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Obesity is associated with increased cancer risk and weak responses to vaccination and sepsis treatment. Although dendritic cells (DCs) are fundamental for the initiation and maintenance of competent immune responses against pathogens and tumors, how obesity alters the normal physiology of these myeloid cells remains largely unexplored. In this study, we report that obesity caused by prolonged high-fat diet feeding disrupts the metabolic and functional status of mouse splenic DCs (SpDCs). High-fat diet–induced obesity drastically altered the global transcriptional profile of SpDCs, causing severe changes in the expression of gene programs implicated in lipid metabolism and mitochondrial function. SpDCs isolated from obese mice demonstrated enhanced mitochondrial respiration provoked by increased fatty acid oxidation (FAO), which drove the intracellular accumulation of reactive oxygen species that impaired Ag presentation to T cells. Accordingly, treatment with the FAO inhibitor etomoxir, or antioxidants such as vitamin E or N-acetyl-l-cysteine, restored the Ag-presenting capacity of SpDCs isolated from obese mice. Our findings reveal a major detrimental effect of obesity in DC physiology and suggest that controlling mitochondrial FAO or reactive oxygen species overproduction may help improve DC function in obese individuals.

Lysophosphatidic acid (LPA) is a bioactive lipid enriched in the tumor microenvironment of immunosuppressive malignancies such as ovarian cancer. Although LPA enhances the tumorigenic attributes of cancer cells, the immunomodulatory activity of this phospholipid messenger remains largely unexplored. Here, we report that LPA operates as a negative regulator of type I interferon (IFN) responses in ovarian cancer. Ablation of the LPA-generating enzyme autotaxin (ATX) in ovarian cancer cells reprogrammed the tumor immune microenvironment, extended host survival, and improved the effects of therapies that elicit protective responses driven by type I IFN. Mechanistically, LPA sensing by dendritic cells triggered PGE2 biosynthesis that suppressed type I IFN signaling via autocrine EP4 engagement. Moreover, we identified an LPA-controlled, immune-derived gene signature associated with poor responses to combined PARP inhibition and PD-1 blockade in patients with ovarian cancer. Controlling LPA production or sensing in tumors may therefore be useful to improve cancer immunotherapies that rely on robust induction of type I IFN.Significance:This study uncovers that ATX–LPA is a central immunosuppressive pathway in the ovarian tumor microenvironment. Ablating this axis sensitizes ovarian cancer hosts to various immunotherapies by unleashing protective type I IFN responses. Understanding the immunoregulatory programs induced by LPA could lead to new biomarkers predicting resistance to immunotherapy in patients with cancer.See related commentary by Conejo-Garcia and Curiel, p. 1841.This article is highlighted in the In This Issue feature, p. 1825

Supplementary Figure from Tumor-Derived Lysophosphatidic Acid Blunts Protective Type I Interferon Responses in Ovarian Cancer

Recognition of pathogen-associated molecular patterns can trigger the IRE1α arm of the endoplasmic reticulum (ER) stress response in immune cells. IRE1α activation has been shown to maintain ER homeostasis while simultaneously coordinating diverse immunomodulatory programs in the setting of bacterial and viral infections. However, the role of IRE1α signaling in innate immune responses to fungal pathogens is unknown. Here we report that systemic infection with the fungusCandida albicanscauses severe renal immunopathology by triggering inflammatory IRE1α hyperactivation in host myeloid cells. Mechanistically, sensing of fungal β-glucans by the C-type lectin receptor Dectin-1 induced Src–Syk–NOX-dependent accumulation of intracellular reactive oxygen species and the ensuing generation of lipid peroxidation byproducts that sustained IRE1α activation. Selective deletion of IRE1α in leukocytes, or treatment with an IRE1α pharmacological inhibitor, reduced detrimental inflammatory responses in the kidney and extended survival in mice systemically infected withC. albicans. Hence, controlling IRE1α overactivation may be useful to impede the fatal immunopathogenic progression of disseminated candidiasis.One sentence summaryInnate IRE1α signaling in disseminated candidiasis

Lysophosphatidic acid (LPA) is a bioactive lipid enriched in the tumor microenvironment of immunosuppressive malignancies such as ovarian cancer. Although LPA enhances the tumorigenic attributes of cancer cells, the immunomodulatory activity of this phospholipid messenger remains largely unexplored. Here, we report that LPA operates as a negative regulator of type I interferon (IFN) responses in ovarian cancer. Ablation of the LPA-generating enzyme autotaxin (ATX) in ovarian cancer cells reprogrammed the tumor immune microenvironment, extended host survival, and improved the effects of therapies that elicit protective responses driven by type I IFN. Mechanistically, LPA sensing by dendritic cells triggered PGE2 biosynthesis that suppressed type I IFN signaling via autocrine EP4 engagement. Moreover, we identified an LPA-controlled, immune-derived gene signature associated with poor responses to combined PARP inhibition and PD-1 blockade in patients with ovarian cancer. Controlling LPA production or sensing in tumors may therefore be useful to improve cancer immunotherapies that rely on robust induction of type I IFN. Significance: This study uncovers that ATX–LPA is a central immunosuppressive pathway in the ovarian tumor microenvironment. Ablating this axis sensitizes ovarian cancer hosts to various immunotherapies by unleashing protective type I IFN responses. Understanding the immunoregulatory programs induced by LPA could lead to new biomarkers predicting resistance to immunotherapy in patients with cancer. See related commentary by Conejo-Garcia and Curiel, p. 1841. This article is highlighted in the In This Issue feature, p. 1825

The transcription factor NFAT1 plays a pivotal role in the homeostasis of T lymphocytes. However, its functional importance in non-CD4+ T cells, especially in systemic immune disorders, is largely unknown. In this study, we report that NFAT1 regulates dendritic cell (DC) tolerance and suppresses systemic autoimmunity using the experimental autoimmune myasthenia gravis (EAMG) as a model. Myasthenia gravis and EAMG are T cell–dependent, Ab-mediated autoimmune disorders in which the acetylcholine receptor is the major autoantigen. NFAT1-knockout mice showed higher susceptibility to EAMG development with enhanced Th1/Th17 cell responses. NFAT1 deficiency led to a phenotypic alteration of DCs that show hyperactivation of NF-κB–mediated signaling pathways and enhanced binding of NF-κB (p50) to the promoters of IL-6 and IL-12. As a result, NFAT1-knockout DCs produced much higher levels of proinflammatory cytokines such as IL-1β, IL-6, IL-12, and TNF-α, which preferentially induce Th1/Th17 cell differentiation. Our data suggest that NFAT1 may limit the hyperactivation of the NF-κB–mediated proinflammatory response in DCs and suppress autoimmunity by serving as a key regulator of DC tolerance.

Inositol-requiring enzyme 1[α] (IRE1[α])–X-box binding protein spliced (XBP1) signaling maintains endoplasmic reticulum (ER) homeostasis while controlling immunometabolic processes. Yet, the physiological consequences of IRE1a–XBP1 activation in leukocytes remain unexplored. We found that induction of prostaglandin-endoperoxide synthase 2 (Ptgs2/Cox-2) and prostaglandin E synthase (Ptges/mPGES-1) was compromised in IRE1a-deficient myeloid cells undergoing ER stress or stimulated through pattern recognition receptors. Inducible biosynthesis of prostaglandins, including the pro-algesic mediator prostaglandin E2 (PGE2), was decreased in myeloid cells that lack IRE1a or XBP1 but not other ER stress sensors. Functional XBP1 transactivated the human PTGS2 and PTGES genes to enable optimal PGE2 production. Mice that lack IRE1α–XBP1 in leukocytes, or that were treated with IRE1a inhibitors, demonstrated reduced pain behaviors in PGE2-dependent models of pain. Thus, IRE1a–XBP1 is a mediator of prostaglandin biosynthesis and a potential target to control pain.

Although probiotics could confer health benefits to host physiology, their efficacy is strain specific. To identify anti-inflammatory probiotics, an ex vivo screening system was developed, and validated effector functions of selected candidates using experimental rheumatoid arthritis as an in vivo model. Lactobacillus helveticus HY7801 was selected as the best candidate. Oral administration of L. helveticus HY7801 prevented the development of collagen-induced experimental arthritis, and diminished disease progression and severity by reducing antigen specific IgG levels and inflammatory immune response. Administration of L. helveticus HY7801 may induce regulatory CD11c+ dendritic cells, which in turn induce a phenotypic alteration of immune cells by reducing pro-inflammatory cytokines (TNF-α, IFN-γ, and IL-17A) while enhancing anti-inflammatory cytokine (IL-10) by CD4+ T cells. This study suggests that screening of probiotic strains based on the IL-10highIL-12low selection criterion may be applicable to identify anti-inflammatory probiotics as an efficacious food for treating inflammatory immune responses including rheumatoid arthritis.

IL-31 is a key mediator of itching in atopic dermatitis (AD) and is preferentially produced by activated CD4+ T cells and Th2 cells. Although pathophysiological functions of IL-31 have been suggested in diverse immune disorders, the molecular events underlying IL-31 gene regulation are still unclear. In this study we identified the transcription start site and functional promoter involved in IL-31 gene regulation in mouse CD4+ T cells. TCR stimulation–dependent IL-31 expression was found to be closely linked with in vivo binding of NFAT1 and JunB to the IL-31 promoter. Although NFAT1 alone enhanced IL-31 promoter activity, it was further enhanced in the presence of JunB. Conversely, knockdown of either NFAT1 or JunB resulted in reduced IL-31 expression. NFAT1-deficient CD4+ T cells showed a significant defect in IL-31 expression compared with wild-type CD4+ T cells. In agreement with these findings, mice subjected to atopic conditions showed much higher levels of IL-31, which were closely correlated with a significant increase in the number of infiltrated NFAT1+CD4+ T cells into the AD ears. Amelioration of AD progression by cyclosporin A treatment was well correlated with downregulation of IL-31 expressions in CD4+ T cells and total ear residual cells. In summary, our results suggest a functional cooperation between NFAT1 and JunB in mediating IL-31 gene expression in CD4+ T cells and indicate that interference with this interaction or their activity has the potential of reducing IL-31–mediated AD symptoms.

Tumours evade immune control by creating hostile microenvironments that perturb T cell metabolism and effector function 1?4 . However, it remains unclear how intra-tumoral T cells integrate and interpret metabolic stress signals. Here we report that ovarian cancer?an aggressive malignancy that is refractory to standard treatments and current immunotherapies 5?8 ?induces endoplasmic reticulum stress and activates the IRE1α?XBP1 arm of the unfolded protein response 9,10 in T cells to control their mitochondrial respiration and anti-tumour function. In T cells isolated from specimens collected from patients with ovarian cancer, upregulation of XBP1 was associated with decreased infiltration of T cells into tumours and with reduced IFNG mRNA expression. Malignant ascites fluid obtained from patients with ovarian cancer inhibited glucose uptake and caused N-linked protein glycosylation defects in T cells, which triggered IRE1α?XBP1 activation that suppressed mitochondrial activity and IFNγ production. Mechanistically, induction of XBP1 regulated the abundance of glutamine carriers and thus limited the influx of glutamine that is necessary to sustain mitochondrial respiration in T cells under glucose-deprived conditions. Restoring N-linked protein glycosylation, abrogating IRE1α?XBP1 activation or enforcing expression of glutamine transporters enhanced mitochondrial respiration in human T cells exposed to ovarian cancer ascites. XBP1-deficient T cells in the metastatic ovarian cancer milieu exhibited global transcriptional reprogramming and improved effector capacity. Accordingly, mice that bear ovarian cancer and lack XBP1 selectively in T cells demonstrate superior anti-tumour immunity, delayed malignant progression and increased overall survival. Controlling endoplasmic reticulum stress or targeting IRE1α?XBP1 signalling may help to restore the metabolic fitness and anti-tumour capacity of T cells in cancer hosts. Fil: Song, Minkyung. Weill Cornell Medicine; Estados Unidos Fil: Sandoval, Tito A.. Weill Cornell Medicine; Estados Unidos Fil: Chae, Chang-Suk. Weill Cornell Medicine; Estados Unidos Fil: Chopra, Sahil. Weill Cornell Medicine; Estados Unidos Fil: Tan, Chen. Weill Cornell Medicine; Estados Unidos Fil: Rutkowski, Melanie R.. University of Virginia; Estados Unidos Fil: Raundhal, Mahesh. Dana Farber Cancer Institute; Estados Unidos. Harvard Medical School; Estados Unidos Fil: Chaurio, Ricardo A.. H. Lee Moffitt Cancer Center & Research Institute; Estados Unidos Fil: Payne, Kyle K.. H. Lee Moffitt Cancer Center & Research Institute; Estados Unidos Fil: Konrad, Csaba. Weill Cornell Medicine; Estados Unidos Fil: Bettigole, Sarah E.. Quentis Therapeutics Inc.; Estados Unidos Fil: Shin, Hee Rae. Quentis Therapeutics Inc.; Estados Unidos Fil: Crowley, Michael J. P.. Weill Cornell Graduate School of Medical Sciences; Estados Unidos Fil: Cerliani, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Kossenkov, Andrew V.. The Wistar Institute; Estados Unidos Fil: Motorykin, Ievgen. Weill Cornell Medicine,; Estados Unidos Fil: Zhang, Sheng. Weill Cornell Medicine,; Estados Unidos Fil: Manfredi, Giovanni. Weill Cornell Medicine,; Estados Unidos Fil: Zamarin, Dmitriy. Memorial Sloan Kettering Cancer Center; Estados Unidos Fil: Holcomb, Kevin. Weill Cornell Medicine,; Estados Unidos Fil: Rodriguez, Paulo C.. H. Lee Moffitt Cancer Center & Research Institute; Estados Unidos Fil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Conejo Garcia, Jose R.. H. Lee Moffitt Cancer Center & Research Institute; Estados Unidos Fil: Glimcher, Laurie H.. Dana Farber Cancer Institute; Estados Unidos. Harvard Medical School; Estados Unidos Fil: Cubillos-Ruiz, Juan R.. Weill Graduate School Of Medical Sciences; Estados Unidos. Weill Graduate School Of Medical Sciences; Estados Unidos

Harnessing the intrinsic ability of our immune system to recognize and eliminate malignanT-cells represents the most promising anticancer strategy since the development of chemotherapy. However, hostile microenvironmental conditions within aggressive solid tumors inhibit the optimal activity of protective immune cells. Targeting immunosuppression and re-programming immune cell function in the tumor microenvironment are thus fundamental requirements for developing successful cancer immunotherapies. Our CRI postdoctoral project aims at identifying, understanding and disabling the molecular mechanisms by which endoplasmic reticulum (ER) stress responses inhibit the natural function of dendritic cells (DCs) in the ovarian cancer microenvironment. The central hypothesis of this study is that ovarian tumors trigger ER stress and aberrant activation of the IRE1-XBP1 pathway in infiltrating DCs to cripple key immuno-metabolic processes and impede the development of protective T-cell responses. To determine how IRE1α-XBP1 overactivation defines regulatory DC phenotypes in the ovarian cancer microenvironment, metastatic ovarian tumors were developed in ER stress-Activated Indicator (ERAI) reporter mice. We found that DCs demonstrating IRE1 activation in the tumor microenvironment overexpress tolerogenic and immunosuppressive molecules. Next, to determine whether tumor-derived factors may affect DC metabolism, we optimized an ex vivo culture system that recreates the tumor microenvironment using malignant ascites samples from ovarian cancer patients. We treated human monocyte-derived DCs from healthy donors with patient-derived ovarian cancer malignant ascites supernatants and assessed the bioenergetic profile of DCs. Oxygen consumption rate (OCR), which measures mitochondrial respiration, ATP production and spare respiratory capacity, was increased in DCs exposed to ascites supernatants. We found that these metabolic changes upon ascites treatment relied on IRE1a-XBP1 pathway. Further, since therapeutic DC-based vaccines have shown limited effects in ovarian cancer patients, we tested the novel translational hypothesis that XBP1-deficient bone marrow-derived DCs (BMDCs) would be better equipped to endure and function in the tumor microenvironment when used as therapeutic vaccines. While adoptive transfer of WT BMDCs did not induce any therapeutic effect, treatment with XBP1-deficient BMDCs elicited a marked increase in overall host survival. This result suggest that compared with WT BMDCs, their XBP1-deficient counterparts were resistant to the immunosuppressive effects of the tumor microenvironment. Our results provide a unique mechanistic rationale for targeting the IRE1-XBP1 arm of the ER stress response as a potent approach to reprogram and enhance antitumor immune cell function in cancer. These findings should also pave the way for devising a new generation of cancer immunotherapies that may improve the dismal survival of >21,000 American women affected by ovarian cancer each year. Citation Format: Chang-Suk Chae. Incessant ER stress responses promote dendritic cell dysfunction in ovarian cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A056.

Immune-based therapies that induce remarkable and durable responses against melanoma and lung cancer have unfortunately demonstrated limited success in ovarian cancer patients. This is likely due to the exceptional immunoregulatory nature of ovarian tumors, which employ numerous strategies to effectively suppress anti-tumor immunity. Here, we summarize a decade of research indicating that ovarian cancers possess an exquisite capacity to subvert the activity of host dendritic cells (DCs) as a key mechanism to impede the development and maintenance of protective T cell-based immune responses. Identifying, understanding, and disabling the precise mechanisms promoting DC dysfunction in ovarian cancer are, therefore, fundamental requirements for devising the next generation of successful immunotherapies against this devastating malignancy.

Allergic contact hypersensitivity (CHS) is an inflammatory skin disease mediated by allergen specific T cells. In this study, we investigated the role of transcription factor NFAT1 in the pathogenesis of contact hypersensitivity. NFAT1 knock out (KO) mice spontaneously developed CHS-like skin inflammation in old age. Healthy young NFAT1 KO mice displayed enhanced susceptibility to hapten-induced CHS. Both CD4+ and CD8+ T cells from NFAT1 KO mice displayed hyper-activated properties and produced significantly enhanced levels of inflammatory T helper 1(Th1)/Th17 type cytokines. NFAT1 KO T cells were more resistant to activation induced cell death (AICD) and regulatory T cells derived from these mice showed a partial defect in their suppressor activity. NFAT1 KO T cells displayed a reduced expression of apoptosis associated BCL-2/BH3 family members. Ectopic expression of NFAT1 restored the AICD defect in NFAT1 KO T cells and increased AICD in normal T cells. Recipient Rag2−/− mice transferred with NFAT1 KO T cells showed more severe CHS sensitivity due to a defect in activation induced hapten-reactive T cell apoptosis. Collectively, our results suggest the NFAT1 plays a pivotal role as a genetic switch in CD4+/CD8+ T cell tolerance by regulating AICD process in the T cell mediated skin inflammation.

IL-9 regulates diverse inflammatory immune responses. Although the functional importance of IL-9 has been investigated in various pathophysiological conditions, molecular mechanisms by which TCR stimulation induced IL-9 gene expression are still unclear. In this study, we investigated the functional importance of the NFAT1 and NF-κB (p65) in IL-9 gene transcription in CD4(+) T cells. In vivo binding of NFAT1 and NF-κB (p65) to the IL-9 promoter was observed. NFAT1 binding induced a transcriptionally active chromatin configuration at the IL-9 promoter locus, whereas NF-κB (p65) binding transactivated the IL-9 promoter. Mouse deficient in NFAT1 shows a significant down-regulation of IL-9 expression that resulted from an inaccessible chromatin configuration at the IL-9 promoter. In parallel, knockdown of NF-κB (p65) also resulted in reduced IL-9 expression. In this process, NFAT1 plays a pivotal role as a core protein that creates an accessible platform for the assembly of transcription activators. The presence of NFAT1 correlates with recruitment of NF-κB (p65), p300, and active histone markers on the IL-9 promoter, resulting in a transcriptionally competent promoter. NFAT1 deficiency significantly reduced the recruitment of the above activation complex to the IL-9 promoter. In summary, our data suggest that functional cooperation of NFAT1 and NF-κB synergistically enhances IL-9 transcription in CD4(+) T cells.

IL-10 is a multifunctional cytokine that plays a crucial role in immunity and tolerance. IL-10 is produced by diverse immune cell types, including B cells and subsets of T cells. Although Th1 produce IL-10, their expression levels are much lower than Th2 cells under conventional stimulation conditions. The potential role of E26 transformation-specific 1 (Ets-1) transcription factor as a negative regulator for Il10 gene expression in CD4+ T cells has been implicated previously. In this study, we investigated the underlying mechanism of Ets-1–mediated Il10 gene repression in Th1 cells. Compared with wild type Th1 cells, Ets-1 knockout Th1 cells expressed a significantly higher level of IL-10, which is comparable with that of wild type Th2 cells. Upregulation of IL-10 expression in Ets-1 knockout Th1 cells was accompanied by enhanced chromatin accessibility and increased recruitment of histone H3 acetylation at the Il10 regulatory regions. Reciprocally, Ets-1 deficiency significantly decreased histone deacetylase 1 (HDAC1) enrichment at the Il10 regulatory regions. Treatment with trichostatin A, an inhibitor of HDAC family, significantly increased Il10 gene expression by increasing histone H3 acetylation recruitment. We further demonstrated a physical interaction between Ets-1 and HDAC1. Coexpression of Ets-1 with HDAC1 synergistically repressed IL-10 transcription activity. In summary, our data suggest that an interaction of Ets-1 with HDAC1 represses the Il10 gene expression in Th1 cells.

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are antibody-mediated autoimmune diseases in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Previously we have revealed that oral treatment with the less native recombinant fragment of the extracellular domain of the human AChR (Halpha1-205) suppressed ongoing EAMG, whereas the more native recombinant Trx-Halpha1-210 exacerbated EAMG. In this study, we speculated on the role of B-cell epitopes in oral tolerogens for the induction of oral tolerance in EAMG. We developed a B-cell epitope-free AChR fragment (BF-AChR) by removing two major B-cell epitopes (67-76 and 129-145) from Trx-Halpha1-210. BF-AChR exhibited a poor response to EAMG sera and to AChR-specific B- and T-cells while its parent fragment, Trx-Halpha1-210, showed much higher reactivity. Oral administration of BF-AChR ameliorated the symptoms in ongoing myasthenic rats accompanied by a significant decrease in AChR-specific humoral and Th1 cellular responses. The underlying mechanism for BF-AChR-induced oral tolerance was mediated by a shift from Th1 to regulatory T-cell (IL-10(+), CD4(+) TGF-beta(+) or Foxp3(+)) responses. This shift was assessed by changes in the cytokine profile and a deviation in the anti-AChR IgG isotypes from IgG2a/IgG2b to IgG1. Our results suggest that the removal of pathogenic B-cell epitopes from AChR fragments increases tolerogenicity by reducing the activation and proliferation of autoreactive B- and T-cells. Collectively, careful consideration of the immunogenicity of a tolerogen is necessary to induce successful oral tolerance in autoimmune disorders.

Probiotics are live bacteria that confer health benefits to the host physiology. Although protective role of probiotics have been reported in diverse diseases, no information is available whether probiotics can modulate neuromuscular immune disorders. We have recently demonstrated that IRT5 probiotics, a mixture of 5 probiotics, could suppress diverse experimental disorders in mice model. In this study we further investigated whether IRT5 probiotics could modulate the progression of experimental autoimmune myasthenia gravis (EAMG). Myasthenia gravis (MG) is a T cell dependent antibody mediated autoimmune disorder in which acetylcholine receptor (AChR) at the neuromuscular junction is the major autoantigen. Oral administration of IRT5 probiotics significantly reduced clinical symptoms of EAMG such as weight loss, body trembling and grip strength. Prophylactic effect of IRT5 probiotics on EMAG is mediated by down-regulation of effector function of AChR-reactive T cells and B cells. Administration of IRT5 probiotics decreased AChR-reactive lymphocyte proliferation, anti-AChR reactive IgG levels and inflammatory cytokine levels such as IFN-gamma, TNF-alpha, IL-6 and IL-17. Downregulation of inflammatory mediators in AChR-reactive lymphocytes by IRT5 probiotics is mediated by the generation of regulatory dendritic cells (rDCs) that express increased levels of IL-10, TGF-beta, arginase 1 and aldh1a2. Furthermore, DCs isolated from IRT5 probiotics-fed group effectively converted CD4(+) T cells into CD4+ Foxp3(+) regulatory T cells compared with control DCs. Our data suggest that IRT5 probiotics could be applicable to modulate antibody mediated autoimmune diseases including myasthenia gravis.

The function and differentiation of induced regulatory T (iTreg) cells are tightly regulated by various signaling cascade. In this study, we have investigated the role of TCR signaling to induce Foxp3 gene expression in cooperation with TGF-β/IL-2 stimulation. Activation of CD4(+) T cells by TCR signaling or TGF-β/IL-2 alone failed to enhance Foxp3 expression. Only when TCR stimulation is coupled together with TGF-β/IL-2, CD4(+) T cells expressed high levels of Foxp3 by maintaining open chromatin structure around its promoter region. Under this condition, stimulation-dependent recruitment of JunB together with c-Rel enhanced Foxp3 expression. Over expression of JunB and c-Rel significantly enhanced Foxp3 promoter activity while treatment of JunB siRNA or inhibition of TCR signaling by MAPK inhibitors significantly reduced Foxp3 expression. Collectively our results suggest that TCR signaling together with TGF-β/IL-2 stimulation cooperatively enhance Foxp3 gene expression by maintaining accessible chromatin structure and by actively recruiting key transcription factors JunB and c-Rel.

Nuclear factor of activated T cells (NFAT) is a family of transcription factors composed of five proteins. Among them, NFAT1 is a predominant NFAT protein in CD4(+) T cells. NFAT1 positively regulates transcription of a large number of inducible cytokine genes including IL-2, IL-4, IL-5 and other cytokines. However, disruption of NFAT1 results in an unexpected increase of IL-4. In this study, we have investigated the role of NFAT1 in regulation of IL-4 gene expression in T helper 2 cells (Th2) from an epigenetic viewpoint. NFAT1 deficient Th2 cells showed a sustained IL-4 expression while wild type (WT) cells reduced its expression. We tested whether epigenetic maintenance and changes in the chromatin architecture of IL-4 promoter locus play a role in differential IL-4 transcription between in WT and NFAT1 deficient Th2 cells. Compared with WT, NFAT1 deficient CD4(+) Th2 cells exhibited enhanced chromatin accessibility with permissive histone modification and DNA demethylation in the IL-4 promoter region. Transcription factors bound to IL-4 promoter region in the absence of NFAT1 were identified by Micro-LC/LC-MS/MS analysis. Among the candidates, preferential recruitment of JUNB to the IL-4 promoter was confirmed by chromatin immunoprecipitation analysis. Overexpression of JUNB together with SATB1 synergistically upregulated IL-4 promoter activity, while knockdown JUNB significantly reduced IL-4 expression. Our results suggest that the prolonged IL-4 expression in NFAT1 deficient Th2 cells is mediated by preferential binding of JUNB/SATB1 to the IL-4 promoter with permissive chromatin architecture.

Aims We previously reported that Lactobacillus casei (L. casei) has beneficial effects in experimental rheumatoid arthritis (RA) by suppressing inflammatory immune responses. The major purpose of this study was to evaluate therapeutic effects of L. casei on pathological responses in experimental rodent model of osteoarthritis (OA). Main methods Experimental OA was induced by intra-articular injection of monosodium iodoacetate (MIA) in Wistar rats. L. casei alone or together with type II collagen (CII) and glucosamine (Gln) was orally administered into OA rats. The pathophysiological aspects of OA were investigated by analyzing mechanical hyperalgesia and histology of articular tissues. Expression of inflammatory molecules was analyzed in CD4+ T cells, synovial fibroblasts, and chondrocytes by quantitative real-time PCR. Key findings Oral administration of L. casei together with CII and Gln more effectively reduced pain, cartilage destruction, and lymphocyte infiltration than the treatment of Gln or L. casei alone. This co-administration also decreased expression of various pro-inflammatory cytokines (interleukin-1β (IL-1β), IL-2, IL-6, IL-12, IL-17, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)) and matrix metalloproteinases (MMP1, MMP3, and MMP13), while up-regulating anti-inflammatory cytokines (IL-4 and IL-10). These results are concomitant with reduced translocation of NF-κB into the nucleus and increased expression of the tissue inhibitor of MMP1 (TIMP1) and CII in chondrocytes. Significance Our study provides evidence that L. casei could act as a potent nutraceutical modulator for OA treatment by reducing pain, inflammatory responses, and articular cartilage degradation.

The beneficial effects of probiotics have been described in many diseases, but the mechanism by which they modulate the immune system is poorly understood. In this study, we identified a mixture of probiotics that up-regulates CD4 + Foxp3 + regulatory T cells (Tregs). Administration of the probiotics mixture induced both T-cell and B-cell hyporesponsiveness and down-regulated T helper (Th) 1, Th2, and Th17 cytokines without apoptosis induction. It also induced generation of CD4 + Foxp3 + Tregs from the CD4 + CD25 − population and increased the suppressor activity of naturally occurring CD4 + CD25 + Tregs. Conversion of T cells into Foxp3 + Tregs is directly mediated by regulatory dendritic cells (rDCs) that express high levels of IL-10, TGF-β, COX-2, and indoleamine 2,3-dioxygenase. Administration of probiotics had therapeutical effects in experimental inflammatory bowel disease, atopic dermatitis, and rheumatoid arthritis. The therapeutical effect of the probiotics is associated with enrichment of CD4 + Foxp3 + Tregs in the inflamed regions. Collectively, the administration of probiotics that enhance the generation of rDCs and Tregs represents an applicable treatment of inflammatory immune disorders.

Probiotics have been shown to exert beneficial effects on modulation of diverse diseases. However, no information is available for the effect of probiotics in the induction of oral tolerance in autoimmune diseases. The main purpose of this study was to elucidate whether Lactobacillus casei (L. casei) affect the induction of oral tolerance in experimental rheumatoid arthritis (RA). Type II collagen (CII) alone or together with L. casei was orally administered into collagen-induced arthritis (CIA) rats, and its effects on the clinical and histopathological aspects of RA were investigated. Co-administration of L. casei with CII more effectively suppressed clinical symptoms, paw swelling, lymphocyte infiltration and destruction of cartilage tissues of experimental arthritis than the rats treated with CII alone. The enhanced therapeutic efficacy was associated with an increase in anti-inflammatory cytokines (IL-10 and TGF-beta) while decreasing pro-inflammatory cytokines (IL-1beta, IL-2, IL-6, IL-12, IL-17, IFN-gamma and TNF-alpha). Co-administration of L. casei with CII more effectively suppressed CII-reactive T cell proliferation and the levels of Th1-type IgG isotypes (IgG2a and IgG2b), while up-regulating Foxp3 expression levels and the population of Foxp3(+) CD4(+) T cells. Our study provides evidence that L. casei could potentiate antigen-specific oral tolerance and suppress Th1-type immune responses of arthritic inflammation.

Hypoxia-inducible factor-2α (HIF-2α) is sufficient to cause experimental rheumatoid arthritis and acts to regulate the functions of fibroblast-like cells from tissue surrounding joints, independent of HIF-1α., Rheumatoid arthritis (RA) is a systemic autoimmune disorder that manifests as chronic inflammation and joint tissue destruction. However, the etiology and pathogenesis of RA have not been fully elucidated. Here, we explored the role of the hypoxia-inducible factors (HIFs), HIF-1α (encoded by HIF1A) and HIF-2α (encoded by EPAS1). HIF-2α was markedly up-regulated in the intimal lining of RA synovium, whereas HIF-1α was detected in a few cells in the sublining and deep layer of RA synovium. Overexpression of HIF-2α in joint tissues caused an RA-like phenotype, whereas HIF-1α did not affect joint architecture. Moreover, a HIF-2α deficiency in mice blunted the development of experimental RA. HIF-2α was expressed mainly in fibroblast-like synoviocytes (FLS) of RA synovium and regulated their proliferation, expression of RANKL (receptor activator of nuclear factor–κB ligand) and various catabolic factors, and osteoclastogenic potential. Moreover, HIF-2α–dependent up-regulation of interleukin (IL)-6 in FLS stimulated differentiation of TH17 cells—crucial effectors of RA pathogenesis. Additionally, in the absence of IL-6 (Il6 −/− mice), overexpression of HIF-2α in joint tissues did not cause an RA phenotype. Thus, our results collectively suggest that HIF-2α plays a pivotal role in the pathogenesis of RA by regulating FLS functions, independent of HIF-1α., Author Summary Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by chronic inflammation in joint tissues leading to destruction of cartilage and bone. Despite some therapeutic advances, the etiology of RA pathogenesis is not yet clear, and effective treatment of RA remains a significant, unmet medical need. Hypoxia is a prominent feature of inflamed tissue within RA-affected joints, and earlier work has implicated limited involvement of hypoxia-inducible factor (HIF)-1 α. We explored the role of a second HIF family member, HIF-2α, in RA pathogenesis. We showed that HIF-2α is markedly increased in the tissue lining the RA-affected joints. Notably and in contrast to HIF-1α, when overexpressed in normal mouse joint tissues, HIF-2α is sufficient to cause RA-like symptoms. Conversely, an HIF-2α deficiency blocks the development of experimental arthritis in mice. We discovered further that HIF-2α regulates RA pathogenesis by modulating various RA-associated functions of joint-specific fibroblast-like cells, including proliferation, expression of cytokines, chemokines, and matrix-degrading enzymes, and bone-remodeling potential. HIF-2α also increases the ability of these cells to promote interleukin-6–dependent differentiation of TH17 cells, a known effector of RA pathogenesis. We thus show that HIF-1α and HIF-2α have distinct roles and act via different mechanisms in RA pathogenesis.

Lee, C.-G., H.-K. Kwon, A. Sahoo, W. Hwang, J.-S. So, J.-S. Hwang, C.-S. Chae, G.-C. Kim, J.-E. Kim, H.-S. So, E. S. Hwang, R. Grenningloh, I-C. Ho, and S.-H. Im. 2012. Interaction of Ets-1 with HDAC1 represses IL-10 expression in Th1 cells. J. Immunol . 188: [2244–2253][1]. An additional funding