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Genome-wide association studies have identified risk loci linked to inflammatory bowel disease (IBD)(1)—a complex chronic inflammatory disorder of the gastrointestinal tract. The increasing prevalence of IBD in industrialized countries and the augmented disease risk observed in migrants who move into areas of higher disease prevalence suggest that environmental factors are also important determinants of IBD susceptibility and severity(2). However, the identification of environmental factors relevant to IBD and the mechanisms by which they influence disease has been hampered by the lack of platforms for their systematic investigation. Here we describe an integrated systems approach, combining publicly available databases, zebrafish chemical screens, machine learning and mouse preclinical models to identify environmental factors that control intestinal inflammation. This approach established that the herbicide propyzamide increases inflammation in the small and large intestine. Moreover, we show that an AHR–NF-κB–C/EBPβ signalling axis operates in T cells and dendritic cells to promote intestinal inflammation, and is targeted by propyzamide. In conclusion, we developed a pipeline for the identification of environmental factors and mechanisms of pathogenesis in IBD and, potentially, other inflammatory diseases.

Therapeutic approaches for the induction of immune tolerance remain an unmet clinical need for the treatment of autoimmune diseases, including multiple sclerosis (MS). Based on its role in the control of the immune response, the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) is a candidate target for novel immunotherapies. Here, we report the development of AhR-activating nanoliposomes (NLPs) to induce antigen-specific tolerance. NLPs loaded with the AhR agonist ITE and a T cell epitope from myelin oligodendrocyte glycoprotein (MOG)(35–55) induced tolerogenic dendritic cells and suppressed the development of experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS, in preventive and therapeutic setups. EAE suppression was associated with the expansion of MOG(35–55)-specific FoxP3(+) regulatory T cells (Treg cells) and type 1 regulatory T cells (Tr1 cells), concomitant with a reduction in central nervous system-infiltrating effector T cells (Teff cells). Notably, NLPs induced bystander suppression in the EAE model established in C57BL/6 × SJL F1 mice. Moreover, NLPs ameliorated chronic progressive EAE in nonobese diabetic mice, a model which resembles some aspects of secondary progressive MS. In summary, these studies describe a platform for the therapeutic induction of antigen-specific tolerance in autoimmune diseases.

For decades, the aryl hydrocarbon receptor (AHR) was studied for its role in environmental chemical toxicity i.e., as a quirk of nature and a mediator of unintended consequences of human pollution. During that period, it was not certain that the AHR had a “normal” physiological function. However, the ongoing accumulation of data from an ever-expanding variety of studies on cancer, cancer immunity, autoimmunity, organ development, and other areas bears witness to a staggering array of AHR-controlled normal and pathological activities. The objective of this review is to discuss how the AHR has gone from a likely contributor to genotoxic environmental carcinogen-induced cancer to a master regulator of malignant cell progression and cancer aggression. Particular focus is placed on the association between AHR activity and poor cancer outcomes, feedback loops that control chronic AHR activity in cancer, and the role of chronically active AHR in driving cancer cell invasion, migration, cancer stem cell characteristics, and survival.

Significance Immune checkpoint inhibitors have emerged as critical therapeutics for several cancer types, including head and neck squamous cell carcinoma. However, enthusiasm remains constrained by the fact that only a minority of patients benefit. Therefore, there is a need to identify new immunotherapy targets. Here, we provide evidence supporting our hypothesis that the aryl hydrocarbon receptor (AhR) influences multiple immune checkpoints in a model of oral squamous cell carcinoma (OSCC). Remarkably, transplant of AhR-deleted OSCC cells generates completely protective tumor immunity characterized by a decrease in multiple immune checkpoints (PD-L1, CD39, CTLA4, PD1, and Lag3) on malignant and/or immune cells. These results have important implications for understanding the biology of cancer immunosuppression and for targeting the AhR for cancer immunotherapy., Immune checkpoint inhibitors represent some of the most important cancer treatments developed in the last 20 y. However, existing immunotherapy approaches benefit only a minority of patients. Here, we provide evidence that the aryl hydrocarbon receptor (AhR) is a central player in the regulation of multiple immune checkpoints in oral squamous cell carcinoma (OSCC). Orthotopic transplant of mouse OSCC cells from which the AhR has been deleted (MOC1AhR-KO) results, within 1 wk, in the growth of small tumors that are then completely rejected within 2 wk, concomitant with an increase in activated T cells in tumor-draining lymph nodes (tdLNs) and T cell signaling within the tumor. By 2 wk, AhR+ control cells (MOC1Cas9), but not MOC1AhR-KO cells up-regulate exhaustion pathways in the tumor-infiltrating T cells and expression of checkpoint molecules on CD4+ T cells (PD-1, CTLA4, Lag3, and CD39) and macrophages, dendritic cells, and Ly6G+ myeloid cells (PD-L1 and CD39) in tdLNs. Notably, MOC1AhR-KO cell transplant renders mice 100% immune to later challenge with wild-type tumors. Analysis of altered signaling pathways within MOC1AhR-KO cells shows that the AhR controls baseline and IFNγ-induced Ido and PD-L1 expression, the latter of which occurs through direct transcriptional control. These observations 1) confirm the importance of malignant cell AhR in suppression of tumor immunity, 2) demonstrate the involvement of the AhR in IFNγ control of PD-L1 and IDO expression in the cancer context, and 3) suggest that the AhR is a viable target for modulation of multiple immune checkpoints.

Mesenchymal stem cells (MSCs) are promising candidates for the development of cell-based drug delivery systems for autoimmune inflammatory diseases, such as multiple sclerosis (MS). Here, we investigated the effect of Ro-31-8425, an ATP-competitive kinase inhibitor, on the therapeutic properties of MSCs. Upon a simple pretreatment procedure, MSCs spontaneously took up and then gradually released significant amounts of Ro-31-8425. Ro-31-8425 (free or released by MSCs) suppressed the proliferation of CD4(+) T cells in vitro following polyclonal and antigen-specific stimulation. Systemic administration of Ro-31-8425-loaded MSCs ameliorated the clinical course of experimental autoimmune encephalomyelitis (EAE), a murine model of MS, displaying a stronger suppressive effect on EAE than control MSCs or free Ro-31-8425. Ro-31-8425-MSC administration resulted in sustained levels of Ro-31-8425 in the serum of EAE mice, modulating immune cell trafficking and the autoimmune response during EAE. Collectively, these results identify MSC-based drug delivery as a potential therapeutic strategy for the treatment of autoimmune diseases.

Tumor-associated macrophages (TAMs) play an important role in the immune response to cancer, but the mechanisms by which the tumor microenvironment controls TAMs and T cell immunity are not completely understood. Here we report that kynurenine produced by glioblastoma cells activates aryl hydrocarbon receptor (AHR) in TAMs to modulate their function and T cell immunity. AHR promotes CCR2 expression, driving TAM recruitment in response to CCL2. AHR also drives the expression of KLF4 and suppresses NF-κB activation in TAMs. Finally, AHR drives the expression of the ectonucleotidase CD39 in TAMs, which promotes CD8(+) T cell dysfunction by producing adenosine in cooperation with CD73. In humans, the expression of AHR and CD39 was highest in grade 4 glioma, and high AHR expression was associated with poor prognosis. In summary, AHR and CD39 expressed in TAMs participate in the regulation of the immune response in glioblastoma and constitute potential targets for immunotherapy.

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the CNS that causes disability in young adults as a result of the irreversible accumulation of neurological deficits. Although there are potent disease-modifying agents for its initial relapsing-remitting phase, these therapies show limited efficacy in secondary progressive MS (SPMS). Thus, there is an unmet clinical need for the identification of disease mechanisms and potential therapeutic approaches for SPMS. Here, we show that the sphingosine 1-phosphate receptor (S1PR) modulator fingolimod (FTY720) ameliorated chronic progressive experimental autoimmune encephalomyelitis in nonobese diabetic mice, an experimental model that resembles several aspects of SPMS, including neurodegeneration and disease progression driven by the innate immune response in the CNS. Indeed, S1PR modulation by FTY720 in murine and human astrocytes suppressed neurodegeneration-promoting mechanisms mediated by astrocytes, microglia, and CNS-infiltrating proinflammatory monocytes. Genome-wide studies showed that FTY720 suppresses transcriptional programs associated with the promotion of disease progression by astrocytes. The study of the molecular mechanisms controlling these transcriptional modules may open new avenues for the development of therapeutic strategies for progressive MS.

After upregulation of AHR in astrocytes by type I interferons, commensal-microbe-derived metabolites of dietary tryptophan act on astrocytes to suppress CNS inflammation.

ObjectiveMS is an autoimmune demyelinating disease of the CNS, which causes neurologic deficits in young adults and leads to progressive disability. The aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, can drive anti-inflammatory functions in peripheral immune cells and also in CNS-resident cells. Laquinimod is a drug developed for the treatment of MS known to activate AHR, but the cellular targets of laquinimod are still not completely known. In this work, we analyzed the contribution of AHR activation in astrocytes to its beneficial effects in the experimental autoimmune encephalomyelitis (EAE) preclinical model of MS.MethodsWe used conditional knockout mice, in combination with genome-wide analysis of gene expression by RNA-seq and in vitro culture systems to investigate the effects of laquinimod on astrocytes.ResultsWe found that AHR activation in astrocytes by laquinimod ameliorates EAE, a preclinical model of MS. Genome-wide RNA-seq transcriptional analyses detected anti-inflammatory effects of laquinimod in glial cells during EAE. Moreover, we established that the Delaq metabolite of laquinimod dampens proinflammatory mediator production while activating tissue-protective mechanisms in glia.ConclusionsTaken together, these findings suggest that AHR activation by clinically relevant AHR agonists may represent a novel therapeutic approach for the treatment of MS.

Genome-wide studies have identified genetic variants linked to neurologic diseases. Environmental factors also play important roles, but no methods are available for their comprehensive investigation. We developed an approach that combines genomic data, screens in a novel zebrafish model, computational modeling, perturbation studies, and multiple sclerosis (MS) patient samples to evaluate the effects of environmental exposure on CNS inflammation. We found that the herbicide linuron amplifies astrocyte pro-inflammatory activities by activating signaling via sigma receptor 1, inositol-requiring enzyme-1α (IRE1α), and X-box binding protein 1 (XBP1). Indeed, astrocyte-specific shRNA- and CRISPR/Cas9-driven gene inactivation combined with RNA-seq, ATAC-seq, ChIP-seq, and study of patient samples suggest that IRE1α-XBP1 signaling promotes CNS inflammation in experimental autoimmune encephalomyelitis (EAE) and, potentially, MS. In summary, these studies define environmental mechanisms that control astrocyte pathogenic activities and establish a multidisciplinary approach for the systematic investigation of the effects of environmental exposure in neurologic disorders.

The complement system is part of the innate immune response responsible for removing pathogens and cellular debris, in addition to helping to refine CNS neuronal connections via microglia-mediated pruning of inappropriate synapses during brain development. However, less is known about the role of complement during normal aging. Here, we studied the role of the central complement component, C3, in synaptic health and aging. We examined behavior as well as electrophysiological, synaptic, and neuronal changes in the brains ofC3-deficient male mice (C3KO) compared with age-, strain-, and gender-matched C57BL/6J (wild-type, WT) control mice at postnatal day 30, 4 months, and 16 months of age. We found the following: (1) region-specific and age-dependent synapse loss in aged WT mice that was not observed inC3KO mice; (2) age-dependent neuron loss in hippocampal CA3 (but not in CA1) that followed synapse loss in aged WT mice, neither of which were observed in agedC3KO mice; and (3) significantly enhanced LTP and cognition and less anxiety in agedC3KO mice compared with aged WT mice. Importantly, CA3 synaptic puncta were similar between WT andC3KO mice at P30. Together, our results suggest a novel and prominent role for complement protein C3 in mediating aged-related and region-specific changes in synaptic function and plasticity in the aging brain.SIGNIFICANCE STATEMENTThe complement cascade, part of the innate immune response to remove pathogens, also plays a role in synaptic refinement during brain development by the removal of weak synapses. We investigated whether complement C3, a central component, affects synapse loss during aging. Wild-type (WT) andC3knock-out (C3KO) mice were examined at different ages. The mice were similar at 1 month of age. However, with aging, WT mice lost synapses in specific brain regions, especially in hippocampus, an area important for memory, whereasC3KO mice were protected. AgedC3KO mice also performed better on learning and memory tests than aged WT mice. Our results suggest that complement C3, or its downstream signaling, is detrimental to synapses during aging.

Our understanding of the pathways that regulate lymphocyte metabolism, as well as the effects of metabolism and its products on the immune response, is still limited. We report that a metabolic program controlled by the transcription factors hypoxia inducible factor-1α (HIF1-α) and aryl hydrocarbon receptor (AHR) supports the differentiation of type 1 regulatory T cell (Tr1) cells. HIF1-α controls the early metabolic reprograming of Tr1 cells. At later time points, AHR promotes HIF1-α degradation and takes control of Tr1 cell metabolism. Extracellular ATP (eATP) and hypoxia, linked to inflammation, trigger AHR inactivation by HIF1-α and inhibit Tr1 cell differentiation. Conversely, CD39 promotes Tr1 cell differentiation by depleting eATP. CD39 also contributes to Tr1 suppressive activity by generating adenosine in cooperation with CD73 expressed by responder T cells and antigen-presenting cells. These results suggest that HIF1-α and AHR integrate immunological, metabolic and environmental signals to regulate the immune response.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor with important functions in the immune response and cancer. AHR agonists are provided by the environment, the commensal flora and the metabolism. Considering AHR physiological functions, AHR agonists may have important effects on health and disease. Thus, the quantification of AHR agonists in biological samples is of scientific and clinical relevance. We compared different reporter systems for the detection of AHR agonists in serum samples of Multiple Sclerosis (MS) patients, and assessed the influence of transfection methods and cell lines in a reporter-based in vitro assay. While the use of stable or transient reporters did not influence the measurement of AHR agonistic activity, the species of the cell lines used in these reporter assays had important effects on the reporter readings. These observations suggest that cell-specific factors influence AHR activation and signaling. Thus, based on the reported species selectivity of AHR ligands and the cell species-of-origin effects that we describe in this manuscript, the use of human cell lines is encouraged for the analysis of AHR agonistic activity in human samples. These findings may be relevant for the analysis of AHR agonists in human samples in the context of inflammatory and neoplastic disorders.

Unconjugated bilirubin (UCB), a product of heme oxidation, has known immunosuppressant properties but the molecular mechanisms, other than antioxidant effects, remain largely unexplored. We note that UCB modulates T helper type 17 (Th17) immune responses, in a manner dependent upon heightened expression of CD39 ectonucleotidase. UCB has protective effects in experimental colitis, where it enhances recovery after injury and preferentially boosts IL-10 production by colonic intraepithelial CD4+ cells. In vitro, UCB confers immunoregulatory properties on human control Th17 cells, as reflected by increased levels of FOXP3 and CD39 with heightened cellular suppressor ability. Upregulation of CD39 by Th17 cells is dependent upon ligation of the aryl hydrocarbon receptor (AHR) by UCB. Genetic deletion of CD39, as in Entpd1–/– mice, or dysfunction of AHR, as in Ahrd mice, abrogates these UCB salutary effects in experimental colitis. However, in inflammatory bowel disease (IBD) samples, UCB fails to confer substantive immunosuppressive properties upon Th17 cells, because of decreased AHR levels under the conditions tested in vitro. Immunosuppressive effects of UCB are mediated by AHR resulting in CD39 upregulation by Th17. Boosting downstream effects of AHR via UCB or enhancing CD39-mediated ectoenzymatic activity might provide therapeutic options to address development of Th17 dysfunction in IBD.

Tumor-associated macrophages (TAMs) play an important role in the immune response to cancer, but the mechanisms by which the tumor microenvironment controls TAMs and T cell immunity are not completely understood. Here we report that kynurenine produced by glioblastoma cells activates aryl hydrocarbon receptor (AHR) in TAMs to modulate their function and T cell immunity. AHR promotes CCR2 expression, driving TAM recruitment in response to CCL2. AHR also drives the expression of KLF4 and suppresses NF-κB activation in TAMs. Finally, AHR drives the expression of the ectonucleotidase CD39 in TAMs, which promotes CD8

Yeste, Ada et al., Type 1 diabetes (T1D) is a T cell-dependent autoimmune disease that is characterized by the destruction of insulin-producing β cells in the pancreas. The administration to patients of ex vivo-differentiated FoxP3 regulatory T (T) cells or tolerogenic dendritic cells (DCs) that promote T cell differentiation is considered a potential therapy for T1D; however, cell-based therapies cannot be easily translated into clinical practice. We engineered nanoparticles (NPs) to deliver both a tolerogenic molecule, the aryl hydrocarbon receptor (AhR) ligand 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), and the β cell antigen proinsulin (NP) to induce a tolerogenic phenotype in DCs and promote T cell generation in vivo. NP administration to 8-week-old nonobese diabetic mice suppressed autoimmune diabetes. NP induced a tolerogenic phenotype in DCs, which was characterized by a decreased ability to activate inflammatory effector T cells and was concomitant with the increased differentiation of FoxP3 T cells. The induction of a tolerogenic phenotype in DCs by NPs was mediated by the AhR-dependent induction of Socs2, which resulted in inhibition of nuclear factor κB activation and proinflammatory cytokine production (properties of tolerogenic DCs). Together, these data suggest that NPs constitute a potential tool to reestablish tolerance in T1D and potentially other autoimmune disorders., This work was supported by grants ES025530, AI075285, and AI093903 from the NIH, 17-2011-371 from the Juvenile Diabetes Research Foundation, the Boston Area Diabetes Endocrinology Research Center (to F.J.Q.), and PI14-1600 from the Spanish Ministry of Economy and Competitiveness (ISCIII) with FEDER cofunding (to D.P.). M.C.T. received support from an educational grant from Mallinckrodt Pharmaceuticals (A113307) and from a postdoctoral fellowship (0571-15-6) from Coordination for the Improvement of Higher Education Personnel, Brazil. I.D.M. received support from an educational grant from Questcor (A219074) and from a postdoctoral fellowship (FG 2036-A1/1) from the National Multiple Sclerosis Society. S.C.K. received support from The Helmsley Charitable Trust.

SummaryThe T cell receptor (TCR) controls the cellular adaptive immune response to antigens, but our understanding of TCR repertoire diversity and response to challenge is still incomplete. For example, TCR clones shared by different individuals with minimal alteration to germline gene sequences (public clones) are detectable in all vertebrates, but their significance is unknown. Although small in size, the zebrafish TCR repertoire is controlled by processes similar to those operating in mammals. Thus, we studied the zebrafish TCR repertoire and its response to stimulation with self and foreign antigens. We found that cross-reactive public TCRs dominate the T cell response, endowing a limited TCR repertoire with the ability to cope with diverse antigenic challenges. These features of vertebrate public TCRs might provide a mechanism for the rapid generation of protective T cell immunity, allowing a short temporal window for the development of more specific private T cell responses.

Objectives: For most adults with initial clinical presentation of multiple sclerosis (MS), biological disease was likely initiated many years prior. Pediatric-onset MS provides an opportunity to study early disease processes. Methods: Using antigen microarrays, including CNS-related proteins, lipids, and other autoantigens, we studied early immunologic events involved in clinical onset of pediatric MS. Serum samples were collected at the time of incident acquired CNS demyelinating syndromes (ADS) in children who, in subsequent prospective follow-up, were ascertained to have either pediatric MS (ADS-MS) or a monophasic illness (ADS-mono). Samples were obtained both at the time of ADS presentation and 3 months into follow-up. We used an initial training set of samples to implicate antibody signatures associated with each group, and then a test set. An additional set of follow-up samples (stability set) was used as a form of internal validation. Results: Children with ADS-MS tended to have distinguishable serum antibody patterns both at the time of ADS presentation and 3 months into follow-up. At the time of ADS, serum samples from patients with ADS-MS or ADS-mono reacted against similar numbers of CNS antigens, although CNS antigens implicated in adult MS were more often targeted in children with ADS-MS. The follow-up ADS-MS samples reacted against a broader panel of CNS antigens, while corresponding ADS-mono samples exhibited a contraction of the initial antibody response. Conclusions: Our findings in this prospective cohort of pediatric-onset CNS demyelinating diseases point to an active process of epitope spreading during early stages of MS, not seen in monophasic CNS inflammatory conditions.