Expression Of Th17 Cells Research Articles

Cigarette Smoke Induction of Interleukin-27/WSX-1 Regulates the Differentiation of Th1 and Th17 Cells in a Smoking Mouse Model of Emphysema.

IFN-γ-producing CD4+ T (Th1) cells and IL-17-producing CD4+ T (Th17) cells play a critical role in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the immune regulation between Th1 and Th17 cells remains unclear. Previous studies have demonstrated that interleukin-27 (IL-27)/WSX-1 exerted pro- or anti-inflammatory effects in many acute inflammatory diseases by modulating T cell-mediated immune response, but little was known about its role in chronic inflammatory disease, especially in smoking-related lung diseases. Considering IL-27 is an important regulator in T lymphocytes immune responses and was found markedly increased in patients with COPD, we hypothesized that IL-27/WSX-1 may exert immuno-regulatory effects on the differentiation of Th1 and Th17 cells in smoking-related COPD. In this study, we aimed to evaluate the expression of IL-27 in patients with COPD and explore the role of IL-27/WSX-1 on Th1 and Th17 cells differentiation in a smoking mouse model of emphysema. We found that elevated expression of IL-27 was associated with increased proportion of Th1 cells and Th17 cells in patients with COPD and demonstrated parallel findings in cigarette smoke-exposed mice. In addition, cigarette smoke exposure upregulated the expression of IL-27R (WSX-1) by naive CD4+ T cells in mice. In vitro, IL-27 significantly augmented the secretion of IFN-γ by naive CD4+ T cells via a T-bet, p-STAT1, and p-STAT3-dependent manner, but inhibited the production of IL-17 by a ROR-γt and p-STAT1-dependent way. Furthermore, anti-IL27 treatment dramatically decreased the expression of IFN-γ-producing CD4+ T cells in cigarette smoke-exposed mice. These findings proposed that IL-27 has functions for promoting the expression of Th1 cells but inhibiting the expression of Th17 cells in vitro and IL-27 neutralization-attenuated Th1-mediated inflammation in vivo, suggesting targeting IL-27/WSX-1 may provide a new therapeutic approach for smoking-related COPD.

Mass Cytometry Reveals PD1 Upregulation Is an Early Step in MDS Disease Progression

BackgroundMDS is characterized by ineffective haematopoiesis and a propensity to leukaemia transformation, with increasing evidence linking immune exhaustion to disease progression. Immune checkpoints are known to be upregulated on T cells in cancer, and inhibitors of CTLA-4 and PD1/PLD1 axis have demonstrated efficacy with likely clinical benefit in MDS. We have previously shown profound changes in both the number and function of components of the adaptive immune system, particularly Tregs, in MDS (Kordasti, Blood 2007). In order to characterise the immune signature in a wider range of T cell subsets simultaneously, with particular emphasis on cells likely to be affected by checkpoint inhibitor therapy (CPI), we analyse CTLA-4 and PD1 expression in MDS by cytometry by time-of-flight (CyTOF). Additionally, we aim to explore whether these differences are accentuated by the absence or presence of somatic mutations, or in morphologically more advanced disease.Materials and MethodsMDS patients (n=56) and age-matched healthy donors (HD, n=6) were stained with two panels of 35 and 34 antibodies for unstimulated and PMA/Ionomycin-stimulated PBMCs, respectively. Samples were run on CyTOF and data analysed using visual stochastic neighbour embedding (viSNE, Cytobank) to generate t-distributed SNE scores by unsupervised multi-dimensional reduction of T cells. Spanning-tree progression analysis of density-normalized events (SPADE), was performed and T cell subsets identified from heat maps based on typical phenotypic markers (Regulatory, Naive, Memory, Effector Memory (EM) Central Memory (CM), Effector, Terminal Effector (TE)). T cells were also clustered based on cytokine secretion (IFN-ϒ, TNF-α, IL-17, IL-2 and IL-10).Somatic mutation analysis was performed on 48 of the MDS patients using our established targeted panel of 24 genes known to be mutated in MDS, for subgroup analysis (Mohamedali, Leukaemia 2015)Results and DiscussionDemographics and subgroups are outlined in figure 1.The number of Tregs was significantly higher in RAEB than non-RAEB MDS (9.6% of total CD4+ cells vs 7.5% p=0.02) and in RAEB versus HD (9.6% vs 5.8% p=0.01). There was a significantly higher proportion of Tregs in MDS patients with somatic mutations compared to those without (8.7% vs 7.06% p<0.05) and HD (8.7% vs 5.9% p<0.05), confirming our previous findings. Amongst two subpopulations of Tregs previously identified (Kordasti, Blood 2016) there was no difference between HD and MDS in terms of their number. However, Tregs A and B have an increased PD1 expression in MDS vs HD (p=0.03 &p=0.003). In addition, CD4+EM and CD4+Memory, CD4+/CD8+ Na•ve T cells and TNF-α secreting cells all expressed more PD1 when compared to HD (p<0.05), see figure 2. However, there were no differences in T cell PD1 expression when comparing low and high risk MDS patients, suggesting immune exhaustion to tumour antigens is a common founder event in MDS.By contrast, CTLA-4 expression was not significantly different in T cell subsets between MDS and HD. Sub-groups based on specific somatic mutations did reveal that CD4+ Memory, CD4+TE and CD8+CM subsets in SF3B1-mutated MDS cases had less CTLA-4 expression compared to MDS cases with other mutations (p<0.05). Furthermore, SF3B1-mutant cases also had less CTLA-4 expression when compared to MDS cases without any detected mutations in CD4+ Memory, CD4+EM and CD8+CM subsets (p<0.05).Taken together this work suggests a possible 3 hit-model for disease progression in MDS. The establishment of MDS is accompanied by increased expression of PD1 most likely in response to increased PDL1 on tumour cells, which argues for early introduction of PD1 inhibitor in the disease course. Increased CTLA-4 expression is likely a later step, dependent on the acquisition of specific mutations by the malignant clone, that further alters the relationship between tumour and immune surveillance. Finally, in disease progression through from non-RAEB to RAEB/AML there is an expansion of suppressive Tregs facilitating tumour outgrowth. Furthermore, pro-inflammatory IL17 secreting T helper cells (Th17) have been shown to be increased in low risk MDS and are known to be associated with autoimmune disease. The trend towards increased PD1 expression in Th17 cells in a group of low risk patients harbouring SF3B1 mutations (p=0.09) is suggestive that acquisition of increased PD1 expression is a key branch point in disease outcome. [Display omitted] [Display omitted] DisclosuresIrish:Incyte: Research Funding; Janssen: Research Funding; Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Upregulated Expression of microRNA-16 Correlates with Th17/Treg Cell Imbalance in Patients with Rheumatoid Arthritis

To explore the correlation between miR-16 expression in T cells of peripheral blood mononuclear cells (PBMCs) and Th17/Treg imbalance in rheumatoid arthritis (RA) patients. Forty RA patients were recruited as the case group and further grouped as active RA and inactive RA groups; 21 healthy individuals were selected as the control group. Th17 and Treg were measured by flow cytometry, and their related cytokines were measured by FlowCytomix. RORγt, FoxP3 mRNA, and miR-16 expression in T cells was determined by real-time quantitative polymerase chain reaction. Western blotting was performed to measure RORγt and FoxP3 protein expression. RA patients showed upregulated Th17 and RORγt mRNA and protein expression compared with the controls (all p < 0.05); active RA patients showed lower Treg and FoxP3 mRNA and protein expression compared with inactive RA patients and controls (all p < 0.05). Secretion levels of Th17-related cytokines were higher in active RA patients than in inactive RA patients and controls (all p < 0.05); whereas those of Treg-related cytokines were lower in active RA patients than in controls (all p < 0.05). Active RA patients showed increased miR-16 expression in Th17 cells and decreased miR-16 expression in Treg cells of PBMCs (both p < 0.05). Pearson's test showed that in the PBMCs of the RA patients, miR-16 expression in the Th17 cells was positively related with RORγt mRNA expression, and miR-16 expression in the Treg cells was positively related with FoxP3 mRNA expression (both p < 0.05). The expression of miR-16 in Th17 and Treg cells of PBMCs in RA patients was closely associated with the expression of RORγt and FoxP3. MiR-16 may be involved in Th17/Treg imbalance of RA patients by affecting the expression of RORγt and FoxP3.

Osteopontin inhibition of miR-129-3p enhances IL-17 expression and monocyte migration in rheumatoid arthritis

BackgroundOsteopontin (OPN) is an important proinflammatory cytokine in rheumatoid arthritis (RA). Levels of OPN have been shown to be significantly correlated with interleukin-17 (IL-17) production and expression of Th17 cells in the synovial fluid of RA patients. Here, we investigated the role of OPN in monocyte migration, IL-17 production and osteoblasts. MethodsOPN and IL-17 expression profiles in osteoarthritis (OA) and RA synovial fluid were determined by enzyme-linked immunosorbent assay (ELISA). The expression of the microRNA, miR-129-3p, in osteoblasts was analyzed by real-time quantitative polymerase chain reaction (qPCR). Immunoreactive proteins were spotted by Western blotting. We used the collagen-induced arthritis (CIA) mouse model to investigate the role of OPN in monocyte migration during RA. ResultsOPN and IL-17 expression were higher in RA synovial fluid as compared to OA samples. We also found that OPN promotes IL-17 expression in osteoblasts and thereby enhances monocyte migration via the Syk/PI3K/Akt signaling pathway. miR-129-3p expression was found to be negatively regulated by OPN via the Syk/PI3K/Akt signal cascade. In contrast, lentiviral vectors expressing short hairpin RNA inhibited OPN expression and ameliorated articular swelling, cartilage erosion and monocyte infiltration in the ankle joints of CIA mice. ConclusionTo our knowledge, our study is the first to describe how OPN promotes monocyte migration by upregulating IL-17 expression in osteoblasts in RA disease. SignificanceThese findings indicate that OPN could serve as a potential therapeutic target for the treatment of RA.

In vivo anti-inflammatory activities of novel cytokine IL-38 in Murphy Roths Large (MRL)/lpr mice

The newly named interleukin (IL)-36 subfamily member IL-38 has been shown to exert anti-inflammatory activity. However, the in vivo immunomodulatory activity of IL-38 was poorly investigated in systemic lupus erythematosus (SLE). We have investigated the expression of CD4+IL-17+ Th17, CD4+IFN-γ+ Th1 and CD3+CD4−CD8− double negative (DN) T cells and the related immunopathological mechanisms in female MRL/lpr mice model of spontaneous lupus-like disease, with or without IL-38 treatment. Intravenous administration of murine recombinant IL-38 into MRL/lpr mice can ameliorate the lupus-like clinical symptoms including proteinuria, leukocyteuria and skin lesions. A remission of histopathology characteristics of skin and nephritis was also observed upon IL-38 treatment. Accordingly, IL-38 receptor was expressed on the cell surface of both CD4+ Th and CD19+ B lymphocytes. The splenic Th17 and DN T lymphocytes, the average mRNA level of epigenetically regulated gene expression of Th17 cells, and serum concentrations of IL-17 and IL-22 were significantly decreased upon the treatment of IL-38 (all p<0.05). The in vivo results suggest that IL-38 can ameliorate skin inflammation and nephritis in SLE mice probably via suppressing the formation of inflammatory cytokines such as IL-17 and IL-22, and pathogenic DN T cells. These findings may provide a biochemical basis for further investigation of the therapeutic mechanisms of IL-38 for the treatment of autoimmune-mediated inflammation.

The paracrine immunomodulatory interactions between the human dental pulp derived mesenchymal stem cells and CD4 T cell subsets

Mesenchymal stem cells (MSCs) have strong immunomodulatory properties, however these properties may show some differences according to the tissue type of their isolate. In this study we investigated the paracrine interactions between human DP derived MSCs (hDP-MSCs) and the CD4+ T helper cell subsets to establish their immunomodulatory mechanisms. We found that the CD4+-Tbet+ (Th1) and CD4+-Gata3+ (Th2) cells were suppressed by the hDP-MSCs, but the CD4+-Stat3+ (Th17) and CD4+-CD25+-FoxP3+ (Treg) cells were stimulated. The expressions of T cell specific cytokines interferon gamma (IFN-g), interleukin (IL)-4 and IL-17a decreased, but IL-10 and transforming growth factor beta-1 (TGF-b1) increased with the hDP-MSCs. The expressions of indoleamine-pyrrole 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), soluble human leukocyte antigen G (sHLA-G) derived from hDP-MSCs slightly increased, but hepatocyte growth factor (HGF) significantly increased in the co-culture groups. According to our findings, the hDP-MSCs can suppress the Th1 and Th2 subsets but stimulate the Th17 and Treg subsets. The Stat3 expression of Th17 cells may have been stimulated by the HGF, and thus the pro-inflammatory Th17 cells may have altered into the immunosuppressive regulatory Th17 cells. Further prospective studies are needed to confirm our findings.

Effect of bacteria lysates and 1, 25 - dihydroxy vitamin D3 on airway inflammation in asthmatic mice models

Objective To discuss the effects of bacteria lysates(OM-85BV), 1, 25 - dihydroxy vitamin D3 [1, 25-(OH)2-VitD3], two immune regulators on airway inflammation in asthmatic mice models, and its pathways of action on airway inflammation were discussed. Methods Forty male BALB/c mice graded 4-6 weeks were randomly divided into 5 groups.Group A: control group; group B: asthmatic model group; group C: 1, 25-(OH)2-VitD3 group; group D: OM-85BV group; group E: combination group.On days 0 to 14, mice in C, D and E groups were given 1, 25-(OH)2-VitD3, OM-85BV and 1, 25-(OH)2-VitD3+ OM-85BV, and mice in A, B groups were given 9 g/L saline instead.On days 15, 22 and 29, mice in B, C, D, E groups were intraperitoneally with injection of ovalbumin(OVA)- aluminum hydroxide [Al(OH)3]. Group A were given 9 g/L saline instead.On days 36 to 40, mice of B, C, D, E groups were given an aerosol challenge of 10 g/L OVA for 0.5 h once a day.Mice in the control group were given the same amount of 9 g/L saline.Animals were sacrificed 24 hours after the final inhalational challenge, and for the recovered bronchoalveolar lavage fluid(BALF) of the left lung was used for differential inflammatory cell counts and for detecting the level of inflammatory cytokine interleukin-17(IL-17). Right lung samples were used for pathological investigation and detecting the expression of the IL-17 mRNA and RORγt mRNA by real time-PCR. Results Compared with the control group, the asthma models expressed more serious expression in bronchospasm contraction, hyperplasia disorders of bronchial epithelial cells, infiltration of inflammatory cells in lung, and so on.Compared with the control group, the total number of inflammatory cells counts[(104.04±5.51) 107/L vs (22.79±1.91) 107/L] and eosinophils proportion[(37.63±3.64)% vs (2.37±1.55)%] in BALF in group B were significantly increased (all P<0.05), the levels of IL-17 [(85.13±5.77) 103 pg/L vs (47.44±4.57) 103 pg/L] in BALF were significantly higher(P<0.05), the relative expressions of IL-17 mRNA(13.68±1.59 vs 1.00±0.00) and RORγt mRNA (4.53±0.51 vs 1.00±0.00)in lung were higher, which had a statistical significance(all P<0.05). The situations of group C, D, E were obviously improved compared with group B, and those of group D were improved remarkably. Conclusions Oral OM-85BV and 1, 25-(OH)2-VitD3 intervention could relieve the airway inflammation of asthmatic mice models, and its effect can be remarkable by oral OM-85BV.The two immune regulators could relieve the degree of airway inflammation on asthmatic mice models by reducing the expression of Th17 cells differentiation.Therefore the two immune regulators could be the choices for preventing the happening and the development of the asthmatic airway inflammation. Key words: Asthma; Bacteria lysates; 1, 25-dihydroxy vitamin D3; Th17 cell

High expression of heme oxygenase-1 in target organs may attenuate acute graft-versus-host disease through regulation of immune balance of TH17/Treg

The high incidence of acute graft-versus-host disease (aGVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Grades III and IV aGVHD are the leading causes of death in allo-HSCT recipients. Heme oxygenase-1(HO-1) has anti-inflammatory and immune-regulatory functions. In this study, we evaluated the none GVHD and grade I–IV patients samples which were collected at the first re-examination after successful allo-HSCT, we found that expressions of HO-1 mRNA in the bone marrow and peripheral blood mononuclear cells of allo-HSCT recipients who had subsequent non-GVHD and grade I aGVHD were significantly higher than those in patients with Grade III–IV aGVHD. We then demonstrated that enhanced expression of HO-1 in target organs by infusing HO-1-gene-modified Mesenchymal stem cells (MSCs) alleviated the clinical and histopathological severity of aGVHD in experimental mice. Flow cytometry revealed a higher expression of Treg cells and a lower expression of TH17 cells in splenic and lymph node tissues of mice with enhanced HO-1 expression, as compared to that in the aGVHD mice. This was further substantiated by lower expression levels of ROR-Υt and IL-17A mRNA, and higher levels of Foxp3 mRNA in the splenic tissue of mice with enhanced HO-1 expression. Our results indicate that high expression of HO-1 may reduce the severity of aGVHD by regulation of the TH17/Treg balance.

Multi-step Th17 differentiation in response to segmented filamentous bacteria in the mouse intestine

Abstract Th17 cells have significant roles in maintaining homeostasis and regulating host defense against various pathogens in our bodies. Our laboratory initially identified segmented filamentous bacteria (SFB) as a unique commensal that is sufficient for Th17 cell differentiation and promotion of Th17-dependent autoimmune diseases such as a mouse model of spontaneous arthritis. The molecular and cellular requirements of SFB-induced Th17 cell differentiation are still unclear. To understand the whole process of Th17 cells differentiation in vivo, we developed SFB-specific T cell receptor transgenic (7B8) mice. We can trace SFB-specific Th17 cell differentiation and response by transferring fluorescently-labeled 7B8 naïve T cells into SFB-gavaged host mice. Using this approach, we have elucidated the requirements for cytokines and antigen presenting cells (APCs) to understand the process of Th17 cell differentiation. Here, we describe that initial induction and expansion of Th17 cells occurs in mesenteric lymph nodes (MLN), and their subsequent migration to intestine is integrin β7-dependent. Although RORγt expression in Th17 cells is mainly dependent on IL-6 signaling in the MLN, IL-23R signaling also contributes to RORγt expression in Th17 cells in the ileum in the absence of IL-6. CD103+ CD11b+ APCs are not important for induction and initial expansion of SFB-specific Th17 cells in MLN, but play important roles in maintenance of SFB-specific Th17 cells in the ileum. Taken together, these results indicate that Th17 cell differentiation proceeds in multiple discrete stages.

Transcriptome profiling unveils the role of cholesterol in IL-17A signaling in psoriasis.

Psoriasis is a chronic inflammatory skin disease characterized by altered proliferation and differentiation of keratinocytes as well as infiltration of immune cells. Increased expression of Th17 cells and cytokines secreted by them provides evidence for its central role in the pathogenesis of psoriasis. IL-17A, signature cytokine of Th17 cells was found to be highly differentially expressed in psoriatic lesional skin. However, cellular and molecular mechanism by which IL-17A exerts its function on keratinocyte is incompletely understood. To understand IL-17A mediated signal transduction pathways, gene expression profiling was done and differentially expressed genes were analysed by IPA software. Here, we demonstrate that during IL-17A signaling total cholesterol levels were elevated, which in turn resulted in the suppression of genes of cholesterol and fatty acid biosynthesis. We found that accumulation of cholesterol was essential for IL-17A signaling as reduced total cholesterol levels by methyl β cyclodextrin (MBCD), significantly decreased IL-17A induced secretion of CCL20, IL-8 and S100A7 from the keratinocytes. To our knowledge this study for the first time unveils that high level of intracellular cholesterol plays a crucial role in IL-17A signaling in keratinocytes and may explain the strong association between psoriasis and dyslipidemia.

Reciprocal regulation of RORγt acetylation and function by p300 and HDAC1.

T helper 17 (Th17) cells not only play critical roles in protecting against bacterial and fungal infections but are also involved in the pathogenesis of autoimmune diseases. The retinoic acid-related orphan receptor (RORγt) is a key transcription factor involved in Th17 cell differentiation through direct transcriptional activation of interleukin 17(A) (IL-17). How RORγt itself is regulated remains unclear. Here, we report that p300, which has histone acetyltransferase (HAT) activity, interacts with and acetylates RORγt at its K81 residue. Knockdown of p300 downregulates RORγt protein and RORγt-mediated gene expression in Th17 cells. In addition, p300 can promote RORγt-mediated transcriptional activation. Interestingly, the histone deacetylase (HDAC) HDAC1 can also interact with RORγt and reduce its acetylation level. In summary, our data reveal previously unappreciated posttranslational regulation of RORγt, uncovering the underlying mechanism by which the histone acetyltransferase p300 and the histone deacetylase HDAC1 reciprocally regulate the RORγt-mediated transcriptional activation of IL-17.

Role of Imbalance between Th17 Cells and Treg Cells in the Pathogenesis of Children with Henoch-Schonlein Purpura

To explore the role of Th17 cells, CD4⁺ CD25⁺ regulatory Treg cells (Treg) and its transcription factor RORγt and FoxP3 in the pathogenesis of children with Henoch-Schonlein purpura (HSP) so as to provide a new strategy for treatment of children with Henoch-Schonlein purpura by regulating the balance of Th17 and Treg cells. Forty children with Henoch-Schonlein purpura in acute phase admitted in our hospital from February 2012 to March 2013 were enrolled in this study, forty healthy children were simultaneously used as controls. The expression of RORγt mRNA and FoxP3 mRNA in peripheral blood mononuclear cells was detected by real-time PCR using SYBR Green I. The levels of IL-17A, TGF-β1, IL-2 and IL-6 in serum were measured by ABC-ELISA. The ratio of Th17 cells to Treg cells in peripheral blood T lymphocytes was detected by flow cytometry. The levels of Th17 cells (2.75 ± 0.60%) and RORγt mRNA (1.11 ± 0.51) in HSP group were significantly higher than levels of Th17 cells (1.41 ± 0.29%) and RORγt mRNA (0.65 ± 0.24) (P < 0.01) in control group, but the levels of Treg cells (4.56 ± 1.26%) and FoxP3 mRNA (1.15 ± 0.45) in HSP group were lower than those of Treg cells (7.85 ± 1.97%) and FoxP3 mRNA (2.32 ± 1.1) (P < 0.01) in the control group. The relative levels of serum IL-17A, IL-6, TGF-β1 (40.40 ± 11.81 pg/ml, 75.38 ± 27.19 pg/ml, 309.41 ± 81.03 pg/ml) in the HSP group were significantly higher than those in the control group [IL-17A (20.32 ± 10.70 pg/ml), IL-6 (25.16 ± 8.31 pg/ml), TGF-β1 (236.34 ± 66.01 pg/ml)] (P < 0.01), but the level of serum IL-2 (25.60 ± 13.19 pg/ml) in the HSP group was lower than that (34.42 ± 11.69 pg/ml) in the control group (P < 0.01). The further detection demonstrated that in the children with acute HSP, the expression of Th17 cells positively correlated with RORγt mRNA, IL-17A and IL-6 with the correlation coefficients of 0.887, 0.938 and 0.934 (P < 0.01), respectively. The positive correlation was also shown between the Treg cells and FoxP3 mRNA, IL-2 with the correlation coefficients of 0.834 and 0.932 (P < 0.01), respectively. There are higher expression levels of Th17 cells, RORγt mRNA and IL-17A, and lower expression levels of Treg cells, FoxP3 mRNA of children with HSP in acute phase, which shows that Th17/Treg imbalance exists in children with HSP in acute phase. The levels of serum IL-6, TGF-β1 increase and the serum IL-2 decrease in children with HSP in acute phase, moreover, there are the positive correlations between the levels of Th17 cells and expression of IL-6, as well as the level of Treg cells and expression of IL-2 in children with HSP in acute phase.

Activation of the Interleukin-34 Inflammatory Pathway in Response to Influenza A Virus Infection

Interleukin 34 (IL-34) is a newly recognized cytokine that functions similarly to macrophage colony-stimulating factor. This study investigated the mechanism by which IL-34 is produced in response to exogenous pathogen infections in humans. The results showed that the IL-34 levels were higher in the serum and peripheral blood mononuclear cells (PBMCs) from 155 influenza A virus (IAV)-infected patients than in those from 145 healthy individuals. The expression level of IL-34 in IAV-infected PBMCs was blocked by IL-22-specific siRNA. This result indicated that IL-34 was induced by IL-22 in the inflammatory cascade. The mRNA and protein expression levels of IL-22 activated by IAV infection were significantly inhibited by IL-34 overexpression but induced by IL-34-specific siRNA. Thus, a feedback system most likely exists between IL-34 and IL-22. The IL-22 expression in T helper type 17 (Th17) cells of PBMCs was higher than IL-34 expression in Th17 cells of PBMCs, and there was IL-34 expression in IL-22+ Th17 cells. This result showed that the production of IL-22 and IL-34 is both from the same and different subset of cells, which indicated that the regulatory mechanism of IL-22/IL-34 is through the autocrine or paracrine systems. In conclusion, IL-34 is induced by IL-22 in the inflammatory cascade in response to IAV infection. Therefore, IL-34 is a promising target for the screening of anti-inflammatory medicines.

Th1/Th17 plasticity is a marker of advanced β cell autoimmunity and impaired glucose tolerance in humans.

Upregulation of IL-17 immunity and detrimental effects of IL-17 on human islets have been implicated in human type 1 diabetes. In animal models, the plasticity of Th1/Th17 cells contributes to the development of autoimmune diabetes. In this study, we demonstrate that the upregulation of the IL-17 pathway and Th1/Th17 plasticity in peripheral blood are markers of advanced β cell autoimmunity and impaired β cell function in human type 1 diabetes. Activated Th17 immunity was observed in the late stage of preclinical diabetes in children with β cell autoimmunity and impaired glucose tolerance, but not in children with early β cell autoimmunity. We found an increased ratio of IFN-γ/IL-17 expression in Th17 cells in children with advanced β cell autoimmunity, which correlated with HbA1c and plasma glucose concentrations in an oral glucose tolerance test, and thus impaired β cell function. Low expression of Helios was seen in Th17 cells, suggesting that Th1/Th17 cells are not converted thymus-derived regulatory T cells. Our results suggest that the development of Th1/Th17 plasticity may serve as a biomarker of disease progression from β cell autoantibody positivity to type 1 diabetes. These data in human type 1 diabetes emphasize the role of Th1/Th17 plasticity as a potential contributor to tissue destruction in autoimmune conditions.

Histone deacetylase inhibitor regulates the balance of Th17/Treg in allergic asthma.

The aim of this study is to investigate the expression pattern of histone deacetylase 9 in peripheral blood of patients with allergic asthma and its regulatory effect on the balance of Th17/Treg cells involved in the pathogenesis of asthma. flap-Ub promoter-GFP-WRE vector was used to construct the Jurkat-HA-FOXP3 cell line. After histone deacetylase inhibitor-trichostatin A (TSA) treatment, FOXP3 and RORγt expression were detected by real-time-polymerase chain reaction (RT-PCR). BALB/c mice were randomly assigned to control group, TSA treatment and the asthma group. Serum Immunoglobulin E (IgE) was detected with enzyme-linked immunosorbent assay (ELISA), airway inflammation in lung tissue evaluated by haematoxylin/eosin staining, bronchoalveolar lavage fluid (BALF) cell number and differential counted, interleukin (IL)-17A and TGF-β concentrations in BALF measured with ELISA, and expression of RORγt and FOXP3 messenger RNA (mRNA)measured by RT-PCR. Forty-seven patients with asthma were recruited and assigned to intermittent, mild and moderate-severe group. GATA3, IL-4, histone deacetylases (HDAC) 9 mRNA expression level were measured by RT-PCR. After TSA treatment, FOXP3 mRNA level was upregulated, while RORγt mRNA level was downregulated. FOXP3 protein level was also upregulated by TSA. In vivo, TSA treatment can inhibit IL-17 but promote transforming growth factor-beta production in the BALF of asthma mice, and inhibited the expression of Th17 cells and RORγt mRNA in lung; also can promote Foxp3 mRNA expression. GATA3, IL-4 mRNA expression levels were upregulated in patients with asthma than the healthy control. HDAC9 mRNA expression level was associated with the severity of disease. The histone deacetylase inhibitor TSA can regulate the balance of Th17/Treg in asthma by regulating the activity of histone deacetylase.

Novel benzenediamine derivative FC99 ameliorates zymosan-induced arthritis by inhibiting RORγt expression and Th17 cell differentiation.

Increased IL-17-producing helper T (Th17) cells have been observed in patients with rheumatoid arthritis (RA). The retinoic-acid-related orphan nuclear receptor (RORγt) is the master regulator of Th17 cells. Our previous research showed that FC99 possesses anti-inflammation activity. However, to date the effects of FC99 on RORγt expression in Th17 cell differentiation have not been investigated yet. In the present study, we found that FC99 significantly attenuated arthritis-like symptoms, i.e., suppressing the development of paw edema in zymosan-induced arthritis (ZIA) mice. H&E staining showed that the infiltration of inflammatory cells in ankle synovial tissues was significantly suppressed. FC99 also reduced the mRNA levels of pro-inflammatory cytokines in ankle synovial tissues as shown by Q-PCR analysis. The protein levels of the pro-inflammatory cytokines in sera were also suppressed after FC99 treatment. Moreover, FC99 decreased the RORγt mRNA level in spleen tissues. Th17 cell percentage was significantly decreased in spleens and draining lymph nodes (dLNs). The mRNA and protein levels of IL-17A and IL-23 were reduced after FC99 treatment in ZIA mice. Furthermore, in vitro experiments showed that FC99 inhibited the expression of IL-6 in LPS-induced RAW264.7 cells and BMDCs. Moreover, FC99 significantly inhibited the RORγt expression in PMA-induced CD4(+) T cells and LPS-induced RAW264.7 cells. These data indicate that FC99 improves arthritis-like pathological symptoms in vivo and in vitro, which might be related to the inhibition of RORγt expression in Th17 cells. Our findings suggest that FC99 may be a potential therapeutic candidate for the treatment of RA and other inflammatory disorders.

The E3 Deubiquitinase USP17 Is a Positive Regulator of Retinoic Acid-related Orphan Nuclear Receptor γt (RORγt) in Th17 Cells

Stable retinoic acid-related orphan nuclear receptor γt (RORγt) expression is pivotal for the development and function of Th17 cells. Here we demonstrate that expression of the transcription factor RORγt can be regulated through deubiquitination, which prevents proteasome-mediated degradation. We establish that USP17 stabilizes RORγt protein expression by reducing RORγt polyubiquitination at its Lys-360 residue. In contrast, knockdown of endogenous USP17 in Th17 cells resulted in decreased RORγt protein levels and down-regulation of Th17-related genes. Furthermore, USP17 expression was up-regulated in CD4(+) T cells from systemic lupus erythematosus patients. Our data reveal a molecular mechanism in which RORγt expression in Th17 cells can be positively regulated by USP17, thereby modulating Th17 cell functions.

TIR-domain-containing adapter-inducing interferon-β (TRIF) regulates Th17-mediated intestinal immunopathology in colitis.

Gastrointestinal mucosa reserves abundant Th17 cells where host response to commensal bacteria maintains Th17-cell generation. Although functional heterogeneity and dynamic plasticity of Th17 cells appear to be involved in chronic inflammatory disorders, how their plasticity is regulated in intestinal mucosa is unknown. Here we show that innate TRIF signaling regulates intestinal Th17-cell generation and plasticity during colitis. Absence of TRIF in mice resulted in increased severity of experimental colitis, which was associated with aberrant generation of Th17 cells especially of interferon (IFN)-γ-expressing Th17 cells in the lamina propria. The abnormal generation and plasticity of Th17 cells involved impaired expression of interleukin (IL)-27p28 by lamina propria macrophages but not dendritic cells. Treatment of TRIF-deficient mice with IL-27p28 during colitis reduced the number and IFN-γ expression of Th17 cells in the intestine. In vitro, TRIF-deficient macrophages induced more Th17 cells than wild-type (WT) macrophages during co-culture with WT naive T cells in response to cecal bacterial antigen. Many of Th17 cells induced by TRIF-deficient macrophages expressed IFN-γ due to impaired expression of IL-27p28 by macrophages and defective activation of STAT1 in T cells. These results outline TRIF-dependent regulatory mechanism by which host response to intestinal bacteria maintains Th17-cell-mediated pathology during colitis.

Activation of α7nAChR by Nicotine Reduced the Th17 Response in CD4+T Lymphocytes

Background: nAChRs play an important role in the regulation and modulation of immune cell proliferation, differentiation, migration and cell-cell interactions. The present study was to characterize the expression of α7nAChR on human peripheral blood mononuclear cells (hPBMC) and CD4+T lymphocytes, and to explore the change of Th17 expression after activation of α7nAChR on human CD4+T lymphocytes.Methods: A Ficoll gradient was used to separate hPBMC from whole blood, and then CD4+T lymphocytes were isolated by magnetic bead separation. The expression of α7nAChR on PBMC and CD4+T lymphocytes was analyzed using flow cytometry before and after stimulation with phytohemagglutinin (PHA). The effect of α7nAChR stimulation by nicotine or inhibition by α-bungarotoxin (α-BTX), as well as Th17 expression on the phenotype of CD4+T cells was evaluated using flow cytometric analysis.Results: The percentage of CD4+T cells in reduced PBMC, while the expression of α7nAChR increased when cells were stimulated by nicotine. This effect vanished when co-treated with nicotine and α-BTX. α7nAChR was found to expressed in about 90% of CD4+T cells. However, α7nAChR expression reduced to 80% on CD4+T cells after stimulation with PHA for 24 h. Stimulation of α7nAChR with nicotine increased the expression of Th17 cells, and this upregulation reduced when AChRα7 was inhibited by α-BTX.Conclusion: α7nAChR was ubiquitously expressed by CD4+T lymphocytes, which was correlated with the cell activation status. Meanwhile, activation of nAChRα7 by nicotine in CD4 cells reduced the Th17 response.

MiR-155 Activates Cytokine Gene Expression in Th17 Cells by Regulating the DNA-Binding Protein Jarid2 to Relieve Polycomb-Mediated Repression

Specification of the T helper 17 (Th17) cell lineage requires a well-defined set of transcription factors, but how these integrate with posttranscriptional and epigenetic programs to regulate gene expression is poorly understood. Here we found defective Th17 cell cytokine expression in miR-155-deficient CD4+ T cells in vitro and in vivo. Mir155 was bound by Th17 cell transcription factors and was highly expressed during Th17 cell differentiation. miR-155-deficient Th17 and T regulatory (Treg) cells expressed increased amounts of Jarid2, a DNA-binding protein that recruits the Polycomb Repressive Complex 2 (PRC2) to chromatin. PRC2 binding to chromatin and H3K27 histone methylation was increased in miR-155-deficient cells, coinciding with failure to express Il22, Il10, Il9, and Atf3. Defects in Th17 cell cytokine expression and Treg cell homeostasis in the absence of Mir155 could be partially suppressed by Jarid2 deletion. Thus, miR-155 contributes to Th17 cell function by suppressing the inhibitory effects of Jarid2.