Retinoic Acid Orphan Receptor Research Articles

ERK/MEK Pathway Regulates Th17 Cell Differentiation in Patients with Pemphigus Vulgaris.

T helper (Th) cells are involved in the pathogenesis of pemphigus vulgaris (PV). However, the mechanism still needs more exploration. This study aimed to evaluate the molecular mechanism of the dysregulation of Th17 cells in the peripheral blood of patients with PV. Serum levels of IL-17 and anti-Dsg3 titres in patients with PV were analysed using ELISA. The mRNA expression of retinoic acid orphan receptor γt (RORγt) in CD4+ T cells was detected using reverse transcription-quantitative PCR (qPCR). The number of Th17 cells was examined using flow cytometry. Western blot analysis and immunofluorescent staining were also performed to investigate the expression levels of ERK/MAPK signalling proteins and Th17 lineage-associated proteins. The proportion of Th17 cells and Th17 spectrum-associated proteins (p-STAT3, RORγt and IL-17) were upregulated in CD4+ cells in PV patients. The increased transcriptional levels of RORγt and IL-17 correlated positively with the severity of PV. Elevated phosphorylation of the ERK signalling factors was found in the collected CD4+ T cells in PV patients. The inhibition of the ERK signalling pathway significantly reduced the differentiation of Th17 cells in PV patients in vitro. Th17 cells are essential in the dysregulation of PV, and ERK signalling is involved in Th17-type immunity and promotes the development of PV. The study here provides us with a potential therapeutic target for PV.

Harnessing chitosan and poly-(γ-glutamic acid)-based biomaterials towards cancer immunotherapy

Cancer-related immunosuppression is one of the most challenging aspects of cancer therapy, being one of the principal causes of disease relapse. Most recent immunotherapeutic strategies have been at the forefront of therapies to overcome this problem through targeting T cell-related pathways. Notwithstanding their remarkable clinical success, current immunotherapies have still benefited a small percentage of patients. As such, considering solid tumors are virtually infiltrated by a high proportion of innate immune cells, frequently mentioned as masters of immunosuppression, targeting these cells may offer a durable immune response. Thus, finding new strategies to reprogram the function of innate immune system in the tumor microenvironment is still an unmet clinical need.Biomaterials-based immunomodulatory therapies have been increasingly explored to achieve better cancer treatments. In particular, chitosan (Ch), a polysaccharide derived from the deacetylation of chitin, as well as poly-γ-glutamic acid (γ-PGA), a polymer bacterially synthetized, that can spontaneously self-assemble by electrostatic interactions in different structures, have been preclinically explored for their immunomodulatory properties. Ch and γ-PGA have been found to activate Toll-like receptors, and trigger common pro-inflammatory immune pathways, such as Nuclear factor kappa B (NF-кB) and Mitogen-activated protein kinase (MAPK). Other pathways and receptors, such as the Retinoic Acid Orphan Receptor γt (RORγt), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT), or the inflammasome have also been shown to be differentially activated by either polymer. Hence the prospective application of Ch/γ-PGA formulations in cancer therapy, in an effort to synergize their effects to heighten the therapeutic response via innate immunity, could constitute a novel immunotherapeutic strategy. This review highlights how Ch and γ-PGA can efficiently reprogram innate immune cells in tumors, and ultimately initiate activation of T cell immunity against advanced cancers. Moreover, the therapeutic potential of Ch/γ-PGA-based biomaterials as adjuvants of conventional cancer therapies and as diagnostic tools is discussed.

IL-23 signaling in Th17 cells is inhibited by HIV infection and is not restored by HAART: Implications for persistent immune activation.

ObjectivesHIV infection causes a profound depletion of gut derived Th17 cells, contributing to loss of mucosal barrier function and an increase in microbial translocation, thus driving systemic immune activation. Despite normalization of circulating CD4+ T cell counts with highly active antiretroviral therapy (HAART), Th17 frequency and function often remain impaired. Given the importance of interleukin (IL)-23 in the generation and stabilization of Th17 cells we hypothesized that impaired IL-23 signaling causes persistent Th17 dysfunction in HIV infection.MethodsThe effects of in vitro HIV infection on responses to IL-23 in Th17 cells were examined. These included the production of IL-17, phosphorylated STAT3 (pSTAT3) and the transcription of retinoic acid orphan receptor C (RORC) gene. Blood derived Th17 cells from untreated and HAART-treated HIV-infected individuals were also examined for the IL-23 induced production of phosphorylated STAT3 (pSTAT3) and the expression of the IL-23 receptors.ResultsIn vitro HIV infection significantly inhibited IL-17 production and IL-23 induced pSTAT3 while expression of RORC RNA was unaffected. Th17 cells isolated from untreated and HAART-treated HIV-infected individuals showed complete loss of IL-23 induced pSTAT3 without a decrease in the expression of the IL-23 receptors.ConclusionsThis study is the first to demonstrate an effect of HIV on the IL-23 signaling pathway in Th17 cells. We show that in vitro and in vivo HIV infection results in impaired IL-23 signaling which is not reversed by HAART nor is it a result of reduced receptor expression, suggesting that HIV interferes with IL-23-activated signaling pathways. These findings may explain the inability of HAART to restore Th17 frequency and function and the resulting persistent chronic immune activation observed in HIV infected individuals.

Musculin inhibits human T-helper 17 cell response to interleukin 2 by controlling STAT5B activity.

We recently demonstrated that human T-helper (Th) 17 cells, unlike Th1 cells, do not proliferate in response to T-cell receptor stimulation, mainly because of their reduced capacity to produce and respond to IL-2. In this study, we show that their lower responsiveness to IL-2 is due to the selective expression of Musculin (MSC), a member of the basic helix-loop-helix transcription factors. We show that MSC expression in human Th17 cells is retinoic acid orphan receptor (ROR)γt-dependent, and allows the upregulation of PPP2R2B, a regulatory member of the protein phosphatase 2A (PP2A) enzyme. High PPP2R2B levels in human Th17 cells were responsible for the reduced STAT5B Ser-193 phosphorylation upon IL-2 signalling and, therefore, impaired STAT5B DNA binding and transcriptional activity on IL-2 target genes. PP2A, observed in Th17 cells, controls also STAT3, dephosphorylating Ser727, thus increasing its activity that plays a crucial role in Th17 development and/or maintenance. Thus, our findings identify an additional mechanism responsible for the limited expansion of human Th17 cells, and could provide a further explanation for the rarity of these cells in inflamed tissues.

Human circulating group 2 innate lymphoid cells can express CD154 and promote IgE production

Protection against helminths consists of adaptive responses by TH2 cells and innate responses by group 2 innate lymphoid cells (ILC2s), with these latter being well characterized in mice but less so in human subjects. We sought to characterize human circulating ILC2s and compare their functional profile with that of autologous TH2 cells. Circulating ILC2s and TH2 cells were isolated by means of fluorescence-activated cell sorting and magnetic cell sorting and expanded invitro. ILC2s were then stimulated with phorbol 12-myristate 13-acetate plus ionomycin, IL-25 plus IL-33 (IL-25/IL-33), or a mixture of Toll-like receptor ligands to evaluate their ability to produce cytokines, express CD154, and induce IgE production by autologous Bcells. Cytokines and transcription factor gene methylation were assessed. ILC2s expressed GATA-3, retinoic acid orphan receptor (RORC) 2, and RORα; were able to produce IL-5, IL-13, and IL-4; and, accordingly, were characterized by demethylation of IL4, IL13, IL5, GATA3, and RORC2, whereas the IFNG, IFNG promoter, and TBX21 regions of interest were methylated. ILC2s expressed TLR1, TLR4, and TLR6, and TLR stimulation induced IL-5 and IL-13 production. Moreover, ILC2s expressed CD154 in response to phorbol 12-myristate 13-acetate plus ionomycin, IL-25/IL-33, or a mixture of TLR ligands. Stimulated ILC2s also induced IgM, IgG, IgA, and IgE production by Bcells. Finally, circulating ILC2s from atopic patients were not different in numbers and frequency but expressed higher IL-4 levels than those from nonatopic subjects. This study provides the first evidence that human ILC2s can express CD154 and stimulate the production of IgE by B lymphocytes through IL-25/IL-33 stimulation or TLR triggering.

Demethylation of the RORC2 and IL17A in human CD4+ T lymphocytes defines Th17 origin of nonclassic Th1 cells.

Th17-derived Th1 lymphocytes, termed nonclassic, differ from classic Th1 cells because of the presence of retinoic acid orphan receptor (ROR)C2 and the surface expression of CD161 and CCR6. We demonstrate in this article that nonclassic Th1 cells, like Th17 cells, have a marked RORC2 and IL17A demethylation, whereas classic Th1 cells exhibit a complete methylation of these genes. The analysis of RORC2 DNA methylation in the CD4(+)CD161(+) and CD4(+)CD161(-) naive Th subsets from umbilical cord blood surprisingly revealed comparable hypermethylation levels. PCR analysis at the single-cell level revealed that RORC2 mRNA was expressed by none of the CD4(+)CD161(-) and present only in a minority of CD4(+)CD161(+) naive Th cells. These findings provide two important novel observations on the physiology of human Th17 cells: 1) they confirm at the epigenetic level the origin of nonclassic Th1 cells from Th17 cells, also identifying in the RORC2 and IL17A methylation status a novel tool for their distinction from classic Th1 cells, and 2) they demonstrate that RORC2-expressing cells are only a minority in the subset of CD4(+)CD161(+) naive Th cells, which are known to contain all Th17 cell precursors.

Human Th1 dichotomy: origin, phenotype and biologic activities.

The great variety of pathogens present in the environment has obliged the immune system to evolve different mechanisms for tailored and maximally protective responses. Initially, two major types of CD4+ T helper (Th) effector cells were identified, and named as type 1 (Th1) and type (Th2) cells because of the different cytokines they produce. More recently, a third type of CD4+ Th effectors has been identified and named as Th17 cells. Th17 cells, however, have been found to exhibit high plasticity because they rapidly shift into the Th1 phenotype in the inflammatory sites. Therefore, in these sites usually there is a dichotomic mixture of classic and non classic (Th17-derived) Th1 cells. In humans, non classic Th1 cells express CD161, as well as the retinoic acid orphan receptor C, IL-17 receptor E, IL-1RI, CCR6, and IL-4-induced gene 1 and Tob-1, which are all virtually absent from classic Th1 cells. The possibility to distinguish these two cell subsets may allow the opportunity to better establish their respective pathogenic role in different chronic inflammatory disorders. In this review, we discuss the different origin, the distinctive phenotypic features and the major biologic activities of classic and non classic Th1 cells. This article is protected by copyright. All rights reserved.

No association between RORA polymorphisms and PTSD in two independent samples.

Logue et al.1 reported genome-wide significant association between a polymorphism (rs8042149) in the RORA gene, encoding the retinoic acid orphan receptor A, and posttraumatic stress disorder (PTSD) in a cohort of trauma-exposed white non-Hispanic US veterans and their partners. The genome-wide association study yielded evidence of association for three additional SNPs at the 10-6 threshold in the same cohort (rs8041061, rs8024133, rs11071561). Amstadter et al.2 reported a significant association between rs8042149 and PTSD symptoms in the 2004 Florida Hurricane Study. The RORA gene encodes a nuclear hormone receptor that regulates the transcription activity of nearby genes. It is widely expressed in the brain, where it protects cortical neurons against oxidative stress-induced apoptosis by increasing the expression of antioxidant proteins.1 Logue et al.1 proposed that genetic variations in RORA may alter its expression, reducing the capacity of neurons to respond to biochemical stressors induced by traumatic stress.

S100A4 regulates the Src-tyrosine kinase dependent differentiation of Th17 cells in rheumatoid arthritis

Objectives: To evaluate the role of S100A4, a calcium-binding regulator of nonmuscle myosin assembly, for T-cell responses in rheumatoid arthritis. Methods: Arthritis was induced in the methylated bovine serum albumin (mBSA)-immunized mice lacking the entire S100A4 protein (S100A4KO) and in wild-type counterparts treated with short hairpin ribonucleic acid (shRNA)-lentiviral constructs targeting S100A4 (S100A4-shRNA). The severity of arthritis was evaluated morphologically. T-cell subsets were characterized by the expression of master transcription factors, and functionally by proliferation activity and cytokine production. The activity of the Scr-kinases Fyn and Lck was assessed by the autophosphorylation of C-terminal thyrosine and by the phosphorylation of the CD5 cytodomain. The interaction between S100A4 and the CD5 cytodomain was analysed by nuclear magnetic resonance spectrophotometry. Results: S100A4-deficient mice (S100A4KO and S100A4-shRNA) had significantly alleviated morphological signs of arthritis and joint damage. Leukocyte infiltrates in the arthritic joints of S100A4-deficient mice accumulated Foxp3+ Treg cells, while the number of RORγt+ and (pTyr705)STAT3+ cells was reduced. S100A4-deficient mice had a limited formation of Th17-cells with low retinoic acid orphan receptor gamma t (RORγt) mRNA and IL17 production in T-cell cultures. S100A4-deficient mice had a low expression and activity of T-cell receptor (TCR) inhibitor CD5 and low (pTyr705)STAT3 (signal transducer and activator of transcription 3), which led to increased (pTyr352)ZAP-70 (theta-chain associated protein kinase of 70kDa), lymphocyte proliferation and production of IL2. In vitro experiments showed that S100A4 directly binds Lck and Fyn and reciprocally regulates their kinase activity towards the CD5 cytodomain. Spectrometry demonstrates an interaction between the CD5 cytodomain and EF2-binding sites of S100A4. Conclusion: The present study demonstrates that S100A4 plays an important part in the pathogenesis of arthritis. It controls CD5-dependent differentiation of Th17 cells by regulating the activity of the Src-family kinases Lck and Fyn.

AB0146 Ursolic Acid Ameliorates Autoimmune Arthritis via Suppression of TH17 and B Cell Differentiation

BackgroundUrsolic acid (UA) is a pentacytclic triterpenoid compound which is ubiquitously present in plants.ObjectivesBased on its known anti-inflammatory and anti-oxidative effect, this study was undertaken to investigate the effect of...

Imbalance of peripheral blood Th17 and Treg responses in patients with chronic obstructive pulmonary disease

Autoimmune responses mediated by cluster of differentiation 4(+) T cells may contribute to the development of chronic obstructive pulmonary disease (COPD). However, little is known about the frequency of peripheral blood Th17 cells and of regulatory T cells (Tregs) in Chinese patients with COPD. This study is aimed at determining the frequency of circulating Th17 and Tregs in patients with moderate and severe COPD, heavy smokers and healthy controls (HC). The percentages of circulating T-helper type (Th)17 cells and Tregs were determined by flow cytometry in 32 patients with moderate COPD, 33 patients with severe COPD, 35 smokers, and 31 HC. The concentrations of serum Th17- and Treg-related cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The levels of retinoic acid orphan receptor (ROR)-γt and Forkhead box p3 (Foxp3) mRNA transcripts in peripheral blood mononuclear cells were determined by real-time polymerase chain reaction. The potential correlation between the percentages of Th17 Tregs, serum cytokines and lung function was evaluated. In comparison with that in the smokers and HC, significantly higher frequencies of Th17 cells and higher levels of ROR-γt mRNA transcripts and serum interleukin (IL)-17A, IL-6, IL-21, IL-22 and IL-23, but lower frequency of Tregs and lower levels of Foxp3 and serum IL-10 were detected in patients with moderate and severe COPD. The increased ratios of Th17 to Tregs were negatively correlated with the values of forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1) and FEV1/FVC. An imbalance of circulating Th17 cells and Tregs is associated with the deterioration of pulmonary function in patients with moderate and severe COPD.

Detection of IL-17A-producing peripheral blood monocytes in Langerhans cell histiocytosis patients.

Langerhans cell histiocytosis (LCH) is a rare disease of unknown cause with manifestations ranging from isolated granulomatous lesions to life-threatening multi-system organ involvement. This disorder is further characterized by infiltration of immune cells in affected tissues and an association with interleukin (IL)-17A has been reported. Here, we investigated the presence of IL-17A-producing cells among peripheral blood mononuclear cells isolated from LCH patients and observed a high percentage of IL-17A(+) monocytes in peripheral blood of LCH patients compared to controls. The IL-17A(+) monocytes were also positive for the transcription factor retinoic acid orphan receptor (ROR) γt and showed increased mRNA levels for both IL-17A and RORγt. Notably, IL-17A was produced by all monocyte subsets and the expression level was positively associated with LCH disease activity. These data support a role for monocytes in the pathogenesis of LCH. Future therapeutic approaches may consider identification of patients who may benefit from IL-17A-targeted interventions.

Interleukin-17 in atherosclerosis and cardiovascular disease: the good, the bad, and the unknown

This editorial refers to ‘Circulating levels of interleukin-17 and cardiovascular outcomes in patients with acute myocardial infarction’, by T. Simon et al. , doi:10.1093/eurheartj/ehs263 Over the past few years our understanding of the importance of adaptive immunity in acute coronary syndromes (ACS) has considerably increased.1 Profound abnormalities have been observed in specific subsets of CD4+T cells, including type 1 helper T (Th1) cells,2,3 CD4+CD28nullT cells,4 and naturally occurring regulatory T cells (Treg cells).5 Recently a new lineage of CD4+T cells, type 17 helper T (Th17) cells producing the signature cytokines interleukin (IL)-17, IL-21, and IL-22, has been identified.6 This has fundamentally changed the Th1/Th2 dichotomy paradigm. The Th1 cell subset is characterized by interferon (IFN)-γ production and T-bet expression, whereas the Th2 cell lineage shows IL-4, IL-5, and IL-10 production and GATA-3 expression. At the other extreme, Treg cells expressing FoxP3 are essential to maintain the homeostasis of cell subsets involved in adaptive immunity by contact-dependent suppression or by releasing anti-inflammatory cytokines, IL-10 and transforming growth factor (TGF)-β1.7 Like Th1, Th2, and Treg cells, specific polarizing cytokines—TGF-β, IL-6, and IL-23—are required for differentiation of Th17 cells which express the transcription factors retinoic acid orphan receptor (ROR) α and RORγt. However, Th17 cells do not represent a homogeneous lineage, as they can be reprogrammed to other T-cell subsets depending on the cytokine environment.6 Accordingly, IL-17 can be co-expressed with a variety of other cytokines including IFN-γ, IL-10, and IL-4. Indeed, a mixed Th1–Th17 subgroup expressing both T-bet and RORγt has been recently identified in the setting of autoimmunity.6 Plasticity …

Distinctive features of classic and nonclassic (Th17 derived) human Th1 cells

T helper17 (Th17) lymphocytes represent a third arm of the CD4(+) T-cell effector responses, in addition to Th1 and Th2 cells. Th17 cells have been found to exhibit high plasticity because they rapidly shift into the Th1 phenotype in inflammatory sites. In humans, Th1 cells derived from Th17 cells express CD161, whereas classic Th1 cells do not; these Th17-derived Th1 cells have been termed nonclassic Th1 cells. In this study, we examined similarities and differences between classic and nonclassic human Th1 cells by assessing a panel of T-cell clones, as well as CD161(+) or CD161(-) CD4(+) T cells derived ex vivo from the circulation of healthy subjects or the synovial fluid of patients with juvenile idiopathic arthritis. The results show that nonclassic Th1 cells can be identified based on CD161 expression, as well as the consistent expression of retinoic acid orphan receptor C, IL-17 receptor E, CCR6, and IL-4-induced gene 1, which are all virtually absent in classic Th1 cells. The possibility to distinguish these two-cell subsets by using such a panel of markers may allow the opportunity to better establish the respective pathogenic roles of classic and nonclassic (Th17 derived) Th1 cells in different chronic inflammatory disorders.

Imbalance of Th17/Treg cells in mice with chronic cigarette smoke exposure

BackgroundRecent studies have revealed that autoimmune responses mediated by CD4+ T cells may contribute to the development of chronic obstructive pulmonary disease (COPD). Meanwhile, imbalance of Th17/Treg has been reported to play a key role in the pathogenesis of autoimmune diseases. However, information on Th17/Treg balance in COPD is relatively limited. MethodWe established a mouse model of COPD induced by chronic cigarette smoke (CS) exposure. Th17 and Treg in lung tissue and peripheral blood were quantified by flow cytometry. The level of the specific transcription factors of both T cell subsets in lung tissue was determined by real-time PCR. The expressions of Th17- and Treg-related cytokines in serum and bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA). ResultsWe found that mice with chronic CS exposure showed significant increase in lung Th17 prevalence, retinoic acid orphan receptor (ROR)-γt mRNA and Th17-related cytokines (IL-17A, IL-6 and IL-23). Meanwhile, there was obvious decrease in Treg cell prevalence, Forkhead box (Fox) p3 mRNA and Treg-related cytokine IL-10, as compared to mice underwent sub-acute CS exposure and air-exposure. Similar tendency was also found for the Th17/Treg ratio in peripheral blood. ConclusionsOur study thus reveals that the Th17/Treg imbalance exists in mice with chronic CS exposure, suggesting its potential role in the breakdown of immune self-tolerance in COPD. Further research on regulation of Th17/Treg balance may provide insights into the development of new therapeutic targets for this disease.

Cutting edge: lymphoid tissue inducer cells maintain memory CD4 T cells within secondary lymphoid tissue.

Phylogeny shows that CD4 T cell memory and lymph nodes coevolved in placental mammals. In ontogeny, retinoic acid orphan receptor (ROR)γ-dependent lymphoid tissue inducer (LTi) cells program the development of mammalian lymph nodes. In this study, we show that although primary CD4 T cell expansion is normal in RORγ-deficient mice, the persistence of memory CD4 T cells is RORγ-dependent. Furthermore, using bone marrow chimeric mice we demonstrate that LTi cells are the key RORγ-expressing cell type sufficient for memory CD4 T cell survival in the absence of persistent Ag. This effect was specific for CD4 T cells, as memory CD8 T cells survived equally well in the presence or absence of LTi cells. These data demonstrate a novel role for LTi cells, archetypal members of the innate lymphoid cell family, in supporting memory CD4 T cell survival in vivo.