CAR directs T cell adaptation to bile acids in the small intestine.

Bile acids are lipid-emulsifying metabolites synthesized in hepatocytes and maintained in vivo through enterohepatic circulation between the liver and small intestine1. As detergents, bile acids can cause toxicity and inflammation in enterohepatic tissues2. Nuclear receptors maintain bile acid homeostasis in hepatocytes and enterocytes3, but it is unclear how mucosal immune cells tolerate high concentrations of bile acids in the small intestine lamina propria (siLP). CD4+ T effector (Teff) cells upregulate expression of the xenobiotic transporter MDR1 (encoded by Abcb1a) in the siLP to prevent bile acid toxicity and suppress Crohn's disease-like small bowel inflammation4. Here we identify the nuclear xenobiotic receptor CAR (encoded by Nr1i3) as a regulator of MDR1 expression in T cells that can safeguard against bile acid toxicity and inflammation in the mouse small intestine. Activation of CAR induced large-scale transcriptional reprogramming in Teff cells that infiltrated the siLP, but not the colon. CAR induced the expression of not only detoxifying enzymes and transporters in siLP Teff cells, as in hepatocytes, but also the key anti-inflammatory cytokine IL-10. Accordingly, CAR deficiency in T cells exacerbated bile acid-driven ileitis in T cell-reconstituted Rag1-/- or Rag2-/- mice, whereas pharmacological activation of CAR suppressed it. These data suggest that CAR acts locally in T cells that infiltrate the small intestine to detoxify bile acids and resolve inflammation. Activation of this program offers an unexpected strategy to treat small bowel Crohn's disease and defines lymphocyte sub-specialization in the small intestine.

The Xenobiotic Transporter Mdr1 Enforces T Cell Homeostasis in the Presence of Intestinal Bile Acids.

CD4+ T cells are tightly regulated by microbiota in the intestine, but whether intestinal T cells interface with host-derived metabolites is less clear. Here, we show that CD4+ T effector (Teff) cells upregulated the xenobiotic transporter, Mdr1, in the ileum to maintain homeostasis in the presence of bile acids. Whereas wild-type Teff cells upregulated Mdr1 in the ileum, those lacking Mdr1 displayed mucosal dysfunction and induced Crohn's disease-like ileitis following transfer into Rag1-/- hosts. Mdr1 mitigated oxidative stress and enforced homeostasis in Teff cells exposed to conjugated bile acids (CBAs), a class of liver-derived emulsifying agents that actively circulate through the ileal mucosa. Blocking ileal CBA reabsorption in transferred Rag1-/- mice restored Mdr1-deficient Teff cell homeostasis and attenuated ileitis. Further, a subset of ileal Crohn's disease patients displayed MDR1 loss of function. Together, these results suggest that coordinated interaction between mucosal Teff cells and CBAs in the ileum regulate intestinal immune homeostasis.

Small-molecule RORγt antagonists inhibit T helper 17 cell transcriptional network by divergent mechanisms.

We identified three retinoid-related orphan receptor gamma t (RORγt)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORγt binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORγt acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the RORα binding motif. RORγt is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORγt-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity.

Aminoacyl-tRNA synthetase inhibition activates a pathway that branches from the canonical amino acid response in mammalian cells.

Signaling pathways that sense amino acid abundance are integral to tissue homeostasis and cellular defense. Our laboratory has previously shown that halofuginone (HF) inhibits the prolyl-tRNA synthetase catalytic activity of glutamyl-prolyl-tRNA synthetase (EPRS), thereby activating the amino acid response (AAR). We now show that HF treatment selectively inhibits inflammatory responses in diverse cell types and that these therapeutic benefits occur in cells that lack GCN2, the signature effector of the AAR. Depletion of arginine, histidine, or lysine from cultured fibroblast-like synoviocytes recapitulates key aspects of HF treatment, without utilizing GCN2 or mammalian target of rapamycin complex 1 pathway signaling. Like HF, the threonyl-tRNA synthetase inhibitor borrelidin suppresses the induction of tissue remodeling and inflammatory mediators in cytokine-stimulated fibroblast-like synoviocytes without GCN2, but both aminoacyl-tRNA synthetase (aaRS) inhibitors are sensitive to the removal of GCN1. GCN1, an upstream component of the AAR pathway, binds to ribosomes and is required for GCN2 activation. These observations indicate that aaRS inhibitors, like HF, can modulate inflammatory response without the AAR/GCN2 signaling cassette, and that GCN1 has a role that is distinct from its activation of GCN2. We propose that GCN1 participates in a previously unrecognized amino acid sensor pathway that branches from the canonical AAR.

Development of a putative Zn2+-chelating but highly selective MMP-13 inhibitor.

Starting from an already known MMP-13 inhibitor, 1, we pursued an SAR-approach focusing on optimizing interactions close to the Zn2+ binding site of the enzyme. We found the oxetane containing compound 32 (MMP-13 IC50 = 42 nM), which exhibited complete inhibition of collagenolysis in in vitro studies and an excellent selectivity profile among the MMP family. Interestingly, docking studies propose that the oxetane ring in 32 is oriented towards the Zn2+ ion for chelating the metal ion. Chelating properties of MMP13-inhibitors are often connected with non-selectivity within the enzyme family. Compound 32 demonstrates a rare example where the selectivity can be explained via combinatorial effects of interactions within the S1' loop and a chelating effect of the oxetane moiety. Furthermore, in vivo pharmacokinetic studies were performed demonstrating a concentration of 1.97 µM of 32 within the synovial fluid of the rat knee joint, which makes the compound a promising lead compound for further optimization and development for osteoarthritis.

Xenobiotic and endobiotic handling by the mucosal immune system.

PURPOSE OF REVIEW: Mucosal immune cells in the intestinal tract are continuously exposed to a barrage of both foreign and endogenously generated metabolites, termed xenobiotics, and endobiotics, respectively. This review summarizes recent insights into the mechanisms by which xenobiotics and endobiotics regulate intestinal immunity and inflammation. RECENT FINDINGS: The community of enteric microbes (i.e., microbiota) has profound impacts on the development and function of the mucosal immune system. The composition and function of gut microbiota is dynamically regulated by diet, and this interplay dictates which and how many immunomodulatory xenobiotics are present in the intestine. Microbiota also regulate the concentration and composition of circulating bile acids, an abundant class of liver-derived endobiotics with pleotropic immunoregulatory activities. A growing body of literature is emerging that sheds new light on the mechanisms by which xenobiotics and endobiotics interact with germline-encoded receptors and transporters to shape mucosal immune function. SUMMARY: The complex and dynamic interplay among xenobiotics, endobiotics, and the mucosal immune system is a new frontier in mucosal immunology that is proving fruitful for the discovery of novel and pharmacologically accessible mechanisms with relevance to human inflammatory diseases.

Orphans against autoimmunity.

In this issue of Immunity, Hermann-Kleiter et al. (2008) identify a nuclear orphan receptor, NR2F6, as a negative regulator of the T helper 17 cell subset and report that NR2F6-deficient mice develop late-onset autoimmune disease.

Identity crisis of Th17 cells: many forms, many functions, many questions.

Th17 cells are a subset of CD4(+) effector T cells characterized by expression of the IL-17-family cytokines, IL-17A and IL-17F. Since their discovery nearly a decade ago, Th17 cells have been implicated in the regulation of dozens of immune-mediated inflammatory diseases and cancer. However, attempts to clarify the development and function of Th17 cells in human health and disease have generated as many questions as answers. On one hand, cytokine expression in Th17 cells appears to be remarkably dynamic and is subject to extensive regulation (both positive and negative) in tissue microenvironments. On the other hand, accumulating evidence suggests that the human Th17 subset is a heterogeneous population composed of several distinct pro- and anti-inflammatory subsets. Clearly, Th17 cells as originally conceived no longer neatly fit the long-standing paradigm of stable and irrepressible effector T cell function. Here we review current concepts surrounding human Th17 cells, with an emphasis on their plasticity, heterogeneity, and their many, tissue-specific functions. In spite of the challenges ahead, a comprehensive understanding of Th17 cells and their relationship to human disease is key to ongoing efforts to develop safer and more selective anti-inflammatory medicines.

Halofuginone-induced amino acid starvation regulates Stat3-dependent Th17 effector function and reduces established autoimmune inflammation.

The IL-23 pathway is genetically linked to autoimmune disease in humans and is required for pathogenic Th17 cell function in mice. However, because IL-23R-expressing mature Th17 cells are rare and poorly defined in mice at steady-state, little is known about IL-23 signaling. In this study, we show that the endogenous CCR6(+) memory T cell compartment present in peripheral lymphoid organs of unmanipulated mice expresses Il23r ex vivo, displays marked proinflammatory responses to IL-23 stimulation in vitro, and is capable of transferring experimental autoimmune encephalomyelitis. The prolyl-tRNA synthetase inhibitor halofuginone blocks IL-23-induced Stat3 phosphorylation and IL-23-dependent proinflammatory cytokine expression in endogenous CCR6(+) Th17 cells via activation of the amino acid starvation response (AAR) pathway. In vivo, halofuginone shows therapeutic efficacy in experimental autoimmune encephalomyelitis, reducing both established disease progression and local Th17 cell effector function within the CNS. Mechanistically, AAR activation impairs Stat3 responses downstream of multiple cytokine receptors via selective, posttranscriptional suppression of Stat3 protein levels. Thus, our study reveals latent pathogenic functions of endogenous Th17 cells that are regulated by both IL-23 and AAR pathways and identifies a novel regulatory pathway targeting Stat3 that may underlie selective immune regulation by the AAR.

In vivo regulation of gene expression and T helper type 17 differentiation by RORγt inverse agonists.

The orphan nuclear receptor, retinoic acid receptor-related orphan nuclear receptor γt (RORγt), is required for the development and pathogenic function of interleukin-17A-secreting CD4(+) T helper type 17 (Th17) cells. Whereas small molecule RORγt antagonists impair Th17 cell development and attenuate autoimmune inflammation in vivo, the broader effects of these inhibitors on RORγt-dependent gene expression in vivo has yet to be characterized. We show that the RORγt inverse agonist TMP778 acts potently and selectively to block mouse Th17 cell differentiation in vitro and to impair Th17 cell development in vivo upon immunization with the myelin antigen MOG35-55 plus complete Freund's adjuvant. Importantly, we show that TMP778 acts in vivo to repress the expression of more than 150 genes, most of which fall outside the canonical Th17 transcriptional signature and are linked to a variety of inflammatory pathologies in humans. Interestingly, more than 30 genes are related with SMAD3, a transcription factor involved in the Th17 cell differentiation. These results reveal novel disease-associated genes regulated by RORγt during inflammation in vivo, and provide an early read on potential disease indications and safety concerns associated with pharmacological targeting of RORγt.

T cell-derived IL-17 mediates epithelial changes in the airway and drives pulmonary neutrophilia.

Th17 cells are a proinflammatory subset of effector T cells that have been implicated in the pathogenesis of asthma. Their production of the cytokine IL-17 is known to induce local recruitment of neutrophils, but the direct impact of IL-17 on the lung epithelium is poorly understood. In this study, we describe a novel mouse model of spontaneous IL-17-driven lung inflammation that exhibits many similarities to asthma in humans. We have found that STAT3 hyperactivity in T lymphocytes causes an expansion of Th17 cells, which home preferentially to the lungs. IL-17 secretion then leads to neutrophil infiltration and lung epithelial changes, in turn leading to a chronic inflammatory state with increased mucus production and decreased lung function. We used this model to investigate the effects of IL-17 activity on airway epithelium and identified CXCL5 and MIP-2 as important factors in neutrophil recruitment. The neutralization of IL-17 greatly reduces pulmonary neutrophilia, underscoring a key role for IL-17 in promoting chronic airway inflammation. These findings emphasize the role of IL-17 in mediating neutrophil-driven pulmonary inflammation and highlight a new mouse model that may be used for the development of novel therapies targeting Th17 cells in asthma and other chronic pulmonary diseases.

Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase.

Febrifugine, the bioactive constituent of one of the 50 fundamental herbs of traditional Chinese medicine, has been characterized for its therapeutic activity, though its molecular target has remained unknown. Febrifugine derivatives have been used to treat malaria, cancer, fibrosis and inflammatory disease. We recently demonstrated that halofuginone (HF), a widely studied derivative of febrifugine, inhibits the development of T(H)17-driven autoimmunity in a mouse model of multiple sclerosis by activating the amino acid response (AAR) pathway. Here we show that HF binds glutamyl-prolyl-tRNA synthetase (EPRS), inhibiting prolyl-tRNA synthetase activity; this inhibition is reversed by the addition of exogenous proline or EPRS. We further show that inhibition of EPRS underlies the broad bioactivities of this family of natural product derivatives. This work both explains the molecular mechanism of a promising family of therapeutics and highlights the AAR pathway as an important drug target for promoting inflammatory resolution.

Quantifying forms and functions of intestinal bile acid pools in mice.

Bile acids (BAs) are gastrointestinal metabolites that serve dual functions in lipid absorption and cell signaling. BAs circulate actively between the liver and distal small intestine (i.e., ileum), yet the dynamics through which complex BA pools are absorbed in the ileum and interact with intestinal cells in vivo remain ill-defined. Through multi-site sampling of nearly 100 BA species in individual wild type mice, as well as mice lacking the ileal BA transporter, Asbt/Slc10a2, we calculate the ileal BA pool in fasting C57BL/6J mice to be ~0.3 µmoles/g. Asbt-mediated transport accounts for ~80% of this pool and amplifies size, whereas passive absorption explains the remaining ~20%, and generates diversity. Accordingly, ileal BA pools in mice lacking Asbt are ~5-fold smaller than in wild type controls, enriched in secondary BA species normally found in the colon, and elicit unique transcriptional responses in cultured ileal explants. This work quantitatively defines ileal BA pools in mice and reveals how BA dysmetabolism can impinge on intestinal physiology.

Clonal Parabacteroides from Gut Microfistulous Tracts as Transmissible Cytotoxic Succinate-Commensal Model of Crohn's Disease Complications.

Crohn's disease (CD) has been traditionally viewed as a chronic inflammatory disease that cause gut wall thickening and complications, including fistulas, by mechanisms not understood. By focusing on Parabacteroides distasonis (presumed modern succinate-producing commensal probiotic), recovered from intestinal microfistulous tracts (cavernous fistulous micropathologies CavFT proposed as intermediate between 'mucosal fissures' and 'fistulas') in two patients that required surgery to remove CD-damaged ilea, we demonstrate that such isolates exert pathogenic/pathobiont roles in mouse models of CD. Our isolates are clonally-related; potentially emerging as transmissible in the community and mice; proinflammatory and adapted to the ileum of germ-free mice prone to CD-like ileitis (SAMP1/YitFc) but not healthy mice (C57BL/6J), and cytotoxic/ATP-depleting to HoxB8-immortalized bone marrow derived myeloid cells from SAMP1/YitFc mice when concurrently exposed to succinate and extracts from CavFT-derived E. coli , but not to cells from healthy mice. With unique genomic features supporting recent genetic exchange with Bacteroides fragilis -BGF539, evidence of international presence in primarily human metagenome databases, these CavFT Pdis isolates could represent to a new opportunistic Parabacteroides species, or subspecies (' cavitamuralis' ) adapted to microfistulous niches in CD.

Hyperactivation of nuclear factor of activated T cells 1 (NFAT1) in T cells attenuates severity of murine autoimmune encephalomyelitis.

Nuclear factor of activated T cells (NFAT) proteins are a group of Ca(2+)-regulated transcription factors residing in the cytoplasm of resting cells. Dephosphorylation by calcineurin results in nuclear translocation of NFAT and subsequent expression of target genes; rephosphorylation by kinases, including casein kinase 1 (CK1), restores NFAT to its latent state in the cytoplasm. We engineered a hyperactivable version of NFAT1 with increased affinity for calcineurin and decreased affinity for casein kinase 1. Mice expressing hyperactivable NFAT1 in their T-cell compartment exhibited a dramatically increased frequency of both IL-17- and IL-10-producing cells after differentiation under Th17 conditions-this was associated with direct binding of NFAT1 to distal regulatory regions of Il-17 and Il-10 gene loci in Th17 cells. Despite higher IL-17 production in culture, the mice were significantly less prone to myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis than controls, correlating with increased production of the immunomodulatory cytokine IL-10 and enhanced accumulation of regulatory T cells within the CNS. Thus, NFAT hyperactivation paradoxically leads to decreased susceptibility to experimental autoimmune encephalomyelitis, supporting previous observations linking defects in Ca(2+)/NFAT signaling to lymphoproliferation and autoimmune disease.

ATP-dependent transporters: emerging players at the crossroads of immunity and metabolism.

Nearly 50 ATP-binding cassette (ABC) transporters are encoded by mammalian genomes. These transporters are characterized by conserved nucleotide-binding and hydrolysis (i.e., ATPase) domains, and power directional transport of diverse substrate classes - ions, small molecule metabolites, xenobiotics, hydrophobic drugs, and even polypeptides - into or out of cells or subcellular organelles. Although immunological functions of ABC transporters are only beginning to be unraveled, emerging literature suggests these proteins have under-appreciated roles in the development and function of T lymphocytes, including many of the key effector, memory and regulatory subsets that arise during responses to infection, inflammation or cancers. One transporter in particular, MDR1 (Multidrug resistance-1; encoded by the ABCB1 locus in humans), has taken center stage as a novel player in immune regulation. Although MDR1 remains widely viewed as a simple drug efflux pump in tumor cells, recent evidence suggests that this transporter fills key endogenous roles in enforcing metabolic fitness of activated CD4 and CD8 T cells. Here, we summarize current understanding of the physiological functions of ABC transporters in immune regulation, with a focus on the anti-oxidant functions of MDR1 that may shape both the magnitude and repertoires of antigen-specific effector and memory T cell compartments. While much remains to be learned about the functions of ABC transporters in immunobiology, it is already clear that they represent fertile new ground, both for the definition of novel immunometabolic pathways, and for the discovery of new drug targets that could be leveraged to optimize immune responses to vaccines and cancer immunotherapies.

Transcriptional Behavior of Regulatory T Cells Predicts IBD Patient Responses to Vedolizumab Therapy.

BACKGROUND: Inflammatory bowel disease (IBD) involves chronic T cell-mediated inflammatory responses. Vedolizumab (VDZ), a monoclonal antibody against α4ß7 integrin, inhibits lymphocyte extravasation into intestinal mucosae and is effective in ulcerative colitis (UC) and Crohn's disease (CD). AIM: We sought to identify immune cell phenotypic and gene expression signatures that related to response to VDZ. METHODS: Peripheral blood (PBMC) and lamina propria mononuclear cells (LPMCs) were analyzed by flow cytometry and Cytofkit. Sorted CD4 + memory (Tmem) or regulatory T (Treg) cells from PBMC and LPMC were analyzed by RNA sequencing (RNA-seq). Clinical response (≥2-point drop in partial Mayo scores [UC] or Harvey-Bradshaw index [CD]) was assessed 14 to 22 weeks after VDZ initiation. Machine-learning models were used to infer combinatorial traits that predicted response to VDZ. RESULTS: Seventy-one patients were enrolled: 37 received VDZ and 21 patients remained on VDZ >2 years. Fourteen of 37 patients (38%; 8 UC, 6 CD) responded to VDZ. Immune cell phenotypes and CD4 + Tmem and Treg transcriptional behaviors were most divergent between the ileum and colon, irrespective of IBD subtype or inflammation status. Vedolizumab treatment had the greatest impact on Treg metabolic pathways, and response was associated with increased expression of genes involved in oxidative phosphorylation. The strongest clinical predictor of VDZ efficacy was concurrent use of thiopurines. Mucosal tissues offered the greatest number of response-predictive biomarkers, whereas PBMC Treg-expressed genes were the best predictors in combinatorial models of response. CONCLUSIONS: Mucosal and peripheral blood immune cell phenotypes and transcriptional profiles can inform VDZ efficacy and inform new opportunities for combination therapies.

Vedolizumab (VDZ) is effective in the treatment of IBD. Immunophenotyping and RNAseq of T cells were used to inform its mechanism of action. Changes in T regulatory cells in the periphery and mucosa have the greatest relationship to VDZ response.

Genetic and pharmacological inhibition of the nuclear receptor RORα regulates TH17 driven inflammatory disorders.

Full development of IL-17 producing CD4+ T helper cells (TH17 cells) requires the transcriptional activity of both orphan nuclear receptors RORα and RORγt. However, RORα is considered functionally redundant to RORγt; therefore, the function and therapeutic value of RORα in TH17 cells is unclear. Here, using mouse models of autoimmune and chronic inflammation, we show that expression of RORα is required for TH17 cell pathogenicity. T-cell-specific deletion of RORα reduces the development of experimental autoimmune encephalomyelitis (EAE) and colitis. Reduced inflammation is associated with decreased TH17 cell development, lower expression of tissue-homing chemokine receptors and integrins, and increased frequencies of Foxp3+ T regulatory cells. Importantly, inhibition of RORα with a selective small molecule antagonist mostly phenocopies our genetic data, showing potent suppression of the in vivo development of both chronic/progressive and relapsing/remitting EAE, but with no effect on overall thymic cellularity. Furthermore, use of the RORα antagonist effectively inhibits human TH17 cell differentiation and memory cytokine secretion. Together, these data suggest that RORα functions independent of RORγt in programming TH17 pathogenicity and identifies RORα as a safer and more selective therapeutic target for the treatment of TH17-mediated autoimmunity.

New twists of T cell fate: control of T cell activation and tolerance by TGF-beta and NFAT.

Protective and pathogenic immune responses were initially thought to be determined by the differentiation of naïve T cells into Th1 and Th2 effector subsets and the immunosuppressive activity of thymic-derived regulatory T cells. It is now clear that naïve T cells can also differentiate into 'induced' regulatory T cells or inflammatory T cells that secrete IL-17. These divergent T-cell subsets have opposing functions in imparting inflammation or tolerance, yet both developmental programs are controlled by the pluripotent cytokine transforming growth factor beta and the transcription factor NFAT. Recent findings have begun to shed light on the mechanisms by which TGF-beta and NFAT integrate multiple signaling inputs to determine the direction of naïve T-cell differentiation.