Pin1 maintains the effector program of pathogenic Th17 cells in autoimmune neuroinflammation.

Th17 cells mediated immune response is the basis of a variety of autoimmune diseases, including multiple sclerosis and its mouse model of immune aspects, experimental autoimmune encephalomyelitis (EAE). The gene network that drives both the development of Th17 and the expression of its effector program is dependent on the transcription factor RORγt. In this report, we showed that Peptidylprolyl Cis/Trans Isomerase, NIMA-Interacting 1 (Pin1) formed a complex with RORγt, and enhanced its transactivation activity, thus sustained the expression of the effector genes as well as RORγt in the EAE-pathogenic Th17 cells. We first found out that PIN1 was highly expressed in the samples from patients of multiple sclerosis, and the expression of Pin1 by the infiltrating lymphocytes in the central nerve system of EAE mice was elevated as well. An array of experiments with transgenic mouse models, cellular and molecular assays was included in the study to elucidate the role of Pin1 in the pathology of EAE. It turned out that Pin1 promoted the activation and maintained the effector program of EAE-pathogenic Th17 cells in the inflammation foci, but had little effect on the priming of Th17 cells in the draining lymph nodes. Mechanistically, Pin1 stabilized the phosphorylation of STAT3 induced by proinflammatory stimuli, and interacted with STAT3 in the nucleus of Th17 cells, which resulted in the increased expression of Rorc. Moreover, Pin1 formed a complex with RORγt, and enhanced the transactivation of RORγt to the +11 kb enhancer of Rorc, which enforced and maintained the expression of both Rorc and the effector program of pathogenic Th17 cells in EAE. Finally, the inhibition of Pin1, by genetic knockdown or by small molecule inhibitor, deceased the population of Th17 cells and the neuroinflammation, and alleviated the symptoms of EAE. These findings suggest that Pin1 is a potential therapeutic target for MS and other autoimmune inflammatory diseases.

Evaluation of the activity of cardiac glycosides on RORγ and RORγT nuclear receptors.

Cardiac glycosides, derived from plants and animals, have been recognized since ancient times. These substances hinder the function of the sodium-potassium pump within eukaryotic cells. Many reports have shown that these compounds influence the activity of nuclear receptors. Thus, we assessed the effects of various cardiac glycosides at nontoxic concentrations on RORγ and RORγT. RORγT is a crucial protein involved in the differentiation of Th17 lymphocytes. Sixteen analyzed cardiac glycosides exhibited varying toxicities in HepG2 cells, all of which demonstrated agonistic effects on RORγ, as confirmed in the RORγ-HepG2 reporter cell line. The overexpression of both the RORγ and RORγT isoforms intensified the effects of these compounds. Additionally, these glycosides induced the expression of G6PC, a gene regulated by RORγ, in HepG2 cells. Subsequently, the effects of two endogenous cardiac glycosides (marinobufagenin and ouabain) and the three most potent glycosides (bufalin, oleandrin, and telecinobufagenin) were evaluated in Th17 primary lymphocytes. All of these compounds increased the expression of the IL17A, IL17F, IFNG, and CXCL10 genes, but they exhibited varying effects on GZMB and CCL20 expression. Molecular docking analysis revealed the robust binding affinity of cardiac glycosides for the ligand binding domain of the RORγ/RORγT receptors. Thus, we demonstrated that at nontoxic concentrations, cardiac glycosides have agonistic effects on RORγ/RORγT nuclear receptors, augmenting their activity. This potential can be harnessed to modulate the phenotype of IL17-expressing cells (e.g., Th17 or Tc17 lymphocytes) in adoptive therapy for combating various types of cancer.

Shifting phenotype and differentiation of CD11b+Gr.1+ immature heterogeneous myeloid derived adjuster cells support inflammation and induce regulators of IL17A in imiquimod induced psoriasis.

OBJECTIVE AND DESIGN: The exact immunological mechanism of widespread chronic inflammatory skin disorder psoriasis has not been fully established. CD11b+Gr.1+ myeloid-derived cells are immature heterogeneous cells with T-cell suppressive property in neoplasia; however, influence of these cells on adaptive immunity is highly contextual; therefore, we dubbed these cells as myeloid-derived adjuster cells (MDAC). We studied imiquimod induced psoriasis in mouse model and evaluated for the first time the RORγt-NFAT1 axis in MDACs and the function, differentiation and interaction of these cells with T cells. MATERIALS AND METHODS: The status of T cells and MDACs; their functionality and differentiation properties, and the roles of RORγt and NFAT1 in MDACs were evaluated using flow cytometry, qRT-PCR and confocal imaging. RESULTS: We found gradual increase in T cells and MDACs and an increase in the number of IL17 -secreting MDACs and T cells in the skin of psoriatic animals. We also noted that MDAC differentiation is biased toward M1 macrophages and DCs which perpetuate inflammation. We found that psoriatic MDACs were unable to suppress T-cell proliferation or activation but seemingly helped these T cells produce more IL17. Inhibition of the RORγt/NFAT1 axis in MDACs increased the suppressive nature of MDACs, allowing these cells to suppress the activity of psoriatic T-cells. CONCLUSION: Our results indicate that altered MDAC properties in psoriatic condition sustains pathological inflammation and RORγt and NFAT1 as promising intervention target for psoriasis management.

Extrathymic AIRE-Expressing Cells: A Historical Perspective.

Since its discovery, Aire has been the topic of numerous studies in its role as a transcriptional regulator in the thymus where it promotes the "promiscuous" expression of a large repertoire of tissue-restricted antigens (TRAs) that are normally expressed only in the immune periphery. This process occurs in specialized medullary thymic epithelial cells (mTECs) and mediates the elimination of self-reactive T cells or promotes their conversion to the Foxp3+ regulatory T cell lineage, both of which are required for the prevention of autoimmunity. In recent years, there has been increasing interest in the role of extrathymic Aire expression in peripheral organs. The focus has primarily been on the identification of the cellular source(s) and mechanism(s) by which extrathymic AIRE affects tolerance-related or other physiological processes. A cadre of OMICs tools including single cell RNA sequencing and novel transgenic models to trace Aire expression to perform lineage tracing experiments have shed light on a phenomenon that is more complex than previously thought. In this chapter, we provide a deeper analysis of how extrathymic Aire research has developed and progressed, how cellular sources were identified, and how the function of AIRE was determined. Current data suggests that extrathymic AIRE fulfills a function that differs from what has been observed in the thymus and strongly argues that its main purpose is to regulate transcriptional programs in a cell content-dependent manner. Surprisingly, there is data that also suggests a non-transcriptional role of extrathymic AIRE in the cytoplasm. We have arrived at a potential turning point that will take the field from the classical understanding of AIRE as a transcription factor in control of TRA expression to its role in immunological and non-immunological processes in the periphery.

USP25 attenuates anti-GBM nephritis in mice by negative feedback regulation of Th17 cell differentiation.

PURPOSE: This study aimed to elucidate the role of USP25 in a mouse model of anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). METHODS: USP25-deficient anti-GBM GN mice were generated, and their nephritis progression was monitored. Naïve CD4+ T cells were isolated from spleen lymphocytes and stimulated to differentiate into Th1, Th2, and Th17 cells. This approach was used to investigate the impact of USP25 on CD4+ T lymphocyte differentiation in vitro. Furthermore, changes in USP25 expression were monitored during Th17 differentiation, both in vivo and in vitro. RESULTS: USP25-/- mice with anti-GBM GN exhibited accelerated renal function deterioration, increased infiltration of Th1 and Th17 cells, and elevated RORγt transcription. In vitro experiments demonstrated that USP25-/- CD4+ T lymphocytes had a higher proportion for Th17 cell differentiation and exhibited higher RORγt levels upon stimulation. Wild-type mice with anti-GBM GN showed higher USP25 levels compared to healthy mice, and a positive correlation was observed between USP25 levels and Th17 cell counts. Similar trends were observed in vitro. CONCLUSION: USP25 plays a crucial role in mitigating renal histopathological and functional damage during anti-GBM GN in mice. This protective effect is primarily attributed to USP25's ability to inhibit the differentiation of naïve CD4+ T cells into Th17 cells. The underlying mechanism may involve the downregulation of RORγt. Additionally, during increased inflammatory responses or Th17 cell differentiation, USP25 expression is activated, forming a negative feedback regulatory loop that attenuates immune activation.

27-hydroxycholesterol causes cognitive deficits by disturbing Th17/Treg balance and the related immune responses in mild cognitive impairment patients and C57BL/6J mice.

BACKGROUND: Cognitive impairment is associated with dysregulated immune responses. Emerging evidence indicates that Th17 cells and their characteristic cytokine-IL-17 are receiving growing interest in the pathogenesis of cognitive decline. Here, we focus on the involvement of Th17 cells in mild cognitive impairment (MCI) and the possible mechanism of cholesterol metabolite-27-hydroxycholesterol (27-OHC). METHODS: 100 individuals were recruited into the nested case-control study who completed cognition assessment and the detection of oxysterols and Th17-related cytokines in serum. In addition, mice were treated with 27-OHC and inhibitors of RORγt and Foxp3 (Th17 and Treg transcription factors), and the factors involved in Th17/Treg balance and amyloidosis were detected. RESULTS: Our results showed there was enhanced 27-OHC level in serum of MCI individuals. The Th17-related cytokines homeostasis was altered, manifested as increased IL-17A, IL-12p70, IL-23, GM-CSF, MIP-3α and TNF-α but decreased IL-13, IL-28A and TGF-ß1. Further, in vivo experiments showed that 27-OHC induced higher immunogenicity, which increased Th17 proportion but decreased Treg cells in peripheral blood mononuclear cells (PBMCs); Th17 proportions in hippocampus, and IL-17A level in serum and brain were also higher than control mice. The fluorescence intensity of amyloid-ß (Aß) and the precursor of amyloid A amyloidosis-serum amyloid A (SAA) was increased in the brain of 27-OHC-treated mice, and worse learning and memory performance was supported by water maze test results. While by inhibiting RORγt in 27-OHC-loaded mice, Th17 proportions in both PBMCs and hippocampus were reduced, and expressions of IL-17A and TGF-ß1 were down- and up-regulated, respectively, along with a decreased amyloidosis in brain and improved learning and memory decline. CONCLUSIONS: Altogether, our results demonstrate that excessive 27-OHC aggravates the amyloidosis and leads to cognitive deficits by regulating RORγt and disturbing Th17/Treg balance.

Pharmacokinetics, Safety, and Tolerability of Cedirogant in Healthy Japanese and Chinese Adults.

Cedirogant is an inverse agonist of retinoic acid-related orphan receptor gamma, thymus (RORγt) developed for treatment of psoriasis. This study aimed to characterize pharmacokinetics, pharmacodynamics, safety, and tolerability of cedirogant following a single oral dose in Japanese participants and multiple oral doses in Japanese and Chinese participants. The single doses evaluated in healthy Japanese participants were 75, 225, and 395 mg. The multiple doses evaluated in both healthy Japanese and Chinese participants was 375 mg once daily for 14 days. Cedirogant plasma exposure increased dose proportionally with administration of single doses. Maximum cedirogant plasma concentration was reached within a median time of 4-5 hours after dosing. The harmonic mean elimination half-life ranged from 19 to 25 hours. Cedirogant pharmacokinetics were similar between Japanese and Chinese participants. Compared with healthy Western participants in a cross-study analysis, steady-state cedirogant plasma exposure was 38%-73% higher in Japanese or Chinese participants. Ex vivo interleukin-17 inhibition increased in a dose-dependent manner and was maximized by 375 mg once-daily doses. The cedirogant regimens tested were generally well tolerated, and no new safety issues were identified. The results supported enrollment of Japanese and Chinese subjects in subsequent clinical trials for cedirogant.

Nrf2 Deficiency Accelerates IL-17-Dependent Neutrophilic Airway Inflammation in Asthmatic Mice.

Asthma is a heterogeneous disease that can be broadly classified into type 2, which is primarily steroid-sensitive and eosinophilic, and non-type 2, which is primarily steroid-resistant and neutrophilic. While the mechanisms leading to the development of molecular-targeted therapies for type 2 asthma are being elucidated, much remains to be learned about non-type 2 asthma. To investigate the role of oxidative stress in refractory allergic airway inflammation, we compared asthma models generated by immunizing wild-type and nuclear factor erythroid-2-related factor 2 (Nrf2)-deficient mice with the house dust mite antigen. Both asthma models had similar levels of airway inflammation and hyperresponsiveness, but the Nrf2-deficient mice had increased oxidative stress and exacerbated neutrophilic airway inflammation compared with the wild-type mice. Type 2 cytokines and the expression of GATA3, a transcription factor that is important for Th2 cell differentiation, had decreased in Nrf2-deficient mice compared with the wild-type mice, whereas helper T (Th) 17 cytokines and the expression of RORγt, which is important for Th17 cell differentiation, had increased. Furthermore, the neutrophilic airway inflammation caused by Nrf2 deficiency was ameliorated by interleukin (IL)-17 neutralization. We have concluded that the disruption of the Nrf2-mediated antioxidant defense system contributed to the induction of Th17 differentiation and exacerbated allergic neutrophilic airway inflammation.

SPH7854, a gut-limited RORγt antagonist, ameliorates TNBS-induced experimental colitis in rat.

Multiple lines of evidence suggest that Retinoic Acid Related Orphan Nuclear Receptor gamma t (RORγt) is a potent therapeutic target for inflammatory bowel disease (IBD). However, systemic blockade of RORγt easily leads to thymic lymphoma and aberrant liver function. Therefore, the development of gut-limited RORγt antagonists may lead to the development of innovative IBD therapeutics that improve safety and retain effectiveness. We discovered SPH7854, a potent and selective RORγt antagonist. The effect of SPH7854 on the differentiation of T helper 1 (Th1)/Th17/regulatory T (Treg) cells was evaluated in mouse and human primary cells. SPH7854 (2-(4-(ethylsulfonyl)phenyl)-N- (6-(2-methyl-2-(pyridin-2-yl) propanoyl)pyridin-3-yl)acetamide) dose-dependently inhibited interleukin-17A (IL-17A) secretion from mouse CD4 + T cells and human peripheral blood mononuclear cells (PBMC). Additionally, SPH7854 strongly suppressed Th17 cell differentiation and considerably promoted Treg cell differentiation while slightly affected Th1 cell differentiation from mouse CD4 + T cells. The pharmacokinetic (PK) studies indicated that SPH7854 was restricted to the gut: the bioavailability and maximal plasma concentration of SPH7854 after oral administration (6 mg/kg) were 1.24 ± 0.33 % and 4.92 ± 11.81 nM, respectively, in rats. Strikingly, oral administration of SPH7854 (5 mg/kg and 15 mg/kg) twice daily significantly alleviated 2, 4, 6-trinitrobenzensulfonic acid (TNBS)-induced colitis in rats. SPH7854, especially at 15 mg/kg, significantly alleviated symptoms and improved macroscopic signs and microscopic structure in rat colitis, with decreased colonic mucosal levels of IL-17A, IL-6, tumor necrosis factor α (TNFα), monocyte chemoattractant protein-1 (MCP-1) and myeloperoxidase (MPO). These evidences indicated that blockade of RORγt activity via a gut-limited antagonist may be an effective and safe therapeutic strategy for IBD treatment.

Eosinophils Contribute to Oral Tolerance via Induction of RORγt-Positive Antigen-Presenting Cells and RORγt-Positive Regulatory T Cells.

Oral tolerance has been defined as the specific suppression of immune responses to an antigen by prior oral administration of the antigen. It has been thought to serve to suppress food allergy. Previous studies have shown that dendritic cells (DCs) and regulatory T cells (Tregs) are involved in the induction of oral tolerance. However, the detailed mechanisms of Treg induction in oral tolerance remain largely unknown. Eosinophils have been recognized as effector cells in allergic diseases, but in recent years, the diverse functions of tissue-resident eosinophils have been reported. Eosinophils in the intestine have been reported to induce Tregs by releasing TGF-ß, but the role of eosinophils in oral tolerance is still controversial. In this study, we analyzed the roles of eosinophils in oral tolerance using eosinophil-deficient ΔdblGATA mice (mice lacking a high-affinity GATA-binding site in the GATA1 promoter). ΔdblGATA mice showed impaired antigen-induced oral tolerance compared to wild-type mice. The induction of RORγt+ Tregs in mesenteric lymph nodes (MLNs) by oral tolerance induction was impaired in ΔdblGATA mice compared to wild-type mice. An increase in RORγt+ antigen-presenting cells (APCs), which are involved in RORγt+ Treg differentiation, in the intestine and MLNs was not seen in ΔdblGATA mice. Notably, the expansion of group 3 innate lymphoid cells (ILC3s), a subset of RORγt+ APCs, by oral tolerance induction was seen in wild-type mice but not ΔdblGATA mice. These results suggest that eosinophils are crucial in the induction of oral tolerance, possibly via the induction of RORγt+ APCs and RORγt+ Tregs.

Ursodeoxycholic acid and 18ß-glycyrrhetinic acid alleviate ethinylestradiol-induced cholestasis via downregulating RORγt and CXCR3 signaling pathway in iNKT cells.

Estrogen-induced intrahepatic cholestasis (IHC) is a mild but potentially serious risk and urges for new therapeutic targets and effective treatment. Our previous study demonstrated that RORγt and CXCR3 signaling pathway of invariant natural killer T (iNKT) 17 cells play pathogenic roles in 17α-ethinylestradiol (EE)-induced IHC. Ursodeoxycholic acid (UDCA) and 18ß-glycyrrhetinic acid (GA) present a protective effect on IHC partially due to their immunomodulatory properties. Hence in present study, we aim to investigate the effectiveness of UDCA and 18ß-GA in vitro and verify the accessibility of the above targets. Biochemical index measurement indicated that UDCA and 18ß-GA presented efficacy to alleviate EE-induced cholestatic cytotoxicity. Both UDCA and 18ß-GA exhibited suppression on the CXCL9/10-CXCR3 axis, and significantly restrained the expression of RORγt in vitro. In conclusion, our observations provide new therapeutic targets of UDCA and 18ß-GA, and 18ß-GA as an alternative treatment for EE-induced cholestasis.

Decreased GATA3 levels cause changed mouse cutaneous innate lymphoid cell fate, facilitating hair follicle recycling.

In mice, skin-resident type 2 innate lymphoid cells (ILC2s) exhibit some ILC3-like characteristics. However, the underlying mechanism remains elusive. Here, we observed lower expression of the ILC2 master regulator GATA3 specifically in cutaneous ILC2s (cILC2s) compared with canonical ILC2s, in line with its functionally divergent role in transcriptional control in cILC2s. Decreased levels of GATA3 enabled the expansion of RORγt fate-mapped (RORγtfm+) cILC2s after postnatal days, displaying certain similarities to ILC3s. Single-cell trajectory analysis showed a sequential promotion of the RORγtfm+ cILC2 divergency by RORγt and GATA3. Notably, during hair follicle recycling, these RORγtfm+ cILC2s accumulated around the hair follicle dermal papilla (DP) region to facilitate the process. Mechanistically, we found that GATA3-mediated integrin α3ß1 upregulation on RORγtfm+ cILC2s was required for their positioning around the DP. Overall, our study demonstrates a distinct regulatory role of GATA3 in cILC2s, particularly in promoting the divergence of RORγtfm+ cILC2s to facilitate hair follicle recycling.

Elevated levels of peripheral Th17 cells and Th17-related cytokines in patients with periampullary adenocarcinoma.

AIM: Periampullary adenocarcinoma (PAC) is a malignant tumor originating at the ampulla of Vater, distal common bile duct, head of the pancreas, ampulla and duodenum. The levels of circulating Th17 cells and Th17-related cytokines in patients with PAC remain unreported. Therefore, the aim of this study was to determine the levels of circulating Th17 cells and Th17-related cytokines in patients with PAC. MATERIALS AND METHODS: Flow cytometry was used to measure Th17 cell proportions in PBMCs from 60 PAC patients and 30 healthy controls. Enzyme-linked immunosorbent assay (ELISA) was used to quantify IL-17A and IL-23 levels in serum samples, while quantitative reverse transcription polymerase chain reaction (qRT-PCR) assessed IL-17A mRNA expression and Th17-related transcription factors (RORγt and STAT3) in tissue samples. RESULTS: The findings showed a substantial increase in Th17 cell percentages, elevated concentrations of IL-17A and IL-23, and higher mRNA expression levels of IL-17A, RORγt, and STAT3 in patients with PAC when compared to healthy controls (HCs). CONCLUSION: Th17 cells play an important role in the pathogenesis of PAC and may represent potential therapeutic targets.

The biotherapeutic Clostridium butyricum MIYAIRI 588 strain potentiates enterotropism of Rorγt+Treg and PD-1 blockade efficacy.

Immune checkpoint inhibitors (ICI) have been positioned as a standard of care for patients with advanced non-small-cell lung carcinomas (NSCLC). A pilot clinical trial has reflected optimistic association between supplementation with Clostridium butyricum MIYAIRI 588 (CBM588) and ICI efficacy in NSCLC. However, it remains to be established whether this biotherapeutic strain may be sufficient to heighten the immunogenicity of the tumor draining lymph nodes to overcome resistance to ICI. Herein, we report that supplementation with CBM588 led to an improved responsiveness to antibody targeting programmed cell death protein 1 (aPD-1). This was statistically associated with a significant decrease in α-diversity of gut microbiota from CBM588-treated mice upon PD-1 blockade. At the level of the tumor-draining lymph node, such combination of treatment significantly lowered the frequency of microbiota-modulated subset of regulatory T cells that express Retinoic Orphan Receptor gamma t (Rorγt+ Treg). Specifically, this strongly immunosuppressive was negatively correlated with the abundance of bacteria that belong to the family of Ruminococcaceae. Accordingly, the colonic expression of both indoleamine 2,3-Dioxygenase 1 (IDO-1) and interleukin-10 (IL-10) were heightened in mice with greater PD-1 blockade efficacy. The CBM588-induced ability to secrete Interleukin-10 of lamina propria mononuclear cells was heightened in tumor bearers when compared with cancer-free mice. Conversely, blockade of interleukin-10 signaling preferentially enhanced the capacity of CD8+ T cells to secrete Interferon gamma when being cocultured with CBM588-primed lamina propria mononuclear cells of tumor-bearing mice. Our results demonstrate that CBM588-centered intervention can adequately improve intestinal homeostasis and efficiently overcome resistance to PD-1 blockade in mice.

Increased gene expression of CCR6 and RORγt in peripheral blood cells of rheumatoid arthritis patients and their correlation with anti-cyclic citrullinated peptide and disease activity.

OBJECTIVES: The significance of T helper 17 (Th17) cells in the pathogenesis of rheumatoid arthritis (RA) has recently been demonstrated in many studies. Retinoic acid receptor-related orphan receptor γt (RORγt) is a transcription factor that is specifically involved in the generation of Th17 cells. Besides, the chemokine receptor CCR6, the receptor for CCL20, is characteristically expressed by these cells. Considering the pivotal roles of Th17 cells in RA pathogenesis, in this study, we assessed the gene expression of CCR6 and RORγt in the peripheral blood leukocytes of new case RA patients. Also, we evaluated their association with anticyclic citrullinated peptide (anti-CCP) antibodies and disease activity. METHODS: Forty-five new case RA patients and 45 healthy persons have been recruited in this investigation. The gene expression of CCR6 and RORγt was evaluated by quantitative real-time PCR (qRT-PCR), and anti-CCP antibodies plasma levels were measured using the enzyme-linked immunosorbent assay (ELISA) technique. Disease activity was measured according to the disease activity score-28 (DAS-28) formula. RESULTS: The gene expression of CCR6 and RORγt increased remarkably in new case RA patients compared to healthy controls (p < .05 and p < .01, respectively). Moreover, there was a positive correlation between RORγt gene expression and parameters, including gene expression of CCR6 (p = .001, r = .461), plasma levels of CCL20 (p = .0009, r = .477), ESR (p = .004, r = .419), DAS-28 (p = .006, r = .402), anti-CCP (p = .019, r = .346), and RF (p = .001, r = .451). Also, CCR6 gene expression was positively associated with the DAS-28 (p = .037, r = .310), plasma levels of anti-CCP (p = .037, r = .312), and ESR (p = .029, r = .327). CONCLUSION: Increased gene expression of CCR6 and RORγt in peripheral blood leukocytes of new case RA patients may contribute to the exacerbation and pathogenesis of RA.