take place, generating new

13

viruses containing segments from both strains. Each segment is transcribed to plus-strand
RNAs, serving as viral mRNAs. Each mRNA has a long open reading frame, ORF. Each
ORF encodes one protein. However, there are 10 RNA segments and the virus encodes
twelve proteins. Eight segments code for one protein each from the mRNAs and two
segments code for two proteins each. The latter segments include 81 and M3 gene. S]
gene encodes the 013 and 01 proteins and M3 gene encodes the uNSC and u3 proteins.
Those extra proteins are encoded from AUG frames within the ORF of the main proteins.
Among the 12 translated proteins, eight are structural (M, 1.2, 91.3, 141, u2, 61, 02 and 03),
and the rest four are non-structural (uNS, uNSC, oNS, and 615) (87, 89) and facilitate

viral survival, infectivity, replication and assembly to new viruses.

The mature structural form of infectious viruses is the virion released from
infected cells. During infection, viral particles undergo a serial of conformational changes
that facilitate their entrance in the cytoplasm. The intact virion consists of two capsids
composed of structural proteins. The outer capsid is made up largely of 03 and pl protein
and is stabilized by interactions with the 72 protein (87). During infection, virions
undergo partial uncoating to yield two types of subvirion particles, infectious subviral

particles (ISVPs) and cores.

Viral particles mediate infection by attachment of the virion to cellular receptors.
Attachment is mediated by binding of the viral 01 protein. The 01 protein contains
receptor binding domains (RBD) that determine cell tropism of the variant reovirus
strains (90). Until now, two cellular receptors have been recognized, but others remain to
be elicited (91). The identified receptors are: a-linked sialic acid and junctional adhesion

molecule (JAM) that can be independently bound by reovirus strains (90).

14

Upon binding, the virion enters the cell by receptor-mediated endocytosis (Fig.
2.1, step 2). In endosomes the virion undergoes acid-dependant proteolytic cleavages and
forms the ISVPs (Fig. 2.1, step 7). When ISVPs are formed in the lumen by intestinal
proteases, entry of ISVPs into the cell does not depend on endocytosis (Fig. 2.1 step 5)
(87, 89). ISVPs are released from the endosome in the cytoplasm as cores (Fig. 2.1, step
8). Replication of reovirus starts within the core particle in the cytoplasm (Fig. 2.1, step
9), with synthesis of 10 capped RNA that serve both as mRNA for translation and as
templates for minus-strand synthesis. These RNA are exported from the core and are
translated in the cytoplasm (Fig. 2.1, step 10). Transcription of reoviral genomes occurs
in two phases: primary transcription occurs within the cores from infecting viral particles
the first 6 to 8 hours of infection, while secondary transcription occurs in particles
assembled by newly formed proteins reaching maximum rates to 12 hours after infection
(Fig. 2.1, step 14-15). Secondary transcription contributes to the majority of RNA
transcript produced during infection (87). Shortly after the onset of infection, synthesis of
reoviral proteins increases at the expense of cellular proteins (87). Moreover, regulation
of the transcription rates occurs with the longer segments to be transcribed at lower rates.
Assortment of the viral RNAs occurs when 10 viral mRNAs are packed within a newly
formed viral particle (Fig. 2.], step 13). Generation of the double-strand segments occurs
within the nascent particles with synthesis of a minus strand for each plus strand viral

RNA that remains associated with each other to form a ds RNA segment (87).

Endosomal and intestinal lumen proteases result to cleavage of the 03 protein of
the intact virion and to generation of ISVPs. Cleavage of the 03 protein promotes

conformational changes of the ol structural protein that becomes like an expanded

15

Figure 2.1. Attachment, endocytosis, replication, assembly and release of reovirus.
(1) infectious viral particles; (2) cell attachment via viral protein, 01 interactions with
sialic acid and/or JAM receptors on target cells and receptor-mediated endocytosis; (3)
intestinal proteases produce intermediate subviral particles (ISVPs); (4) ISVPs; (5) ISVPS
enter target cell via endocytosis; (6) endosomes and lysosomes become acidified; (7) the
virion undergoes acid-dependant proteolytic cleavages and forms the ISVPs; (8) release
of reovirus ISVPs in the cytoplasm; (9) reoviral cores in the cytoplasm; (10) ISVPS
penetrate directly through the cell membrane; (11) transcription occurs within reovirus
cores and capped mRNAs are released; (12) primary translation of all 10 mRNA using
host translation machinery; (13) viral mRNAs are packed within newly formed viral
proteins where new cores assemble; (14) secondary transcription of viral genomes and
viral mRNA occurs in particles assembled by newly formed proteins; (15) translation of
the secondary viral RNA; (16) assembly of the viral particle with preformed self-
assembled complexes of outer-capsid proteins added to subviral particles; (17) mature
viral particles are released from the cell by lysis. (Figure reproduced from Flint et al.,
Principles of Virology, second ed., ASM Press, Washington, 2004 [87]).

16

 

 

 

 

flexible fiber. The 01 protein is considered as the reovirus cell-attachment protein and its
conformational change is believed to mediate interaction with cellular receptors and
increase infectivity. The other protein of outer capsid, pl, facilitates interaction with the
membrane bilayers and thus mediates release of ISPVs from endosomes into cytoplasm.
Cores are produced by further release of the ol fibers and p1 subunits and are
mostly composed by 71.1 and 02 proteins. In the transition from ISVP to core, parts of the
12 protein change conformation and rotate to form a turret-like structure, which facilitates
entry of nucleotides into the core and exit of newly synthesized viral mRNAs (87, 89).
Assembly of reovirus particles occurs at different stages. In each stage the
particles have different functional properties. Before the formation of viral particles,
simple complexes are formed by the assembly of RNA with proteins. In more advanced
stages, structured particles can be recognized, and transcription of the minus strand
occurs. Further, more stable viral particles are formed and finally complete viral particles
(87). In the final steps of capsid assembly, preformed self-assembled complexes of outer-
capsid proteins are added to subviral particles (Fig. 2.1, step 16) and mature viral
particles are released from the cell by lysis (Fig. 2.1, step 17) (87). It is believed that
reovirus assembly takes place in specific cytoplasmic inclusions observed during

infection, named viral factories (87).

Reovirus effects on host cells

Reovirus infection induces a number of structural and fimctional changes in the

host cell. Part of these changes facilitate viral survival, while others mediate host cell

18

efforts to alert immune system for the ongoing infection, or to protect the body from
systemic spread of the infection. Numerous cell structural changes facilitate viral (89)
replication and assembly while others initiate sophisticated signaling cascade to attract
immune cells to the focus of infection and evoke responses to eliminate the invading
pathogen.

One of the changes that reovirus induces in host cells is the development of
cytoplasmic inclusions, called viral factories. Viral factories contain dsRNA, viral
proteins and nascent viral particles that are associated with cytoskeleton. These viral
factories do not associate with cellular membranes. These inclusions appear as dense
granules in the cytoplasm and during infection, increase in size and move towards the
nucleus (87). Viral factories do not contain ribosomes, thus, active translation of viral
RNA does not occur there. Viral factories are associated with microtubules that play a
role in transportation of the viral particles. When cells get infected by reovirus, their
cytoskeleton is continuously altered, disrupted and reorganized, changing the shape of the
cell constantly (87). Overall, there is agreement that viral factories have a role in viral
assembly and maturation of infectious particles (87).

Virus-infected cells undergo further changes that put them into an antiviral state.
This state is elicited by a complex cellular signal transduction and gene expression
initiated with secretion of interferons (IFNs) (89, 92). The various strains have different
capacities to induce IFNs, with reovirus T3 to be the most effective. IFNs promote the
activation of more than 300 genes participating in the antiviral response that results in
cessation of protein synthesis. Inhibition is selective for reovirus proteins and not for

cellular proteins (92, 93). Among the key components of this antiviral response is the

19

RNA-dependent protein kinase, PKR, which inhibits the elF-2 protein synthesis initiation
factor by phosphorylation. Reovirus has however evolved mechanisms to override this
cellular defense. Reoviral 03 protein, which has been demonstrated to bind to ds RNA
(87, 94), can prevent activation of PKR in vitro (87). Activation of PKR has been related
to host cell shut off and enhanced viral translation (87). Another important IFN inducible
factor, RNase-L, degrades most of cellular and viral RNA (89). PKR together with
RNase-L activation contribute to immediate host defense response as evidenced by a
general host shutoff. However, reovirus replication is not inhibited neither by PKR nor
RN ase-L (87). In contrast, reovirus replicates to higher yields in the presence of PKR and
RNase-L. Because deletion of these proteins results in impaired reovirus replication,
reovirus seems to have evolved effective mechanisms to avoid the actions of these classic
IFN-stimulated antiviral responses possibly by benefiting from their expression (94).
Another consequence of reovirus infection is inhibition of cellular DNA synthesis
and induction of apoptosis. T3 serotype typically is a more potent inducer of apoptosis
than other strains. Reovirus replication is not required for the cell to go to apoptotic death
(90, 95). Binding of reovirus outer capsid to cellular receptors is efficient to activate a
number of signaling cascades, including c-Jun N terminal kinase (JN K) and extracellular
signal related kinase (ERK) that result to apoptotic pathways (90, 95). These signaling
cascades activate NF -KB and c-Jun transcription factors, both of which are involved in
inducing apoptosis in infected cells, particularly those infected with reovirus T3 (89, 90).
Moreover, increased NF -KB activation is linked with increased production of
chemokines, cytokines and possibly surface markers signaling the immune cells for the

ongoing infection.

20

Enteric reovirus infection

The various reovirus serotypes differ in their tropism, pathogenesis and potency
of the infection. TIL and T3D strains are both able to infect host via the intestine but
their capacities to spread and cause disease are different (96). After infection by the oral
route, both types bind to M-cells that overlay Peyer’s Patch (PP) -dome in the small
intestine (96) but only reovirus TIL can replicate in intestinal tissue (95). T3D serotype
spreads from the intestinal tract into mononuclear cells of PP, is transmitted to the
adjacent neurons and reaches the brain through the vagus nerve (95). TIL serotype
spreads to mononuclear cells of PP and mesenteric lymph nodes and concludes in the
spleen. Differences occur both in the regions of the intestine they infect, with T3D
infecting intestinal intraepithelial lymphocytes and TIL epithelial cells, and on the

disease induced with T3D reovirus to be the only one that can cause encephalitis (95).

TIL reovirus has been widely used as a model to study interactions between the
mucosal immune system and gut infection. The mucosal immune system is mainly
comprised of the gut associated lymphoid tissues (GALT). GALT consists of discrete
populations of cells located in PP and gut lamina propria. PP contain B-cells in germinal
centers surrounded by mantles of T-cells. Enteric reovirus infection specifically targets
PP, ensuring high exposure of virus to mucosal immune cells (34-37). Virus-specific T-
and B-cells are primed in the patch and migrate via the afferent lymphatics to the
mesenteric lymph node and spleen. From the spleen, primed effectors disseminate via the
blood stream to other mucosal sites and the intestine, where they home to the lamina

propria. Mature plasma cells secrete antibodies that are transported into the lumen, while

21

viral-specific cytotoxic T-cells are deveIOped among the intraepithelial lymphocyte
populations (97). Migration and specific homing of the effector cells in the intestinal
tissues and the sites of infection is mediated with tissue-specific adhesion molecules and
chemokine interactions (98).

The kinetics of oral enteric reovirus infection have been well characterized (99).
The part of the intestine affected the most is the distal small intestine. Immediately after
oral administration, reovirus antigens are found at the dome of M cells, and within 2 (1
they are detected in PP, where reovirus replication occurs while an immune response is
initiated. By the 5th d after inoculation, viral antigens are mostly found in enterocytes of
the crypts. Productive infection is limited to immature crypt enterocytes, due to a
requirement for an activated RAS signaling pathway that promotes translation of reovirus
mRNAs and viral replication (94). Virus is released from cells in the crypts and infects
adjacent epithelial cells or is shed in the lumen. By the sixth day after infection, viral titer
decreases dramatically in all segments of the intestine except the ileum, where the viral
titer can be 2 to 3 log higher compared to other tissues. In neonate mice, the infection
spreads systemically, but in adult mice the infection is limited to the mucosa, although
viral antigens and antigen-activated cells appear later in the mesenteric lymph nodes and
the spleen. Twenty days after infection, the intestine seems to have normal morphology
and viral antigens are only detected in mononuclear cells residing in lamina propria.

Reovirus infection triggers significant antibody and cytotoxic responses. The
antibody responses are characterized by increased secretion of mucosal IgA and elevation
of serum IgG (100, 101). Secretory IgA has a major protective role for the mucosal

system. It is a polymeric antibody that is secreted on the mucosal surfaces and neutralizes

22

the invading pathogens (89, 97, 102, 103). The role of IgA in reovirus infection becomes
important for protection against secondary infections (102). While an initial infection can
be cleared by mice that lack IgA by other antibodies in the mucosa, upon a secondary
infection, the same mice become infected again whereas wild mice were protected (102).
Reovirus infection increases the polymeric immunoglobulin receptor (plgR) mRNA and
protein in mucosal epithelial cells, facilitating the transport of IgA to the lumen (104).
Thus an intact viral-specific IgA response is essential for protection and clearance of the

virus in the guts.

Activation of B-cells by CD4+ T-helper cells with the secretion of cytokines,
activation of BI cells by T-cell independent mechanisms and a pleiotropic redundancy
among many cytokines in the intestine are among the mechanisms involved in mucosal
IgA production (105). Other thymus-independent mechanisms through 75 T-cells are
equally important for the induction of IgA (105). Almost up to 40% of IgA production in
the murine gastrointestinal tract is derived by Bl cells (105). Induction of IgA is also
upregulated by cytokines as IFN-y, IL-4, TNF-a and IL-1 and the transcription factor
IRF-l (105, 106). Other cytokines such as IL-5, IL-6 and IL-10 also regulate production
of IgA in the gut (103, 106). The biological role of all these cytokines on the secretion of
IgA is not clearly defined. lL-6 is considered a major regulator of mucosal IgA induction,
however genetic deficiency in IL-6 does not abrogate antigen specific IgA in the gut,
demonstrating the complexity of the regulatory mechanisms for mucosal IgA generation
(107). Retinoic acid potentially synergizes with IL-6 or IL-5 in the mucosal compartment
to regulate IgA secretion (105). The organism has developed many complementary ways

to maintain intact IgA production indicating its critical role for the protection (105).

23

In addition to mucosal IgA, specific IgG is induced upon enteric reovirus
infection. The majority of reovirus-specific serum IgG is IgGZa and IgG2b, while levels
of virus-specific IgGI are low (97, 106, 108). Reovirus-specific IgG prevents viral
infection by inhibiting virus-cell interactions, penetration and uncoating of the virus. In
addition to being produced in systemic lymphoid tissue and found in serum, antigen
specific IgG is also detected in mucosal tissues (97). In the absence of virus-specific IgA,
passive transfer of specific IgG facilitates clearance of reovirus in severe
immunodeficient mice (SCID) and immunoglobulin knockouts (101). However, in
immunocompetent mice, systemic administration of reovirus-specific IgG cannot inhibit
viral replication in the intestine (101). So the role of IgG antibodies seems to be

important in preventing viral systemic spread rather than within the gut mucosal surfaces.

After oral reovirus infection, a robust cellular response is induced. CD4+ T helper
cells are differentiated mostly towards Thl cells while a weaker Th2 response is
promoted (109). Th1 cells produce cytokines such as IFN-y, TNF-a, TNF-B and IL-2 that
activate cell-mediated immune responses, whereas Th2 cell produce cytokines such as
IL-4, IL-5, IL-10 and TGF-B that mediate antibody production. Reovirus infection
induces IFN-y in PP and intraepithelial lymphocytes (IEL), synthesis of IL-5, IL-6 and
IFN-y in PP cultures (109) and synthesis of IL-12 and IFN-y in mesenteric lymph nodes
(110). In IEL, 4 (I post infection (PI), the strong inflammatory cytokines IL-6, IL-12 and
IFN-y that lead to T-cell activation are expressed. Additionally, IEL express IL-17 and
stem cell factor (SCF), the colony-stimulating factors GSCF and GM-CSF, and the
proteins MCP-l and MCP-S, which contribute to the mobilization and/or cell

proliferation of dendritic cells, monocytes and macrophages (1 l 1). IEL from reovirus

24

infected mice have been shown to have elevated levels of IL-10 (111), while PP have less
IL-10 expression (110) suggesting differential expression of cytokines in different
tissues. IL-lO has been suggested to delay viral clearance (112-114). Other cytokines
such as IL-2 and IL-4 were shown to be only modestly increased in reovirus infected
mice at 4 d PI compared to non-infected mice (106, 109, 11]), suggesting that these two
cytokines may be expressed at earlier time points or may be part of an ongoing process of
lymphocyte proliferation in non-infected mice to maintain a homeostatic process of T-

cell and/or B-cell expansion in the small intestine (111).

Enteric infection with reovirus induces strong cell-mediated immune responses in
the gut that are characterized by the appearance of CD8Jr virus specific cytotoxic
lymphocytes in PP and IEL. Virus-specific precursor cytotoxic T-cells are found in a
frequency of 200 every 106 CD8+ IEL, and they express d/B TCR (115). Their role is to
lyse virus-infected cells in an MHC-restricted manner (98). Reovirus infection induces
monoclonal or oligoclonal expansion of TCR subpopulations (115). Additionally,
thymus-independent cytotoxic T-cells contribute to reovirus clearance from the gut.
Thymus-deficient mice are resistant to reovirus infection, indicating a role for thymus-
independent lymphocytes, natural killer cells and macrophages (97, 115). SCID mice,
which do not have any T and B-cells but still have natural killer cells, are not able to
overcome reovirus infection and die from the induced disease, confirming the role of

thymus-independent T-lymphocytes in protection (115).

In general, the complexity of the immune responses in the mucosa is
demonstrated by the synergistic action of different concurrent mechanisms and

intracellular signaling events. Apart from the adaptive responses, innate mucosal

25

responses are equally important upon a viral infection. Secretion of antibodies of low
specificity, complement activation, expression and activation of Toll-like Receptors
(TLR), production of type I IFNs and cell mediated innate responses seem to play a role
in dealing with the infection (92). Innate cell response includes the first cells to arrive at
the site of infection and involves neutrophils, macrophages, natural killer cells and other
granulocytes. Innate responses in the mucosa include also peristalsis of mucosal smooth
muscle, mucus and enzyme secretion, and defensins; these mucosal functions are
considered to have a contribution to protection against pathogens. However, we know
that specifically for reovirus infection, defensins do not play important role since they are
only effective against enveloped viruses or bacteria (116).

Among the innate responses responsible for viral clearance, induction of the type
I IFNs, IFN-a and IFN-B, has been shown to be critical. These cytokines put the cell into
an antiviral state and help evoke further adaptive responses. A recent study on the role of
type I IFNs on reovirus infection showed that these cytokines are indispensable for
reovirus clearance in the intestine (73). The study suggested that the major source of type
I IFNs is PP, the first site of the intestine that encounters the virus (117), were resident
dendritic cells take up antigens. In contrast, epithelial cells, natural killer cells or TLR
signaling were shown not to be as critical as IFN signaling for reovirus clearance (74).
Based on these findings, more extensive studies need to be done focusing on how
nutritional modifications such as those of n-3 PUFA supplementation shape the innate
immune responses.

Overall, studies on reovirus-induced immunity have been well-characterized, and

thus reovirus can be a good tool to study alterations of the immune system in response to

26

external factors. Thus, it is possible to assess modifications of normal responses to
interventions with nutrients, drugs and toxicants. Furthermore, because quantitative
judgments about overall immune firnction are very difficult, a common practice is to
measure aspects of immunity (certain cell functions, cytokine production, phagocytosis,
etc) to have a possible insight for the status of the immune system. Since reovirus is the
subject of ongoing research about the innate and adaptive immune responses, it is feasible
to employ the appropriate tests and measurements to study immune modulation with
nutrients, xenobiotics and drugs.

In the present study, we contribute to a growing field of research focusing on
targeting the immune system with functional foods or nutritional supplements. The
possibility that with selective nutritional choices we can enhance the immune system is
particular interesting for science and attractive for the food industry. Several nutrients
have been shown to be essential for a sufficient immune function, such as essential amino
acids, the essential fatty acid linoleic acid, vitamin A, folic acid, vitamin B6, vitamin
B12, vitamin C, vitamin E, Zn, Cu, Fe, and Se (118). Their deficiency leads to decreased
immune responses to pathogens and increased susceptibility. Increased intakes of some
nutrients above habitual and recommended levels can enhance certain aspects of immune
function. However, excess amounts of other nutrients might impair immune functions. It
is not clear, however, whether a normal immune function can be “improved”. Yet, it is
possible to measure aspects of the immune function and thus, we can define the status of
the immune system. This study describes the effects of a dietary manipulation on the in

vivo response to an enteric viral challenge, and the antibodies in peripheral blood and

27

fecal secretions in response to the virus. Also we determined the severity of the infection

in the mouse, tracking the eradication of the infectious agent (30).

28

CHAPTER 3
DHA SUPPLEMENTATION MODULATES THE LIPID PROFILE OF

LYMPHOID ORGANS AND PERITONEAL MACROPHAGES

3.1 ABSTRACT

Consumption of n-3 polyunsaturated fatty acid- (PUFA-) enriched diet for an
extended period alters the lipid profile of cells within human and animal tissue. Such a
change might impact the anti-iflammatory effects n-3 PUFA. The purpose of this study
was to test the hypothesis that consumption of fish oil will result in elevation of n-3
PUFA membrane lipid profile of lymphoid organs and peritoneal macrophages. Female
B6C3F1 mice were fed AIN-93G semipurified diets containing 1% corn oil and either
6% fish oil enriched in docosahexaenoic acid (DHA diet) or 6% safflower enriched in
oleic acid (control diet). To determine if these diets resulted in elevated n-3 PUFA in
spleen, PP and the peritoneal macrophage cell population, these lymphoid tissues were
isolated after 2, 4, and 6 wk feeding. Membrane phospholipids were extracted, esterified
and analyzed with flame ionization gas chromatography. Fatty acid profiles from the
experimental samples reflected the compositions of the diets. The DHA diet increased n-
3 PUF A concentration in all tissues at the expense of n-6 PUFA but the kinetics of these
changes varied among the various tissues. From this study it was determined that 4 wk
feeding was sufficient for incorporation of n-3 PUFA and reduction of arachidonic acid

in all excised tissues.

29

3.2 RATIONALE

Consumption of a n-3 PUFA-enriched diet for an extended period alters the lipid
profile of cells within human and animal tissue. Since it is assumed that an alteration in
the immune function must be preceded by a change in fatty acid composition of tissues of
interest, it is necessary to determine the appropriate feeding period required to attain
maximal n-3 PUFA incorporation. In this study we fed mice with DHA enriched diet for
various time periods to track the extent of n-3 PUFA incorporation into two lymphoid
tissues, spleen and Peyer’s Patches. (PP), and into peritoneal macrophages. The goal of
this study was to determine the kinetics of possible changes within cell membrane lipid

profiles.

3.3 INTRODUCTION

Lipids, as macronutrients, are rapidly taken up from food and distributed to every
cell membrane in the body. Thus, the type of fat consumed with diet can influence the
lipid profile of cell membranes. Eicosapentanoic acid (EPA) and docosahexaenoic acid
(DHA) are among the longest unsaturated n-3 PUFA derived from fish oil. EPA has 20
carbons and 5 double bonds and DHA has 22 carbons and 6 double bonds. These n-3
PUFA have beneficial effects on several inflammatory responses in cells. While the n-6
PUFA content in tissues is typically high, consumption of diets rich in EPA and DHA

significantly increase n—3 PUFA (119-124). In most tissues, these fatty acids are mainly

30

esterified in phosphatidylcholine and phosphatidylethanolamine classes of membrane
phospholipids (125, 126). There are great differences among tissues and organs relative
to lipid level and type. Some tissues are more resistant than others in changes imposed by

the diet fatty acid composition (127).

Among different cell types, neuronal cells are characterized by endogenously high
PUFA content. This high level of PUFA is assumed to contribute to the unique properties
of neuronal membranes, which include increased ability to fuse or exfoliate membrane
vesicles at high speed (127, 128). It is thus suggested that increased incorporation of n-3
PUFA in other cell types can change their membrane properties towards those of
neuronal cells. In in vitro studies, membranes rich in DHA are thinner, more permeable
and less stable, a fact that enhances vesicle exfoliation, fusion and flip-flop (129).
Moreover, enrichment of membranes with DHA induces unique packing arrangements of
the membrane lipids that can affect protein trafficking to membranes and therefore cell
signaling (130). Thus, n-3 PUFA content of cell membranes influences major aspects of
cell functionality and may have important implications to immune responses.

A well-accepted mechanism for the anti-inflammatory properties of n-3 PUF A is
their ability to incorporate into cell membrane phospholipids. It is known that upon
stimulation, arachidonic acid, (ARA) is released from cell membranes and is metabolized
enzymatically to eicosanoids, potent proinflammatory factors (67). Cyclooxygenase
(COX) and S-Iipoxygenase (5-LOX) enzymes will convert free arachidonic acid to give
rise to the 2-series prostaglandins (P65) and thromboxanes (TXs), hydroxyl- and
hydroperoxy derivatives (S-HETE and 5-HPETE) and the 4-series leukotrienes (LTs)

(68). Both PGEz and LTB4 promote production of cytokines like TNF-a, IL-1 and IL-6

31

(30, 68, 69). Increased consumption of n-3 PUFA results in high incorporation of those
fatty acids in cell membrane at the expense of ARA reducing the proportion of ARA as
an available substrate for synthesis of proinflammatory eicosanoids (67, 68).
Additionally, EPA and DHA incorporated into membrane phospholipids can
competitively gain access to the same enzymes that produce eicosanoids from ARA and
give rise to alternative classes of eicosanoids. EPA results to the 3-series PGs and TXs,
the 5-series LTs and the E series resolvins (68) and DHA to D-series resolvins,
docosatrienes and neuroprotectins (71). These alternative series of lipid mediators have
less proinflammatory properties and in certain cases exert anti-inflammatory effects (67,
71). Thus, incorporation of n-3 PUF A can differentially impact inflammation.

Immune cells are distributed through the body and can be found in higher
numbers concentrated in the lymphoid tissues. One of the secondary lymphoid organs of
the immune system is the spleen. Spleen contains a variety of immune cells concentrated
in the white pulp region. In the spleen, T- and B - lymphocytes contact antigen presenting
cells, macrophages and dendritic cells to survey for possible evidence of infectious agents
or other contaminants. It has been reported altered splenic fatty acid composition with
diets rich n-3 PUFA characterized by substantial increases of n-3 PUFA and reductions
of n-6 PUFA (121, 131). Additionally, functional modifications have been reported such
as suppression of lymphocyte proliferation and Il-2 secretion (132).

Peyer’s Patches (PP) are lymphoid tissue aggregations associated with the mucosa
within the small intestine. They are composed mostly of B-cells that are organized in
large, domed follicles. T-cells occupy the areas between follicles. A specialized

epithelium made up of M cells covers the surface of the follicle. Antigen presenting cells,

32

macrophages and lymphocytes are the main immune cells in PP. To our knowledge, there

is little information about n-3 PUFA incorporation in this tissue.

While spleen and PP are organized aggregations of lymphocytes, macrophages,
are another important immune cell population distributed throughout all the body.
Macrophages are generated from circulating monocytes in the blood and upon
stimulation they migrate to the tissues. Macrophages play a key role in host responses
against pathogens with their ability to phagocytose, release important cytokines and
produce cytotoxic mediators. Moreover, macrophages are significant message-producing
cells characterized by an intense oxidative and lipid metabolism generating reactive
oxygen species and lipid mediators; thus, their functions partly depend on their lipid
composition (133). Dietary n-3 PUFA can potentially modulate many of these functions.
Feeding n-3 PUFA diets results in macrophages that produce less eicosanoid upon LPS
stimulation (134). Macrophages from mice fed with fish oil diet produce significantly
lower levels of proinflammatory cytokines such as TNF-o, IL-IB (135) and IL-6 (134). In
addition higher levels of IL-10, an anti-inflammatory cytokine, are observed upon fish oil

feeding (I 35).

This study was conducted to test the hypothesis that a DHA-enriched diet alters
the fatty acid profile of major lymphoid tissues. Specifically, a diet enriched in DHA was
administered to mice for different time periods and the fatty acid profiles of spleen, PP
and peritoneal macrophages were determined using flame ionization gas chromatography.
We observed that feeding with DHA diet enriched these tissues in n-3 PUFA at the
expense of n-6 PUFA, but the extent of the change varied among the three different

tissues.

33

3.4 MATERIALS AND METHODS

Materials. All chemicals were purchased from Sigma (St. Louis, MO) unless
otherwise noted. To identify fatty acid composition among isolated phospholipids, the
following standards were used: PUFA-1 marine source (Supelco, PA), PUFA-2 animal
source (Supelco, PA), GLC #538 which consisted from 2] fatty acid methyl esters
(FAMEs) and GLC#463 which consisted from 52 FAMEs (Nu-CHECK, MN). All
included EPA, DHA and ARA. Detailed description of all methyl esters for each standard

is provided at the appendix.

Animals. Female B6C3F 1 mice (4 wk old) were purchased from Charles River
Laboratories (Portage, MI). Housing, handling and sample collection procedures
conformed to the policies and recommendations of MSU Laboratory Animal Research
Committee and were in accordance with guidelines established by the National Institutes
for Health. Mice were housed three per cage in a humidity (45-55%)- and temperature
(23-25°C) controlled- university animal care facility room with a 12 hr light and dark
cycle. Mice were acclimated for 1 wk prior to experiment initiation and fed experimental

diets until they were terminated.

Diets. Experimental diets were based on the AIN-93G formulation (Dyets Inc.,
Bethlehem, PA) of Reeves (136) with minor modifications. Both diets contained 920 g/kg
AIN-93G formulation (except for oil), 10 g/kg AIN-93VX Vitamin Mix, and 10 g/kg
corn oil (Dyets Inc., Bethlehem, PA). In control diets, 60 g/kg high oleic acid safflower
oil (Hain Celestial Group, Inc) were added, whereas in DHA diets 60 g/kg MEG-3TM

DHA-enriched oil (min 55 % DHA and 3 % EPA, Ocean Nutrition Canada Ltd,

34

Darrnouth, Nova Scotia) were added. Fatty acid compositions of the diets, based on
supplier data, are summarized in Table 3.1 Diets were prepared every 2 wk, stored in

aliquots at -20°C. Fresh diet was fed to mice daily.

Experimental design and sample preparation. Mice were fed control or DHA
diets for 2, 4, or 6 wk prior to termination. Four days before the completion of the feeding
period, mice were injected intraperitoneally with 1.5 ml of 3% (w/v) aged Brewer’s
thioglycollate broth to elicit macrophage migration to the peritoneal cavity. Mice were
anesthetized and killed by cervical dislocation at intervals. Peritoneal exudates were
collected by washing out the peritoneal cavity with 10 ml cold HBBS buffer. Spleen and
PP were excised. Peritoneal exudates were concentrated by centrifugation and all
experimental samples were stored under N2 atmosphere in microtubes at -80°C until

further analysis.

Lipid extraction, phospholipid isolation and FAME formation. Lipid extraction
was performed as described by Hasler et al. (199]) with slight modifications (137). The
frozen peritoneal exudate pellet suspended in 0.6 ml PBS. One hundred micrograms (100
pg) of spleen, seven PP and the peritoneal exudate suspension in PBS, were homogenized
with 2.4 ml of a 2:1 mixture (v/v) of chloroform-methanol. A second extraction was
further performed with 86:14:] mixture (v/v) of chlorofonn-methanol-saline. To remove
remaining water, anhydrous sodium sulfate was added in the extracts for overnight
incubation at —20°C, and filtered with #1 filter paper. Phospholipids were then separated
from the lipid extract using solid-phase extraction. First, the extract was applied to Maxi-

clean Silica cartridges, 600 mg (Alltech Associates, IL) and neutral lipids and free fatty

35

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acids were eluted with 10 ml chloroform followed by 5m] chloroforrn-methanol 20:]
(v/v). Phospholipids were then eluted with 10 m1 methanol and collected to clean, screw-
capped, glass tubes. Following evaporation under streaming N2, fatty acid methyl esters
were prepared by incubation with 1.5 ml 14% trifluoride boron in methanol for 1 hour at
90°C. Fatty acid methyl esters (FAME) were isolated with hexane extraction, dried and

resuspended to 50 pl of chloroform before the performance of the analysis.

Fatty acid analysis. The fatty acid profile compositions of the experimental
samples were analyzed by capillary gas chromatography (GC-2010-FID, Shimanzu
Scientific Instruments, Chicago IL). An ECTM- WAX capillary column 30 m length and
0.25 mm inside diameter (Alltech, IL) was used for separation of FAME. Oven
parameters included a 10°C/min increase from 40 to 190°C with a 5 min hold and a
further 10°C/min increase from 190 to 240°C for a 20 min hold. Peaks were assigned
based upon retention times of commercial standards. The results are reported as
percentage of total phospholipids and they were calculated by dividing the integrated area
of each peak by the total area of all identified peaks x 100. Percentages less than 0.5%

were considered minimum level of detection.

Statistics. Data was analyzed using the Sigma Stat Software version 9 (Jandel
Scientific, San Rafael, CA). Data was subjected to two-way ANOVA for time and diet
factors. Differences were considered significant at p<0.05. All pairwise multiple

conparisons were done using Holm-Sidak method.

37

3.5 RESULTS

Fatty acid composition of spleen

The effect of the diets on splenic fatty acid composition is shown in Table 3.2.
Among the monounsaturated fatty acids, oleic acid, 18:1n9, (0A), was detected in high
levels in spleen. Feeding with DHA diet had no significant impact on the levels of OA in
spleens compared to those at the start of the experiment. However, consumption of
control diet increased OA incorporation in the splenic tissue by almost 61% by the 6‘h wk
of feeding. 0A was incorporated in a time-dependant way with maximum levels reached
at 6 wk.

The PUFA profile in spleen was modified significantly for both n-6 and n-3 fatty
acids. Of the n-6 PUFA, ARA underwent the greatest changes. DHA diet decreased ARA
content by almost 60% within the first 2 wk, a stable reduction that was not firrther
augmented significantly with longer feeding time. In contrast, ARA content in control-
fed mice increased in the spleen in a time-dependent way, reaching a final increase of
33% within 6 wk. Other n-6 PUFA affected by the diets include linoleic acid, 18:2n-6,
(LA), and gamma-linolenic acid, 18:3n-6, (GLA). Both diets reduced LA levels
compared to 0 wk. However, the DHA diet contributed to smaller LA reduction (~4]%)
than control diet (~54%) at 4 wk feeding. For both diets, the magnitude of LA reduction
became smaller with longer feeding time. GLA also was reduced compared to 0 wk, and
DHA diet contributed to a greater reduction than the control diet after 4 wk feeding.

Consumption of the DHA-enriched diet, greatly, modified the n-3 PUFA profile

in the spleen. It is observed that n-3 PUFA extensively incorporate upon DHA

38

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39

supplementation in as little as 2 wk. EPA concentrations were increased to 4-5% in
DHA-fed mice, while at all times it was undetectable in control-fed mice.
Docosapentaenoic acid, 22:5n—3, (DPA) increased from 0.6 to 2.1- 2.6% within 4 to 6 wk.
DHA was endogenously present at 1.5 — 3.0% at 0 wk and reached final levels of almost
9.5% of total phospholipids. Maximum incorporation of total n-3 PUFA and significant

reduction of total n-6 PUFA was observed within 4 wk of DHA feeding.

Fatty acid composition of Peyer ’5 patches (PP)

Fatty acid composition of PP is shown in Table 3.3. The DHA diet had no effect
on CA concentration comparable to 0 wk. However, the control diet increased CA by
39% after 6 wk feeding reaching 21% of total phospholipids final concentration.

Concerning n-6 PUFA, ARA levels changed significantly upon feeding with the
experimental diets. PP from DHA-fed mice had lower concentrations of ARA compared
to PP from control-fed mice. Levels of ARA reduced by almost 52% with the DHA diet
but they were unchanged with the control diet. Furthermore, LA was reduced in both
groups compared to 0 wk, but there were no differences between the two diets relative to

the magnitude of reduction. Incorporation of GLA was also not affected by the diets.

Among n-3 PUFA, EPA was detected only in the DHA-fed mice to 3.8 - 4.2%
levels. DHA was increased from 2% at 0 wk to almost 8.2% within 2 wk, however, the
final concentration was around 5.9% of total phospholipids after 6 wk feeding with the
DHA diet. In control-fed mice, DHA was between 1-2%. Additionally, DHA diet

increased DPA from undetectable levels to 0.6-1.5% after 4 wk of supplementation.

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41

Fatty acid composition of peritoneal macrophages

The fatty acid composition of peritoneal macrophage phospholipids is shown in
Table 3.4. Feeding with experimental diets altered the composition of cell membranes of
the major fatty acids. After 6 wk feeding, 0A was unaffected in the DHA- and was

increased by almost 43% in the control-fed mice.

Peritoneal macrophages from mice fed DHA had also significantly modified n-6
PUFA profile. DHA diet resulted in lower ARA at all time points compared to control-
fed mice. The magnitude of reduction reached almost 40%. Additionally, both diets
reduced LA content in peritoneal macrophages compared to 0 wk. DHA diet resulted in a
smaller reduction of LA than control diet, similarly to what observed in the spleen.

Incorporation of n-3 PUFA in peritoneal macrophages was slower compared to
other tissues. Levels of total n-3 PUFA reached maximum (15%) only after 6 wk feeding.
Moreover, EPA and DHA were detected in significantly higher levels in the DHA-fed
mice compared to control-fed only after 4 wk feeding. While initially in undetectable or
very low levels, EPA and DPA reached 4% at 6 wk with the administration of the DHA

diet. DHA increased from 3% to a final concentration of 7.4 %.

3.6 DISCUSSION

Gas chromatography was used for fatty acid analysis. This method of fatty acid analysis

is robust, precise, linear over a wide range of fatty acid concentrations, and is considered

the gold standard for measuring fatty acids (138). Compounds are identified mainly by

comparison of retention time with those of purchased standards. Four different standards

42

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43

containing a diverge variety of fatty acid methyl-esters (FAME) were used to identify
peaks within the samples (Appendix).

The results of the present study suggest that by modifying the lipid components of
a diet, we can alter the fatty acid composition of those immune tissues. First of all, we
observed that DHA diet increased n-3 PUFA concentrations in all tissues at the expense
of n-6 PUFA. Additionally, we observed differences in the extent and rate of these
changes among the various tissues. Finally, administration of the diets affected cell
membrane levels of only those fatty acids that were present in higher amounts in the
diets.

Lipids are among the main components of cell membranes, and affect cells’
fluidity, architecture, and possibly cell signaling. Translocation of long chain fatty acids
across the cell membranes is achieved by passive diffusion, or by certain membrane
proteins. In general, membrane transport involves the following steps: absorption,
membrane translocation and desorption of fatty acids in the cytoplasm. Translocation
within the membrane might occur by diffusion or might involve a membrane associated
protein (139). Free fatty acids in the cytoplasm are coupled with fatty acid binding
proteins (FABP) that solubilize the fatty acids from membranes. Fatty acids are then
esterified by long chain fatty acyl CoA synthetases. Fatty acid binding proteins
differentially target fatty acids to phospholipids, triacylglycerols or cholesterol esters,

according to cell type, and its function (139).

In the present study we observed that experimental supplementation with DHA
enriched n-3 PUFA content in tissue membrane phospholipids. DHA diet increased total

n-3 PUFA incorporation in all tissues examined and decreased ARA levels, and

44

subsequently total n-6 PUF A. Displacement of ARA from the tissue and replacement by
EPA or DHA suggests a competition for incorporation of PUFA in membrane
phospholipids. The displacement of ARA from membranes has important implications
for the anti-inflammatory properties of fish oil by reducing the production of ARA-
derived eicosanoids (67). Thus, incorporation of n-3 PUFA in membranes impacts
inflammation and supports the contention that a change in the lipid profile of immune

cells has a role in induction of inflammation.

Our results further suggest that the type of fat in the diet was reflected in the
changes of the fatty acid profile of the tissues. DHA diet contained 6% fish oil, which
consisted of 58% of DHA and 10% EPA. Not surprisingly, this diet increased mainly
incorporation of DHA in membrane phospholipids and in a smaller degree incorporation
of EPA. Control diet contained no n-3 PUFA and its consumption resulted in tissues with
unchanged DHA levels. Additionally, control diet contained 6% high oleic acid safflower
and resulted in significant increases of 0A in all tissues. Although control diet contained
higher amounts of LA than DHA diet, only in PP fatty acid profile was this differential

incorporation observed.

Our study suggests that not all tissues exchange their fatty acids to the same
extent. Spleen and PP were more responsive to changes in their fatty acid profile than
peritoneal macrophages. Although peritoneal macrophages reached a maximum
incorporation of n-3 PUFA comparable to spleen, this was apparent after 6 wk feeding,
while it was apparent at 2 wk for spleen and PP. Moreover, ARA was reduced in all
tissues studied, with PP and spleen being more responsive to changes than peritoneal

macrophages. One possible explanation might be that these experimental samples are

45

consisted of different cell populations. Spleen contains lymphocytes and a great amount
of erythrocytes that are not present in PP or in peritoneal macrophages. Each of these
populations has different turnover rate. Thus, different cells might be more or less
resistant to changes in their fatty acid profiles accordingly to their turnover rate (140).

In summary, our results suggest that the type of fat in a diet affects membrane
fatty acid composition. PP and spleen had rapid changes of their n-3 PUFA within 2 wk,
while peritoneal macrophages incorporated long chain fatty acids in a slower rate. Based
on these results, we could say that 4 wk feeding period is sufficient for a good
incorporation of n-3 PUFA into membrane phospholipids of major secondary lymphoid
organs. Future studies on the impact of n-3 PUFA on the structural and functional
characteristics of cell membranes are likely to increase our understanding of n-3 PUFA

immunomodulatory effects.

46

CHAPTER 4. EFFECTS OF DOCOSAHEXAENOIC ACID CONSUMPTION ON
ENTERIC REOVIRUS INFECTION: IMMUNOGLOBULIN RESPONSES AND

VIRUS CLEARANCE

4.1 ABSTRACT

In addition to their well-known anti-inflammatory properties, n-3 polyunsaturated
fatty acids (PUF A) have been reported to impair host resistance to infectious agents. We
hypothesized that docosahexaenoic acid (DHA) consumption may reduce
immunoglobulin responses to reovirus infection and therefore may reduce viral clearance.
Mice were fed with either control AIN-93G diets or AIN-93G diets containing 3% DHA
for 4 wk. They were then, orally gavaged with reovirus serotype 1, strain Lang (Tl/L).
Reovirus induced robust IgA and IgGZa responses within 7 to 14 days ((1). Reovirus-
specific IgA in fecal pellets increased similarly in control and DHA-fed infected mice
from 2 to 8 (1 post infection (PI); however, at 10 (1 PI, DHA-fed mice secreted
significantly more reovirus-specific IgA. Furthermore, specific IgA and IgG production
did not differ in Peyer's patch (PP) and spleen cultures derived from the two groups at 14
d post-infection (PI). Reovirus-specific IgA and IgG2a titers were also not significantly
different in sera collected from mice at 7 and 14 d PI. When plaque-forming units (PFU)
were assessed in PP-depleted intestinal tissues, no significant differences were detected
between the two groups. However, real-time PCR indicated that, reovirus (L2 gene)
RNng feces in infected DHA-fed mice was higher at day 2, 4 and 6 (1 PI than in

infected control-fed mice but by 8 d PI, the virus was completely cleared in both groups.

47

Additionally, more viral RNA was detected in PP throughout the time course of the
infection in DHA-fed mice. Taken together, these data suggest that in this murine model,
DHA consumption transiently interfered with enteric reovirus clearance, but did not

affect the humoral response to the virus or overall resolution of the intestinal infection.

4.2 RATIONALE

Dietary exposure to the mycotoxin deoxynivalenol (DON) induces upregulation
of IgA production in the mouse and causes clinical signs analogous to early stages of
human IgA nephropathy (IgAN) (25, 141, 142). Consumption of n-3 polyunsaturated
fatty acids (PUFA)—enriched fish oil attenuates DON induced murine IgA nephropathy
(IgAN) by suppressing the induction of proinflammatory cytokines involved in IgA
dysregulation (13, 14, 143). In this study we investigated whether n-3 PUFA attenuate
IgA production only in pathological situations, such as defined in the DON — IgAN
model, or whether they also impair IgA and other mucosal responses to common
pathogens. We used enteric reovirus infection model to examine the effects of a n-3

PUFA, docosahexaenoic acid (DHA), on immunoglobulin responses and viral clearance.

4.3 INTRODUCTION

Among the dietary lipids, n-3 PUFA derived from fish oil are considered to be

particularly beneficial for human health. Nutritionists have long endorsed fish as part of a

heart-healthy diet. In 2004 the Food and Drug Administration (FDA) gave "qualified

48

health claim" status to two n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic
acid (DHA). Specifically they stated that "supportive but not conclusive research shows
that consumption of EPA and DHA omega-3 fatty acids may reduce the risk of coronary
heart diseases” (1). Besides heart-related diseases (2-4), there is experimental and clinical
evidence that other inflammatory diseases can be prevented by or treated with n-3 PUFA
administration. These include rheumatoid arthritis (5), inflammatory bowel disease
(IBD), colitis (6, 7) and immunoglobulin A nephropathy (IgAN) (8-15). There is also
some indication that n-3 PUFA contribute to colon cancer prevention by increasing the
cytotoxicity of several anticancer drugs and by reducing angiogenesis and induction of
inflammation and metastasis (16, 144). Moreover, studies have shown that DHA
promotes the normal brain development of the fetus and the newborn (1 7-19). The role of
n-3 PUFA in neurological and psychological diseases has been another recent research

focus (20-23).

Studies in our laboratory have used an experimental mouse model of human IgA
nephropathy (IgAN) to examine the mechanisms of the beneficial effects of n-3 PUFA in
retarding renal disease progression. In this model, a mycotoxin, deoxynivalenol (DON),
specifically modulates mucosal immune responses of the mouse, targeting the
development, differentiation, and homing of IgA-producing plasma cells in mucosal sites
(24). DON dramatically increases IgA in the systemic compartment, where IgA is
normally found in small amounts. Dietary supplementation with fish oil retards the
progression of DON-induced IgAN- like disease in the mouse by reducing IgA immune
complexes in blood and mesangial IgA deposition in kidney (14). The mechanisms by

which n-3 PUFA mediate these effects are likely to involve altered balance of n-6 and n-3

49

derived eicosanoids and reduced inflammatory responses. Among the inflammatory
responses generated by DON, interleukin 6 (IL-6), a critical cytokine in promoting
differentiation and proliferation of plasma cell precursors, is reduced significantly in n-3
PUFA-fed mice (26). Therefore n-3 PUFA reduce the progression of the disease.
However, it is not known whether fish oil reduces IgA production during normal

responses to mucosal pathogens.

Numerous models have been used to evaluate immunosuppressive effects of fish
oil and whether n-3 PUFA interfere with the clearance of infectious microorganisms (31).
The type of n-3 PUFA, the dose and time period of supplementation, the nature of the
pathogen, the route of administration, and genetic variation of the host can be factors
determining whether n-3 PUFA impair, enhance, or have no effect on host resistance.
Only a few studies with viral agents have been done to assess possible
immunomodulatory effects of n-3 PUFA and to our knowledge, no studies have

employed an enteric viral agent.

The viral models used to estimate possible modulatory effects of n-3 PUFA on
host resistance include cytomegalovirus (CMV), murine retrovirus (MAIDS), and
influenza virus. Mice supplemented with fish oil and then infected with CMV were just
as susceptible to the infection as control mice (80). Mice fed with fish oil for 8 wk and
then infected with MAIDS had significant enhanced survival compared to control mice
(85). Using the same model Xi et al. (63) demonstrated that serum levels of IgG and IgM
were reduced in mice fed fish oil. Additionally, fish oil consumption reduced virus
induction of TNF—a, ILl-B, IL-2 and IFN-y during the infection. In studies with influenza

virus, Byleveld et al. (41) reported that fish oil feeding interfered with viral clearance

50

from lungs and reduced IgA secretion in the respiratory mucosal surfaces. Furthermore,
they reported that IFN-y mRNA expression was down regulated by fish oil
supplementation. These studies suggest that fish oil can enhance or impair immune

responses to viral infections depending on the virus used.

Respiratory enteric orphan virus (reovirus) belongs to the Reoviridae family, the
largest of the six families of viruses with double stranded RNA genome. Type 1 L
reovirus (Tl/L) has been widely used as a model for an enteric infection (34-37, 86).
Among other genera included in the same family is rotavirus, a human pathogen, which is
leading cause of gastroenteritis among children and infants (108). Reovirus infection

often occurs in humans, but most cases are mild or subclinical.

Intestinal infection by reovirus in the mouse is well-studied. Briefly, reovirus
infects the small intestine with preference for the ileum (99). Reovirus entry is mediated
through attachment to receptors on M cells residing on the dome of Peyer’s patches (PP)
(96). M cells conduct vesicular transport of the antigen from the mucosal surface into PP,
a site where antigen presentation takes place and immune responses are initiated (90,
145). Reovirus infection induces robust antibody reponses, as evidenced by mucosal IgA
production, as well as serum IgA and IgG production (100, 108, 115). Furthermore,
reovirus induces strong cell-mediated responses in the gut as evidenced by the
appearance of reovirus-specific CD4+ and CD8+ cytotoxic lymphocytes (CTLs) in PP
and intraepithelial lymphocytes (IEL) (97, 100, 115). Oral exposure to reovirus induces a
robust Th1 response mixed with some Th2 cytokines that is characterized by increased
IFN-y, IL-5 and IL-6 production in the PP (109, 111). Intestinal infection of wild type

mice is a self-limited infection that is cleared within 7 to 10 days.

51

Reovirus has been successfully used to evaluate intestinal immunotoxicity (34-
37). By using as infection model this well-characterized enteric virus in the mouse it
should be possible to investigate nutritional modulation of normal immune responses.
The objective of this study was to test the hypothesis that n-3 PUF A consumption will
result to reduced immunoglobulin responses to reovirus infection and will interfere with
viral clearance. The results suggest that n-3 PUFA did not markedly alter mucosal or
systemic immunoglobulin production to reovirus and only transiently delayed clearance

of the virus from the intestine.

4.4 MATERIALS AND METHODS

Chemicals and virus. All chemicals were purchased from Sigma (St. Louis, MO)
unless otherwise noted. Reovirus serotype 1, strain Lang (Tl/L) was used for all the
experiments. The virus was grown in L929 fibroblast cells at 34°C in DMEM medium
with 5% (v/v) fetal bovine serum (FBS; Atlanta Biologics, Atlanta, GA), 100 U/ml of
penicillin and 100 ug/ml of streptomycin. Third-passage plaque-purified virions were
used for mouse infection and prepared by extraction with 1,1,2-trichloro-l,2,2-
trifluoroethane followed by discontinuous CsCl gradient centrifugation (36, 102). Titers
of the purified virus were determined by plaque assay as described by Cuff et al. (146).

Animals. Female B6C3F 1 mice (4 wk old) were purchased from Charles River
Laboratories (Portage, MI, USA). Housing, handling and sample collection procedures
conformed to the policies and recommendations of MSU’s Laboratory Animal Research

Committee and were in accordance with guidelines established by the National Institutes

52

for Health. Mice were housed three per cage in a humidity (45-55%) and temperature
(23-25°C) controlled university animal care facility room with a 12 hr light and dark
cycle. Mice were acclimated for 1 wk prior to experiment initiation and fed experimental
diets (detailed description provided later) until they were terminated. Reovirus-exposed
mice were held in microisolator cages under negative pressure laminar flow in a

Biosafety Level 2 room at the Michigan State University Research Containment Facility.

Diets. Experimental diets were based on the AIN-93G formulation (without oil)
(Dyets Inc., Bethlehem, PA) of Reeves (136) with minor modifications. Both diets
contained 920 g/kg AIN-93G, 10 g/kg AIN-93VX Vitamin Mix, and 10 g/kg corn oil
(Dyets Inc., Bethlehem, PA). In control diets, 60 g/kg high oleic acid safflower oil (The
Hain Celestial Group, Inc) were added, whereas in DHA diets 60 g/kg MEG-3TM DHA-
enriched oil (min 55 % DHA and 3 % EPA, Ocean Nutrition Canada Ltd.) were added.
Diet composition of the main fatty acids as provided by the suppliers, is summarized in
Table 3.1. Diets were prepared every 2 wk, stored in aliquots at -20°C. Fresh diet was fed
to mice daily.

Experimental design and sample preparation. Mice were fed control and DHA
diets for 4 wk prior to reovirus infection. They were then infected by oral gavage with
3x107 plaque-forming units (PFU) in a total volume of 100 pl borate-buffered saline (pH
7.4) containing 2% (w/v) of gelatin (100). Mice were fed control or DHA diets unitl
experimental termination. At specific time intervals, mice were bled from the lateral
saphenous vein into heparinized tubes and fecal pellets were collected. Plasma was
separated from blood samples and stored at 4°C for later analysis. Fecal pellets were

stored at -20°C until analysis. Pellets were weighed and suspended in PBS to a 10%

53

(w/v) final concentration, held on ice for 2 h, and sonicated for 15 sec. Suspencions were
centrifuged at 1,600 x g for 10 min at 4°C and 1.4 ml of the supernatant was cleared by
centrifugation at 18,000 x g for 10 min at 4°C. Supernatants fractions were used directly
for specific antibody detection by ELISA or for total RNA isolation.

Upon experiment termination, mice were euthanized with isoflurane, and
exsanguinated via the caudal vena cava. PP, spleen and intestines were collected from the
euthanized mice and were processed as described below.

Lymphoid fragment cultures. To assess ex vivo immunoglobulin secretion,
cultures of PP, spleen and lamina propria were used (36). Briefly, 7 PP per mouse were
removed from the intestine, pooled and washed three times in sterile HBSS (Gibco,
Grand Island, NY) with 5 ug/ml of gentamicin and twice with tissue culture medium
consisting of RPMI 1640 medium supplemented with 10% (v/v) FBS (Atlanta
Biologicals, Lawrenceville, GA), 2 mM L-glutamine, 0.5 uM 2-mercaptoethanol, 5 11le
of gentamicin, 100 U/ml penicillin, and 100 ug/ml streptomycin. Individual patches were
cut in half and incubated in 6-well plates with 2 ml of culture media for 5 days at 37°C
under 5% C02 without additional stimulation.

Spleen cultures were washed with tissue culture media, cut into small pieces (2 x
2 mm) and incubated in 6-well plates with 2 ml of culture media for 5 days at 37°C under
5% C02 without additional stimulation. Culture supematants were harvested and stored at
4°C for antibody detection by ELISA without further dilution.

Lamina propria cultures were established after removing PP, by cutting the
intestine longitudinally into 2—3 cm long pieces. Fragments were washed three times with

HBSS with 0.2% NaHCO3, 0.1M HEPES and 5 ug/ml gentamicin. Half of the fragments

54

were stored in 1 ml of sterile PBS containing 0.5% gelatin (w/v) (gel saline) at —80°C to
be used for viral titer detection. The other half of the fragments was incubated for 30 min
twice in 5 mM EDTA in HBSS to remove the epithelial cells. Fragments were washed
two more times with culture media and finally incubated in 6-well plates with 2 ml of
culture media for 5 d at 37°C under 5% CO2. Culture supematants were harvested and
stored at 4°C for antibody detection by ELISA without further dilution.

Virus plaque assay. Viral titers were measured by standard viral plaque assay
(147). Briefly, tissues were placed in 1 ml of sterile gel saline, freeze-thawed 3 times,
homogenized, and then sonicated. Serial dilutions (100 pl) in gel saline were incubated
on monolayers of L929 fibroblasts in 12-well tissue culture plates for 45 min at 34°C and
thereafter overlaid with 3 ml of 1% (w/v) agar in Medium 199 with 5% FBS (v/v) and
cultured at 34°C. Plaques were visualized with neutral red and counted after 7 d
incubation.

Real-time PCR. For viral RNA detection in fecal pellets, total RNA was extracted
from 200 pl supernatant of 10% (w/v) fecal suspension using Trizol reagent (Invitrogen,
Carlsbad, CA). PCR primers were selected from published sequence of 72 core spike (L2
gene) of reovirus Tl/L (88) as follows: forward, 5’ CTG ACG TCG ATC AGG TCG
TTG 3’ and reverse, 5’ GAT GTG GCA TGC ATG CAT GAG 3’. The size of the
expected amplicon was 97 bp. To denature dsRNA, 1 pg of the template was incubated
for 5 min at 95°C with 250 pM of random primers (Promega, Madison, WI) in a total
volume of 12 pl and then cooled on ice. Reverse transcriptase reaction was performed by
adding 4 pl of RT reaction buffer, 2 pl of 0.1M DTT, 1 pl of 10 mM dNTP, and 1 pl of

Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) and incubating at 42°C for

55

60 min. Purified reoviruses were added into 10% (w/w) of fecal pellet suspension from
vehicle mice at concentrations of 0, 10', 102, 103, 104,105 10°, 107 PFU/ml for standard
curve and sensitivity determination (36).

IFN-y mRNA was detected in total RNA isolated from PP with Trizol reagent and
treated with the Turbo DNA-free kit (Ambion, Austin, TX) according to the
manufacturer’s protocol. Probe and primers for mouse IFN-y mRNA and 18S rRNA
(endogenous control) were purchased from Taqman gene expression assays (Applied
Biosystems, Foster City, CA). PCR reactions for IFN-y mRNA and 18S rRNA
quantification were performed on the ABI PRISM 7700 Sequence Detector System using
the One-Step RT-PCR Master Mix Reagents kit according to manufacturer’s protocol. Ct
values were related to RNA concentrations using standard curves derived from serial
dilutions of total RNA and normalized by dividing the IFN-y mRNA Ct values by 188
rRNA Ct for each well. All the results were expressed relative to vehicle, control fed
group, which was mock-infected group with PBS and sacrificed at the end of the
experiment.

ELISA. Serum, culture fluids and supernatant of fecal suspensions were assayed
for virus-specific antibodies by ELISA using a modification of the procedure of Major
and Cuff (147). Briefly, 96—well plates were coated overnight at 4°C with 50 pl /well of
purified reovirus Tl/L, diluted to 1x108 particles/m1 in 0.1 M NaHCO3 coating buffer
(pH 9.6). Plates were washed four times with PBS with 0.1% Tween 20 (PBS-T) and
blocked with 100 pl of blocking buffer (3% w/v bovine serum albumin, BSA in PBS-T)
for 2 h at room temperature. Serial dilutions (100 pl) of serum, culture fluids, or fecal

supematants were added to each well in duplicates and incubated at 4°C for 16 h. The

56

plates were washed seven times with PBS-T, and 100 pl of goat anti-mouse IgG2a-, or
IgA-HRP conjugates (123000) in blocking buffer was added to each well. Plates were
incubated at room temperature for 2 h. Following seven washes with PBS-T, 100 pl of K-
Blue Max TMB substrate (N eogen, Lansing, MI) was added to each well, and plates were
incubated at room temperature for 5 min. Reactions were terminated by adding 50 pl of
2N H2SO4 and the absorbance was read at 450 nm on Vmax Microplate Reader
(Molecular Devices Corp. Sunnyvale, CA). Absorbance at 450 nm was used as endpoint
for fecal, PP, spleen and intestinal fragments cultures. Plasma antibody titers were
defined as the last dilution yielded absorbance of 0.2 or higher at 450nm; a log of the
dilutions was used to calculate the geometric means.

Statistics. We used 2-way ANOVA to determine the effects of time and diet.
Multiple pairwise comparisons were performed using the Bonferroni corrected t test.
Data were reported as means :t SEM. The critical level for normality and equal variance
test was a = 0.01 and the critical level for the rest of the comparisons was a = 0.05. When
used for statistical analysis, nondetectable (ND) data were assigned the limit of detection
value. Data are representative of 2 separate experiments. Statistical analysis performed

with the Sigma Stat Software version 9.0 (Jandel Scientific, San Rafael, CA).

4.5 RESULTS

DHA consumption does not affect intestinal response to oral reovirus challenge

Following oral reovirus infection, reovirus-specific IgA is produced in the lamina

propria and is secreted into the lumen, contributing to early clearance of the virus from

57

the gut (100, 146) as well as protection against reinfection (102, 146). The reovirus-
specific IgA responses in control and DHA-fed mice were compared over 10 d. In
infected control and DHA-fed mice, reovirus-specific IgA antibody was detectable
beginning 6 d post infection (PI) and was highly elevated at 8 (1 PI and 10 (1 PI (Fig. 4.1).
Both infected control and DHA groups exhibited robust and similar induction of specific
IgA. At 10 d PI, DHA-fed mice, secreted statistically significant levels of specific IgA
compared to mice fed control diet.

Ex vivo antibody secretion by isolated PP was compared in the two groups after
reovirus infection 14 d PI. Similar levels of reovirus-specific IgA (Fig. 4.2A) and IgG2a
(Fig. 4.28) were induced in both PP cultures from infected control- and DHA-fed mice.
Ex vivo IgA secretion was also compared in lamina propria cultures. Both control and
DHA-fed mice showed a similar robust induction of specific IgA at 5 and 10 (1 PI (Fig.
4.3). Secretion of reovirus-specific IgA from lamina propria cultures resembled the
secretion of reovirus-IgA into lumen as evidenced by ELISA of the feces. In feces, IgA
and IgG2a were also similar in control- and DHA- fed mice with the only difference
being observed at 10 (1 PI, where the response was higher in DHA-fed mice. Overall,
these data suggest that the induction of mucosal IgA was not significantly affected by

DHA supplementation in the diet.

DHA consumption does not affect the systemic humoral responses to reovirus
Reovirus infection by the oral route results not only in production of mucosal
antibodies, but also in circulating specific IgA, IgG and IgM in serum (108). These

antibodies enhance protection against systemic spread of the infection and also contribute

58

A c
- —0— control
_ —0— control+Reovirus

—v— DHA
. —0— DHA +Reovirus

.0.0
woo
x.

  
 
   

.0.0
010)

Reovirus specific
IgA (A450)
0
A

 

9.0.0.0
O—KNOO

 

days post infection

Figure 4.1. Effects of DHA on fecal IgA levels in response to reovirus. Mice were fed
with DHA and control diet for 4 wk before infection with 3x107 PFU of reovirus. ELISA
absorbances for reovirus-specific IgA in feces collected at different time points PI.
Values are means :t SEM, n = 4. Within a group, means without a common letter differ, P
< 0.05. * Different from infected mice fed a control diet at that time, P < 0.05.

59

.0
oo

.9

to:

8A

a. §0.6*
“<1:
gv
h<
390.4-
0

a:

DC

 

Reovirus spesific
lgGZa (A450)

a a
1"! 1:1 ..
Diet Con DHA Con DHA
Virus — - + +

 

 

Figure 4.2. Effects of DHA on the production of virus-specific antibodies in ex vivo
culture of PP. Mice were fed with DHA and control diet for 4 wk before infection with
3x107 PFU of reovirus. PP were collected at 14 (1 PI and production of reovirus-specific
IgA (A) and IgG2a (B) were determined in ex vivo cultures. Values are means :t SEM, n
= 6. Within a group, means without a common letter differ, P < 0.05.

60

95:9
“#0!

Reovirus specific
IgA (A450)
9
N

00000000000000.0000...
0 0.....0.0.0.0.0.0...0.0.0.0.0.0.0.0.0...0.0‘

  
  

000.000.00.000000000004

P
.3.
OJ

0

   

0
0

 

00
0

    
      

0 :0
0.

   
 

0
0

 

P
o

 

non-infected post infection

Figure 4.3. Effects of DHA on the production of reovirus-specific IgA in lamina
propria cultures. Mice were fed with DHA and control diet for 4 wk before infection
with 3x107 PFU of reovirus. Intestines were removed at 0, 5 and 10 (1 PI and processed to
ex vivo cultures that were incubated at 37°C under 5% CO2 for 5 d with no additional
stimulation. Values are means :1: SEM, n = 6. Within a group, means without a common
letter differ, P < 0.05. "' Different from infected mice fed a control diet at that time, P <
0. 05.

61

to immunological memory (101, 102, 108). When reovirus-specific responses were
assessed at 7 and 14 d PI, both serum IgA and IgG2a antibodies were induced after
infection but were not affected by DHA supplementation (Fig. 4.4). Similar results were
observed for reovirus-specific IgA and IgG2a production in spleen cultures obtained from
control and DHA- fed mice at 14 d PI (Fig. 4.5). Thus, as observed for mucosal Ig
responses, antibody induction in the systemic compartment was not significantly affected

by DHA supplementation.

Effects of DHA on viral presence and clearance from intestinal tissues

Reovirus infection causes a self-limited infection that immunocompetent, adult
mice are able to overcome within 10 d (87). Reovirus is shed into the intestinal lumen
during infection and it is not carried asymptomatically after the course of the infection. It
thus, can be continuously monitored in fecal pellets by real time PCR during the course
of the disease. Mice fed DHA diet had a significantly higher fecal viral burden than
control-fed mice as evidenced by nearly one log more viral RNA in their feces at 2, 4,
and 6 d P] (Fig. 4.6). However, by 8 (1 PI viral RNA was undetectable in both groups
suggesting that the ability to ultimately clear the infection was not affected.

To determine if DHA interfered with viral replication in PP, PP were collected at
1, 3, and 7 d PI and total RNA assessed for reovirus RNA. At 1 d PI, both groups had
same load of viral RNA in their PP, indicating no differences in the entry and uptake of
reovirus. However, at 3 d PI, significantly more viral RNA was detected in the DHA-fed
group. While there was no detectable viral RNA in the infected control group at 7 (1 PI,

the infected DHA group had nearly 6x104 copies of viral RNA/pg total RNA (Fig. 4.7).

62

Reovirus specific
IgA (log titer)

0» s»

0 0|

.N
on

 

Reovirus specific
lgGZa (log titer)

 

 

Virus _:__-__*'__'."_ - - + 4’
7days 14 days

Figure 4.4. Effects of DHA on generation of plasma virus-specific antibody
responses. Mice were fed with DHA and control diet for 4 wk before infection with
3x107 PFU of reovirus. Blood was collected at 7 and 14 (1 PI and specific IgA (A) and
IgG2a (B) titers determined in plasma by ELISA, n = 6. Values are means 3: SEM, n = 6.
Within a group, means without a common letter differ, P < 0. 05.

63

>

0.8
.9.
$12-$06
0) (V
w v
2 <2: 0.4
'5 2’
O
(D
0:

.0
N

.0
or

.0
4:.

Reovirus specific
IgGZa (A450)

a a

 

Diet Con DHA Con DHA
Virus - - + +

Figure 4.5. Effects of DHA on the production of reovirus-specific IgA and IgG2a in
ex vivo spleen cultures. Mice were fed with DHA and control diet for 4 wk before
infection with 3x107 PFU of reovirus. Spleens were collected at 14 d PI and production
reovirus-specific IgA (A) and IgG2a (B) were determined in ex vivo cultures. Values are
means :1: SEM, n =6. Within a group, means without a common letter differ, P < 0. 05.

64

104 -

    

  
 
 

    

      
  
     

  

      

 

 

 

 

 

A m Control
\ * — DHA
a) 3 c
.9 .910 2
82
U .55 C 6" *
N o. ;.; C
—| 78102 2:1
(I) 1;:
.3 ~93 2:3 b
0, E10 ~ 5:3 :5: :5 ,
0: $1 izi :;:
100 ND'ND 3:1 :3: l :2: a a a a
ConDHA 2d 4d 6d 8d 10d
uninfected post infection

Figure 4.6. Effect of DHA diet on reovirus L2 RNA in feces. Mice were fed with DHA
and control diet for 4 wk before infection with 3x107 PFU of reovirus. . Fecal pellets
were collected at intervals and total RNA analyzed by PCR, n = 4. Values are means i
SEM, n = 6. ND, not detectable. Within a group, means without a common letter differ, P
< 0. 05 . *Different from infected mice fed a control diet at that time, P < 0. 05.

65

 

 

 

106 -
B

E m Control 6'
.9 — DHA b
0- <
0 2105 l b b
o m ,,
N _ 3:3
_l m :5 53
a) "" 2’2 5::
a 9 $1 '91 'k
__ .‘o‘ :°:‘ a
> on 04 °I°i 1:9:
o 1 1:15 ;.;.
D: :3: :3

103 ND ND S:f :21 3

Con DHA 1 d E 7 d__
uninfected post infection

Figure 4.7. Effect of DHA diet on reovirus L2 RNA in PP. Mice were fed with DHA
and control diet for 4 wk before infection with 3x107 PF U of reovirus. PP were collected
at 0, l, 3, and 7 (1 PI and total RNA analyzed by PCR. Values are means :1: SEM, n = 6.
ND, not detectable. Within a group, means without a common letter differ, P < 0. 05.
*Different from infected mice fed a control diet at that time, P < 0. 05.

66

A plaque assay was used to determine if viral RNA levels in feces corresponded
with higher numbers of viral particles in intestinal tissues after the removal of PP. Both
infected control and DHA groups carried a virus load of almost 105 PFU/intestine at 5 d
PI, which was reduced by one log at 10 (1 PI (Fig. 4.8). No statistical differences were
detected between control and DHA-fed mice. These data suggest that while more viral
RNA was detected into the intestinal lumen of DHA-fed mice, the high DHA diet did not
increase the number of reovirus infectious units in the intestine.

To determine if the expression of IFN-y must have a role on the differences
observed for viral RNA in PP, we assessed IFN-y mRNA expression in PP at 1, 3, and 7
d PI. No significant differences on the IFN-y relative expression were detected although
there was a trend for the DHA fed mice to have lowered IFN-y expression at 3d PI (p =

0.173) and at 7d PI (p= 0.193) (Fig. 4.9).

4.6 DISCUSSION

Nutritional interventions can potentially shape the immune responses, increasing
or decreasing susceptibility to infections. In this study we tested the hypothesis that n-3
PUFA consumption will result to reduced immunoglobulin responses to reovirus
infection and will interfere with viral clearance. The gut mucosal lymphoid tissue
responds to reovirus infection with both secretion of antibodies and activation of

cytotoxic T-cells. In the gut, PP have key role in the induction of immune responses.

67

6 .
10 m Control

— DHA

_s

O
01
CT

 

 

 

 

 

 

 

(D
.E
'2}; 104 .
(D
...—a
E
\ 103 ‘
3
“a
102 .
ND ND
1 _ _
10 Con DHA 5 d
non- Infected post infection

Figure 4.8. Effect of DHA diet on infectious reovirus in intestinal tissues. Mice were
fed with DHA and control diet for 4 wk before infection with 3x107 PFU of reovirus.
Intestines were removed at 0, 5 and 10 (1 PI and processed to determine virus titers.
Values are means :1: SEM, n = 6. ND, not detectable. Within a group, means without a
common letter differ, P < 0. 05.

68

—0— Control
4 . —o— DHA

 

 

 

 

relative IF N-y mRNA expression

VH 1 3 5 7
days post infection

Figure 4.9. Effect of DHA diet on IFN-y mRNA expression in PP. Mice were fed with
DHA and control diet for 4 wk before infection with 3x107 PFU of reovirus. PP were
collected at 0, 1, 3 and 7 (1 PI. Total RNA isolated and IFNy mRNA was detected by RT-
PCR. Data are means i SEM, n=6 of IFN-y mRNA copies per pg RNA. The data were
normalized to the uninfected control diet group.

69

They are the first site of reovirus infection, contain aggregated lymphocytes, elicit
important innate immune responses and alert the immune system for reovirus invasion.
Reovirus antigens are taken up in this site and transfer to spleen where activation of B
and T-cells takes place. Thus, in PP both humoral and cytotoxic responses are initiated.
During reovirus infection, IgA producing cells are increased and robust specific-IgA
responses are elicited in PP (100). Reovirus-specific IgA contributes to clearance of the
virus from the gut (100, 146) and protects against reinfection (102). Additionally,
production of circulating specific IgA, IgG and IgM, enhance protection against systemic
spread of the infection and contribute to immunological memory (101, 102, 108).

The effects of n-3 PUFA on the induction of antibodies at the mucosal sites are
not well understood. Supplementation with n-3 PUFA significantly attenuated DON—
induced IgA elevation in serum resulted from dysregulation of the mucosal immune
responses (13, 14). Furthermore, induction of influenza-specific lung IgA and serum IgG
were reduced in mice fed fish oil (41). However, some studies imply that
supplementation with fish oil has no effect on antibody secretion. For example, fish oil
supplementation had no significant effect on secretion of IgA in breast milk, yet, it was
positively correlated with increased IgA (42). Additionally, patients suffering from distal
proctocolitis have similar percentages of IgA and IgG containing cells in the rectal
mucosa with administration of n-3 PUFA, while a significant reduction in IgM containing
cells was observed (7).

The present study demonstrates that fish oil did not deleteriously impact the
kinetics of virus-specific antibody secretion in the mucosal compartment. This was

consistent with our inability to see differences in antibody production from PP, lamina

70

propria and spleen cultures. However, DHA-fed mice secreted statistically significant
more reovirus-specific IgA at 10 (1 P1 in the intestinal lumen as detected in fecal samples
(Fig. 4.1). This extended elevation of antibody secretion for the DHA group could be
related to increased shedding of reovirus during the infection among the DHA-fed mice.
It was notably that more viral RNA was detected in feces and PP during the
infection in DHA-fed mice. However, we did not observe differences on the infectious
particles detected in intestinal tissues by plaque assay. It could be speculated that due to
reduced antiviral responses, DHA permitted higher viral RNA replication but there was
insufficient assembly of mature infectious viruses in the intestinal epithelial cells.
Another possibility could be related to epithelial cells which are not a mere passive
barrier but produce many cytokines potentially affecting the balance between crypt cell
proliferation and epithelial cell death (148). A Th1 cytokine response induces epithelia
cell proliferation while a Th2 response induces apoptosis (149, 150). Since n-3 PUFA
have been shown to suppress Th1 responses (151), they might result in enhanced turn-
over rates of differentiated epithelial cells shed into the lumen causing more viral RNA to
be detected in feces. Both of these hypotheses need firrther investigation. Overall though,
the results suggest only a transient effect of n-3 PUFA on viral clearance and no effect on

the overall host’s ability to resolve the infection.

In addition to antibody secretion and viral clearance, the profile of cytokines
induced upon infection determines the outcome of the disease. Here we only detected
IFN-y mRNA expression in PP to see if a polarized Th1 response is altered by the DHA
diet. We observed a trend towards lower IFN-y expression in DHA-fed mice that was not

proven statistically. It is known that supplementation with n-3 PUFA reduces expression

71

of Th1 cytokines, such as IL-1, IL-2, TNF-a, and IFN-y ex vivo (41, 44, 60-62). More
specifically, mice fed with fish oil and infected with influenza virus exhibit impaired
IFN-y production in the lung (41). Furthermore, mice fed with fish oil and infected with
MAIDS had reduced TNF-a, IL-lB, IL-2 and IFN-y in spleen cultures (63). Reduced
IFN-y production was shown also in studies employing fish oil with Listeria
monocytogenes (64, 65), sometimes even before the infection (65). Overall these studies

are consistent with the trend toward reduction in IFN-y observed here.

Viral clearance in the intestine is facilitated by several immune responses elicited
by multiple cells. The classic view of the immune responses focuses on the production of
antibodies and cell cytotoxicity. Reovirus—specific IgA production protects against
reinfection but its role in the clearance of the initial infection might not be indispensable
(102). Thus, n-3 PUFA effects on IgA secretion in the mucosa might not directly affect
early clearance of reovirus. In contrast, a diminished cytotoxic T-cell response might be
detrimental for the clearance of reovirus infection. Besides, n-3 PUFA has been

previously related to reduced cytotoxic responses (46, 48).

Specifically for the mucosal system, cytotoxic responses involve cells that are
activated by both thymus-dependent and thymus-independent mechanism. In vitro studies
show that treatment with n-3 PUFA reduces the expression of major histocompatibility
complex (MHC) molecules on antigen presenting cell (APC) surfaces, a key mechanism
for thymus-dependant activation of cytotoxic T-cells (53-55, 102). Treatment with DHA
results also in B-cells with reduced levels of MHC-I on their surface, possible interfering
with their ability to present antigens (55). Furthermore, n-3 PUF A can alter critical

intracellular signaling pathways and cytokine production patterns and thus direct essential

72

cell-differentiation steps involving in dendritic cell maturation (56). Such interference
with antigen presentation could impair the cellular immune responses to an infection and

possibly delay viral clearance.

Byleveld et al, (2000) showed that with fish oil supplementation, the cytotoxicity
of lung influenza-specific T-cells significantly reduced (46). However, in the mucosal
system thymus-independent cytotoxic cells contribute equally if not in a higher degree in
reovirus clearance (115). These cells are activated in a MHC-I independent way, and
lowered expression of those molecules on APC might not be reflected as impairment of
the cellular cytotoxic responses. Thus, fish oil might not be as potent suppressor of viral
clearance in the intestine as is considered for bacteria in agreement with our study, where
only a transient delay was observed for reovirus clearance. Moreover, reovirus infection
in mice with non-functional MHC-I molecules generates a robust reovirus-specific
antibody production (147), further suggesting that possible reduction of MHC-I molecule
expression by fish oil might not be affect the antibody responses, in agreement again with
our results. However, the effect of fish oil on antigen presentation is not clearly

understood and in vivo experiments need to be done.

In addition to the classical adaptive immune responses described earlier, focus
lately has been given to the contribution of the innate immune responses to host
resistance to infection. Among the innate responses type I IFNs seem to be indispensable
for reovirus clearance in the intestine in contrast to natural killer cell activation or TLR
signaling {Johansson, 2007 355 /id}. IFN-a and IFN-B are produced within the first hours
of the infection, drive the infected cell into an antiviral state and direct an effective

adaptive response to clear the pathogen. Upon reovirus infection, type I IFNs are mainly

73

produced in PP, where reovirus is encountered for the first time (117). Fish oil
supplementation and infection with influenza virus had no adverse effects on IFNa/B
expression in the lung (41), but the impact of n-3 PUF A on IFN type I cytokines in the
gut mucosal is not well-known. The effect of fish oil on this type of innate responses in
the intestine is another possible mechanism that needs further investigation to understand
the observed higher viral load for the DHA-fed mice.

The amount of n-3 PUFA consumed by mice in this study exceeded that normally
consumed by humans. Typical n-3 PUF A intake recommendations for healthy people are
0.3 - 0.5 g/d for DHA and EPA along with 0.8 - 1.1 g/d for a- linolenic acid, while 3 g/d
is recommended for disease prophylaxis and therapy (15). The dose used in the study
corresponds almost five times the maximum recommended consumption levels of n-3
PUFA for humans and it would be similar to a 16 g/d consumption of DHA and EPA
combined (15). Nevertheless, the observation that this high dose did not affect antibody
production and overall viral clearance suggests that immunosuppression of the mucosal
immune responses would not be a major safety concern.

Overall our studies suggest that while DHA consumption transiently delayed viral
clearance, robust reovirus-specific antibody responses were generated and thus, virus
shedding ultimately cleared in both the groups. Future studies with enteric viral models
are essential to understand how nutritional interventions exert immunomodulatory effects
on the mucosal system and how the immune system compensates for any alteration

among its normal functions.

74

APPENDIX

75

Table l. The following reference standard was purchased from Supelco, PA. The
information presented here is what the company provided us.

 

 

 

GLC REFERENCE STANDARD
PUFA-1, marine source
Cat #47033
CHAIN ITEM
C 14:0 METHYL MYRI STATE
C16: 1 METHYL PALMITOLEATE
C 1 8 :0 METHYL STEARATE
C l 8: 1 n9 METHYL OLEATE
C l 8: 1n7 METHYL VACCENATE
C18z2n6 METHYL LINOLEATE
C 1 8 :4n3 METHYL STEARIDON ATE
C20: 1n9 METHYL l l-EICOSENOATE
C20z5n3 METHYL ElCOSAPENTAENOATE
C22: lnll METHYL 13-DOCOSENOATE
C22: 1 n9 METHYL 1 l-DOCOSENOATE
C22:5n3 METHYL DOCOSAPENTAENOATE
C22:6n3 METHYL DOCOSAHEXANEONATE
C24: 1 n9 METHYL NERVONATE

 

76

Table 2. The following reference standard was purchased from Supelco, PA. The
information presented here is what the company provided us.

 

 

 

GLC REFERENCE STANDARD
PUFA-2, animal source
Cat #47015-U
CHAIN ITEM
C14:0 METHYL MYRISTATE
C1620 METHYL PALMITATE
C16: ln7 METHYL PALMITOLEONATE
C 1 8:0 METHYL STEARATE
C 1 8: 1 n9 METHYL OLEATE
C18: ln7 METHYL VACCENATE
C 1 8:2n6 METHYL LINOLEATE
C18z3n6 METHYL G-LINOLENATE
C18:3n3 METHYL LINOLENATE
C20: 1n9 METHYL 1 l-ElCOSENOATE
C20z4n6 METHYL ARACHIDONATE
C20:5n3 METHYL EICOSAPENTAENOATE
C2224n6 METHYL ADRENATE
C22:6n3 METHYL DOCOSAHEXANEONATE

 

77

Table 3. The following reference standard was purchased from Nu-CHECK, MN. The
information presented here is what the company provided us.

 

 

 

GLC REFERENCE STANDARD
SPECIAL PREPARATION
Cat # 538
CHAIN ITEM
C 1420 METHYL MYRI STATE
C14: 1 METHYL MYRISTOLEATE
C1620 METHYL PALMITATE
C 1 6:1 METHYL PALMITOLEATE
C1820 METHYL STEARATE
C18: 1 METHYL OLEATE
C 18:1 METHYL VACCENATE
C1822 METHYL LINOLEATE
C 1 8:3 METHYL LINOLENATE
C20:0 METHYL ARACHIDATE
C20: 1 METHYL l l-EICOSENOATE
C20:2 METHYL 11-14 EICOSADIENOATE
C20:3 METHYL 11-14-17 EICOSATRIENOATE
C20:4 METHYL ARACHIDONATE
C2025 METHYL EICOSAPENTAENOATE
C2220 METHYL BEHENATE
C22: 1 METHYL ERUCATE
C22:6 METHYL DOCOSAHEXAENOATE
C2320 METHYL TRICOSANOATE
C2420 METHYL LIGNOCERATE
C24: 1 METHYL NERVONATE

 

78

Table 4. The following reference standard was purchased from Nu-CHECK, MN. The
information presented here is what the company provided us.

 

GLC REFERENCE STANDARD
SPECIAL PREPARATION

 

 

Cat # 463
CHAIN ITEM
C420 METHYL BUTYRATE
(25:0 METHYL PENTANOATE
C620 METHYL CAPRONATE
C7:0 METHYL HEPTANOATE
C820 METHYL CAPRYLATE
C920 METHYL NONANOATE
c10:o METHYL CAPRATE
or 1:0 METHYL UNDECANOATE
c11:1 METHYL UNDECENOATE
C12:o METHYL LAURATE
C1221 METHYL DODECENOATE
c13:o METHYL TRIDECANOATE
(313:1 METHYL TRIDECENOATE
C1420 METHYL MYRISTATE
c14:1 METHYL MYRISTOLEATE
C1520 METHYL PENTADECANOATE
(215:1 METHYL PENTADECENOATE
C1620 METHYL PALMITATE
C16:1 METHYL PALMITOLEATE
C16:1T METHYL PALMITELAIDATE
c17:o METHYL HEPTADECANOATE
c17:1 METHYL 10-HEPTADECENOATE
Cl8:0 METHYL STEARATE
C1821 METHYL OLEATE
C1821T METHYL EALIDATE
C18: 1 METHYL PETROSELINATE
C1821 METHYL VACCENATE
C1821T METHYL TRANSVACCENATE
C1822 METHYL LINOLEATE
C1822T METHYL LINOELAIDATE
C1823 METHYL GAMMA-LINOLENATE
C19:0 METHYL NONADECANOATE
C19: 1 METHYL 7-NONADECENOATE

79

Table 4. Continued

 

C1823
C2020
C2021
C2021
C2021
C2022
C20:3
C20:4
C2023
C2220
C2221
C2025
C2222
C2223
C2224
C2420
C2225
C2226
C2421

METHYL
METHYL
METHYL
METHYL
METHYL
METHYL
METHYL
METHYL
METHYL
METHYL
METHYL
METHYL
METHYL
METHYL
METHYL
METHYL
METHYL
METHYL
METHYL

LINOLENATE
ARACHIDATE
5-EICOSENOATE
8-EICOSENOATE

l l-EICOSENOATE

11-14 EICOSADIENOATE
HOMOGAMMA LINOLENATE
ARACHIDONATE
EICOSATRIENOATE
BEHENATE

ERUCATE
EICOSAPENTAENOATE
DOCOSADIENOATE
DOCOSATRIENOATE
DOCOSATETRAENOATE
LIGNOCERATE
DOCOSAPENTAENOATE
DOCOSAHEXAENOATE
NERVONATE

 

80

10.

11.

12.

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