Inhibited Th17 Differentiation Research Articles

Ras homolog gene family H (RhoH) deficiency induces psoriasis-like chronic dermatitis by promoting TH17 cell polarization

Ras homolog gene family H (RhoH) is a membrane-bound adaptor protein involved in proximal T-cell receptor signaling. Therefore RhoH plays critical roles in the differentiation of T cells; however, the function of RhoH in the effecter phase of the T-cell response has not been fully characterized. We sought to explore the role of RhoH in inflammatory immune responses and investigated the involvement of RhoH in the pathogenesis of psoriasis. We analyzed effector T-cell and systemic inflammation in wild-type and RhoH-null mice. RhoH expression in T cells in human PBMCs was quantified by using RT-PCR. RhoH deficiency in mice induced TH17 polarization during effector T-cell differentiation, thereby inducing psoriasis-like chronic dermatitis. Ubiquitin protein ligase E3 component N-recognin 5 (Ubr5) and nuclear receptor subfamily 2 group F member 6 (Nr2f6) expression levels decreased in RhoH-deficient T cells, resulting in increased protein levels and DNA binding activity of retinoic acid-related orphan receptor γt. The consequential increase in IL-17 and IL-22 production induced T cells to differentiate into TH17cells. Furthermore, IL-22 binding protein/Fc chimeric protein reduced psoriatic inflammation in RhoH-deficient mice. Expression of RhoH in Tcells was lower in patients with psoriasis with very severe symptoms. Our results indicate that RhoH inhibits TH17 differentiation and thereby plays a role in the pathogenesis of psoriasis. Additionally, IL-22 binding protein has therapeutic potential for the treatment of psoriasis.

NLRP3 inflammasome regulates Th17 differentiation in rheumatoid arthritis

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases. Th17 has been shown to play am important role in the pathogenesis of RA. Accumulating data suggest the involvement of NLRP3 inflammasome in Th17 differentiation in autoimmune diseases. In the current study, we found that NLRP3 inflammasome is activated in CD4 T cells from RA patients. The activation of NLRP3 inflammasome was correlated with disease activities and IL-17A concentration in RA sera. Knockdown of NLRP3 suppressed Th17 differentiation. In addition, caspase-1 or IL-1 receptor inhibitor inhibits Th17 differentiation significantly. Further, ROS production is increased in CD4 T cells from RA patients. The inhibition of ROS production decreased NLRP3 inflammasome activation and IL-1β production in CD4 T cells, leading to the suppression of Th17 differentiation. These findings suggest a pathogenic role of NLRP3 inflammasome in RA by promoting Th17 cell differentiation. NLRP3 inflammasome could be a potential therapeutic target for the treatment of RA.

Pyruvate dehydrogenase phosphatase catalytic subunit 2 limits Th17 differentiation.

Th17 cells favor glycolytic metabolism, and pyruvate dehydrogenase (PDH) is the key bifurcation enzyme, which in its active dephosphorylated form advances the oxidative phosphorylation from glycolytic pathway. The transcriptional factor, inducible cAMP early repressor/cAMP response element modulator (ICER/CREM), has been shown to be induced in Th17 cells and to be overexpressed in CD4+ T cells from the patients with systemic lupus erythematosus (SLE). We found that glycolysis and lactate production in in vitro Th17-polarized T cells was reduced and that the expression of pyruvate dehydrogenase phosphatase catalytic subunit 2 (PDP2), an enzyme that converts the inactive PDH to its active form, and PDH enzyme activity were increased in Th17 cells from ICER/CREM-deficient animals. ICER was found to bind to the Pdp2 promoter and suppress its expression. Furthermore, forced expression of PDP2 in CD4+ cells reduced the in vitro Th17 differentiation, whereas shRNA-based suppression of PDP2 expression increased in vitro Th17 differentiation and augmented experimental autoimmune encephalomyelitis. At the translational level, PDP2 expression was decreased in memory Th17 cells from patients with SLE and forced expression of PDP2 in CD4+ T cells from lupus-prone MRL/lpr mice and patients with SLE suppressed Th17 differentiation. These data demonstrate the direct control of energy production during Th17 differentiation in health and disease by the transcription factor ICER/CREM at the PDH metabolism bifurcation level.

Intestinal host defense outcome is dictated by PGE2 production during efferocytosis of infected cells

Inflammatory responses are terminated by the clearance of dead cells, a process termed efferocytosis. A consequence of efferocytosis is the synthesis of the antiinflammatory mediators TGF-β, PGE2, and IL-10; however, the efferocytosis of infected cells favors Th17 responses by eliciting the synthesis of TGF-β, IL-6, and IL-23. Recently, we showed that the efferocytosis of apoptotic Escherichia coli-infected macrophages by dendritic cells triggers PGE2 production in addition to pro-Th17 cytokine expression. We therefore examined the role of PGE2 during Th17 differentiation and intestinal pathology. The efferocytosis of apoptotic E. coli-infected cells by dendritic cells promoted high levels of PGE2, which impaired IL-1R expression via the EP4-PKA pathway in T cells and consequently inhibited Th17 differentiation. The outcome of murine intestinal Citrobacter rodentium infection was dependent on the EP4 receptor. Infected mice treated with EP4 antagonist showed enhanced intestinal defense against C. rodentium compared with infected mice treated with vehicle control. Those results suggest that EP4 signaling during infectious colitis could be targeted as a way to enhance Th17 immunity and host defense.

IL-2 Inhibition of Th17 Generation Rather Than Induction of Treg Cells Is Impaired in Primary Sjögren's Syndrome Patients.

To investigate the role of IL-2 in the balance of Th17 and Tregs and elucidate the underlying mechanisms of enhanced Th17 differentiation in primary Sjögren's syndrome (pSS) patients. This study involved 31 pSS patients, 7 Sicca patients, and 31 healthy subjects. Th17 and Treg cells were determined by flow cytometry, and IL-17A was detected by immunohistochemistry. IL-2 and IL-6 levels were assessed by ELISA and qPCR. p-STAT5 and p-STAT3 in salivary glands (SGs) were evaluated by immunohistochemistry and flow cytometry. The binding of STAT5 and STAT3 to the Il17a gene locus was measured by chromatin immunoprecipitation. We found that the percentage of Th17 cells was increased in the periphery and SG of pSS patients when compared with healthy subjects, but the Treg cells was unchanged. Meanwhile, the IL-2 level was reduced, and the IL-6 and IL-17A level was increased in the plasma of pSS patients. The ratio of IL-2 and IL-6 level was also decreased and IL-2 level was negatively correlated with the level of IL-17A. The expression of Il6 and Il17a mRNA was significantly increased, whereas Foxp3, Tgfb1, Tnfa, and Ifng mRNA were comparable. Furthermore, the level of STAT5 phosphorylation (p-STAT5) was reduced and p-STAT3 was enhanced in the SGs and in peripheral CD4+ T cells of pSS patients. In vitro IL-2 treatment-induced STAT5 competed with STAT3 binding in human Il17a locus, leading to decreased Th17 differentiation, which was associated with the reduced transcription activation marker H3K4me3. Our findings demonstrated a Treg-independent upregulation of Th17 generation in pSS, which is likely due to a lack of IL-2-mediated suppression of Th17 differentiation. This study identified a novel mechanism of IL-2-mediated immune suppression in pSS.

Presenilin1 regulates Th1 and Th17 effector responses but is not required for experimental autoimmune encephalomyelitis.

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) where pathology is thought to be regulated by autoreactive T cells of the Th1 and Th17 phenotype. In this study we sought to understand the functions of Presenilin 1 (PSEN1) in regulating T cell effector responses in the experimental autoimmune encephalomyelitis (EAE) murine model of MS. PSEN1 is the catalytic subunit of γ-secretase a multimolecular protease that mediates intramembranous proteolysis. γ-secretase is known to regulate several pathways of immune importance. Here we examine the effects of disrupting PSEN1 functions on EAE and T effector differentiation using small molecule inhibitors of γ-secretase (GSI) and T cell-specific conditional knockout mice (PSEN1 cKO). Surprisingly, blocking PSEN1 function by GSI treatment or PSEN1 cKO had little effect on the development or course of MOG35-55-induced EAE. In vivo GSI administration reduced the number of myelin antigen-specific T cells and suppressed Th1 and Th17 differentiation following immunization. In vitro, GSI treatment inhibited Th1 differentiation in neutral but not IL-12 polarizing conditions. Th17 differentiation was also suppressed by the presence of GSI in all conditions and GSI-treated Th17 T cells failed to induce EAE following adoptive transfer. PSEN cKO T cells showed reduced Th1 and Th17 differentiation. We conclude that γ-secretase and PSEN1-dependent signals are involved in T effector responses in vivo and potently regulate T effector differentiation in vitro, however, they are dispensable for EAE.

The Supercarbonate Apatite-MicroRNA Complex Inhibits Dextran Sodium Sulfate-Induced Colitis

The incidence of inflammatory bowel disease (IBD) is increasing. Nucleic acid-based medicine has potential as a next-generation treatment, but it is rarely successful with IBD. The aim of this study was to establish a microRNA-based therapy in an IBD model. For this purpose, we used microRNA-29 (miR-29) and a supercarbonate apatite (sCA) nanoparticle as a drug delivery system. Injection of sCA-miR-29a-3p or sCA-miR-29b-3p into mouse tail veins markedly prevented and restored inflammation because of dextran sulfate sodium (DSS)-induced colitis. RNA sequencing analysis revealed that miR-29a and miR-29b could inhibit the interferon-associated inflammatory cascade. Subcutaneous injection of sCA-miR-29b also potently inhibited inflammation, and it efficiently targeted CD11c+ dendritic cells (DCs) among various types of immune cells in the inflamed mucosa. RT-PCR analysis indicated that the miR-29 RNAs in CD11c+ DCs suppressed the production of interleukin-6 (IL-6), transforming growth factor β (TGF-β), and IL-23 subunits in DSS-treated mice. This may inhibit Th17 differentiation and subsequent activation, which is critical in IBD pathogenesis. In vivo experiments using a non-natural artificial microRNA sequence revealed that targeting of DCs in the inflamed colon is an exceptional feature of sCA. This study suggests that sCA-miR-29s may open a new avenue in nucleic acid-based medicine for IBD treatment.

Janus Kinase Inhibitor Baricitinib Modulates Human Innate and Adaptive Immune System.

The purpose of this study was to elucidate the mechanism of action of baricitinib on Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling, which involves in human innate and adaptive immune system. The effects of baricitinib were evaluated using human monocyte-derived dendritic cells (MoDCs), plasmacytoid dendritic cells (pDCs), B cells, and T cells. Baricitinib concentration-dependently suppressed the expression of CD80/CD86 on MoDCs and the production of type-I interferon (IFN) by pDCs. Baricitinib also suppressed the differentiation of human B cells into plasmablasts by B cell receptor and type-I IFN stimuli and inhibited the production of interleukin (IL)-6 from B cells. Human CD4+ T cells proliferated after T cell receptor stimulation with anti-CD3 and anti-CD28 antibody; however, such proliferation was suppressed by baricitinib in a concentration-dependent manner. In addition, baricitinib inhibited Th1 differentiation after IL-12 stimulation and Th17 differentiation by TGF-β1, IL-6, IL-1β, and IL-23 stimulation. Tofacitinib showed similar effects in these experiments. In naive CD4+ T cells, IFN-α and IFN-γ induced phosphorylation of STAT1, which was inhibited by baricitinib and tofacitinib. Furthermore, IL-6-induced phosphorylation of STAT1 and STAT3 was also inhibited by JAK inhibitors. In conclusion, the results indicated that baricitinib suppresses the differentiation of plasmablasts, Th1 and Th17 cells, as well as innate immunity, such as the T cell stimulatory capacity of dendritic cells. Thus, JAK inhibitors can be potentially clinically effective not only in rheumatoid arthritis but other immune-related diseases.

Carbon monoxide ameliorates murine T-cell-dependent colitis through the inhibition of Th17 differentiation

Recent studies have identified carbon monoxide (CO) as a potential therapeutic molecule for the treatment of inflammatory diseases including intestinal inflammation. In the present study, we explored the efficacy and the mechanisms of action of CO-releasing molecule (CORM)-A1 in T-cell transfer induced colitis model in mice. In addition, the impact of CORM-A1 on the T helper (Th) cell differentiation was evaluated using naïve CD4+ T cells isolated from the spleens in Balb/c mice. The results showed that CORM-A1 conferred protection against the development of intestinal inflammation and attenuated Th17 cell differentiation. Hence, the observed immunomodulatory effects of CORM-A1 could be useful for developing novel therapeutic approaches for managing intestinal inflammation through the regulation of Th17 differentiation.

RTA 1701 is an orally-bioavailable, potent, and selective RORγt inhibitor that suppresses Th17 differentiation in vitro and is efficacious in mouse models of autoimmune disease.

Abstract Retinoic acid-related orphan receptor γt (RORγt) is a transcription factor that directs the differentiation of Th17 cells, a subset of CD4+ helper T cells involved in autoimmune disorders. Once differentiated, Th17 cells secrete interleukin-17A (IL-17A) and other key cytokines implicated in autoimmune and inflammatory disease. Antibodies that target Th17 cells are approved for several autoimmune disorders, validating the important role of Th17 cells in disease. RTA 1701 is a potent and selective RORγt inhibitor in preclinical development for autoimmune disorders. It inhibits the activity of RORγt, as assessed using ligand binding domain and full-length RORγt reporters. Moreover, RTA 1701 is selective for RORγt and does not inhibit the activity of two closely-related receptors, RORα and RORβ. By inhibiting RORγt, RTA 1701 blocks Th17 differentiation of naïve CD4+ T cells and IL-17A production by both Th17 polarized CD4+ T cells and activated peripheral blood mononuclear cells (PBMCs). The expression of other RORγt target genes and Th17 signature genes (eg, IL-17F, IL-21, CCL20) is also reduced following RTA 1701 treatment of Th17-polarized naïve CD4+ T cells, as assessed by qPCR. RTA 1701 is orally bioavailable with excellent pharmacokinetic properties in rodents and monkey. Furthermore, this compound is efficacious in animal models of autoimmune disease, including the collagen-induced arthritis (CIA) mouse model of rheumatoid arthritis and the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Together, these results suggest that RORγt inhibition by RTA 1701 may have therapeutic potential in Th17-associated autoimmune disorders.

Phosphatase PTEN-mediated blockade of IL-2 production is essential for Th17 cell differentiation

Abstract T helper 17 (Th17) cells are one of CD4+ T cell subsets that mediate inflammation and autoimmunity. Unlike other subsets, interleukin 2 (IL-2) inhibits differentiation of Th17 cells but the mechanism that regulates Th17 differentiation is poorly understood. Here, we show that phosphatase and tensin homologue (PTEN) plays key role in Th17 cell differentiation by regulating IL-2 expression. We generated Th17-specific Pten knock-out mice (Pten fl/fl Il17a CRE) to investigate intrinsic role of PTEN. Th17-specific Pten deletion inhibited Th17 differentiation by inducing excessive expression of IL-2 in vitro. Over-expressed IL-2 recognized by IL-2 receptor and induced phosphorylation of STAT5, eventually triggered Foxp3 expression in Th17 cells. Next, we used experimental autoimmune encephalomyelitis (EAE) as in vivo model, Th17-specific deficiency of Pten ameliorated symptoms of EAE. Additionally, we used SF1670 which is PTEN-specific inhibitor for therapeutic approach. Treatment of SF1670 to EAE induced mice showed ameliorated symptoms of EAE, therefore PTEN may be a potential therapeutic target for Th17-mediated diseases.

Regulatory effect of nicotine on the differentiation of Th1, Th2 and Th17 lymphocyte subsets in patients with rheumatoid arthritis

Previous research has demonstrated that nicotine have protective role in rheumatoid arthritis(RA). However, the immunologic mechanisms of nicotine's effect have not been fully elucidated. Herein, the effects of nicotine on the differentiation of Th1, Th2, and Th17 cells were assessed.Peripheral blood mononuclear cells (PBMCs) and CD4+T cells were separated from patients with RA. PBMCs were stimulated with anti-CD3/anti-CD28 in the absence or presence of nicotine. CD4+T cells were cultured in the Th cell differentiation condition in the absence of nicotine or nicotine and alpha- bungarotoxin (αBgt) (the antagonist of nicotine) combined. Levels of T cell cytokines were detected with ELISA and flow cytometry. The expression of specific transcription factors (retinoic orphan re- ceptor c (RORc), T-box transcription factor (T-bet), and GATA Binding Protein 3 (GATA-3)) and signaling molecules (P-ERK1/2 and T-ERK1/2) were determined by Western blot. The results showed nicotine reduced IL-17A and increased IL-4 produced by stimulated PBMCs. During Th17 differentiation conditions, nicotine reduced the levels of IL-17A and RORc, induced the phosphorylation of ERK1/2. Meanwhile, nicotine increased the levels of IL-4 and GATA3 during Th2 differentiation. α-Bgt blocked the effects of nicotine on Th2 and Th17 differentiation. However, nicotine had no effect on the expression of IFN-γ and T-bet in CD4+T cells during Th1differentiation. These results demonstrate that nicotine suppresses Th17 differentiation, promotes Th2 differentiation and improves Th1/Th2 imbalance in RA patients, providing a new justification for its application in the treatment of rheumatoid arthritis.

PR-957 mediates neuroprotection by inhibiting Th17 differentiation and modulating cytokine production in a mouse model of ischaemic stroke.

Acute ischaemic stroke can induce secondary brain injury by activating an inflammatory response that contributes to clinical impairment. As a specificinhibitor of the immunoproteasome subunit low molecular weightpolypeptide 7 (LMP7), PR-957 may participate in regulating pathophysiological and inflammatory responses in multiple diseases of the central nervous system (CNS). We investigated the neuroprotective properties of PR-957 in a mouse model of stroke, induced by middle cerebral artery occlusion (MCAO). After MCAO and injections of PR-957 or vehicle, we evaluated mice behaviourally using modified Neurological Severity Scores (mNSS) and sensorimotor tests, including the adhesive-removal test, a foot-fault test and an inclined plane test. Infarct volume was measured 24 and 72h after MCAO. Infiltration by different lymphocyte subpopulations was evaluated by flow cytometry and immunofluorescent staining of brain tissue from the penumbral area. Quantitative real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay were used to measure the expression of proinflammatory cytokines: interkeukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17A, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, granulocyte colony-stimulating factor (GCSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of phosphorylated signal transducer and activator of transcription 3 (pSTAT-3) protein levels in brain was measured by immunoblot. MCAO mice treated with PR-957 showed a significant decrease in infarct volume and had mild neurological deficits compared to vehicle-treated mice. PR-957 administration also significantly decreased IL-1β, IL-6, IL-12, IL-17A and TNF-α. PR-957 provides neuroprotection via inhibiting T lymphocyte infiltration and decreasing T helper type 17 (Th17) cell differentiation in MCAO mice, which may result from the reduced expression of pSTAT-3. The neuroprotective effect of PR-957 indicates its potential utility as anti-inflammatory therapy for ischaemic stroke.

Demyelination in Multiple Sclerosis: Reprogramming Energy Metabolism and Potential PPARγ Agonist Treatment Approaches.

Demyelination in multiple sclerosis (MS) cells is the site of several energy metabolic abnormalities driven by dysregulation between the opposed interplay of peroxisome proliferator-activated receptor γ (PPARγ) and WNT/β-catenin pathways. We focus our review on the opposing interactions observed in demyelinating processes in MS between the canonical WNT/β-catenin pathway and PPARγ and their reprogramming energy metabolism implications. Demyelination in MS is associated with chronic inflammation, which is itself associated with the release of cytokines by CD4+ Th17 cells, and downregulation of PPARγ expression leading to the upregulation of the WNT/β-catenin pathway. Upregulation of WNT/β-catenin signaling induces activation of glycolytic enzymes that modify their energy metabolic behavior. Then, in MS cells, a large portion of cytosolic pyruvate is converted into lactate. This phenomenon is called the Warburg effect, despite the availability of oxygen. The Warburg effect is the shift of an energy transfer production from mitochondrial oxidative phosphorylation to aerobic glycolysis. Lactate production is correlated with increased WNT/β-catenin signaling and demyelinating processes by inducing dysfunction of CD4+ T cells leading to axonal and neuronal damage. In MS, downregulation of PPARγ decreases insulin sensitivity and increases neuroinflammation. PPARγ agonists inhibit Th17 differentiation in CD4+ T cells and then diminish release of cytokines. In MS, abnormalities in the regulation of circadian rhythms stimulate the WNT pathway to initiate the demyelination process. Moreover, PPARγ contributes to the regulation of some key circadian genes. Thus, PPARγ agonists interfere with reprogramming energy metabolism by directly inhibiting the WNT/β-catenin pathway and circadian rhythms and could appear as promising treatments in MS due to these interactions.

Betaine Ameliorates Experimental Autoimmune Encephalomyelitis by Inhibiting Dendritic Cell-Derived IL-6 Production and Th17 Differentiation.

IL-17-secreting T cells (Th17 cells) play a pathogenic role in multiple autoimmune diseases, including multiple sclerosis (MS), and dendritic cell (DC)-derived cytokines play pivotal roles in promoting the differentiation of naive CD4+ T cells into Th cell subsets (Th1 and Th17). Therefore, small molecules blocking the key cytokines produced by DCs will be beneficial in MS. In this article, we report that betaine treatment ameliorates MS pathogenesis by inhibiting DC-derived IL-6 production and Th17 differentiation. Using experimental autoimmune encephalomyelitis, a widely used mouse model of MS, we found that, compared with the vehicle-treated group, betaine-treated mice exhibited less severe experimental autoimmune encephalomyelitis symptoms, including lower clinical scores, reduced leukocyte infiltration, and less extensive demyelination in the CNS. Moreover, a significantly lower percentage of Th17 cells, one of the major pathogenic effector cells in MS progression, was observed in the peripheral immune system and in the CNS. Interestingly, in the in vitro Th17-differentiation assay, no significant change in Th17 cells was observed between the vehicle- and betaine-treated groups, whereas in the in vitro DC culture experiment, betaine treatment significantly decreased DC-derived IL-6 production. In the DC-T cell coculture experiment, a significantly decreased Th17 differentiation was observed upon betaine treatment. All of these data demonstrated that betaine inhibited Th17 differentiation indirectly by reducing IL-6 production by DCs. In brief, our findings demonstrated the pivotal roles of betaine in modulating MS pathogenesis and suggested that it may serve as a potential novel drug candidate for the treatment of MS.

Relationship between intestinal microbiota and ulcerative colitis: Mechanisms and clinical application of probiotics and fecal microbiota transplantation.

Ulcerative colitis (UC) is an inflammatory disease that mainly affects the colon and rectum. It is believed that genetic factors, host immune system disorders, intestinal microbiota dysbiosis, and environmental factors contribute to the pathogenesis of UC. However, studies on the role of intestinal microbiota in the pathogenesis of UC have been inconclusive. Studies have shown that probiotics improve intestinal mucosa barrier function and immune system function and promote secretion of anti-inflammatory factors, thereby inhibiting the growth of harmful bacteria in the intestine. Fecal microbiota transplantation (FMT) can reduce bowel permeability and thus the severity of disease by increasing the production of short-chain fatty acids, especially butyrate, which help maintain the integrity of the epithelial barrier. FMT can also restore immune dysbiosis by inhibiting Th1 differentiation, activity of T cells, leukocyte adhesion, and production of inflammatory factors. Probiotics and FMT are being increasingly used to treat UC, but their use is controversial because of uncertain efficacy. Here, we briefly review the role of intestinal microbiota in the pathogenesis and treatment of UC.

IFN-β regulates Th17 differentiation partly through the inhibition of osteopontin in experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) and the corresponding animal model, experimental autoimmune encephalomyelitis (EAE), are chronic neuroinflammatory autoimmune diseases. Increased activation of CD4+T cells, especially the Th1 and Th17 subsets, is thought to play a causal role in this disease. IFN-β is widely used in the treatment of MS and is found to decrease IL-17 and OPN production in MS patients and EAE mice. However, a definitive molecular mechanism has not yet been fully elucidated. In this study, we investigated the immunomodulatory effect of IFN-β on the EAE model. We observed disease progression and determined the percentage of Th1/Th17 cells in the peripheral immune organs, brain, and spinal cord of mice. Furthermore, the levels of related cytokines and transcription factors were measured in splenocytes, and the effects of IFN-β on Th17 differentiation were assessed in vitro. Compared to the control group, IFN-β treatment significantly reduced the incidence of EAE and the associated pathological damage. Th1 and Th17 cells in IFN-β-treated mice were significantly reduced, and the levels of cytokines, such as IFN-γ, IL-17, and OPN, were significantly decreased in splenocyte supernatants as well as the levels of corresponding transcription factors. IFN-β inhibited downstream inflammatory cytokines through the inhibition of PI3K/AKT/NF-κB axis and p38, JNK-MAPK, as well as the regulation of mTOR complexes. Moreover, IFN-β inhibited Th17 differentiation and neutralizing OPN antibodies offset the inhibitory effect of IFN-β on Th17 cells. Meanwhile, IFN-β influenced the acetylation of the Il17a and Opn gene promoters. The findings described herein provide novel evidence for the role of IFN-β in Th17 differentiation partly through the inhibition of OPN.

Anti-sepsis protection of Xuebijing injection is mediated by differential regulation of pro- and anti-inflammatory Th17 and T regulatory cells in a murine model of polymicrobial sepsis

Ethnopharmacological relevanceXuebijing injection (XBJ), a Chinese herbal medicine containing extracts from 5 herbs, is frequently used as an add-on with standard therapies to treat sepsis or septic shock with fewer side effects in China. Nonetheless, its mechanism of action on septic shock remains to be unveiled. We explored the differential effects of XBJ on subtypes of CD4+ T cell differentiation and septic shock protection in a murine model to understand the contribution of XBJ to regulation of the inflammation-immune axis function. Materials and methodsIn vitro T cell differentiation assays were performed to determine the effect of XBJ on CD4+ regulatory T cell and T helper cell differentiation. Besides, 2ml/kg, 6ml/kg- and 18ml/kg of XBJ were administered to different groups of septic mice once/day for 5 days after cecal ligation and puncture (CLP) surgeries. 36h after CLP, serum levels of pro-inflammatory cytokine TNF-α and IL-6 were determined with Elisa. Frequencies of CD4+ T cells were analyzed after staining with Tregs and T helper cell lineage specific antibodies by flow cytometer. ResultsXBJ at 18ml/kg stimulated Treg differentiation and moderately inhibited Th17 differentiation in vitro. Accordingly, 18ml/kg XBJ facilitated the expansion of IL-10+ Tregs and normalized pro-inflammatory Th17 population in septic mice. This regimen also significantly reduced serum levels of inflammatory cytokines TNF-α and IL-6 in septic mice. Additionally, 18ml/kg XBJ injection effectively prevented neutrophil infiltration into the lung and kidney and improved survival in this septic shock model. ConclusionsIn summary, XBJ improves survival in septic shock partially through preventing cytokine storm, inhibiting inflammation and regulating the balance of Tregs and Th17 cells. Thus, higher dose of XBJ is a potential regimen to benefit septic shock patients.

Regulation of Th17 Differentiation by IKKα-Dependent and -Independent Phosphorylation of RORγt.

Transcription factor retinoid acid-related orphan receptor (ROR)γt transcriptionally regulates the genes required for differentiation of Th17 cells that mediate both protective and pathogenic immunity. However, little is known about the function of posttranslational modifications in the regulation of RORγt activity. Mass spectrometric analysis of immunoprecipitated RORγt from Th17 cells identified multiple phosphorylation sites. Systematic mutation analysis of the identified phosphorylation sites found that phosphorylation of S376 enhances whereas phosphorylation of S484 inhibits Th17 differentiation. IκB kinase (IKK)α binds and phosphorylates RORγt at S376 but not S484. Knockdown of IKKα, dominant-negative IKKα, and RORγt mutants incapable of interacting with IKKα all decrease Th17 differentiation. Furthermore, nonphosophorylatable RORγt mutant (S376A) impairs whereas phosphomimetic mutant (S376E) stimulates Th17 differentiation independent of IKKα. Therefore, IKKα-dependent phosphorylation of S376 stimulated whereas IKKα-independent phosphorylation of S484 inhibited RORγt function in Th17 differentiation.

PD-L1 up-regulation restrains Th17 cell differentiation in STAT3 loss- and STAT1 gain-of-function patients.

Patients with hypomorphic mutations in STAT3 and patients with hypermorphic mutations in STAT1 share several clinical and cellular phenotypes suggesting overlapping pathophysiologic mechanisms. We, therefore, examined cytokine signaling and CD4+ T cell differentiation in these cohorts to characterize common pathways. As expected, differentiation of Th17 cells was impaired in both cohorts. We found that STAT1 was hyperphosphorylated in response to cytokine stimulation in both cohorts and that STAT1-dependent PD-L1 up-regulation-known to inhibit Th17 differentiation in mouse models-was markedly enhanced as well. Overexpression of SOCS3 strongly inhibited phosphorylation of STAT1 and PD-L1 up-regulation, suggesting that diminished SOCS3 expression may lead to the observed effects. Defects in Th17 differentiation could be partially overcome in vitro via PD-L1 inhibition and in a mouse model of STAT3 loss-of-function by crossing them with PD-1 knockout mice. PD-L1 may be a potential therapeutic target in several genetic diseases of immune deficiency affecting cytokine signaling.