The imbalance of pulmonary Th17/Treg cells in BALB/c suckling mice infected with respiratory syncytial virus-mediated intestinal immune damage and gut microbiota changes.

The immune response induced by respiratory syncytial virus (RSV) infection is closely related to changes in the composition and function of gastrointestinal microorganisms. However, the specific mechanism remains unknown and the pulmonary-intestinal axis deserves further study. In this study, the mRNA levels of ROR-γt and Foxp3 in the lung and intestine increased first and then decreased. IL-17 and IL-22 reached the maximum on the third day after infection in the lung, and on the second day after infection in the small intestine and colon, respectively. RegⅢγ in intestinal tissue reached the maximum on the third day after RSV infection. Moreover, the genus enriched in the RSV group was Aggregatibacter, and Proteus was reduced. RSV infection not only causes Th17/Treg cell imbalance in the lungs of mice but also leads to the release of excessive IL-22 from the lungs through blood circulation which binds to IL-22 receptors on the intestinal surface, inducing RegⅢγ overexpression, impaired intestinal Th17/Treg development, and altered gut microbiota composition. Our research reveals a significant link between the pulmonary and intestinal axis after RSV infection. IMPORTANCE: RSV is the most common pathogen causing acute lower respiratory tract infections in infants and young children, but the complex interactions between the immune system and gut microbiota induced by RSV infection still requires further research. In this study, it was suggested that RSV infection in 7-day-old BALB/c suckling mice caused lung inflammation and disruption of Th17/Treg cells development, and altered the composition of gut microbiota through IL-22 induced overexpression of RegⅢγ, leading to intestinal immune injury and disruption of gut microbiota. This research reveals that IL-22 may be the link between the lung and gut. This study may provide a new insight into the intestinal symptoms caused by RSV and other respiratory viruses and the connection between the lung and gut axis, as well as new therapeutic ideas for the treatment of RSV-infected children.

Tumor mitochondrial oxidative phosphorylation stimulated by the nuclear receptor RORγ represents an effective therapeutic opportunity in osteosarcoma.

Osteosarcoma (OS) is the most common malignant bone tumor with a poor prognosis. Here, we show that the nuclear receptor RORγ may serve as a potential therapeutic target in OS. OS exhibits a hyperactivated oxidative phosphorylation (OXPHOS) program, which fuels the carbon source to promote tumor progression. We found that RORγ is overexpressed in OS tumors and is linked to hyperactivated OXPHOS. RORγ induces the expression of PGC-1ß and physically interacts with it to activate the OXPHOS program by upregulating the expression of respiratory chain component genes. Inhibition of RORγ strongly inhibits OXPHOS activation, downregulates mitochondrial functions, and increases ROS production, which results in OS cell apoptosis and ferroptosis. RORγ inverse agonists strongly suppressed OS tumor growth and progression and sensitized OS tumors to chemotherapy. Taken together, our results indicate that RORγ is a critical regulator of the OXPHOS program in OS and provides an effective therapeutic strategy for this deadly disease.

Downregulation of Mirlet7 miRNA family promotes Tc17 differentiation and emphysema via de-repression of RORγt.

Environmental air irritants including nanosized carbon black (nCB) can drive systemic inflammation, promoting chronic obstructive pulmonary disease (COPD) and emphysema development. The let-7 microRNA (Mirlet7 miRNA) family is associated with IL-17-driven T cell inflammation, a canonical signature of lung inflammation. Recent evidence suggests the Mirlet7 family is downregulated in patients with COPD, however, whether this repression conveys a functional consequence on emphysema pathology has not been elucidated. Here, we show that overall expression of the Mirlet7 clusters, Mirlet7b/Mirlet7c2 and Mirlet7a1/Mirlet7f1/Mirlet7d, are reduced in the lungs and T cells of smokers with emphysema as well as in mice with cigarette smoke (CS)- or nCB-elicited emphysema. We demonstrate that loss of the Mirlet7b/Mirlet7c2 cluster in T cells predisposed mice to exaggerated CS- or nCB-elicited emphysema. Furthermore, ablation of the Mirlet7b/Mirlet7c2 cluster enhanced CD8+IL17a+ T cells (Tc17) formation in emphysema development in mice. Additionally, transgenic mice overexpressing Mirlet7g in T cells are resistant to Tc17 and CD4+IL17a+ T cells (Th17) development when exposed to nCB. Mechanistically, our findings reveal the master regulator of Tc17/Th17 differentiation, RAR-related orphan receptor gamma t (RORγt), as a direct target of Mirlet7 in T cells. Overall, our findings shed light on the Mirlet7/RORγt axis with Mirlet7 acting as a molecular brake in the generation of Tc17 cells and suggest a novel therapeutic approach for tempering the augmented IL-17-mediated response in emphysema.

RORγt inverse agonist TF-S14 inhibits Th17 cytokines and prolongs skin allograft survival in sensitized mice.

Chronic antibody mediated rejection (AMR) is the major cause of solid organ graft rejection. Th17 contributes to AMR through the secretion of IL17A, IL21 and IL22. These cytokines promote neutrophilic infiltration, B cell proliferation and donor specific antibodies (DSAs) production. In the current study we investigated the role of Th17 in transplant sensitization. Additionally, we investigated the therapeutic potential of novel inverse agonists of the retinoic acid receptor-related orphan receptor gamma t (RORγt) in the treatment of skin allograft rejection in sensitized mice. Our results show that RORγt inverse agonists reduce cytokine production in human Th17 cells in vitro. In mice, we demonstrate that the RORγt inverse agonist TF-S14 reduces Th17 signature cytokines in vitro and in vivo and leads to blocking neutrophilic infiltration to skin allografts, inhibition of the B-cell differentiation, and the reduction of de novo IgG3 DSAs production. Finally, we show that TF-S14 prolongs the survival of a total mismatch grafts in sensitized mice. In conclusion, RORγt inverse agonists offer a therapeutic intervention through a novel mechanism to treat rejection in highly sensitized patients.

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.

[Xixin Decoction regulates Th17/Treg balance and transcription factor expression in senescence-accelerated mouse-prone].

This study aims to investigate the effect of Xixin Decoction on the T helper 17 cell(Th17)/regulatory T cell(Treg) ba-lance of intestinal mucosa and the expression of related transcription factors in the senescence-accelerated mouse-prone 8(SAMP8) model. Fifty 14-week male mice of SAMP8 were randomized by the random number table method into model group, probiotics group, and high-, medium-, and low-dose Xixin Decoction groups, with 10 mice in each group. Ten 14-week male mice of senescence-acce-lerated mouse-resistant 1(SAMR1) served as control group. After 10 weeks of feeding, the mice were administrated with correspon-ding drugs for 10 weeks. Morris water maze test was carried out to examine the learning and memory abilities of mice. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the content of secretory immunoglobulin A(SIgA) in the intestinal mucosa, and flow cytometry to detect the percentage content of Th17 and Treg in the intestinal mucosa. Western blot was performed to determine the protein levels of retinoid-related orphan receptor gamma t(RORγt) and forkhead box p3(Foxp3) in the mouse colon tissue. Compared with control group, the escape latency of mice in model group was significantly prolonged(P<0.01), and the number of times of crossing the platform and the residence time in the target quadrant were significantly reduced within 60 s(P<0.01), intestinal mucosal SIgA content was significantly decreased(P<0.01), Th17 content was increased(P<0.05), Treg content was decreased(P<0.01), the expression of RORγt protein was increased and Foxp3 protein was decreased in colon(P<0.01). Compared with the model group, high-dose Xixin Decoction group improved the learning and memory ability(P<0.05 or P<0.01). Probiotics group and high-and medium-dose Xixin Decoction group increased the content of SIgA in intestinal mucosa(P<0.05 or P<0.01), decreased percentage content of Th17 and increased the percentage content of Treg in intestinal mucosa(P<0.05 or P<0.01). Furthermore, they down-regulated the protein level of RORγt and up-regulated the protein level of Foxp3 in the intestinal mucosa(P<0.01). In conclusion, Xixin Decoction may act on intestinal mucosal immune barrier, affect gut-brain information exchange, and improve the learning and memory ability of SAMP8 by promoting SIgA secretion and regulating the Th17/Treg balance and the expression of RORγt and Foxp3.

Exploring the immune-modulating properties of boswellic acid in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a condition where the central nervous system loses its myelin coating due to autoimmune inflammation. The experimental autoimmune encephalomyelitis (EAE) simulates some aspects of human MS. Boswellic acids are natural compounds derived from frankincense extract, known for their anti-inflammatory properties. The purpose of this research was to investigate therapeutic potential of boswellic acids. Mice were divided into three groups: low-dose (LD), high-dose (HD), and control groups (CTRL). Following EAE induction, the mice received daily doses of boswellic acid for 25 days. Brain tissue damage, clinical symptoms, and levels of TGF-ß, IFN-γ, and IL-17 cytokines in cell cultured supernatant of lymphocytes were assessed. Gene expression of transcription factors in brain was measured using real-time PCR. The levels of brain demyelination were significantly lower in the treatment groups compared to the CTRL group. Boswellic acid reduced the severity and duration of EAE symptoms. Furthermore, boswellic acid decreased the amounts of IFN-γ and IL-17, also the expression of T-bet and ROR-γt in brain. On the contrary, it increased the levels of TGF-ß and the expression FoxP3 and GATA3. Our findings suggest that boswellic acids possess therapeutic potential for EAE by modulating the immune response and reducing inflammation.

Dysregulation of Wnt/ß-catenin signaling contributes to intestinal inflammation through regulation of group 3 innate lymphoid cells.

RORγt+ group 3 innate lymphoid cells (ILC3s) are essential for intestinal homeostasis. Dysregulation of ILC3s has been found in the gut of patients with inflammatory bowel disease and colorectal cancer, yet the specific mechanisms still require more investigation. Here we observe increased ß-catenin in intestinal ILC3s from inflammatory bowel disease and colon cancer patients compared with healthy donors. In contrast to promoting RORγt expression in T cells, activation of Wnt/ß-catenin signaling in ILC3s suppresses RORγt expression, inhibits its proliferation and function, and leads to a deficiency of ILC3s and subsequent intestinal inflammation in mice. Activated ß-catenin and its interacting transcription factor, TCF-1, cannot directly suppress RORγt expression, but rather alters global chromatin accessibility and inhibits JunB expression, which is essential for RORγt expression in ILC3s. Together, our findings suggest that dysregulated Wnt/ß-catenin signaling impairs intestinal ILC3s through TCF-1/JunB/RORγt regulation, further disrupting intestinal homeostasis, and promoting inflammation and cancer.

[The role of microbiota and flavonoids in maintaining the balance of helper and regulatory T-lymphocytes associated with the intestinal immune barrier].

The gastrointestinal tract is a barrier, represented by dynamic and mutually regulating components (microbial, chemical, physical and immune) for the selective penetration of luminal contents into the internal environment. From the point of view of immunologists, even in a physiological condition, the epithelium of the intestinal wall is in a state of mild inflammation, which is explained by the constant invasion of antigens (food, microbial) and, in turn, the constant readiness of the immune system to respond. The purpose of this review was to analyze information about the formation of microbial and immunological barriers, immunological tolerance to microbiota and the possible role of flavonoids in this. Material and methods. The literature search was carried out using PubMed, ResearchGate, Elibrary databases mainly for the last 10 years, using the following keywords: flavonoid, gut microbiome/microbiota, Th17, Treg, RORγt, immunity, segmented filamentous bacteria. Results. During the immune response, a significant role in maintaining the intestinal barrier function is assigned to helper T lymphocytes type 17 (Th17). The intestinal microbiome is a key element in the formation of the immune barrier. Th17 differentiation in the intestine is fully triggered by commensals (apparently, the main role belongs to segmented filamentous bacteria) after weaning and the start of complementary feeding. Pro-inflammatory Th17 effectors in the gut are controlled by anti-inflammatory regulatory T-cells (Treg). In recent years, it has been established that despite the opposing functions of regulatory cells and effector Th17 cells, their differentiation is similar and is characterized by the expression of the common transcription factor RORγt. The main part of the peripheral regulatory lymphocytes of the intestine is a population that stably expresses not only FOXP3, but also RORγt. Flavonoids, which are plant secondary metabolites of the polyphenolic structure, are able to inhibit intracellular kinases and, as a result, influence the activation and implementation of effector functions of immunocompetent cells. Some flavonoids promote RORγt expression and appear to be able to reprogram the effector phenotype of Th17 cells, reducing their pathogenicity. Conclusion. Understanding the interactions between the microbiota, immune cells, and factors involved in their regulation, which are critical for the maintenance of tolerance, may facilitate progress in the prevention and therapeutic approaches to treat immunoinflammatory and autoimmune diseases.

Suppressor T helper type 17 cell responses in intestinal transplant recipients with allograft rejection.

BACKGROUND: Intestinal transplant (ITx) rejection is associated with memory T helper type 17 cell (Th17) infiltration of grafted tissues. Modulation of Th17 effector cell response is facilitated by T regulatory (Treg) cells, but a phenotypic characterization of this process is lacking in the context of allograft rejection. METHODS: Flow cytometry was performed to examine the expression of surface receptors, cytokines, and transcription factors in Th17 and Treg cells in ITx control (n = 34) and rejection patients (n = 23). To elucidate key pathways guiding the rejection biology, we utilized RNA sequencing (RNAseq) and assessed epigenetic stability through pyrosequencing of the Treg-specific demethylated region (TSDR). RESULTS: We found that intestinal allograft rejection is characterized by Treg cellular infiltrates, which are polarized toward Th17-type chemokine receptor, ROR-γt transcription factor expression, and cytokine production. These Treg cell subsets have maintained epigenetic stability, as defined by FoxP3-TSDR methylation status, but displayed upregulation of functional Treg and purinergic signaling genes by RNAseq analysis such as CD39, in keeping with suppressor Th17 properties. CONCLUSION: We show that ITx rejection is associated with increased polarized cells that express a Th17-like phenotype concurrent with regulatory purinergic markers.

Core fucosylation of maternal milk N-glycans imparts early-life immune tolerance through gut microbiota-dependent regulation in RORγt+ Treg cells.

Milk glycans play key roles in shaping and maintaining a healthy infant gut microbiota. Core fucosylation catalyzed by fucosyltransferase (Fut8) is the major glycosylation pattern on human milk N-glycan, which was crucial for promoting the colonization and dominant growth of Bifidobacterium and Lactobacillus spp. in neonates. However, the influence of core-fucose in breast milk on the establishment of early-life immune tolerance remains poorly characterized. In this study, we found that the deficiency of core-fucose in the milk of maternal mice caused by Fut8 gene heterozygosity (Fut8+/-) resulted in poor immune tolerance towards the ovalbumin (OVA) challenge, accompanied by a reduced proportion of intestinal RORγt+ Treg cells and the abundance of Lactobacillus spp., especially L. reuteri and L. johnsonii, in their breast-fed neonates. The administration of the L. reuteri and L. johnsonii mixture to neonatal mice compromised the OVA-induced allergy and up-regulated the intestinal RORγt+ Treg cell proportions. However, Lactobacillus mixture supplementation did not alleviate allergic responses in RORγt+ Treg cell-deficient mice caused by Rorc gene heterozygosity (Rorc+/-) post OVA challenge, indicating that the intervention effects depend on the RORγt+ Treg cells. Interestingly, instead of L. reuteri and L. johnsonii, we found that the relative abundance of another Lactobacillus spp., L. murinus, in the gut of the offspring mice was significantly promoted by intervention, which showed enhancing effects on the proliferation of splenic and intestinal RORγt+ Treg cells in in vitro studies. The above results indicate that core fucosylation of breast milk N-glycans is beneficial for the establishment of RORγt+ Treg cell mediated early-life immune tolerance through the manipulation of symbiotic bacteria in mice.

Retinoid orphan receptor gamma t (rorγt) promotes inflammatory eosinophilia but is dispensable for innate immune-mediated colitis.

Inflammatory bowel diseases (IBD) result from uncontrolled inflammation in the intestinal mucosa leading to damage and loss of function. Both innate and adaptive immunity contribute to the inflammation of IBD and innate and adaptive immune cells reciprocally activate each other in a forward feedback loop. In order to better understand innate immune contributions to IBD, we developed a model of spontaneous 100% penetrant, early onset colitis that occurs in the absence of adaptive immunity by crossing villin-TNFAIP3 mice to RAG1-/- mice (TRAG mice). This model is driven by microbes and features increased levels of innate lymphoid cells in the intestinal mucosa. To investigate the role of type 3 innate lymphoid cells (ILC3) in the innate colitis of TRAG mice, we crossed them to retinoid orphan receptor gamma t deficient (Rorγt-/-) mice. Rorγt-/- x TRAG mice exhibited markedly reduced eosinophilia in the colonic mucosa, but colitis persisted in these mice. Colitis in Rorγt-/- x TRAG mice was characterized by increased infiltration of the intestinal mucosa by neutrophils, inflammatory monocytes, macrophages and other innate cells. RNA and cellular profiles of Rorγt-/- x TRAG mice were consistent with a lack of ILC3 and ILC3 derived cytokines, reduced antimicrobial factors, increased activation oof epithelial repair processes and reduced activation of epithelial cell STAT3. The colitis in Rorγt-/- x TRAG mice was ameliorated by antibiotic treatment indicating that microbes contribute to the ILC3-independent colitis of these mice. Together, these gene expression and cell signaling signatures reflect the double-edged sword of ILC3 in the intestine, inducing both proinflammatory and antimicrobial protective responses. Thus, Rorγt promotes eosinophilia but Rorγt and Rorγt-dependent ILC3 are dispensable for the innate colitis in TRAG mice.

RORγt up-regulates RAG gene expression in DP thymocytes to expand the Tcra repertoire.

Recombination activating gene (RAG) expression increases as thymocytes transition from the CD4-CD8- double-negative (DN) to the CD4+CD8+ double-positive (DP) stage, but the physiological importance and mechanism of transcriptional up-regulation are unknown. Here, we show that a DP-specific component of the recombination activating genes antisilencer (DPASE) provokes elevated RAG expression in DP thymocytes. Mouse DP thymocytes lacking the DPASE display RAG expression equivalent to that in DN thymocytes, but this supports only a partial Tcra repertoire due to inefficient secondary Vα-Jα rearrangement. These data indicate that RAG up-regulation is required for a replete Tcra repertoire and that RAG expression is fine-tuned during lymphocyte development to meet the requirements of distinct antigen receptor loci. We further show that transcription factor RORγt directs RAG up-regulation in DP thymocytes by binding to the DPASE and that RORγt influences the Tcra repertoire by binding to the Tcra enhancer. These data, together with prior work showing RORγt to control Tcra rearrangement by regulating DP thymocyte proliferation and survival, reveal RORγt to orchestrate multiple pathways that support formation of the Tcra repertoire.

IRF4-mediated Treg phenotype switching can aggravate hyperoxia-induced alveolar epithelial cell injury.

Bronchopulmonary dysplasia (BPD) is characterized by alveolar dysplasia, and evidence indicates that interferon regulatory factor 4 (IRF4) is involved in the pathogenesis of various inflammatory lung diseases. Nonetheless, the significance and mechanism of IRF4 in BPD remain unelucidated. Consequently, we established a mouse model of BPD through hyperoxia exposure, and ELISA was employed to measure interleukin-17 A (IL-17 A) and interleukin-6 (IL-6) expression levels in lung tissues. Western blotting was adopted to determine the expression of IRF4, surfactant protein C (SP-C), and podoplanin (T1α) in lung tissues. Flow cytometry was utilized for analyzing the percentages of FOXP3+ regulatory T cells (Tregs) and FOXP3+RORγt+ Tregs in CD4+ T cells in lung tissues to clarify the underlying mechanism. Our findings revealed that BPD mice exhibited disordered lung tissue structure, elevated IRF4 expression, decreased SP-C and T1α expression, increased IL-17 A and IL-6 levels, reduced proportion of FOXP3+ Tregs, and increased proportion of FOXP3+RORγt+ Tregs. For the purpose of further elucidating the effect of IRF4 on Treg phenotype switching induced by hyperoxia in lung tissues, we exposed neonatal mice with IRF4 knockout to hyperoxia. These mice exhibited regular lung tissue structure, increased proportion of FOXP3+ Tregs, reduced proportion of FOXP3+RORγt+ Tregs, elevated SP-C and T1α expression, and decreased IL-17 A and IL-6 levels. In conclusion, our findings demonstrate that IRF4-mediated Treg phenotype switching in lung tissues exacerbates alveolar epithelial cell injury under hyperoxia exposure.

Unraveling the Impact of Blood Transfusion on Transcription Factors Regulating T Helper 1, 2, 17 and Regulatory T cells.

T helper 1 (TH1) and TH2 lymphocytes are the most important components of the immune system affected by blood transfusion. This study aimed`` to evaluate the effect of blood transfusion on gene expression of transcription factors related to the development of TH1, TH2, TH17 and regulatory T cells (Tregs). In this cross-sectional study, 20 patients diagnosed with abdominal aortic aneurysms requiring surgical repair were studied from January 2018 to August 2020. We utilized real-time PCR to evaluate the expression of transcription factor genes associated with TH1, TH2, TH17, and Treg, namely T-box-expressed-in-T-cells (T-bet), GATA-binding protein 3 (GATA-3), retinoid-related orphan receptor (RORγt), and fork head box protein 3 (Foxp3), respectively. The sampling occurred before anesthesia, 24- and 72 hours post-transfusion, and at the time of discharge. The results showed that the T-bet gene expression, compared to the time before transfusion, was significantly decreased 24 hours after blood transfusion and upon discharge while GATA3 genes exhibited a significant reduction both 24 and 72 hours after the transfusion, as compared to the pre-transfusion levels and the time of patient discharge. The Foxp3 gene demonstrated an increase at all study stages, with a notable surge, particularly 72 hours after red blood cell (RBC) transfusion. Conversely, the expression of RORγt gene, consistently decreased throughout all stages of the study. RBC transfusion in abdominal aortic aneurysm patients altered the balance of transcription gene expression of TH1, TH2, TH17, and Treg cells.

Discovery of RORγ Allosteric Fluorescent Probes and Their Application: Fluorescence Polarization, Screening, and Bioimaging.

Retinoic acid receptor-related orphan receptor γ (RORγ) acts as a crucial transcription factor in Th17 cells and is involved in diverse autoimmune disorders. RORγ allosteric inhibitors have gained significant research focus as a novel strategy to inhibit RORγ transcriptional activity. Leveraging the high affinity and selectivity of RORγ allosteric inhibitor MRL-871 (1), this study presents the design, synthesis, and characterization of 11 allosteric fluorescent probes. Utilizing the preferred probe 12h, we established an efficient and cost-effective fluorescence polarization-based affinity assay for screening RORγ allosteric binders. By employing virtual screening in conjunction with this assay, 10 novel RORγ allosteric inhibitors were identified. The initial SAR studies focusing on the hit compound G381-0087 are also presented. The encouraging outcomes indicate that probe 12h possesses the potential to function as a powerful tool in facilitating the exploration of RORγ allosteric inhibitors and furthering understanding of RORγ function.

Differential Upregulation of Th1/Th17-Associated Proteins and PD-L1 in Granulomatous Mycosis Fungoides.

Granulomatous Mycosis Fungoides (GMF) is a rare form of mycosis fungoides (MF) characterized by a granulomatous infiltrate associated with the neoplastic lymphoid population and is considered to have a worse prognosis compared with regular MF. The upregulation of the T helper (Th) axis, especially Th17, plays an important role in the pathogenesis of several inflammatory/infectious granulomatous cutaneous diseases, but its role in GMF is still not elucidated to date. In this study, we evaluated the immunohistochemical expression of Th1 (Tbet), Th2 (GATA-3), Th17 (RORγT), T regulatory (Foxp3), and immune checkpoint (IC) (PD-1 and PD-L1) markers in a cohort of patients with GMF and MF with large cell transformation (MFLCT). Skin biopsies from 49 patients (28 GMF and 21 MFLCT) were studied. Patients with GMF were associated with early clinical stage (p = 0.036) and lower levels of lactate dehydrogenase (p = 0.042). An increased percentage of cells positive for Tbet (p = 0.017), RORγT (p = 0.001), and PD-L1 (p = 0.011) was also observed among the GMF specimens, while a stronger PD-1 intensity was detected in cases of MFLCT. In this cohort, LCT, RORγT < 10%, Foxp3 < 10%, age, and advanced stage were associated with worse overall survival (OS) in univariate analysis. GMF demonstrated Th1 (cellular response) and Th17 (autoimmunity) phenotype, seen in early MF and granulomatous processes, respectively, which may be related to the histopathological appearance and biological behavior of GMF. Further studies involving larger series of cases and more sensitive techniques are warranted.

Early life imprinting of intestinal immune tolerance and tissue homeostasis.

Besides its canonical role in protecting the host from pathogens, the immune system plays an arguably equally important role in maintaining tissue homeostasis. Within barrier tissues that interface with the external microenvironment, induction of immune tolerance to innocuous antigens, such as commensal, dietary, and environmental antigens, is key to establishing immune homeostasis. The early postnatal period represents a critical window of opportunity in which parallel development of the tissue, immune cells, and microbiota allows for reciprocal regulation that shapes the long-term immunological tone of the tissue and subsequent risk of immune-mediated diseases. During early infancy, the immune system appears to sacrifice pro-inflammatory functions, prioritizing the establishment of tissue tolerance. In this review, we discuss mechanisms underlying early life windows for intestinal tolerance with a focus on newly identified RORγt+ antigen-presenting cells-Thetis cells-and highlight the role of the intestinal microenvironment in shaping intestinal immune system development and tolerance.

Electroacupuncture of scalp acupoint alleviates cerebral ischemic inflammatory injury by down-regulating RORγt and promoting balance of IL-17A+Th17/FOXP3+Treg in MCAO rats. / 电头针调控RORγt促进IL-17A+Th17/FOXP3+Treg细胞平衡缓解MCAOå¤§é¼ è„‘ç¼ºè¡€ç‚Žæ€§æŸä¼¤çš„ç ”ç©¶.

OBJECTIVES: To observe the effect of electroacupuncture (EA) of scalp acupoint (Dingnieqian-xiexian, MS6) on expression of retinoid-related orphan receptor γT (ROR γ t), interleukin (IL)-17A, IL-10, transfor-ming growth factor-ß1 (TGF-ß1), IL-6, IL-21, and IL-17A+ Thelper cells(Th) 17 and forkhead transcription factor P3 (FOXP3)+ regulatory T cells (Treg) differentiation of ischemic cortex in ischemic stroke rats, so as to explore its molecular mechanisms underlying relief of inflammatory injury of ischemic stroke. METHODS: A total of 120 male SD rats were randomly assigned to sham operation, model, EA, inhibitor, agonist and EA+agonist groups, with 15 rats in each group. The ischemic stroke model was established by occlusion of the left middle cerebral artery according to Longa's methods. For rats of the EA group and EA+agonist group, EA (2 Hz/100 Hz, 1 mA) was applied to bilateral MS6 for 30 min, once daily for 7 days. Rats of the inhibitor group received intraperitoneal injection of solution of SR1001 (RORγt inhibitor) (2.5 mg/mL, 10 mg/kg), once daily for 7 days. Rats of the agonist and EA+agonist groups received intraperitoneal injection of solution of SR1078 (RORγt agonist) (5 mg/mL, 5 mg/kg) before EA, once daily for 7 days. Rats of the sham operation and model groups were grabbed and fixed in the same way with the other groups. The Zea-longa's score, modified neurological severity score (mNSS) and the neurobehavioral score were assessed before and after the intervention. At the end of experiments, the ischemic cortex tissue was collected. The 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining was used to detect the volume of cerebral infarction. The expression of RORγt mRNA was detected by real-time quantitative PCR；the protein expression levels of RORγt, IL-17A, IL-10 and TGF-ß1 were detected by Western blot；the immunoactivity of IL-6 and IL-21 were detected by immunohistochemistry；the fluorescence areas of IL-17A+Th17 and FOXP3+Treg cells were measured by immunofluorescence and their ratio was calculated in the tissue of ischemic cortex. RESULTS: Relevant to the sham operation group, the model group had a significant increase in the Zea-Longa's score, mNSS score, neurobehavioral score, cerebral infarct volume, expression levels of RORγt mRNA and protein, IL-17A protein, IL-6 and IL-21 immunoactivity, IL-17A+Th17 immunofluorescence intensity, and the ratio of IL-17A+Th17/FOXP3+Treg (P<0.01), and an obvious decrease in the expression levels of TGF-ß1 and IL-10 proteins and FOXP3+Treg immunofluorescence intensity (P<0.01). In contrast to the model group, both EA and inhibitor groups had a significant decrease in the Zea-Longa's score, mNSS score, neurobehavioral score, cerebral infarct volume, expression levels of RORγt mRNA and protein, IL-17A protein, IL-6 and IL-21 immunoactivity, IL-17A+Th17 immunofluorescence intensity, and the ratio of IL-17A+Th17/FOXP3+Treg (P<0.01, P<0.05), and a marked increase in the expression levels of TGF-ß1 and IL-10 proteins and FOXP3+Treg immunofluorescence intensity (P<0.05, P<0.01), while the above indicators of the agonist group were all reversed (P<0.01, P<0.05). Comparison between the agonist and EA+agonist groups showed that the Zea-Longa's score, mNSS score, neurobehavioral score, cerebral infarct volume, expression levels of RORγt mRNA and protein, IL-17A protein, IL-6 and IL-21 immunoactivity, IL-17A+Th17 immunofluorescence intensity, and the ratio of IL-17A+Th17/FOXP3+Treg were significantly lower (P<0.01, P<0.05), and the expression of TGF-ß1 and IL-10 proteins and FOXP3+Treg immunofluorescence intensity were obviously higher (P<0.01, P<0.05) in the EA+agonist group than in the agonist group, suggesting that EA intervention can effectively weaken the effects of RORγt agonist. CONCLUSIONS: EA of scalp acupoint MS6 can effectively improve the neurological function, behavior reaction and reduce cerebral infarct volume in ischemic stroke rats, which may be associated with its functions in down-regulating the expression of RORγt and promoting the balance of IL-17A+Th17/FOXP3+Treg to alleviate inflammatory injury after ischemic stroke.

Down-Regulation of INSR Restores Th17/Treg Immune Balance and Alleviates Airway Hyperviscosity in Asthmatic Mice via Inactivation of STAT3 Pathway.

BACKGROUND: Allergic asthma (AA) is a prevalent chronic airway inflammation disease. In this study, this study aims to investigate the biological functions and potential regulatory mechanisms of the insulin receptor (INSR) in the progression of AA. METHODS: BALB/c mice (n = 48) were randomly divided into the following groups: control group, AA group, AA+Lentivirus (Lv)-vector short hairpin RNA (shRNA) group, AA+Lv-vector group, AA+Lv-INSR shRNA group, and AA+Lv-INSR group. The pulmonary index was calculated. mRNA and protein expression levels of INSR, signal transducer and activator of transcription 3 (STAT3), Janus kinase 2 (JAK2), phosphorylated-STAT3 (p-STAT3), phosphorylated-JAK2 (p-JAK2), alpha-smooth muscle actin (α-SMA), febrile neutropenia (FN), mucin 5AC (MUC5AC), and mucin 5B (MUC5B) were examined using reverse-transcription quantitative PCR (RT-qPCR) and western blot assays. Positive expressions of INSR, retinoic acid-related orphan receptor gamma-t (RORγt), and forkhead box protein P3 (Foxp3) were quantified by immunohistochemistry. Fluorescence intensities of α-SMA and FN were detected by immunofluorescence. Pathological morphology was observed through hematoxylin-eosin (H&E) staining, Masson staining, and Periodic Acid-Schiff (PAS) staining. Contents of immunoglobulin E (IgE), interleukin-6 (IL-6), eotaxin, interleukin-4 (IL-4), interleukin-13 (IL-13), interferon-γ (IFN-γ), interleukin-17 (IL-17), and interleukin-10 (IL-10) were quantified using enzyme-linked immunosorbent assay (ELISA). The percentage of T helper 17 (Th17) and regulatory T (Treg) cells was determined through flow cytometry. RESULTS: Compared to the control group, expression levels of INSR, p-STAT3, p-JAK2, α-SMA, FN, MUC5AC, MUC5B, RORγt, and Foxp3, as well as IgE, IL-6, eotaxin, IL-4, IL-13, and IL-17 contents, pulmonary index, glycogen-positive area (%), and Th17 cell percentage significantly increased (p < 0.05). Additionally, pulmonary histopathological deterioration and collagen deposition were aggravated, while Treg cell percentage and IFN-γ and IL-10 contents remarkably decreased (p < 0.05). The overexpression of INSR further exacerbated the progression of allergic asthma, but the down-regulation of INSR reversed the trends of the above indicators. CONCLUSIONS: The down-regulation of INSR alleviates airway hyperviscosity, inflammatory infiltration, and airway remodeling, restoring Th17/Treg immune balance in AA mice by inactivating the STAT3 pathway.