Jonathan F. Clinthorne

Dietary Fish Oil Alters T Lymphocyte Cell Populations and Exacerbates Disease in a Mouse Model of Inflammatory Colitis

Inflammatory bowel diseases (IBD) increase the risk of developing colorectal cancer. Dietary components that reduce inflammation are associated with lower cancer risk. The long-chain omega-3 fatty acid docosahexaenoic acid (DHA) is present in fish oil and has potent anti-inflammatory properties. The objective of this study is to determine whether dietary fish oil enriched with DHA (DFO) could reduce experimentally induced colitis and colon cancer risk in a mouse model. When SMAD3-/- mice are exposed to Helicobacter hepaticus, mild colitis is observed 4 weeks postinfection. Mice were fed isocaloric diets modified to include corn oil, safflower oil, or DFO (doses ranging from 0.75% to 6.00%) as the fatty acid source for 8 weeks. Mice were gavaged with H. hepaticus; DFO feeding was continued; and mice were sacrificed 4 weeks after infection. The colon and cecum were collected for histopathology. Spleens and mesenteric lymph nodes were collected and analyzed for T-cell populations using flow cytometry. Contrary to expectations, DFO induced severe colitis and adenocarcinoma formation. DFO consumption was associated with decreased CD8(+) cell frequency and diminished CD69 expression on CD4(+) and CD8(+) T-cell populations. Mice consuming DFO also exhibited higher FoxP3(+) CD25(+) CD4(+) T regulatory cell frequency, FoxP3 expression, and altered L-selectin expression during infection. We concluded that DFO-fed mice may be less equipped to mount a successful response to H. hepaticus infection, increasing colon cancer risk. These results support the need to establish a tolerable upper limit for DHA intake particularly in the context of chronic inflammatory conditions such as IBD.

DHA-enriched fish oil targets B cell lipid microdomains and enhances ex vivo and in vivo B cell function

DHA is a n‐3 LCPUFA in fish oil that generally suppresses T lymphocyte function. However, the effect of fish oil on B cell function remains relatively understudied. Given the important role of B cells in gut immunity and increasing human fish oil supplementation, we sought to determine whether DFO leads to enhanced B cell activation in the SMAD−/− colitis‐prone mouse model, similar to that observed with C57BL/6 mice. This study tested the hypothesis that DHA from fish oil is incorporated into the B cell membrane to alter lipid microdomain clustering and enhance B cell function. Purified, splenic B cells from DFO‐fed mice displayed increased DHA levels and diminished GM1 microdomain clustering. DFO enhanced LPS‐induced B cell secretion of IL‐6 and TNF‐α and increased CD40 expression ex vivo compared with CON. Despite increased MHCII expression in the unstimulated ex vivo B cells from DFO‐fed mice, we observed no difference in ex vivo OVA‐FITC uptake in B cells from DFO or CON mice. In vivo, DFO increased lymphoid tissue B cell populations and surface markers of activation compared with CON. Finally, we investigated whether these ex vivo and in vivo observations were consistent with systemic changes. Indeed, DFO‐fed mice had significantly higher plasma IL‐5, IL‐13, and IL‐9 (Th2‐biasing cytokines) and cecal IgA compared with CON. These results support the hypothesis and an emerging concept that fish oil enhances B cell function in vivo.

Natural killer cell function is altered during the primary response of aged mice to influenza infection.

Influenza is a public health concern, especially for the elderly. While influenza vaccination is efficacious in the young, it offers only limited protection in the elderly. Thus, it becomes imperative to understand age-related changes in the primary response to influenza infection. This study identified potential age-related defects in natural killer (NK) cell function during influenza infection. We showed that NK cells from aged mice were reduced and had impaired function and altered phenotype in lungs during influenza infection. Aged NK cells demonstrated decreased IFN-γ production, but not degranulation, after influenza infection. However, after ex vivo activation with YAC-1 cells, aged NK cells demonstrated both reduced IFN-γ production and degranulation. IFN-γ was also reduced in aged NK cells after activation with anti-NKp46 and soluble cytokines. IFN-β, and IL-12p40 mRNA expression was not significantly different from that observed in adult mice. Analysis of NK cell subsets indicated that aged mice had more immature and less terminally mature NK cells. These data suggest that aging affects the numbers, function and phenotype of NK cells. Thus, these defects in NK cell function could impair the ability of aged mice to induce a strong antiviral immune response during the early stages of the infection.

NK Cell Maturation and Function in C57BL/6 Mice Are Altered by Caloric Restriction

NK cells are a heterogenous population of innate lymphocytes with diverse functional attributes critical for early protection from viral infections. We have previously reported a decrease in influenza-induced NK cell cytotoxicity in 6-mo-old C57BL/6 calorically restricted (CR) mice. In the current study, we extend our findings on the influence of CR on NK cell phenotype and function in the absence of infection. We demonstrate that reduced mature NK cell subsets result in increased frequencies of CD127+ NK cells in CR mice, skewing the function of the total NK cell pool. NK cells from CR mice produced TNF-α and GM-CSF at a higher level, whereas IFN-γ production was impaired following IL-2 plus IL-12 or anti-NK1.1 stimulation. NK cells from CR mice were highly responsive to stimulation with YAC-1 cells such that CD27−CD11b+ NK cells from CR mice produced granzyme B and degranulated at a higher frequency than CD27−CD11b+ NK cells from ad libitum fed mice. CR has been shown to be a potent dietary intervention, yet the mechanisms by which the CR increases life span have yet to be fully understood. To our knowledge, these findings are the first in-depth analysis of the effects of caloric intake on NK cell phenotype and function and provide important implications regarding potential ways in which CR alters NK cell function prior to infection or cancer.

Supplementation with Galacto-Oligosaccharides Increases the Percentage of NK Cells and Reduces Colitis Severity in Smad3-Deficient Mice

The gut microbiota plays an essential role in intestinal immunity. Prebiotics, including galacto-oligosaccharides (GOS), are fermentable fibers that beneficially affect the host by stimulating the growth of specific microbial populations. We investigated the effect of GOS on colitis development and on immune variables in Smad3-deficient mice treated with the pathogen Helicobacter hepaticus. Mice were supplemented daily with 5000 mg GOS/kg body weight 2 wk prior to infection and 4 wk postinfection, a time period during which colitis severity peaks in this model. Mice (n = 4-8/treatment at each time) were killed preinfection (0 d) and at 3, 7, and 28 d postinfection to evaluate immune variables in the spleen and in mesenteric lymph nodes (MsLN) by flow cytometry. Colon and cecum samples were collected for histopathologic analysis. Fecal pellets (n = 8-9/treatment) were collected prior to infection to measure relative changes in Bifidobacterium ssp. and Lactobacillum ssp. by real-time PCR. GOS significantly reduced colitis severity in response to H. hepaticus (P < 0.0001). This was associated with a significant increase in the percentage of NK cells in the spleen (P < 0.001) and in MsLN (P < 0.001) at 3 d postinfection and a 1.5-fold increase in fecal Bifidobacterium ssp. (P = 0.003). GOS stimulated NK expression of CCR9, a chemokine receptor involved in lymphocyte trafficking to the gut preinfection (0 d) in the blood (P = 0.02), spleen (P = 0.033), and MsLN (P = 0.017). In addition, GOS stimulated colonic IL-15 production 3 d postinfection (P < 0.001). These data suggest that GOS reduces colitis by modulating the function and trafficking of NK cells and may provide a novel therapeutic strategy for individuals with inflammatory bowel disease.

Short-Term Re-Feeding of Previously Energy-Restricted C57BL/6 Male Mice Restores Body Weight and Body Fat and Attenuates the Decline in Natural Killer Cell Function after Primary Influenza Infection

A hallmark of energy restriction (ER) is a decrease in total body fat, which is thought to increase lifespan and maintain immune function. However, we have shown that during primary influenza infection, ER induces rapid weight loss, impairs natural killer (NK) cell function, and increases mortality in young and aged mice. To determine whether influenza-induced NK cell function could be restored in ER mice, young adult (6 mo) male C57BL/6 mice were fed an ER diet or re-fed (RF) control diet ad libitum for 2 wk before infection with PR8 influenza A. An initial hyperphagic response was observed in RF mice, characterized by increased food intake, rapid weight gain, and restoration of body fat and fat depots by 5-7 d of re-feeding to levels comparable to control ad libitum (AL) mice. Re-feeding improved survival and attenuated the decline in NK cell function during infection, evidenced by increased numbers, percentages, and CD69 expression by d 3 postinfection in RF mice. Interestingly, an altered metabolic phenotype was observed during infection of RF mice, with plasma leptin concentrations greater than in ER mice but less than in AL mice. In contrast, adiponectin concentrations of RF mice were lower than those of both ER and AL mice. These data suggest that re-feeding for a defined period before, and perhaps throughout, influenza season may provide the energy needed to counter the deleterious effects of ER on NK cell function, especially during exposure to newly emerging strains of influenza, for which vaccines are limited or unavailable.

Energy Intake and Response to Infection with Influenza

Influenza is a worldwide public health concern, particularly with emerging new strains of influenza to which vaccines are ineffective, limited, or unavailable. In addition, the relationship between adequate nutrition and immune function has been repeatedly demonstrated. Mouse models provide strong evidence that energy extremes, including energy restriction (ER) and diet-induced obesity (DIO), have deleterious effects on the immune response to influenza infection. Both ER and DIO mice demonstrate increased susceptibility and mortality to influenza infection. The effects of ER are more pronounced during innate responses to influenza infection, whereas the effects of DIO are evidenced during innate and adaptive responses to both primary and secondary infection. There are striking similarities between ER and DIO during influenza infection, including impaired natural killer cell function and altered inflammation. Future studies must develop effective nutritional paradigms to offset the effects of these energy extremes on the immune response to an acute infection.

Natural killer cell development and maturation in aged mice.

The effect of aging on natural killer cell homeostasis is not well studied in humans or in animal models. We compared natural killer (NK) cells from young and aged mice to investigate age-related defects in NK cell distribution and development. Our findings indicate aged mice have reduced NK cells in most peripheral tissues, but not in bone marrow. Reduction of NK cells in periphery was attributed to a reduction of the most mature CD11b(+) CD27(-) NK cells. Apoptosis was not found to explain this specific reduction of mature NK cells. Analysis of NK cell development in bone marrow revealed that aged NK cells progress normally through early stages of development, but a smaller percentage of aged NK cells achieved terminal maturation. Less mature NK cells in aged bone marrow correlated with reduced proliferation of immature NK cells. We propose that advanced age impairs bone marrow maturation of NK cells, possibly affecting homeostasis of NK cells in peripheral tissues. These alterations in NK cell maturational status have critical consequences for NK cell function in advanced age: reduction of the mature circulating NK cells in peripheral tissues of aged mice affects their overall capacity to patrol and eliminate cancerous and viral infected cells.

Marine fish oils are not equivalent with respect to B-cell membrane organization and activation

We previously reported that docosahexaenoic-acid (DHA)-enriched fish oil (DFO) feeding altered B-cell membrane organization and enhanced B-cell function. The purpose of this study was to evaluate whether menhaden oil (MO) and eicosapentaenoic-acid (EPA)-enriched fish oil (EFO) alters B-cell function/phenotype similarly. Mice were fed control (CON), MO, EFO or DFO diets for 5weeks. We evaluated the fatty acid composition of B-cell phospholipids, membrane microdomain organization, ex vivo B-cell functionality and in vivo B-cell subsets. Red blood cells and B cells were found to be strongly (r>0.85) and significantly (P<.001) correlated for major n-3 and n-6 long-chain polyunsaturated fatty acids (LCPUFAs). Compared to CON, MO and DFO resulted in decreased clustering of membrane microdomains, whereas EFO increased clustering. All fish oil treatments had 1.12-1.60 times higher CD40 expression following stimulation; however, we observed 0.86 times lower major histocompatibility complex class II expression and 0.7 times lower interleukin (IL)-6 production from EFO, but 3.25 times higher interferon-γ from MO and 1.5 times higher IL-6 from DFO. By 90min of incubation, MO had 1.11 times higher antigen uptake compared to CON, whereas EFO was 0.86 times lower. All fish oil treatments resulted in decreasingly mature splenic and bone marrow B-cell subsets. We conclude that diets high in n-3 LCPUFAs may elicit similar B-cell phenotypes but different organizational and functional outcomes. More specifically, these data suggest that the EPA and DHA content of a diet influences immunological outcomes, highlighting the importance of understanding how specific n-3 LCPUFAs modulate B-cell development and function.

Increased presence of effector lymphocytes during Helicobacter hepaticus -induced colitis

AIM To identify and characterize drosophila mothers against decapentaplegic (SMAD)3-dependent changes in immune cell populations following infection with Helicobacter hepaticus (H. hepaticus). METHODS SMAD3(-/-) (n = 19) and colitis-resistant SMAD3(+/-) (n = 24) mice (8-10 wk of age) were infected with H. hepaticus and changes in immune cell populations [T lymphocytes, natural killer (NK) cells, T regulatory cells] were measured in the spleen and mesenteric lymph nodes (MsLNs) at 0 d, 3 d, 7 d and 28 d post-infection using flow cytometry. Genotype-dependent changes in T lymphocytes and granzyme B(+) cells were also assessed after 28 d in proximal colon tissue using immunohistochemistry. RESULTS As previously observed, SMAD3(-/-), but not SMAD3(+/-) mice, developed colitis, peaking at 4 wk post-infection. No significant changes in T cell subsets were observed in the spleen or in the MsLNs between genotypes at any time point. However, CD4(+) and CD8(+)/CD62L(lo) cells, an effector T lymphocyte population, as well as NK cells (NKp46/DX5(+)) were significantly higher in the MsLNs of SMAD3(-/-) mice at 7 d and 28 d post-infection. In the colon, a higher number of CD3(+) cells were present in SMAD3(-/-) compared to SMAD3(+/-) mice at baseline, which did not significantly change during infection. However, the number of granzyme B(+) cells, a marker of cytolytic lymphocytes, significantly increased in SMAD3(-/-) mice 28 d post-infection compared to both SMAD3(+/-) mice and to baseline values. This was consistent with more severe colitis development in these animals. CONCLUSION Data suggest that defects in SMAD3 signaling increase susceptibility to H. hepaticus-induced colitis through aberrant activation and/or dysregulation of effector lymphocytes.