Trivalent arsenic impairs the effector response of human CD4+ and CD8+ T cells to influenza A virus ex vivo.

Arsenic is a persistent environmental contaminant that humans are exposed to primarily through contaminated water supplies. Arsenic has been shown to have numerous immunomodulatory effects, including deleterious effects on T cell function. However, the effect of arsenic on human T cell function in the context of influenza infection remains poorly characterized. The goal of this study was to determine the effects of arsenic on T cell activation and effector function in a human-relevant ex vivo model with influenza challenge. Flow cytometric analysis of T cells following the treatment of primary human peripheral blood mononuclear cells with environmentally relevant concentrations of arsenic trioxide and subsequent challenge with influenza A virus showed reduced viability, alterations in activation, a reduction in the population of memory cells, and reduced effector function evidenced by decreased IFNγ and granzyme B production. Overall, these studies suggest that arsenic impairs the human T cell response to influenza which corroborates epidemiological findings and could have further implications for antiviral immunity and vaccine efficacy.

The Nrf2 activator tBHQ inhibits the activation of primary murine natural killer cells.

Tert-butylhydroquinone (tBHQ) is a commonly used food preservative with known immunomodulatory activity; however, there is little information regarding its role on natural killer (NK) cell activation and function. tBHQ is a known activator of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which results in induction of cytoprotective genes. Activation of Nrf2 has been shown to modulate immune responses in a number of different models. In addition, studies in our laboratory have shown that tBHQ inhibits numerous early events following T cell activation. In the current study, we investigated whether activated NK cells are impacted by tBHQ, since many signaling cascades that control NK cell effector function also contribute to T cell function. Splenocytes were isolated from female, wild-type C57Bl/6J mice and treated with 1 µM or 5 µM tBHQ. NK cell function was assessed after activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 24 h. Activation of NK cells in the presence of tBHQ decreased total NK cell percentage, production of intracellular interferon gamma (IFNÉ£), granzyme B, and perforin, and induction of the cell surface proteins CD25 and CD69, which are markers of NK cell activation. In addition to NK cell effector function, NK cell maturation was also altered in response to tBHQ. Notably, this is the first study to demonstrate that the Nrf2 activator, tBHQ, negatively impacts effector function and maturation of NK cells.

Age, calorie restriction, and age of calorie restriction onset reduce maturation of natural killer cells in C57Bl/6 mice.

Calorie restriction (CR), also known as energy restriction, has been shown to have a deleterious impact on both adult and aged mouse survival during influenza virus infection. Natural killer (NK) cell phenotypic differences contribute to increased susceptibility of adult CR mice. We hypothesized NK cell phenotype from adult and aged C57Bl/6 mice fed NIH-31 diet ad libitum (AL) would be different from NK cell phenotype from adult and aged mice fed NIH-31/NIA fortified diet at 40% CR. We hypothesized NK cell phenotype from mice consuming 40% CR initiated at 20 months of age would not be different from 40% CR initiated at 3 months of age. We initiated the 40% restriction either at the standard 12 weeks of age or at 78 weeks of age. NK cells were isolated and quantified from various tissues using flow cytometry. Aged CR mice had significantly reduced levels of terminally mature (CD27-CD11b+) NK cells, increased expression of the immature marker CD127, and decreased expression of the mature marker DX5. Total number of NK cells among cells was significantly lower in the lung and spleen of old-onset aged CR mice compared to aged AL mice, while there was no significant difference between young-onset aged CR and aged AL mice. Old-onset aged CR mice had significantly less early mature (DX5+ and CD27+CD11b+) NK cells compared to young-onset aged CR and aged AL fed mice. Overall, we found that CR in aged mice is detrimental to maturation of NK cells, which is exacerbated when CR is initiated in old age.

Energy restriction impairs dendritic cell development in C57BL/6J mice.

Dendritic cells (DC) are antigen-presenting cells known for stimulating naïve T lymphocytes. The sequential stages of DC development from common myeloid progenitors have been elucidated in murine bone marrow. Energy-restriction (ER) is a pro-longevity dietary intervention with mixed immunological outcomes. The objective of this study was to examine the development of DC in adult C57Bl6J mice fed a 40% ER diet. We observed increased myeloid progenitors, but decreased common DC progenitors, precursor conventional DC and plasmacytoid DC. Furthermore, we observed increased macrophages and cells expressing CD169 in the bone marrow of ER mice. There was no significant difference in DC subsets from unfractionated ER and ad libitum-fed murine bone marrow samples cultured in GM-CSF-supplemented media or Flt3L-supplemented media. Examining rates of proliferation with 6h BrdU incorporation and Ki-67 staining showed these DC progenitor populations have different proliferation rates in ER compared with AL mice. We show here, for the first time, ER results in altered myelopoiesis resulting in reduced DC development but enhanced monocyte/macrophage development in steady-state C57Bl6J mice. In conclusion, these data may partially explain prior observations of impaired early innate immune responses to primary infection such as influenza in ER mice.

High Sensitivity of Aged Mice to Deoxynivalenol (Vomitoxin)-Induced Anorexia Corresponds to Elevated Proinflammatory Cytokine and Satiety Hormone Responses.

Deoxynivalenol (DON), a trichothecene mycotoxin that commonly contaminates cereal grains, is a public health concern because of its adverse effects on the gastrointestinal and immune systems. The objective of this study was to compare effects of DON on anorectic responses in aged (22 mos) and adult (3 mos) mice. Aged mice showed increased feed refusal with both acute i.p. (1 mg/kg and 5 mg/kg) and dietary (1, 2.5, 10 ppm) DON exposure in comparison to adult mice. In addition to greater suppression of food intake from dietary DON exposure, aged mice also exhibited greater but transient body weight suppression. When aged mice were acutely exposed to 1 mg/kg bw DON i.p., aged mice displayed elevated DON and DON3GlcA tissue levels and delayed clearance in comparison with adult mice. Acute DON exposure also elicited higher proinflammatory cytokine and satiety hormone responses in the plasma of the aged group compared with the adult group. Increased susceptibility to DON-induced anorexia in aged mice relative to adult mice suggests that advanced life stage could be a critical component in accurate human risk assessments for DON and other trichothecenes.

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.

Short-term supplementation with active hexose correlated compound improves the antibody response to influenza B vaccine.

Administration of bioactive nutritional supplements near or at the time of immunization has been a recent approach to stimulate human immune response to vaccination. Active hexose correlated compound (AHCC), a mushroom extract, has been shown to protect mice against lethal primary influenza infection. Moreover, when AHCC was administered pre-vaccination in mice, they showed improved protection from lethal avian flu infection when compared to mice vaccinated alone. In this study, we hypothesized that AHCC will also improve the immune responses of healthy individuals to influenza vaccine. A randomized controlled study was performed with 30 healthy adults to evaluate the effects of AHCC supplementation on the immune response to the 2009-2010 seasonal influenza vaccine. Blood was drawn pre-vaccination and 3 wk post-vaccination. Immediately post-vaccination, the AHCC group began supplementation with AHCC (3 g/d). Flow cytometric analysis of lymphocyte subpopulations revealed that AHCC supplementation increased NKT cells (P < .1), and CD8 T cells (P < .05) post-vaccination compared to controls. Analysis of antibody production 3 weeks post-vaccination revealed that AHCC supplementation significantly improved protective antibody titers to influenza B, while the improvement was not significant in the control group. Overall, our study showed that AHCC supplementation improved some lymphocyte percentages and influenza B antibody titers over the control. Future studies are required to determine the kinetics of AHCC supplementation to improve the overall response to influenza vaccination.

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.

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.

Differential effects of stimulatory factors on natural killer cell activities of young and aged mice.

Age-associated influences on natural killer (NK) cell functions following cytokine stimulation were examined in splenocytes from C57BL/6 mice. NK cells of both young and aged mice exhibited significantly increased: interferon-γ production after interleukin (IL)-12 or IL-15 alone or any combination of IL-12, IL-18, and IL-2; cytotoxicity after IL-2 or IL-15; and granzyme B expression after IL-15. The only significant age-associated differences were observed in interferon-γ production after IL-15 or IL-12 + 18 + 2 and in granzyme B expression following IL-2 or IL-15. Perforin expression did not increase following stimulation; however, NK cells from aged mice expressed significantly higher levels than young mice. These results underscore the complexity of the cytokine-induced functional activities of NK cells and illustrate the differential response of NK cells from young and aged mice to cytokine stimulation.

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-ß, 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.

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.

Potential for the detection of molecular complexes and determination of interaction geometry by 2DIR: application to protein sciences.

The ability to detect molecular complexes and determine their geometries is crucial to our understanding of all biological phenomena, including protein structures and functions. We recently demonstrated that a novel 2DIR technique, EVV 2DIR spectroscopy, can be used for this purpose. In this paper, we evaluate the potential utility of the method for the analysis of protein composition, structure and function. In order to do this we apply computational tools to a group of selected biological systems, for which our calculated spectra all showed features that can in principle be detected with existing sensitivities. We also investigate the possibility of using our technique to detect and analyse hydrogen-bonded systems through a tyrosine-water model.

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.

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.

Detection of complex formation and determination of intermolecular geometry through electrical anharmonic coupling of molecular vibrations using electron-vibration-vibration two-dimensional infrared spectroscopy.

Electrical interactions between molecular vibrations can be non-linear and thereby produce intermolecular coupling even in the absence of a chemical bond. We use this fact to detect the formation of an intermolecular complex using electron-vibration-vibration two-dimensional infrared spectroscopy (EVV 2DIR) and also to determine the distance and angle between the two molecular species.

Biological and biomedical applications of two-dimensional vibrational spectroscopy: proteomics, imaging, and structural analysis.

In the last 10 years, several forms of two-dimensional infrared (2DIR) spectroscopy have been developed, such as IR pump-probe spectroscopy and photon-echo techniques. In this Account, we describe a doubly vibrationally enhanced four-wave mixing method, in which a third-order nonlinear signal is generated from the interaction of two independently tunable IR beams and an electron-polarizing visible beam at 790 nm. When the IR beams are independently in resonance with coupled vibrational transitions, the signal is enhanced and cross-peaks appear in the spectrum. This method is known as either DOVE (doubly vibrationally enhanced) four-wave mixing or EVV (electron-vibration-vibration) 2DIR spectroscopy. We begin by discussing the basis and properties of EVV 2DIR. We then discuss several biological and potential biomedical applications. These include protein identification and quantification, as well as the potential of this label-free spectroscopy for protein and peptide structural analysis. In proteomics, we also show how post-translational modifications in peptides (tyrosine phosphorylation) can be detected by EVV 2DIR spectroscopy. The feasibility of EVV 2DIR spectroscopy for tissue imaging is also evaluated. Preliminary results were obtained on a mouse kidney histological section that was stained with hematoxylin (a small organic molecule). We obtained images by setting the IR frequencies to a specific cross-peak (the strongest for hematoxylin was obtained from its analysis in isolation; a general CH(3) cross-peak for proteins was also used) and then spatially mapping as a function of the beam position relative to the sample. Protein and hematoxylin distribution in the tissue were measured and show differential contrast, which can be entirely explained by the different tissue structures and their functions. The possibility of triply resonant EVV 2DIR spectroscopy was investigated on the retinal chromophore at the centre of the photosynthetic protein bacteriorhodopsin (bR). By putting the visible third beam in resonance with an electronic transition, we were able to enhance the signal and increase the sensitivity of the method by several orders of magnitude. This increase in sensitivity is of great importance for biological applications, in which the number of proteins, metabolites, or drug molecules to be detected is low (typically pico- to femtomoles). Finally, we present theoretical investigations for using EVV 2DIR spectroscopy as a structural analysis tool for inter- and intramolecular interaction geometries.

Sub-mitogenic phorbol myristate acetate co-stimulation rescues the PHA-induced activation of both naïve and memory T cells cultured in the rotating-wall vessel bioreactor.

T lymphocytes are unresponsive to T cell receptor (TCR) stimulation during culture in spaceflight or ground-based microgravity analogs such as the rotating-wall vessel (RWV) bioreactor. The TCR-induced activation of a subset of T cells can be rescued in the RWV by co-stimulation with sub-mitogenic doses of phorbol ester (PMA). We report that PMA co-stimulation of primary human T cells cultured in the RWV rescues the phytohemagglutinin (PHA)-induced activation of the CD8+ and CD4+ T cell subsets as well as naïve and memory CD4+ T cells. Importantly, T cells activated in the RWV by PHA+PMA contained these subsets in proportions strikingly similar to control cultures activated with PHA alone. The data indicate that rescuing T cell activation with PMA co-stimulation does not significantly perturb the heterogeneity of the responding cells, and represent an important proof of principle for the design of immune-boosting agents for use in spaceflight.

Dendritic cells are required for optimal activation of natural killer functions following primary infection with herpes simplex virus type 1.

Natural killer (NK) cells play an important role in the optimal clearance of herpes simplex virus type 1 (HSV-1) infection in mice. Activated NK cells function via cytokine secretion or direct cytolysis of target cells; dendritic cells (DCs) are thought to make critical contributions in the activation of both of these functions. Yet, the magnitude and physiological relevance of DC-mediated NK cell activation in vivo is not completely understood. To examine the contribution of DC help in regulating NK cell functions after infection with HSV-1, we utilized a transgenic mouse model that allows the transient ablation of DCs. Using this approach, it was found that the gamma interferon (IFN-gamma) expression potential of NK cells is quantitatively and qualitatively impaired in the absence of DCs. With regard to priming of NK cytolytic functions, the ablation of DCs did not significantly affect cytotoxic protein expression by NK cells. An in vivo cytolytic assay did, however, reveal impairments in the magnitude of NK cell cytotoxicity. Overall, this study provides direct evidence that functional DCs are required for optimal IFN-gamma expression and cytolytic function by NK cells following infection with HSV-1.