Anthony T. Cao

Th17 Cells Upregulate Polymeric Ig Receptor and Intestinal IgA and Contribute to Intestinal Homeostasis

Although CD4+ Th17 cells are enriched in normal intestines, their role in regulation of the host response to microbiota, and whether and how they contribute to intestinal homeostasis, is still largely unknown. It is also unclear whether Th17 cells regulate intestinal IgA production, which is also abundant in the intestinal lumen and has a crucial role as the first defense line in host response to microbiota. In this study, we found that intestinal polymeric Ig receptor (pIgR) and IgA production was impaired in T cell-deficient TCR-βxδ−/− mice. Repletion of TCR-βxδ−/− mice with Th17 cells from CBir1 flagellin TCR transgenic mice, which are specific for a commensal Ag, increased intestinal pIgR and IgA. The levels of intestinal pIgR and IgA in B6.IL-17R (IL-17R−/−) mice were lower than wild type mice. Treatment of colonic epithelial HT-29 cells with IL-17 increased pIgR expression. IL-17R−/− mice demonstrated systemic antimicroflora Ab response. Consistently, administering dextran sulfate sodium (DSS) to C57BL/6 mice after treatment with IL-17–neutralizing Ab resulted in more severe intestinal inflammation compared with control Ab. Administering DSS to IL-17R−/− mice resulted in increased weight loss and more severe intestinal inflammation compared with wild type mice, indicating a protective role of Th17 cells in intestinal inflammation. Individual mice with lower levels of pIgR and intestinal-secreted IgA correlated with increased weight loss at the end of DSS administration. Collectively, our data reveal that microbiota-specific Th17 cells contribute to intestinal homeostasis by regulating intestinal pIgR expression and IgA secretion.

Interleukin-12 Converts Foxp3+ Regulatory T Cells to Interferon–γ-Producing Foxp3+ T Cells That Inhibit Colitis

BACKGROUND & AIMS Regulatory T (Treg) cells are plastic, but the in vivo mechanisms by which they are converted into foxhead box p3 (Foxp3+) interferon (IFN)-γ+ T cells and whether these converted cells retain the ability to inhibit colitis are not clear. METHODS Foxp3+ Treg cells were generated by culture of naïve CD4+ T cells from Foxp3GFP CBir1 T-cell receptor (TCR) transgenic (Tg) (CBir1-Tg) mice, which are specific for CBir1 flagellin (an immunodominant microbiota antigen), with transforming growth factor-β. Foxp3GFP+ CBir1-Tg Treg cells were isolated by fluorescence-activated cell sorting and transferred into TCRβxδ-/- mice. Colitis was induced by transfer of naïve CBir1-Tg CD4+ T cells into immunodeficient mice. RESULTS Microbiota antigen-specific Foxp3+ Treg cells were converted, in the intestine, to IFN-γ+ T-helper (Th)1 cells, interleukin (IL)-17+ Th17 cells, and Foxp3+ T cells that coexpress IFN-γ and/or IL-17. Conversion of Treg cells into IFN-γ-producing Th1 cells and Foxp3+IFN-γ+ T cells required innate cell production of IL-12 in the intestine; blocking IL-12 with an antibody inhibited their conversion to Th1 and Foxp3+IFN-γ+ T cells in the intestines of mice that were recipients of Treg cells. Addition of IL-12, but not IL-23, promoted conversion of Treg cells into Th1 and Foxp3+IFN-γ+ T cells, in vitro. Foxp3+IFN-γ+ T cells had regulatory activity because they suppressed proliferation of naïve T cells, in vitro, and inhibited induction of colitis by microbiota antigen-specific T cells. IFN-γ+ Th1 cells were not converted into Treg cells; Foxp3+IFN-γ+ T cells differentiated into IFN-γ+ but not Foxp3+ T cells. CONCLUSIONS IL-12 promotes conversion of Treg cells into IFN-γ-expressing cells; Foxp3+IFN-γ+ T cells retain their regulatory functions and develop during the transition of Foxp3+ Treg cells into IFN-γ+ Th1 cells.

miR-10a inhibits dendritic cell activation and Th1/Th17 cell immune responses in IBD

Objective Although both innate and adaptive responses to microbiota have been implicated in the pathogenesis of IBD, it is still largely unknown how they are regulated during intestinal inflammation. In this report, we investigated the role of microRNA (miR)-10a, a small, non-coding RNA, in the regulation of innate and adaptive responses to microbiota in IBD. Methods miR-10a expression was analysed in the inflamed mucosa of IBD patients treated with or without antitumour necrosis factor (anti-TNF) monoclonal antibodies (mAb) (infliximab) by qRT-PCR. Human monocyte-derived dendritic cells (DC) and IBD CD4+ T cells were transfected with miR-10a precursor to define their effect on the function of DC and CD4+ T cells. Results The expression of miR-10a was markedly decreased, while NOD2 and interleukin (IL)-12/IL-23p40 were significantly increased, in the inflamed mucosa of IBD patients compared with those in healthy controls. Commensal bacteria, TNF and interferon-γ inhibited human DC miR-10a expression in vitro. Anti-TNF mAb treatment significantly promoted miR-10a expression, whereas it markedly inhibited NOD2 and IL-12/IL-23p40 in the inflamed mucosa. We further identified NOD2, in addition to IL-12/IL-23p40, as a target of miR-10a. The ectopic expression of the miR-10a precursor inhibited IL-12/IL-23p40 and NOD2 in DC. Moreover, miR-10a was found to markedly suppress IBD T helper (Th)1 and Th17 cell responses. Conclusions Our data indicate that miR-10a is decreased in the inflamed mucosa of IBD and downregulates mucosal inflammatory response through inhibition of IL-12/IL-23p40 and NOD2 expression, and blockade of Th1/Th17 cell immune responses. Thus, miR-10a could play a role in the pathogenesis and progression of IBD.

Microbiota metabolite short-chain fatty acid acetate promotes intestinal IgA response to microbiota which is mediated by GPR43

Intestinal IgA, which is regulated by gut microbiota, has a crucial role in maintenance of intestinal homeostasis and in protecting the intestines from inflammation. However, the means by which microbiota promotes intestinal IgA responses remain unclear. Emerging evidence suggests that the host can sense gut bacterial metabolites in addition to pathogen-associated molecular patterns and that recognition of these small molecules influences host immune response in the intestines and beyond. We reported here that microbiota metabolite short-chain fatty acid acetate promoted intestinal IgA responses, which was mediated by “metabolite-sensing” GPR43. GPR43−/− mice demonstrated lower levels of intestinal IgA and IgA+ gut bacteria compared with those in wild type (WT) mice. Feeding WT but not GPR43−/− mice acetate but not butyrate promoted intestinal IgA response independent of T cells. Acetate promoted B-cell IgA class switching and IgA production in vitro in the presence of WT but not GPR43−/− dendritic cells (DCs). Mechanistically, acetate-induced DC expression of Aldh1a2, which converts Vitamin A into its metabolite retinoic acid (RA). Moreover, blockade of RA signaling inhibited the acetate induction of B-cell IgA production. Our studies thus identified a new pathway by which microbiota promotes intestinal IgA response through its metabolites.

Interleukin (IL)-21 promotes intestinal IgA response to microbiota

Commensal microbiota-specific T helper type 17 (Th17) cells are enriched in the intestines, which can convert into T follicular helper (Tfh) in Peyer’s patches, and are crucial for production of intestinal immunoglobulin A (IgA) against microbiota; however, the role of Th17 and Tfh cytokines in regulating the mucosal IgA response to enteric microbiota is still not completely known. In this study, we found that intestinal IgA was impaired in mice deficient in interleukin (IL)-17 or IL-21 signaling. IL-21, but not IL-17, is able to augment B-cell differentiation to IgA+ cells as mediated by transforming growth factor β1 (TGFβ1) and accelerate IgA class switch recombination (CSR). IL-21 and retinoic acid (RA) induce IgA+ B-cell development and IgA production and drives autocrine TGFβ1 production to initiate IgA CSR. Repletion of T-cell-deficient TCRβxδ−/− mice with Th17 cells specific for commensal bacterial antigen increased the levels of IgA+ B cells and IgA production in the intestine, which was blocked by neutralizing IL-21. Thus IL-21 functions to strongly augment IgA production under intestinal environment. Furthermore, IL-21 promotes intestinal B-cell homing through α4β7 expression, alone or with TGFβ and RA. Together, IL-21 from microbiota-specific Th17 and/or Tfh cells contributes to robust intestinal IgA levels by enhancing IgA+ CSR, IgA production and B-cell trafficking into the intestine.

TGF-β converts Th1 cells into Th17 cells through stimulation of Runx1 expression

Differentiated CD4+ T cells preserve plasticity under various conditions. However, the stability of Th1 cells is unclear, as is whether Th1 cells can convert into Th17 cells and thereby contribute to the generation of IFN‐γ+IL‐17+CD4+ T cells, the number of which correlates with severity of colitis. We investigated whether IFN‐γ+Th1 cells can convert into Th17 cells under intestinal inflammation and the mechanisms involved. IFN‐γThy1.1+ Th1 cells were generated by culturing naïve CD4+ T cells from IFN‐γThy1.1 CBir1 TCR‐Tg reporter mice, whose TCR is specific for an immunodominant microbiota antigen, CBir1 flagellin, under Th1 polarizing conditions. IFN‐γThy1.1+ Th1 cells induced colitis in Rag−/− mice after adoptive transfer and converted into IL‐17+Th17, but not Foxp3+Treg cells in the inflamed intestines. TGF‐β and IL‐6, but not IL‐1β and IL‐23, regulated Th1 conversion into Th17 cells. TGF‐β induction of transcriptional factor Runx1 is crucial for the conversion, since silencing Runx1 by siRNA inhibited Th1 conversion into Th17 cells. Furthermore, TGF‐β enhanced histone H3K9 acetylation but inhibited H3K9 trimethylation of Runx1‐ and ROR‐γt‐binding sites on il‐17 or rorc gene in Th1 cells. We conclude that Th1 cells convert into Th17 cells under inflammatory conditions in intestines, which is possibly mediated by TGF‐β induction of Runx1.

Downregulation of microRNA-107 in intestinal CD11c(+) myeloid cells in response to microbiota and proinflammatory cytokines increases IL-23p19 expression.

Commensal flora plays an important role in the development of the mucosal immune system and in maintaining intestinal homeostasis. However, the mechanisms involved in regulation of host‐microbiota interaction are still not completely understood. In this study, we examined how microbiota and intestinal inflammatory conditions regulate host microRNA expression and observed lower microRNA‐107 (miR‐107) expression in the inflamed intestines of colitic mice, compared with that in normal control mice. miR‐107 was predominantly reduced in epithelial cells and CD11c+ myeloid cells including dendritic cells and macrophages in the inflamed intestines. We demonstrate that IL‐6, IFN‐γ, and TNF‐α downregulated, whereas TGF‐β promoted, miR‐107 expression. In addition, miR‐107 expression was higher in the intestines of germ‐free mice than in mice housed under specific pathogen‐free conditions, and the presence of microbiota downregulated miR‐107 expression in DCs and macrophages in a MyD88‐ and NF‐κB‐dependent manner. We determined that the ectopic expression of miR‐107 specifically repressed the expression of IL‐23p19, a key molecule in innate immune responses to commensal bacteria. We concluded that regulation of miR‐107 by intestinal microbiota and proinflammatory cytokine serve as an important pathway for maintaining intestinal homeostasis.

Microbiota regulation of inflammatory bowel disease and colorectal cancer

The host and microbiota have evolved mechanisms for coexistence over millions of years. Accumulating evidence indicates that a dynamic mutualism between the host and the commensal microbiota has important implications for health, and microbial colonization contributes to the maintenance of intestinal immune homeostasis. However, alterations in communication between the mucosal immune system and gut microbial communities have been implicated as the core defect that leads to chronic intestinal inflammation and cancer development. We will discuss the recent progress on how gut microbiota regulates intestinal homeostasis and the pathogenesis of inflammatory bowel disease and colorectal cancer.

IL-17A promotes protective IgA responses and expression of other potential effectors against the lumen-dwelling enteric parasite Giardia

Giardia lamblia is a leading protozoan cause of diarrheal disease worldwide. It colonizes the lumen and epithelial surface of the small intestine, but does not invade the mucosa. Acute infection causes only minimal mucosal inflammation. Effective immune defenses exist, yet their identity and mechanisms remain incompletely understood. Interleukin (IL)-17A has emerged as an important cytokine involved in inflammation and antimicrobial defense against bacterial pathogens at mucosal surfaces. In this study, we demonstrate that IL-17A has a crucial function in host defense against Giardia infection. Using murine infection models with G. muris and G. lamblia, we observed marked and selective induction of intestinal IL-17A with peak expression after 2 weeks. Th17 cells in the lamina propria and innate immune cells in the epithelial compartment of the small intestine were responsible for the IL-17A response. Experiments in gene-targeted mice revealed that the cytokine, and its cognate receptor IL-17RA, were required for eradication of the parasite. The actions of the cytokine were mediated by hematopoietic cells, and were required for the transport of IgA into the intestinal lumen, since IL-17A deficiency led to marked reduction of fecal IgA levels, as well as for increased intestinal expression of several other potential effectors, including β-defensin 1 and resistin-like molecule β. In contrast, intestinal hypermotility, another major antigiardial defense mechanism, was not impacted by IL-17A loss. Taken together, these findings demonstrate that IL-17A and IL-17 receptor signaling are essential for intestinal defense against the important lumen-dwelling intestinal parasite Giardia.

mTOR Mediates IL-23 Induction of Neutrophil IL-17 and IL-22 Production.

It has been shown recently that neutrophils are able to produce IL-22 and IL-17, which differentially regulate the pathogenesis of inflammatory bowel disease. However, it is still largely unknown how the neutrophil production of IL-22 and IL-17 is regulated, and their role in the pathogenesis of inflammatory bowel disease. In this study, we found that IL-23 promoted neutrophil production of IL-17 and IL-22. IL-23 stimulated the neutrophil expression of IL-23R as well as rorc and ahr. Retinoid acid receptor–related orphan receptor γ t and aryl-hydrocarbon receptor differentially regulated IL-23 induction of neutrophil IL-17 and IL-22. In addition, IL-23 induced the activation of mTOR in neutrophils. Blockade of the mTOR pathway inhibited IL-23–induced expression of rorc and ahr, as well as IL-17 and IL-22 production. By using a microbiota Ag-specific T cell–mediated colitis model, we demonstrated that depletion of neutrophils, as well as blockade of IL-22, resulted in a significant increase in the severity of colitis, thereby indicating a protective role of neutrophils and IL-22 in chronic colitis. Collectively, our data revealed that neutrophils negatively regulate microbiota Ag-specific T cell induction of colitis, and IL-23 induces neutrophil production of IL-22 and IL-17 through induction of rorc and ahr, which is mediated by the mTOR pathway.