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Background In a mouse model of colitis, we previously reported the protective role of the adenosine axis on effector T (Teff) cell responses and disease progression. Inflammatory reactions are paired with cell metabolic reprogramming from oxidative phosphorylation (OXPHOS) to aerobic glycolysis which is also a hallmark of T cell activation. Thus, we sought to evaluate the role of the adenosine 2A receptor (A2AR) signaling in the regulation of T cell metabolism. Methods Teff cells were reactivated in the presence or absence of A2AR agonists. Gene expression of mTOR, HIF-1α and cMyc (transcriptional factors of glycolysis-related genes) were quantified by qPCR. T cell metabolic phenotype was evaluated by transcriptomic profiling (Nanostring nCounter® Metabolic Pathway Panel), as well as real-time extracellular flux analysis, glucose uptake, and ATP production assays. Results mTOR, HIF-1α and cMyc gene expression increased as soon as 2hr post stimulation. At this time point, transcriptomic analysis revealed an A2AR agonist-induced downregulation of pathways related to T cell activation (i.e. mTOR, MAPK, PI3K, NFκB and cMyc pathways) and energy metabolism (i.e glycolysis, autophagy, fatty acid synthesis) whereas oxidative metabolism pathways (OXPHOS, fatty acid oxidation, glutamine metabolism) were upregulated (Pathway Score q-value Conclusion A2AR signaling provokes a rewiring of the activated T cell metabolism towards an oxidative state making adenosine a potential T cell metabolic regulator that can be exploited in the treatment of IBD. Supported by NIH R01AI079145

Background Inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), affect ~1% of the people in the United States. SAMP1/YitFcs mice are a model of CD with terminal ileitis which is rare in other animal models of IBD. Switch of cell energy metabolism towards aerobic glycolysis is a hallmark of inflammation and has been implicated in immune-mediated diseases. Thus, we sought to determine how energy metabolism is altered in the SAMP mice. Methods Glucose metabolism in vivo was determined by 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET). Tissue taking up the 18F-FDG were collected for histology. Metabolic phenotype of activated B and CD4+ T lymphocytes isolated from mesenteric lymph nodes of control and SAMP mice was evaluated by extracellular flux analysis (SeaHorse). To relate the SAMP model to human disease, normal and inflamed ileum from mice and humans were assayed by RNAseq focusing on metabolism-related genes. Results PET scans showed a varying tissue distribution of 18F-FDG uptake in SAMP mice, including ileum, salivary glands and kidneys which all had histological evidence of inflammation. Compared to control, both SAMP B and T cells displayed an increase in glycolysis and oxidative phosphorylation (OXPHOS) rates which is supported by an overall increase in genes related to glucose metabolism, TCA cycle, OXPHOS and fatty acid oxidation in the ileum. Conclusion SAMP mice have a multi-organ immune-mediated disease relying on an increase in cell energy metabolism. Therefore, targeting aerobic glucose metabolism to limit inflammation emerges as a new strategy for CD treatment.

Increased apoptotic death of gastric epithelial cells is a hallmark of H. pylori infection, and altered epithelial cell turnover is an important contributor to gastric carcinogenesis. To address the fate of apoptotic gastric epithelial cells and their role in H. pylori mucosal disease, we investigated phagocyte clearance of apoptotic gastric epithelial cells in H. pylori infection. Human gastric mononuclear phagocytes were analyzed for their ability to take up apoptotic epithelial cells in vivo using immunofluorescence analysis. We then used primary human gastric epithelial cells induced to undergo apoptosis by exposure to live H. pylori to study apoptotic cell uptake by autologous monocyte-derived macrophages. We show that HLA-DR+ mononuclear phagocytes in human gastric mucosa contain cytokeratin-positive and TUNEL-positive apoptotic epithelial cell material, indicating that gastric phagocytes are involved in apoptotic epithelial cell clearance. We further show that H. pylori both increased apoptosis in primary gastric epithelial cells and decreased phagocytosis of the apoptotic epithelial cells by autologous monocyte-derived macrophages. Reduced macrophage clearance of apoptotic cells was mediated in part by H. pylori-induced macrophage TNF-α, which was expressed at higher levels in H. pylori-infected, compared to uninfected, gastric mucosa. Importantly, we show that H. pylori-infected gastric mucosa contained significantly higher numbers of apoptotic epithelial cells and higher levels of non-phagocytosed TUNEL-positive apoptotic material, consistent with a defect in apoptotic cell clearance. Thus, as shown in other autoimmune and chronic inflammatory diseases, insufficient phagocyte clearance may contribute to the chronic and self-perpetuating inflammation in human H. pylori infection.

Adenosine is an endogenous metabolite produced during hypoxia or inflammation. Previously implicated as an anti-inflammatory mediator in CD4+ T cell regulation, we report that adenosine acts via dendritic cell (DC) A2B adenosine receptor (A2BAR) to promote the development of Th17 cells. Mouse naive CD4+ T cells cocultured with DCs in the presence of adenosine or the stable adenosine mimetic 5′-(N-ethylcarboximado) adenosine resulted in the differentiation of IL-17– and IL-22–secreting cells and elevation of mRNA that encode signature Th17-associated molecules, such as IL-23R and RORγt. The observed response was similar when DCs were generated from bone marrow or isolated from small intestine lamina propria. Experiments using adenosine receptor antagonists and cells from A2BAR−/− or A2AAR−/−/A2BAR−/− mice indicated that the DC A2BAR promoted the effect. IL-6, stimulated in a cAMP-independent manner, is an important mediator in this pathway. Hence, in addition to previously noted direct effects of adenosine receptors on regulatory T cell development and function, these data indicated that adenosine also acts indirectly to modulate CD4+ T cell differentiation and suggested a mechanism for putative proinflammatory effects of A2BAR.

The endogenous purine nucleoside adenosine is an important antiinflammatory mediator that contributes to the control of CD4+ T cell responses. While adenosine clearly has direct effects on CD4+ T cells, it remains to be determined whether actions on APC such as dendritic cells (DC) are also important. In this report we characterize DC maturation and function in BMDC stimulated with LPS in the presence or absence of the nonselective adenosine receptor agonist NECA (5′-N-ethylcarboxamidoadenosine). We found that NECA inhibited TNF-α and IL-12 in a concentration-dependent manner, whereas IL-10 production was increased. NECA-treated BMDC also expressed reduced levels of MHC class II and CD86 and were less effective at stimulating CD4+ T cell proliferation and IL-2 production compared with BMDC exposed to vehicle control. Based on real-time RT-PCR, the A2A adenosine receptor (A2AAR) and A2BAR were the predominant adenosine receptors expressed in BMDC. Using adenosine receptor subtype selective antagonists and BMDC derived from A2AAR−/− and A2BAR−/−mice, it was shown that NECA modulates TNF-α, IL-12, IL-10, and CD86 responses predominantly via A2BAR. These data indicate that engagement of A2BAR modifies murine BMDC maturation and suggest that adenosine regulates CD4+ T cell responses by selecting for DC with impaired immunogencity.

Recruitment of lymphocytes to sites of inflammation requires the sequential engagement of adhesion molecules and chemokine receptors. Of these, the lectin-like molecule CD44 has been particularly implicated in inflammatory trafficking. Using a TNF-driven model of chronic ileitis (i.e., B6.129P-TnfΔARE mice) that recapitulates many features of Crohn’s disease, we demonstrate dynamic changes in the expression and functional state of CD44 on CD8+ T cells. These cells coexpress CD44 and L-selectin, giving them a surface phenotype similar to that of central memory T cells. Yet functionally they exhibit the phenotype of effector T cells, because they produce IFN-γ. Unexpectedly, depletion of the CD8+ population had no effect on the severity of ileitis. Further analyses showed a second CD8+ population that lacked CD44, but expressed CD103, produced TGF-β, inhibited the proliferation of CD4+ in vitro, and attenuated adoptively transferred ileitis in vivo, most likely counteracting the proinflammatory role of the CD44high subset. Collectively, these data suggest that the presence or absence of CD44 and CD103 on the CD8+ lymphocyte surface defines functionally distinct subsets of CD8+ T cells in vivo. These inflammation-driven populations exert distinct roles during the development of chronic ileitis, and influence the balance of effector and regulatory functions in the chronically inflamed small intestine.

A2A adenosine receptors (A2AAR) inhibit inflammation, although the mechanisms through which adenosine exerts its effects remain unclear. Although the transfer of regulatory Th cells blocks colitis induced by pathogenic CD45RBhigh Th cells, we show that CD45RBlow or CD25+ Th cells from A2AAR-deficient mice do not prevent disease. Moreover, CD45RBhigh Th cells from A2AAR-deficient mice were not suppressed by control CD45RBlow Th cells. A2AAR agonists suppressed the production of proinflammatory cytokines by CD45RBhigh and CD45RBlow T cells in association with a loss of mRNA stability. In contrast, anti-inflammatory cytokines, including IL-10 and TGF-β, were minimally affected. Oral administration of the A2AAR agonist ATL313 attenuated disease in mice receiving CD45RBhigh Th cells. These data suggest that A2AAR play a novel role in the control of T cell-mediated colitis by suppressing the expression of proinflammatory cytokines while sparing anti-inflammatory activity mediated by IL-10 and TGF-β.

Infection by Helicobacter pylori leads to injury of the gastric epithelium and a cellular infiltrate that includes CD4+ T cells. H. pylori binds to class II MHC molecules on gastric epithelial cells and induces their apoptosis. Because urease is an abundant protein expressed by H. pylori, we examined whether it had the ability to bind class II MHC and induce apoptosis in class II MHC-bearing cells. Flow cytometry revealed the binding of PE-conjugated urease to class II MHC+ gastric epithelial cell lines. The binding of urease to human gastric epithelial cells was reduced by anti-class II MHC Abs and by staphylococcal enterotoxin B. The binding of urease to class II MHC was confirmed when urease bound to HLA-DR1-transfected COS-1 (1D12) cells but not to untransfected COS-1 cells. Urease also bound to a panel of B cell lines expressing various class II MHC alleles. Recombinant urease induced apoptosis in gastric epithelial cells that express class II MHC molecules, but not in class II MHC− cells. Also, Fab from anti-class II MHC and not from isotype control Abs blocked the induction of apoptosis by urease in a concentration-dependent manner. The adhesin properties of urease might point to a novel and important role of H. pylori urease in the pathogenesis of H. pylori infection.

T cell responses to self Ags and normal microbial flora are carefully regulated to prevent autoreactivity. Because IL-10-deficient mice develop colitis, and this response is triggered by luminal flora, we investigated whether IL-10 regulates the ability of microbial Ags to induce autoreactive T cells that could contribute to intestinal inflammation. T cells from wild-type mice were primed with staphylococcal enterotoxin B (SEB) in vitro, which induced an autoreactive proliferative response to syngeneic feeder cells. The cells were predominately CD3+ and CD4+. T cells from IL-10-deficient mice were constitutively autoreactive, and SEB priming enhanced this further. The autoreactive, proliferative response of T cells from wild-type mice was suppressed by IL-10 in the primary or secondary culture, and this effect was inhibited by neutralizing Abs to the IL-10R. To confirm that an autoreactive repertoire was expanded after SEB priming, we used CBA/J mice (Mls-1a) in which autoreactive T cells recognizing the endogenous viral superantigen are depleted (Vβ6, 7, 8.1 TCR-bearing cells). However, SEB rescued these autoreactive T cell repertoires. Adding anti-MHC class II Ab blocked the autoreactive response. SEB-primed splenic or colonic T cells also induced apoptosis in syngeneic intestinal epithelial cells that was blocked significantly by IL-10. Thus, microbial Ags have the potential to abrogate self tolerance by stimulating autoreactive T cells that become cytolytic to target cells. IL-10 plays a protective role in maintaining self tolerance after microbial stimulation by preventing the activation of T cells that contribute to epithelial cell damage.

Background: Adenosine accumulates in inflamed tissue and can suppress proliferation and cytokine production in helper T cells (Th). Th cells from mice lacking the A2A Adenosine Receptor (A2AAR-/-) do not function properly in the CD45RB transfer model of colitis. Methods: To examine the role of A2AAR in regulating colitis, we tested colonic myeloperoxidase (MPO) activity after adoptive transfer of Th cell susbsets from wildtype or A2AAR-/- mice. Th cells from mesenteric lymph nodes (MLN) of colitic mice were treated with an A2AAR agonist to assess effects on cytokine production. To investigate the role of A2AAR on myeloid cells in the CD45RB model of colitis, we adoptively transferred wildtype Th cells into RAG1/A2AAR double knockout (DKO) mice. Results: Colons of mice that received A2AAR-/- CD45RBhi cells with wildtype or A2AAR-/- CD45RBlo Treg had significantly greater MPO activity than mice that received wildtype CD45RBhi and CD45RBlo cells. Cytokine production by MLN Th cells from colitic mice was suppressed by an A2AAR agonist. Adoptive transfer of CD45RBhi Th cells into either RAG1-/- or DKO mice induced colitis but co-transfer of CD45RBlo Tregs attenuated colitis only in RAG1-/- mice. Conclusions: These data indicate that adenosine signaling through A2AAR plays an important suppressive role in colitis and that this is mediated through effects on both T cells and myeloid cells. CK supported by a CCFA Fellowship.