Targeting aryl hydrocarbon receptor functionally restores tolerogenic dendritic cells derived from patients with multiple sclerosis.

Multiple sclerosis (MS) is a chronic disease characterized by dysregulated self-reactive immune responses that damage the neurons' myelin sheath, leading to progressive disability. The primary therapeutic option, immunosuppressants, inhibits pathogenic anti-myelin responses but depresses the immune system. Antigen-specific monocyte-derived autologous tolerogenic dendritic cells (tolDCs) offer alternative therapeutic approaches to restore tolerance to autoantigens without causing generalized immunosuppression. However, immune dysregulation in MS could impact the properties of the monocytes used as starting material for this cell therapy. Here, we characterized CD14+ monocytes, mature dendritic cells, and vitamin D3-tolDCs (VitD3-tolDCs) from active, treatment-naive MS patients and healthy donors (HDs). Using multiomics, we identified a switch in these cell types toward proinflammatory features characterized by alterations in the aryl hydrocarbon receptor (AhR) and NF-κB pathways. MS patient-derived VitD3-tolDCs showed reduced tolerogenic properties compared with those from HDs, which were fully restored through direct AhR agonism and by use of in vivo or in vitro dimethyl fumarate (DMF) supplementation. Additionally, in the experimental autoimmune encephalomyelitis mouse model, combined therapy of DMF and VitD3-tolDCs was more efficient than monotherapies in reducing the clinical score of mice. We propose that a combined therapy with DMF and VitD3-tolDCs offers enhanced therapeutic potential in treating MS.

Aryl hydrocarbon receptor (AhR)-mediated immune responses to degeneration of the retinal pigment epithelium.

Injuries to the retinal pigment epithelium (RPE) trigger immune responses, orchestrating interactions within the innate and adaptive immune systems in the outer retina and choroid. We previously reported that interleukin 17 (IL-17) is a pivotal signaling molecule originating from choroidal γδ T cells, exerting protective effects by mediating functional connections between the RPE and subretinal microglia. In this current study, we generated mice with aryl hydrocarbon receptor (AhR) knockout specifically in IL-17-producing cells. These animals had deficiency in IL-17 production from γδ T cells, and exhibited increased sensitivity to both acute and chronic insults targeting the RPE. These findings imply that IL-17 plays a crucial role as a signaling cytokine in preserving the homeostasis of the outer retina and choroid.

BHLHE40 Regulates Myeloid Cell Polarization through IL-10-Dependent and -Independent Mechanisms.

Better understanding of the host responses to Mycobacterium tuberculosis infections is required to prevent tuberculosis and develop new therapeutic interventions. The host transcription factor BHLHE40 is essential for controlling M. tuberculosis infection, in part by repressing Il10 expression, where excess IL-10 contributes to the early susceptibility of Bhlhe40-/- mice to M. tuberculosis infection. Deletion of Bhlhe40 in lung macrophages and dendritic cells is sufficient to increase the susceptibility of mice to M. tuberculosis infection, but how BHLHE40 impacts macrophage and dendritic cell responses to M. tuberculosis is unknown. In this study, we report that BHLHE40 is required in myeloid cells exposed to GM-CSF, an abundant cytokine in the lung, to promote the expression of genes associated with a proinflammatory state and better control of M. tuberculosis infection. Loss of Bhlhe40 expression in murine bone marrow-derived myeloid cells cultured in the presence of GM-CSF results in lower levels of proinflammatory associated signaling molecules IL-1ß, IL-6, IL-12, TNF-α, inducible NO synthase, IL-2, KC, and RANTES, as well as higher levels of the anti-inflammatory-associated molecules MCP-1 and IL-10 following exposure to heat-killed M. tuberculosis. Deletion of Il10 in Bhlhe40-/- myeloid cells restored some, but not all, proinflammatory signals, demonstrating that BHLHE40 promotes proinflammatory responses via both IL-10-dependent and -independent mechanisms. In addition, we show that macrophages and neutrophils within the lungs of M. tuberculosis-infected Bhlhe40-/- mice exhibit defects in inducible NO synthase production compared with infected wild-type mice, supporting that BHLHE40 promotes proinflammatory responses in innate immune cells, which may contribute to the essential role for BHLHE40 during M. tuberculosis infection in vivo.

ISGylation directly modifies hypoxia-inducible factor-2α and enhances its polysome association.

The hypoxia-inducible factors (HIF)-1α and HIF-2α are central regulators of transcriptional programmes in settings such as development and tumour expansion. HIF-2α moonlights as a cap-dependent translation factor. We provide new insights into how the interferon-stimulated gene 15 (ISG15), a ubiquitin-like modifier, and the HIFs regulate one another in hypoxia and interferon-induced cells. We show that upon ISGylation induction and HIF-α stabilization, both HIFs promote protein ISGylates through transcriptional and/or post-transcriptional pathways. We show the first evidence of HIF-2α modification by ISG15. ISGylation induces system-level alterations to the HIF transcriptional programme and increases the cytoplasmic/nuclear fraction and translation activity of HIF-2α. This work identifies ISG15 as a regulator of hypoxic mRNA translation, which has implications for immune processes and disease progression.

Regulation of the Signal-Dependent E Protein HEBAlt Through a YYY Motif Is Required for Progression Through T Cell Development.

The E protein transcription factors E2A and HEB are critical for many developmental processes, including T cell development. We have shown that the Tcf12 locus gives rise to two distinct HEB proteins, with alternative (HEBAlt) and canonical (HEBCan) N-terminal domains, which are co-expressed during early T cell development. While the functional domains of HEBCan have been well studied, the nature of the HEBAlt-specific (Alt) domain has been obscure. Here we provide compelling evidence that the Alt domain provides a site for the molecular integration of cytokine signaling and E protein activity. Our results indicate that phosphorylation of a unique YYY motif in the Alt domain increases HEBAlt activity by 10-fold, and that this increase is dependent on Janus kinase activity. To enable in vivo studies of HEBAlt in the T cell context, we generated ALT-Tg mice, which can be induced to express a HA-tagged HEBAlt coding cassette in the presence of Cre recombinases. Analysis of ALT-Tg mice on the Vav-iCre background revealed a minor change in the ratio of ISP cells to CD8+ SP cells, and a mild shift in the ratio of T cells to B cells in the spleen, but otherwise the thymus, spleen, and bone marrow lymphocyte subsets were comparable at steady state. However, kinetic analysis of T cell development in OP9-DL4 co-cultures revealed a delay in early T cell development and a partial block at the DN to DP transition when HEBAlt levels or activity were increased. We also observed that HEBCan and HEBAlt displayed significant differences in protein stability that were resolved in the thymocyte context. Finally, a proteomic screen identified STAT1 and Xpo1 as potential members of HEBAlt-containing complexes in thymocytes, consistent with JAK-induced activation of HEBAlt accompanied by translocation to the nucleus. Thus, our results show that the Alt domain confers access to multiple layers of post-translational control to HEBAlt that are not available to HEBCan, and thus may serve as a rheostat to tune E protein activity levels as cells move through different thymic signaling environments during T cell development.

Nanovesicles From Lactobacillus johnsonii N6.2 Reduce Apoptosis in Human Beta Cells by Promoting AHR Translocation and IL10 Secretion.

L. johnsonii N6.2 releases nano-sized vesicles (NVs) with distinct protein and lipid contents. We hypothesized that these NVs play a central role in the delivery of bioactive molecules that may act as mechanistic effectors in immune modulation. In this report, we observed that addition of NVs to the human pancreatic cell line ßlox5 reduced cytokine-induced apoptosis. Through RNAseq analyses, increased expression of CYP1A1, CYP1B1, AHRR, and TIPARP genes in the aryl hydrocarbon receptor (AHR) pathways were found to be significantly induced in presence of NVs. AHR nuclear translocation was confirmed by confocal microscopy. The role of NVs on beta cell function was further evaluated using primary human pancreatic islets. It was found that NVs significantly increased insulin secretion in presence of high glucose concentrations. These increases positively correlated with increased GLUT6 and SREBF1 mRNA and coincided with reduced oxidative stress markers. Furthermore, incubation of NVs with THP-1 macrophages promoted the M2 tolerogenic phenotype through STAT3 activation, expression of AHR-dependent genes and secretion of IL10. Altogether, our findings indicate that bacterial NVs have the potential to modulate glucose homeostasis in the host by directly affecting insulin secretion by islets and through the induction of a tolerogenic immune phenotype.

Role of differentiated embryo-chondrocyte expressed gene 1 (DEC1) in immunity.

Differentiated embryo-chondrocyte expressed gene 1 (DEC1) belongs to the family of basic helix-loop-helix (bHLH)-type transcription factors. DEC1 is expressed in various mammalian cells, but early studies focused on its roles outside the immune system. In recent years, relevant studies have found that DEC1 plays an important role in the immunotherapy of tumors, the functional regulation of the immune system, and the onset of autoimmune diseases. DEC1 promotes interferon (IFN)-γand granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion through the production of CD4+ T cells, which promotes inflammatory defense responses and autoimmune diseases. Additionally, DEC1 can inhibit the expression of interleukin (IL)-10 to further strengthen the immune response. In this review, we summarized recent advances in our understanding of the roles of DEC1 in animal models and human cells, including regulating immune cell differentiation, controlling cytokine production, and maintaining the balance of pro- and anti-inflammatory signals.

Recognition of Tumor-Associated Antigens and Immune Subtypes in Glioma for mRNA Vaccine Development.

Background: Although mRNA vaccines have been efficient for combating a variety of tumors, their effectiveness against glioma remains unclear. There is growing evidence that immunophenotyping can reflect the comprehensive immune status and microenvironment of the tumor, which correlates closely with treatment response and vaccination potency. The purpose of this research was to screen for effective antigens in glioma that could be used for developing mRNA vaccines and to further differentiate the immune subtypes of glioma to create an selection criteria for suitable patients for vaccination. Methods: Gene expression profiles and clinical data of 698 glioma samples were extracted from The Cancer Genome Atlas, and RNA_seq data of 1018 glioma samples was gathered from Chinese Glioma Genome Atlas. Gene Expression Profiling Interactive Analysis was used to determine differential expression genes and prognostic markers, cBioPortal software was used to verify gene alterations, and Tumor Immune Estimation Resource was used to investigate the relationships among genes and immune infiltrating cells. Consistency clustering was applied for consistent matrix construction and data aggregation, Gene oncology enrichment was performed for functional annotation, and a graph learning-based dimensionality reduction method was applied to describe the subtypes of immunity. Results: Four overexpressed and mutated tumor antigens associated with poor prognosis and infiltration of antigen presenting cells were identified in glioma, including TP53, IDH1, C3, and TCF12. Besides, four immune subtypes of glioma (IS1-IS4) and 10 immune gene modules were identified consistently in the TCGA data. The immune subtypes had diverse molecular, cellular, and clinical features. IS1 and IS4 displayed an immune-activating phenotype and were associated with worse survival than the other two subtypes, while IS2 and IS3 had lower levels of tumor immune infiltration. Immunogenic cell death regulators and immune checkpoints were also diversely expressed in the four immune subtypes. Conclusion: TP53, IDH1, C3, and TCF12 are effective antigens for the development of anti-glioma mRNA vaccines. We found four stable and repeatable immune subtypes of human glioma, the classification of the immune subtypes of glioma may play a crucial role in the predicting mRNA vaccine outcome.

Mist1 promoted inflammation in colitis model via K+-ATPase NLRP3 inflammasome by SNAI1.

Ulcerative colitis (UC) is a chronic inflammatory intestinal disease. Genetic susceptibility, gut microbiota and mucosal immune dysfunction play important roles in the pathogenesis and development of UC. We investigate the effect of Mist1 in model of colitis and its underlying mechanism. The expressions of Mist1 in patients with colitis tissue were up-regulated. Meanwhile, Mist1 mRNA and protein expressions in DSS-induced colitis mice model were also induced and Mist1 mRNA and protein expressions of LPS induced THP-1 cell were also up-regulated. we found Mist1 human protein promoted inflammation in DSS-induced colitis mice by NLRP3. So, we up-regulated Mist1 expression and over-expression of Mist1 promoted IL-1ß and NLRP3 protein expression levels in vitro model. However, down-regulation of Mist1 suppressed IL-1ß and NLRP3 protein expression levels in vitro model. Next, SNAI1 is a shooting point of Mist1 in the effects of Mist1 in colitis. The inhibition of SNAI1 reduced the effects of Mist1 on NLRP3 inflammasome in vitro model. Activation of SNAI1 induced the effects of Mist1 on NLRP3 inflammasome in vitro model. Lastly, anti-SNAI1 human protein lowered the effects of Mist1 human protein on NLRP3 inflammasome in DSS-induced colitis mice. We demonstrated that Mist1 promoted inflammation in colitis model via NLRP3 inflammasome by SNAI1, whereas the absence of these macrophages led to a significant improvement in colitis treatment.

Herbal Plants: The Role of AhR in Mediating Immunomodulation.

The prevalence of chronic inflammatory diseases including inflammatory bowel disease (IBD), autoimmunity and cancer have increased in recent years. Herbal-based compounds such as flavonoids have been demonstrated to contribute to the modulation of these diseases although understanding their mechanism of action remains limited. Flavonoids are able to interact with cellular immune components in a distinct way and influence immune responses at a molecular level. In this mini review, we highlight recent progress in our understanding of the modulation of immune responses by the aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor whose activity can be regulated by diverse molecules including flavonoids. We focus on the role of AhR in integrating signals from flavonoids to modulate inflammatory responses using in vitro and experimental animal models. We also summarize the limitations of these studies. Medicinal herbs have been widely used to treat inflammatory disorders and may offer a valuable therapeutic strategy to treat aberrant inflammatory responses by modulation of the AhR pathway.

A Comprehensive Pan-Cancer Analysis of 33 Human Cancers Reveals the Immunotherapeutic Value of Aryl Hydrocarbon Receptor.

Background: Previous studies have reported the potential of aryl hydrocarbon receptor (AhR) in cancer immunotherapy. However, the mechanisms underpinning its therapeutic value have yet to be comprehensively investigated. Thus, this research aimed to explore the underlying association between AhR and cancer immunotherapy in 33 human cancers. Methods: The gene expression data and clinical characteristics of 33 cancers were retrieved from The Cancer Genome Atlas database. The immunotherapeutic cohorts included GSE67501 and GSE78220 as well as IMvigor210, which were obtained from the Gene Expression Omnibus database and included in a previously published study respectively. Clinical parameters, including patient age, gender, survival, and tumor stage were analyzed to assess the prognostic value of AhR. The activity of AhR was generated by single sample gene set enrichment analysis and used to evaluate the difference between the AhR transcriptome and protein expression level. To better understand the role of AhR in cancer immunotherapy, the correlation between AhR and tumor microenvironment, as well as its relation to immune processes/elements, such as immune cell infiltration, immune inhibitors and stimulators, and the major histocompatibility complex were analyzed. The relevant underlying pathways associated with AhR signaling in cancer were also explored. Furthermore, the correlation between AhR and two immunotherapeutic biomarkers (tumor mutational burden and microsatellite instability) was investigated. Finally, the relationship between AhR and immunotherapeutic response was explored using three independent immunotherapeutic cohorts. Results: Although AhR was not closely associated with age (5/33), gender (3/33), or tumor stage (3/21) in any of the studied human cancers, it exhibited potential prognostic value for predicting patient survival. Consistency has been observed between AhR activity and expression in some cancers (7/33). Generally, AhR presented a robust correlation with immune cell infiltration, immune modulators, and immunotherapeutic markers. Moreover, high AhR expression was significantly related to immune-relevant pathways. However, no significant correlation was observed between AhR and the immunotherapeutic response. Conclusions: This research investigated the immunotherapeutic value of AhR in 33 human cancers, providing evidence regarding the function of AhR and its role in clinical treatment. However, considering that a bioinformatics approach was adopted, the current results are preliminary and require further validation.

Modulation of Immune Responses by Nutritional Ligands of Aryl Hydrocarbon Receptor.

Accumulating evidence indicates that nutrition can modulate the immune system through metabolites, either produced by host digestion or by microbiota metabolism. In this review, we focus on dietary metabolites that are agonists of the Aryl hydrocarbon Receptor (AhR). AhR is a ligand-activated transcription factor, initially characterized for its interaction with xenobiotic pollutants. Numerous studies have shown that AhR also recognizes indoles and tryptophan catabolites originating from dietary compounds and commensal bacteria. Here, we review recent work employing diet manipulation to address the impact of nutritional AhR agonists on immune responses, both locally in the intestine and at distant sites. In particular, we examine the physiological role of these metabolites in immune cell development and functions (including T lymphocytes, innate-like lymphoid cells, and mononuclear phagocytes) and their effect in inflammatory disorders.

Benzo(a)pyrene Enhanced Dermatophagoides Group 1 (Der f 1)-Induced TGFß1 Signaling Activation Through the Aryl Hydrocarbon Receptor-RhoA Axis in Asthma.

We have previously demonstrated that benzo(a)pyrene (BaP) co-exposure with dermatophagoides group 1 allergen (Der f 1) can potentiate Der f 1-induced airway inflammation. The underlying mechanism, however, remains undetermined. Here we investigated the molecular mechanisms underlying the potentiation of BaP exposure on Der f 1-induced airway inflammation in asthma. We found that BaP co-exposure potentiated Der f 1-induced TGFß1 secretion and signaling activation in human bronchial epithelial cells (HBECs) and the airways of asthma mouse model. Moreover, BaP exposure alone or co-exposure with Der f 1-induced aryl hydrocarbon receptor (AhR) activity was determined by using an AhR-dioxin-responsive element reporter plasmid. The BaP and Der f 1 co-exposure-induced TGFß1 expression and signaling activation were attenuated by either AhR antagonist CH223191 or AhR knockdown in HBECs. Furthermore, AhR knockdown led to the reduction of BaP and Der f 1 co-exposure-induced active RhoA. Inhibition of RhoA signaling with fasudil, a RhoA/ROCK inhibitor, suppressed BaP and Der f 1 co-exposure-induced TGFß1 expression and signaling activation. This was further confirmed in HBECs expressing constitutively active RhoA (RhoA-L63) or dominant-negative RhoA (RhoA-N19). Luciferase reporter assays showed prominently increased promoter activities for the AhR binding sites in the promoter region of RhoA. Inhibition of RhoA suppressed BaP and Der f 1 co-exposure-induced airway hyper-responsiveness, Th2-associated airway inflammation, and TGFß1 signaling activation in asthma. Our studies reveal a previously unidentified functional axis of AhR-RhoA in regulating TGFß1 expression and signaling activation, representing a potential therapeutic target for allergic asthma.

Hypoxia-inducible factor activity promotes antitumor effector function and tissue residency by CD8+ T cells.

Adoptive T cell therapies (ACTs) hold great promise in cancer treatment, but low overall response rates in patients with solid tumors underscore remaining challenges in realizing the potential of this cellular immunotherapy approach. Promoting CD8+ T cell adaptation to tissue residency represents an underutilized but promising strategy to improve tumor-infiltrating lymphocyte (TIL) function. Here, we report that deletion of the HIF negative regulator von Hippel-Lindau (VHL) in CD8+ T cells induced HIF-1α/HIF-2α-dependent differentiation of tissue-resident memory-like (Trm-like) TILs in mouse models of malignancy. VHL-deficient TILs accumulated in tumors and exhibited a core Trm signature despite an exhaustion-associated phenotype, which led to retained polyfunctionality and response to αPD-1 immunotherapy, resulting in tumor eradication and protective tissue-resident memory. VHL deficiency similarly facilitated enhanced accumulation of chimeric antigen receptor (CAR) T cells with a Trm-like phenotype in tumors. Thus, HIF activity in CD8+ TILs promotes accumulation and antitumor activity, providing a new strategy to enhance the efficacy of ACTs.

E2A-regulated epigenetic landscape promotes memory CD8 T cell differentiation.

During an acute viral infection, CD8 T cells encounter a myriad of antigenic and inflammatory signals of variable strength, which sets off individual T cells on their own differentiation trajectories. However, the developmental path for each of these cells will ultimately lead to one of only two potential outcomes after clearance of the infection-death or survival and development into memory CD8 T cells. How this cell fate decision is made remains incompletely understood. In this study, we explore the transcriptional changes during effector and memory CD8 T cell differentiation at the single-cell level. Using single-cell, transcriptome-derived gene regulatory network analysis, we identified two main groups of regulons that govern this differentiation process. These regulons function in concert with changes in the enhancer landscape to confer the establishment of the regulatory modules underlying the cell fate decision of CD8 T cells. Furthermore, we found that memory precursor effector cells maintain chromatin accessibility at enhancers for key memory-related genes and that these enhancers are highly enriched for E2A binding sites. Finally, we show that E2A directly regulates accessibility of enhancers of many memory-related genes and that its overexpression increases the frequency of memory precursor effector cells and accelerates memory cell formation while decreasing the frequency of short-lived effector cells. Overall, our results suggest that effector and memory CD8 T cell differentiation is largely regulated by two transcriptional circuits, with E2A serving as an important epigenetic regulator of the memory circuit.

Targeted inhibition of EPAS1-driven IL-31 production by a small-molecule compound.

BACKGROUND: IL-31 is a major pruritogen associated with atopic dermatitis (AD). Although a specific antibody for IL-31 receptor has been shown to alleviate pruritus in patients with AD, therapeutic approaches to inhibition of IL-31 production remain unexploited. IL-31 production by TH cells critically depends on the transcription factor EPAS1, which mediates IL31 promoter activation in collaboration with SP1. OBJECTIVE: We aimed at developing small-molecule inhibitors that selectively block IL-31 production by TH cells. METHODS: We generated the reporter cell line that inducibly expressed EPAS1 in the presence of doxycycline to mediate Il31 promoter activation, and we screened 9600 chemical compounds. The selected compounds were further examined by using TH cells from a spontaneous mouse model of AD and TH cells from patients with AD. RESULTS: We have identified 4-(2-(4-isopropylbenzylidene)hydrazineyl)benzoic acid (IPHBA) as an inhibitor of IL31 induction. Although IPHBA did not affect nonspecific T-cell proliferation, IPHBA inhibited antigen-induced IL-31 production by TH cells from both an AD mouse model and patients with AD without affecting other cytokine production and hypoxic responses. In line with this, itch responses induced by adoptive transfer of IL-31-producing TH cells were attenuated when mice were orally treated with IPHBA. Mechanistically, IPHBA inhibited the association between EPAS1 and SP1, resulting in defective recruitment of both transcription factors to the specific sites of the IL31 promoter. We also determined the structure-activity relationship of IPHBA by synthesizing and analyzing 201 analogous compounds. CONCLUSION: IPHBA could be a potential drug leading to inhibition of EPAS1-driven IL-31 production.

Trajectory Shifts in Interdisciplinary Research of the Aryl Hydrocarbon Receptor-A Personal Perspective on Thymus and Skin.

Identifying historical trajectories is a useful exercise in research, as it helps clarify important, perhaps even "paradigmatic", shifts in thinking and moving forward in science. In this review, the development of research regarding the role of the transcription factor "aryl hydrocarbon receptor" (AHR) as a mediator of the toxicity of environmental pollution towards a link between the environment and a healthy adaptive response of the immune system and the skin is discussed. From this fascinating development, the opportunities for targeting the AHR in the therapy of many diseases become clear.

Aryl Hydrocarbon Receptor is Involved in the Proinflammatory Cytokine Response to Cadmium.

OBJECTIVE: To investigate involvement of the aryl hydrocarbon receptor (AhR) in the immunomodulatory effects of cadmium (Cd). METHODS: The effect of Cd on AhR activation ( CYP1A1 and CYP1B1 mRNA expression) was examined in lung leukocytes of Cd-exposed rats (5 and 50 mg/L, 30 d orally) and by in vitro leukocyte exposure. The involvement of AhR signaling in the effects of Cd on the interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF) lung leukocyte response was investigated in vitro using the receptor antagonist CH-223191. RESULTS: Cd increased CYP1B1 ( in vivo and in vitro) and CYP1A1 ( in vitro) mRNA, indicating AhR involvement in the action of Cd. In response to Cd, lung leukocytes increased IL-6 and decreased TNF at the gene expression and protein levels, but decreased IL-1ß production due to reduced NLRP3. The AhR antagonist CH-223191 abrogated the observed effects of Cd on the cytokine response. The absence of AhR reactivity and cytokine response to Cd of leukocytes from the lungs of a rat strain that is less sensitive to Cd toxicity coincided with a high AhR repressor mRNA level. CONCLUSION: AhR signaling is involved in the lung leukocyte proinflammatory cytokine response to Cd. The relevance of the AhR to the cytokine response to Cd provides new insight into the mechanisms of Cd immunotoxicity.