Microbial metabolite butyrate modulates granzyme B in tolerogenic IL-10 producing Th1 cells to regulate intestinal inflammation.

CD4+ T cells play a critical role in regulating autoimmune diseases, and intestinal microbial metabolites control various immune responses. Granzyme B (GzmB)-producing CD4+ T cells have been recently reported to participate in the pathogenesis of autoimmune diseases. Here, we found that GzmbB-deficient CD4+ T cells induced more severe colitis in Rag1-/- mice than wild-type (WT) CD4+ T cells. Germ-free (GF) mice exhibited a lower expression of GzmB in intestinal CD4+ T cells compared to specific pathogen-free (SPF) mice. Intestinal microbial metabolite butyrate increased GzmB expression in CD4+ T cells, especially in IL-10-producing Th1 cells, through HDAC inhibition and GPR43, but not GPR41 and GPR109a. Butyrate-treated GzmB-deficient CD4+ T cells demonstrated more severe colitis compared to butyrate-treated WT CD4+ T cells in the T cell transfer model. Butyrate altered intestinal microbiota composition, but altered microbiota did not mediate butyrate induction of intestinal CD4+ T cell expression of GzmB in mice. Blimp1 was involved in the butyrate induction of GzmB in IL-10-producing Th1 cells. Glucose metabolism, including glycolysis and pyruvate oxidation, mediated butyrate induction of GzmB in Th1 cells. In addition, we found that IKZF3 and NR2F6 regulated GzmB expression induced by butyrate. Together, our studies underscored the critical role of GzmB in mediating gut bacterial metabolite butyrate regulation of T cell tolerance at the mucosal surface.

GPR120 promotes neutrophil control of intestinal bacterial infection.

G-protein coupled receptor 120 (GPR 120) has been implicated in anti-inflammatory functions. However, how GPR120 regulates the neutrophil function remains unknown. This study investigated the role of GPR120 in the regulation of neutrophil function against enteric bacteria. 16S rRNA sequencing was used for measuring the gut microbiota of wild-type (WT) mice and Gpr120-/- mice. Citrobacter rodentium infection and dextran sulfate sodium (DSS)-induced colitis models were performed in WT and Gpr120-/- mice. Mouse peritoneal-derived primary neutrophils were used to determine the neutrophil functions. Gpr120-/- mice showed altered microbiota composition. Gpr120-/- mice exhibited less capacity to clear intestinal Citrobacter rodentium and more severe intestinal inflammation upon infection or DSS insults. Depletion of neutrophils decreased the intestinal clearance of Citrobacter rodentium. GPR120 agonist, CpdA, enhanced WT neutrophil production of reactive oxygen species (ROS) and extracellular traps (NETs), and GPR120-deficient neutrophils demonstrated a lower level of ROS and NETs. CpdA-treated neutrophils showed an enhanced capacity to inhibit the growth of Citrobacter rodentium, which was abrogated by the inhibition of either NETs or ROS. CpdA promoted neutrophil inhibition of the growth of commensal bacteria Escherichia coli O9:H4 and pathobiont Escherichia coli O83:H1 isolated from a Crohn's disease patient. Mechanically, mTOR activation and glycolysis mediated GPR120 induction of ROS and NETs in neutrophils. Additionally, CpdA promoted the neutrophil production of IL-17 and IL-22, and treatment with a conditioned medium of GPR120-activated neutrophils increased intestinal epithelial cell barrier functions. Our study demonstrated the critical role of GPR120 in neutrophils in protection against enteric bacterial invasion.

Intrinsic STING Switches off Pathogenetic Programs of Th1 Cells to Inhibit Colitis.

BACKGROUND & AIMS: T helper 1 (Th1) effector cells are implicated in inflammatory bowel disease. The stimulator of interferon genes (STING), an intracellular DNA sensor, has been shown to regulate infection and various cancers. However, whether and how intrinsic STING signaling in Th1 cells regulates colitis is still unknown. METHODS: Dextran sodium sulfate-induced colitis and wild-type/STING-deficient CD4+T cell adoptive transfer models were used to analyze the role of STING in regulating colitis. The effect of STING on Th1 cells was determined by flow cytometry, RNA sequencing, metabolic assays, and mitochondrial functions. 16S ribosomal RNA sequencing and germ-free mice were used to investigate whether the microbiota were involved. The in vivo effect of STING agonist in murine colitis was determined. The expression and role of STING in human T cells were also determined. RESULTS: Activation of STING transformed proinflammatory IFNγ+Th1 cells into IL-10+IFNγ+Th1 cells, which were dramatically less pathogenic in inducing colitis. STING promoted Th1 interleukin (IL)-10 production by inducing STAT3 translocation into nuclear and mitochondria, which promoted Blimp1 expression and mitochondrial oxidation, respectively. Blockade of glucose or glutamine-derived oxidation, but not lipid-derived oxidation, suppressed STING induction of IL-10. Gut microbiota were changed in STING-/- mice, but the altered microbiota did not mediate STING effects on intestinal CD4+T cell production of IL-10. Translationally, STING agonists suppressed both acute and chronic colitis. Intestinal STING+ CD4+T cells were increased in inflammatory bowel disease patients, and STING agonists upregulated IL-10 production in human CD4+T cells. CONCLUSIONS: These findings establish a crucial role of T cell-intrinsic STING in switching off the pathogenic programs of Th1 cells in intestinal inflammation.

STING Promotes Intestinal IgA Production by Regulating Acetate-producing Bacteria to Maintain Host-microbiota Mutualism.

BACKGROUND: Intestinal Immunoglobulin A (IgA) is crucial in maintaining host-microbiota mutualism and gut homeostasis. It has been shown that many species of gut bacteria produce cyclic dinucleotides, along with an abundance of microbiota-derived DNA present within the intestinal lumen, which triggers the tonic activation of the cytosolic cGAS-STING pathway. However, the role of STING in intestinal IgA remains poorly understood. We further investigated whether and how STING affects intestinal IgA response. METHODS: Intestinal IgA was determined between wild-type (WT) mice and Sting-/- mice in steady conditions and upon enteric Citrobacter rodentium infection. STING agonists were used to stimulating B cells or dendritic cells in vitro. Gut microbiota composition was examined by 16S ribosomal RNA gene sequencing. Bacteria metabolomics functional analyses was performed by PICRUSt2. Fecal short-chain fatty acid (SCFA) was determined by Mass spectrometry and Cedex Bio Analyzer. Gut bacteria from WT mice and Sting-/- mice were transferred into germ-free mice and antibiotic-pretreated mice. RESULTS: Intestinal IgA response was impaired in Sting-/- mice. However, STING agonists did not directly stimulate B cells or dendritic cells to induce IgA. Interestingly, Sting-/- mice displayed altered gut microbiota composition with decreased SCFA-producing bacteria and downregulated SCFA fermentation pathways. Transfer of fecal bacteria from Sting-/- mice induced less IgA than that from WT mice in germ-free mice and antibiotic-pretreated mice, which is mediated by GPR43. Acetate, the dominant SCFA, was decreased in Sting-/- mice, and supplementation of acetate restored intestinal IgA production in Sting-/- mice. CONCLUSIONS: STING promotes intestinal IgA by regulating acetate-producing gut bacteria.

STING pathway contributes to maintaining a group of acetate-producing bacteria. STING regulates through these bacteria in a GPR43-dependent manner.

Th17 Cell-Derived Amphiregulin Promotes Colitis-Associated Intestinal Fibrosis Through Activation of mTOR and MEK in Intestinal Myofibroblasts.

BACKGROUND & AIMS: Intestinal fibrosis is a significant complication of Crohn's disease (CD). Gut microbiota reactive Th17 cells are crucial in the pathogenesis of CD; however, how Th17 cells induce intestinal fibrosis is still not completely understood. METHODS: In this study, T-cell transfer model with wild-type (WT) and Areg-/- Th17 cells and dextran sulfate sodium (DSS)-induced chronic colitis model in WT and Areg-/- mice were used. CD4+ T-cell expression of AREG was determined by quantitative reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. The effect of AREG on proliferation/migration/collagen expression in human intestinal myofibroblasts was determined. AREG expression was assessed in healthy controls and patients with CD with or without intestinal fibrosis. RESULTS: Although Th1 and Th17 cells induced intestinal inflammation at similar levels when transferred into Tcrßxδ-/- mice, Th17 cells induced more severe intestinal fibrosis. Th17 cells expressed higher levels of AREG than Th1 cells. Areg-/- mice developed less severe intestinal fibrosis compared with WT mice on DSS insults. Transfer of Areg-/- Th17 cells induced less severe fibrosis in Tcrßxδ-/- mice compared with WT Th17 cells. Interleukin (IL)6 and IL21 promoted AREG expression in Th17 cells by activating Stat3. Stat3 inhibitor suppressed Th17-induced intestinal fibrosis. AREG promoted human intestinal myofibroblast proliferation, motility, and collagen I expression, which was mediated by activating mammalian target of rapamycin and MEK. AREG expression was increased in intestinal CD4+ T cells in fibrotic sites compared with nonfibrotic sites from patients with CD. CONCLUSIONS: These findings reveal that Th17-derived AREG promotes intestinal fibrotic responses in experimental colitis and human patients with CD. Thereby, AREG might serve as a potential therapeutic target for fibrosis in CD.

Glucose promotes regulatory T cell differentiation to maintain intestinal homeostasis.

Glucose, the critical energy source in the human body, is considered a potential risk factor in various autoimmune diseases when consumed in high amounts. However, the roles of glucose at moderate doses in the regulation of autoimmune inflammatory diseases and CD4+ T cell responses are controversial. Here, we show that while glucose at a high concentration (20% w/v) promotes intestinal inflammation, it suppresses colitis at a moderate dose (6% w/v), which increases the proportion of intestinal regulatory T (Treg) cells but does not affect effector CD4+ T cells. Glucose treatment promotes Treg cell differentiation but it does not affect Treg stability. Feeding glucose alters gut microbiota compositions, which are not involved in the glucose induction of Treg cells. Glucose promotes aryl hydrocarbon receptor (AhR) activation to induce Treg polarization. These findings reveal the different effects of glucose at different doses on the intestinal immune response.

GPR120 Inhibits Colitis Through Regulation of CD4+ T Cell Interleukin 10 Production.

BACKGROUND & AIMS: G protein-coupled receptor (GPR) 120 has been implicated in regulating metabolic syndromes with anti-inflammatory function. However, the role of GPR120 in intestinal inflammation is unknown. Here, we investigated whether and how GPR120 regulates CD4+ T cell function to inhibit colitis development. METHODS: Dextran sodium sulfate (DSS)-induced colitis model, Citrobacter rodentium infection model, and CD4+ T cell adoptive transfer model were used to analyze the role of GPR120 in regulating colitis development. The effect of GPR120 on CD4+ T cell functions was analyzed by RNA sequencing, flow cytometry, and Seahorse metabolic assays. Mice were administered GPR120 agonist for investigating the potential of GPR120 agonist in preventing and treating colitis. RESULTS: Deficiency of GPR120 in CD4+ T cells resulted in more severe colitis in mice upon dextran sodium sulfate insult and enteric infection. Transfer of GPR120-deficient CD4+CD45Rbhi T cells induced more severe colitis in Rag-/- mice with lower intestinal interleukin (IL) 10+CD4+ T cells. Treatment with the GPR120 agonist CpdA promoted CD4+ T cell production of IL10 by up-regulating Blimp1 and enhancing glycolysis, which was regulated by mTOR. GPR120 agonist-treated wild-type, but not IL10-deficient and Blimp1-deficient, T helper 1 cells induced less severe colitis. Furthermore, oral administration of GPR120 agonist protected mice from intestinal inflammation in both prevention and treatment schemes. Gpr120 expression was positively correlated with Il10 expression in the human colonic mucosa, including patients with inflammatory bowel diseases. CONCLUSIONS: Our findings show the role of GPR120 in regulating intestinal CD4+ T cell production of IL10 to inhibit colitis development, which identifies GPR120 as a potential therapeutic target for treating inflammatory bowel diseases.

MicroRNA-10a Negatively Regulates CD4+ T Cell IL-10 Production through Suppression of Blimp1.

An uncontrolled CD4+ T cell response is a critical hallmark of autoimmune diseases. IL-10, which can be produced by both effector and regulatory CD4+ T cells, plays an essential role in the inhibition of autoimmunity. MicroRNAs are key molecules involved in regulating immune responses. However, how miR-10a regulates CD4+ T cell function in the pathogenesis of intestinal immune responses is not fully understood. In this study, we show that the mice with deficient miR-10a in CD4+ T cells were more resistant to intestinal inflammation upon inflammatory insult. miR-10a-deficient CD4+CD45Rbhi T cells were less colitogenic in Rag -/- mice, in which CD4+ T cell production of IL-10 was increased. miR-10a-deficient CD4+ T cells expressed a higher expression of IL-10 in vitro. Blocking the IL-10/IL-10R pathway in vivo aggravated colitis induced by miR-10a-deficient CD4+CD45Rbhi T cells. Mechanically, miR-10a suppressed CD4+ T cell production of IL-10 through targeting Prdm1, which encodes Blimp1. We further show that that CD4+ T cells lacking Blimp1 produced lower levels of IL-10 and induced more severe colitis in Rag -/- mice. These data thus establish the role of miR-10a in the inhibition of IL-10 production in CD4+ T cells to regulate intestinal homeostasis.

Propionate Enhances Cell Speed and Persistence to Promote Intestinal Epithelial Turnover and Repair.

BACKGROUND AND AIMS: Gut bacteria-derived short-chain fatty acids (SCFAs) play crucial roles in the maintenance of intestinal homeostasis. However, how SCFAs regulate epithelial turnover and tissue repair remain incompletely understood. In this study, we investigated how the SCFA propionate regulates cell migration to promote epithelial renewal and repair. METHODS: Mouse small intestinal epithelial cells (MSIE) and human Caco-2 cells were used to determine the effects of SCFAs on gene expression, proliferation, migration, and cell spreading in vitro. Video microscopy and single cell tracking were used to assess cell migration kinetically. 5-bromo-2'-deoxyuridine (BrdU) and hydroxyurea were used to assess the effects of SCFAs on migration in vivo. Lastly, an acute colitis model using dextran sulfate sodium (DSS) was used to examine the effects of SCFAs in vivo. RESULTS: Using video microscopy and single cell tracking, we found that propionate promoted intestinal epithelial cell migration by enhancing cell spreading and polarization, which led to increases in both cell speed and persistence. This novel function of propionate was dependent on inhibition of class I histone deacetylases (HDAC) and GPR43 and required signal transducer and activator of transcription 3 (STAT3). Furthermore, using 5-bromo-2'-deoxyuridine (BrdU) and hydroxyurea in vivo, we found that propionate enhanced cell migration up the crypt-villus axis under homeostatic conditions, while also protecting against ulcer formation in experimental colitis. CONCLUSION: Our results demonstrate a mechanism by which propionate stimulates cell migration in an HDAC inhibition, GPR43, and STAT3 dependent manner, and suggest that propionate plays an important role in epithelial migration independent of proliferation.

Intestinal microbiota-derived short-chain fatty acids regulation of immune cell IL-22 production and gut immunity.

Innate lymphoid cells (ILCs) and CD4+ T cells produce IL-22, which is critical for intestinal immunity. The microbiota is central to IL-22 production in the intestines; however, the factors that regulate IL-22 production by CD4+ T cells and ILCs are not clear. Here, we show that microbiota-derived short-chain fatty acids (SCFAs) promote IL-22 production by CD4+ T cells and ILCs through G-protein receptor 41 (GPR41) and inhibiting histone deacetylase (HDAC). SCFAs upregulate IL-22 production by promoting aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1α (HIF1α) expression, which are differentially regulated by mTOR and Stat3. HIF1α binds directly to the Il22 promoter, and SCFAs increase HIF1α binding to the Il22 promoter through histone modification. SCFA supplementation enhances IL-22 production, which protects intestines from inflammation. SCFAs promote human CD4+ T cell IL-22 production. These findings establish the roles of SCFAs in inducing IL-22 production in CD4+ T cells and ILCs to maintain intestinal homeostasis.

STING controls intestinal homeostasis through promoting antimicrobial peptide expression in epithelial cells.

Stimulator of interferon genes (STING) has been shown to play a critical role in orchestrating immune responses to various pathogens through sensing cyclic dinucleotides. However, how STING regulates intestinal homeostasis is still not completely understood. In this study, we found that STING-/- mice were more susceptible to enteric infection with Citrobacter rodentium compared to wild-type (WT) mice evidenced by more severe intestinal inflammation and impaired bacterial clearance. STING-/- mice demonstrated lower expression of REG3γ but not ß-defensins and Cramp in IECs. Consistently, STING-/- IECs showed reduced capacity to inhibit bacterial growth. STING agonists, both 10-carboxymethyl-9-acridanone (CMA) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA), promoted REG3γ expression IECs. Furthermore, STING agonists promoted WT but not REG3γ-deficient IEC bacterial killing. Mechanistically, STING agonists activated STAT3 and promoted glycolysis in IECs. Inhibition of STAT3 pathway and glycolysis suppressed STING-induced REG3γ production in IECs, and abrogated STING-mediated IEC killing of C. rodentium. Additionally, treatment with the STING ligand, 2,3-cGAMP, inhibited C. rodentium-induced colitis in vivo. Overall, STING promotes IEC REG3γ expression to inhibit enteric infection and intestinal inflammation, thus, maintaining the intestinal homeostasis.

IL-21 Promotes Intestinal Memory IgA Responses.

The role of IL-21, produced mainly by Th17 cells and T follicular helper cells, has been intensively investigated in B cell differentiation and Ab class switch. However, how IL-21 regulates memory IgA+ B cell development and memory IgA responses in the intestines is still not completely understood. In this study, we found the total IgA+ B cells as well as CD38+CD138-IgA+ memory B cells were significantly increased in intestinal lamina propria (LP) of TCRßxδ-/- mice after transfer of microbiota Ag-specific Th17 cells but not Th1 cells. Although IL-21R-/- mice or IL-17R-/- mice showed decreased Ag-specific memory IgA production in the intestines upon infection with Citrobacter rodentium, the percentage of IgA+CD38+CD138- memory B cells in Peyer's patches and LP was decreased only in IL-21R-/- mice, but not in IL-17R-/- mice, after reinfection with C. rodentium compared with wild-type mice. Blockade IL-21 in vivo suppressed intestinal C. rodentium-specific IgA production as well as IgA+CD38+CD138- memory B cells in Peyer's patches and LP. Furthermore, IL-21 significantly induced B cell IgA production in vitro, with the increased expression of genes related with class-switching and memory B cell development, including Aicda, Ski, Bmi1, and Klf2. Consistently, Aicda and Ski expression was decreased in B cells of IL-21R-/- mice after C. rodentium reinfection. In conclusion, our study demonstrated that IL-21 promotes intestinal memory IgA B cell development, possibly through upregulating differentiation-related and class switching-related genes, indicating a potential role of IL-21 in memory IgA+ B cell responses in the intestines.

Microbiota Metabolite Short-Chain Fatty Acids Facilitate Mucosal Adjuvant Activity of Cholera Toxin through GPR43.

The gut microbiota has been shown critical for mucosal adjuvant activity of cholera toxin (CT), a potent mucosal adjuvant. However, the mechanisms involved remain largely unknown. In this study, we report that depletion of gut bacteria significantly decreased mucosal and systemic Ab responses in mice orally immunized with OVA and CT. Feeding mice short-chain fatty acids (SCFAs) promoted Ab responses elicited by CT, and, more importantly, rescued Ab responses in antibiotic-treated mice. In addition, mice deficient in GPR43, a receptor for SCFAs, showed impaired adjuvant activity of CT. Administering CT did not promote SCFA production in the intestines; thus, SCFAs facilitated but did not directly mediate the adjuvant activity of CT. SCFAs promoted B cell Ab production by promoting dendritic cell production of BAFF and ALDH1a2, which induced B cell expression of IFN regulatory factor 4, Blimp1, and XBP1, the plasma B cell differentiation-related genes. Furthermore, when infected with Citrobacter rodentium, GPR43-/- mice exhibited decreased Ab responses and were more susceptible to infection, whereas the administration of SCFAs promoted intestinal Ab responses in wild-type mice. Our study thereby demonstrated a critical role of gut microbiota and their metabolite SCFAs in promoting mucosal adjuvant activity of CT through GPR43.

Antimicrobial resistance and genetic characterization of Shigella spp. in Shanxi Province, China, during 2006-2016.

BACKGROUND: Shigella spp., facultative anaerobic bacilli of the family Enterobacteriaceae, are one of the most common causes of diarrheal diseases in human worldwide which have become a significant public health burden. So, we aimed to analyze the antimicrobial phenotypes and to elucidate the molecular mechanisms underlying resistance to cephalosporins and fluoroquinolones in Shigella isolates from patients with diarrhea in Shanxi Province. RESULTS: During 2006-2016, we isolated a total of 474 Shigella strains (including 337 S. flexneri and 137 S. sonnei). The isolates showed high rates of resistance to traditional antimicrobials, and 26, 18.1 and 3.0% of them exhibited resistance to cephalosporins, fluoroquinolones and co-resistance to cephalosporins and fluoroquinolones, respectively. Notably, 91.1% of these isolates, including 22 isolates that showed an ACTSuT profile, exhibited multidrug resistance (MDR). The resistance rates to cephalosporins in S. sonnei isolates were higher than those in S. flexneri. Conversely, the resistance rates to fluoroquinolones were considerably higher in S. flexneri isolates. Among the 123 cephalosporins-resistant isolates, the most common extended-spectrum beta-lactamase gene was blaTEM-1, followed by blaCTX-M, blaOXA-1, and blaSHV-12. Six subtypes of blaCTX-M were identified, blaCTX-M-14 (n = 36) and blaCTX-M-55 (n = 26) were found to be dominant. Of all the 86 isolates with resistance to fluoroquinolones and having at least one mutation (Ser83Leu, His211Tyr, or Asp87Gly) in the the quinolone resistance-determining regions of gyrA, 79 also had mutation of parC (Ser80Ile), whereas 7 contained plasmid-mediated quinolone resistance genes including qnrA, qnrB, qnrS, and aac(60)-Ib-cr. Furthermore, pulsed-field gel electrophoresis analysis (PFGE) showed a considerable genetic diversity in S. flexneri isolates. However, the S. sonnei isolates had a high genetic similarity. CONCLUSIONS: Coexistence of diverse resistance genes causing the emergence and transmission of MDR might render the treatment of shigellosis difficult. Therefore, continuous surveillance might be needed to understand the actual disease burden and provide guidance for shigellosis.

Interleukin-33 Promotes REG3γ Expression in Intestinal Epithelial Cells and Regulates Gut Microbiota.

BACKGROUND & AIMS: Regenerating islet-derived protein (REG3γ), an antimicrobial peptide, typically expressed by intestinal epithelial cells (IEC), plays crucial roles in intestinal homeostasis and controlling gut microbiota. However, the mechanisms that regulate IEC expression of REG3γ are still largely unclear. In this study, we investigated whether and how interleukin (IL) 33, an alarmin produced by IEC in response to injury, regulates REG3γ expression in IEC, thus contributing to intestinal homeostasis. METHODS: IEC were isolated from wild-type and IL33-/- mice to determine expression of REG3γ and other antimicrobial peptides by quantitative real-time polymerase chain reaction and Western blot. IEC cell lines were used for mechanistic studies. 16S rRNA pyrosequencing analysis was used for measuring gut microbiota. Citrobacter rodentium was used for enteric infections. RESULTS: The expression of REG3γ, but not ß-defensins, in IECs of IL33-/- mice was significantly lower than wild-type mice. IL33 treatment induced IEC expression of REG3γ in both mice and human cell lines. Mechanistically, IL33 activated STAT3, mTOR, and ERK1/2 in IEC. Inhibition of these pathways abrogated IL33-induction of REG3γ. IL33-/- mice demonstrated higher bacteria loads and altered microbiota composition. IL33 did not directly inhibit bacterial growth, but promoted wild-type, not REG3γKO, IECs to kill bacteria in vitro. Consistently, C rodentium infection induced IEC IL33 expression, and IL33-/- mice demonstrated an impaired bacterial clearance with C rodentium infection. CONCLUSIONS: Our study demonstrated that IL33, which is produced by IEC in response to injury and inflammatory stimulation, in turn promotes IEC expression of REG3γ, and controls the gut microbiota of the host.

Microbiota Metabolite Butyrate Differentially Regulates Th1 and Th17 Cells' Differentiation and Function in Induction of Colitis.

BACKGROUND: How the gut microbiota regulates intestinal homeostasis is not completely clear. Gut microbiota metabolite short-chain fatty acids (SCFAs) have been reported to regulate T-cell differentiation. However, the mechanisms underlying SCFA regulation of T-cell differentiation and function remain to be investigated. METHODS: CBir1, an immunodominant microbiota antigen, transgenic T cells were treated with butyrate under various T-cell polarization conditions to investigate butyrate regulation of T-cell differentiation and the mechanism involved. Transfer of butyrate-treated CBir T cells into Rag1-/- mice was performed to study the in vivo role of such T cells in inducing colitis. RESULTS: Although butyrate promoted Th1 cell development by promoting IFN-γ and T-bet expression, it inhibited Th17 cell development by suppressing IL-17, Rorα, and Rorγt expression. Interestingly, butyrate upregulated IL-10 production in T cells both under Th1 and Th17 cell conditions. Furthermore, butyrate induced T-cell B-lymphocyte-induced maturation protein 1 (Blimp1) expression, and deficiency of Blimp1 in T cells impaired the butyrate upregulation of IL-10 production, indicating that butyrate promotes T-cell IL-10 production at least partially through Blimp1. Rag1-/- mice transferred with butyrate-treated T cells demonstrated less severe colitis, compared with transfer of untreated T cells, and administration of anti-IL-10R antibody exacerbated colitis development in Rag-/- mice that had received butyrate-treated T cells. Mechanistically, the effects of butyrate on the development of Th1 cells was through inhibition of histone deacetylase but was independent of GPR43. CONCLUSIONS: These data indicate that butyrate controls the capacity of T cells in the induction of colitis by differentially regulating Th1 and Th17 cell differentiation and promoting IL-10 production, providing insights into butyrate as a potential therapeutic for the treatment of inflammatory bowel disease.

RORγt Represses IL-10 Production in Th17 Cells To Maintain Their Pathogenicity in Inducing Intestinal Inflammation.

The role of retinoid-related orphan receptor γ t (RORγt) in Th17 cell differentiation has been well established; however, how it regulates other T cell lineages is still not clearly understood. In this study, we report that in mice, while promoting Th17 cell differentiation, RORγt inhibited IL-10 production by T cells, thereby preserving the pathogenicity of Th17 cells. Treatment with RORγt-specific inhibitor suppressed Th17 cell signature cytokines, but promoted IL-10 production. RORγt inhibitor-treated Th17 cells induce less severe colitis compared with control Th17 cells. Mechanistically, the RORγt inhibitor induced T cell expression of Blimp-1 (encoded by Prdm1). Prdm1-/- T cells produced significantly fewer IL-10 when treated with RORγt inhibitor compared with wild-type T cells. Furthermore, RORγt inhibitor-treated Prdm1-/- Th17 cells induce more severe colitis compared with RORγt inhibitor-treated wild-type Th17 cells. Collectively, our studies reveal a novel mechanism by which RORγt drives and maintains pathogenic Th17 cell development by inhibiting IL-10 production.

Microbiota-derived short-chain fatty acids promote Th1 cell IL-10 production to maintain intestinal homeostasis.

T-cells are crucial in maintanence of intestinal homeostasis, however, it is still unclear how microbiota metabolites regulate T-effector cells. Here we show gut microbiota-derived short-chain fatty acids (SCFAs) promote microbiota antigen-specific Th1 cell IL-10 production, mediated by G-protein coupled receptors 43 (GPR43). Microbiota antigen-specific Gpr43-/- CBir1 transgenic (Tg) Th1 cells, specific for microbiota antigen CBir1 flagellin, induce more severe colitis compared with wide type (WT) CBir1 Tg Th1 cells in Rag-/- recipient mice. Treatment with SCFAs limits colitis induction by promoting IL-10 production, and administration of anti-IL-10R antibody promotes colitis development. Mechanistically, SCFAs activate Th1 cell STAT3 and mTOR, and consequently upregulate transcription factor B lymphocyte-induced maturation protein 1 (Blimp-1), which mediates SCFA-induction of IL-10. SCFA-treated Blimp1-/- Th1 cells produce less IL-10 and induce more severe colitis compared to SCFA-treated WT Th1 cells. Our studies, thus, provide insight into how microbiota metabolites regulate Th1 cell functions to maintain intestinal homeostasis.

Neutrophils Promote Amphiregulin Production in Intestinal Epithelial Cells through TGF-ß and Contribute to Intestinal Homeostasis.

Neutrophils are the first responders to sites of inflammation when the intestinal epithelial barrier is breached and the gut microbiota invade. Despite current efforts in understanding the role of neutrophils in intestinal homeostasis, the complex interactions between neutrophils and intestinal epithelial cells (IECs) is still not well characterized. In this study, we demonstrated that neutrophils enhanced production of amphiregulin (AREG), a member of the EGFR ligand family, by IECs, which promoted IEC barrier function and tissue repair. Depletion of neutrophils resulted in more severe colitis in mice because of decreased AREG production by IECs upon dextran sodium sulfate (DSS) insult. Administration of AREG restored epithelial barrier function and ameliorated colitis. Furthermore, neutrophil-derived TGF-ß promoted AREG production by IECs. Mechanistically, TGF-ß activated MEK1/2 signaling, and inhibition of MEK1/2 abrogated TGF-ß-induced AREG production by IECs. Collectively, these findings reveal that neutrophils play an important role in the maintenance of IEC barrier function and homeostasis.