Promoted Treg Cell Differentiation Research Articles

Bone marrow mesenchymal stem cells-derived exosomal miR-381 alleviates lung ischemia-reperfusion injury by activating Treg differentiation through inhibiting YTHDF1 expression

AimOur study aimed to investigate whether BMSCs-derived exosomal miR-381 promotes Treg cell differentiation in lung ischemia-reperfusion injury (LIRI), and the underlying mechanism. MethodsThe in vitro and in vivo models of LIRI were established by hypoxia/reoxygenation (H/R) treatment and lung ischemia/reperfusion (I/R) surgery, respectively. BMSCs-derived exosomes were isolated and identified by western blot, nanoparticle tracking analysis, and transmission electron microscopy. Cell viability, proliferation, and apoptosis were assessed by CCK-8, EdU, and flow cytometry assay, respectively. IL-18 secretion level in lung microvascular endothelial cells (LMECs) and lung tissue homogenate was examined by ELISA. Treg cell differentiation was determined using flow cytometry. The relationships between miR-381, YTHDF1, and IL-18 were investigated using dual-luciferase reporter gene, RIP, and/or RNA pull-down assays. MeRIP assay was employed to determine m6A modification of IL-18 mRNA in LMECs. The ubiquitination level of Foxp3 protein in CD4+ T cells was analyzed by Co-IP assay. ResultsBMSCs-derived exosomes reduced LMECs injury and increased Treg cell differentiation in LIRI, whereas miR-381 inhibition in BMSCs weakened these impacts. Mechanistically, miR-381 inhibited IL-18 translation in LMECs by inhibiting YTHDF1 expression via binding to its 3’-UTR. As expected, YTHDF1 overexpression in LMECs abolished the effects of miR-381-overexpressed exosomes on LMECs injury and Treg cell differentiation. Moreover, LMECs-secreted IL-18 inhibited Treg cell differentiation by promoting the ubiquitination degradation of Foxp3 protein. ConclusionBMSCs-derived exosomal miR-381 suppressed IL-18 translation in LMECs through binding to YTHDF1 3’-UTR, thus suppressing the ubiquitination degradation of Foxp3 in CD4+ T cells, which promoted Treg cell differentiation and mitigated LIRI development.

Restoring Treg/Th17 cell balance in ulcerative colitis through HRas silencing and MAPK pathway inhibition

This study investigates HRas-dependent mechanisms in the disruption of regulatory T (Treg) cells and T helper 17 (Th17) cells balance in ulcerative colitis (UC). Comprehensive RNA sequencing and bioinformatics analyses revealed elevated HRas and MAPK pathway-related protein expression in UC samples. Using a murine UC model induced by dextran sulfate sodium (DSS), HRas silencing was found to promote Treg cell differentiation and suppress Th17 cell production, effectively restoring balance. Inactivation of the MAPK pathway played a pivotal role in this rebalancing effect. In vivo experiments further confirmed that HRas silencing mitigated colon tissue damage in DSS-induced mice, emphasizing its potential as a therapeutic strategy for UC.

Resveratrol inhibits AhR/Notch axis and reverses Th17/Treg imbalance in purpura by activating Foxp3.

Resveratrol has been reported to reverse the imbalance of T helper 17/regulatory T (Th17/Treg) by inhibiting the aryl hydrocarbon receptor pathway to treat immune thrombocytopenia. However, the regulation mechanism of the Notch signaling pathway by resveratrol has not been reported in purpura. This study is aimed to explore the mechanism of resveratrol ultrafine nanoemulsion (Res-mNE) in immune thrombocytopenia. The immune thrombocytopenia mouse model was constructed to explore the effect of RES-mNE on immune thrombocytopenia. Cluster of differentiation 4 (CD4+) T cells were isolated and treated with different medications. CD4+ T cells were induced to differentiate into Th17 cells and Treg cells. Flow cytometry was used to detect the proportion of Th17 cells and Treg cells. The secretion was measured by the enzyme-linked immunosorbent assay (ELISA). Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blot were used to detect the mRNA and protein levels. Th17 cells, IL-17A and IL-22 increased in the immune thrombocytopenia mouse model, and the Treg cells and IL-10 decreased. Res-mNE promoted Treg cell differentiation and IL-10 secretion in CD4+ T cells while inhibiting Th17 cell differentiation and IL-17A and IL-22 levels. The AhR activator 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reversed the effect of Res-mNE. Notch inhibitors reduced the ratio of Th17/Treg differentiation. Res-mNE activated the expression of Foxp3 by mediating AhR/Notch signaling to reverse the imbalance of Th17/Treg differentiation in immune thrombocytopenia. Taken together, our findings demonstrated that RES-mNE inhibited the AhR/Notch axis and reversed Th17/Treg imbalance by activating Foxp3.

Immunomodulatory effects of umbilical mesenchymal stem cell-derived exosomes on CD4+ T cells in patients with primary Sjögren's syndrome

BackgroundPrimary Sjögren's syndrome (pSS) is an autoimmune disease that leads to the destruction of exocrine glands and multisystem lesions. Abnormal proliferation, apoptosis, and differentiation of CD4+ T cells are key factors in the pathogenesis of pSS. Autophagy is one of the important mechanisms to maintain immune homeostasis and function of CD4+ T cells. Human umbilical cord mesenchymal stem cell-derived exosomes (UCMSC-Exos) may simulate the immunoregulation of MSCs while avoiding the risks of MSCs treatment. However, whether UCMSC-Exos can regulate the functions of CD4+ T cells in pSS, and whether the effects via the autophagy pathway remains unclear.MethodsThe study analyzed retrospectively the peripheral blood lymphocyte subsets in pSS patients, and explored the relationship between lymphocyte subsets and disease activity. Next, peripheral blood CD4+ T cells were sorted using immunomagnetic beads. The proliferation, apoptosis, differentiation, and inflammatory factors of CD4+ T cells were determined using flow cytometry. Autophagosomes of CD4+ T cells were detected using transmission electron microscopy, autophagy-related proteins and genes were detected using western blotting or RT-qPCR.ResultsThe study demonstrated that the peripheral blood CD4+ T cells decreased in pSS patients, and negatively correlated with disease activity. UCMSC-Exos inhibited excessive proliferation and apoptosis of CD4+ T cells in pSS patients, blocked them in the G0/G1 phase, inhibited them from entering the S phase, reduced the Th17 cell ratio, elevated the Treg ratio, inhibited IFN-γ, TNF-α, IL-6, IL-17A, and IL-17F secretion, and promoted IL-10 and TGF-β secretion. UCMSC-Exos reduced the elevated autophagy levels in the peripheral blood CD4+ T cells of patients with pSS. Furthermore, UCMSC-Exos regulated CD4+ T cell proliferation and early apoptosis, inhibited Th17 cell differentiation, promoted Treg cell differentiation, and restored the Th17/Treg balance in pSS patients through the autophagy pathway.ConclusionsThe study indicated that UCMSC-Exos exerts an immunomodulatory effect on the CD4+ T cells, and maybe as a new treatment for pSS.

The N6-methyladenosine writer WTAP contributes to the induction of immune tolerance post kidney transplantation by targeting regulatory T cells

AbstractN6-methyladenosine (m6A) modification is involved in diverse immunoregulation, while the relationship between m6A modification and immune tolerance post kidney transplantation remains unclear. Expression of Wilms tumor 1-associating protein (WTAP), an m6A writer, was firstly detected in tolerant kidney transplant recipients (TOL). Then the role of WTAP on regulatory T (Treg) cell differentiation and function in CD4+ T cells from kidney transplant recipients with immune rejection (IR) was investigated. The potential target of WTAP and effect of WTAP on immune tolerance in vivo were subsequently verified. WTAP was upregulated in CD4+ T cells of TOL and positively correlated with Treg cell proportion. In vitro, WTAP overexpression promoted Treg cell differentiation and enhanced Treg cell-mediated suppression toward naïve T cells. Forkhead box other 1 (Foxo1) was identified as a target of WTAP. WTAP enhanced m6A modification of Foxo1 mRNA in coding sequence (CDS) region, leading to up-regulation of Foxo1. Overexpression of m6A demethylase removed the effect of WTAP overexpression, while Foxo1 overexpression reversed these effects. WTAP overexpression alleviated allograft rejection in model mice, as evidenced by reduced inflammatory response and increased Treg population. Our study suggests that WTAP plays a positive role in induction of immune tolerance post kidney transplant by promoting Treg cell differentiation and function.

HDAC6-specific inhibitor alleviates hashimoto's thyroiditis through inhibition of Th17 cell differentiation

ObjectiveHashimoto's thyroiditis (HT) is one of the commonest autoimmune disorders. This study was performed to investigate the potential effect of histone deacetylase 6-specific inhibitor (HDAC6i) on Th17 cell differentiation in animal model and the underlying mechanism. MethodsExperimental autoimmune thyroiditis (EAT) mouse model was established by subcutaneously immunization of porcine thyroglobulin (pTg) and adjuvant, and the HDACi Tubastatin A (TSA) or HDAC6i (ACY-1215) was intraperitoneally injected into mice in the following. The histological examination and immune analysis in EAT mice were carried out. Next, the CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) of EAT mice followed by Th17 cell differentiation assay. The associated factor levels, and the protein interaction between HDAC6 and PKM2 were examined. Subsequently, the effect of STAT3 activation on Th17 cell differentiation was explored. ResultsACY-1215 or TSA treatment reduced lymphocytic infiltration and alleviated thyroid tissue injury in EAT mice. Correspondingly, either ACY-1215 or TSA treatment reduced the levels of anti-thyroglobulin (Tg), anti-thyroid peroxidase (TPO), IL-17A, and IFN-γ in the serum, decreased Th17 cell differentiation, but enhanced TGF-β level and promoted Treg cell differentiation. In vitro, after induction of Th17 cell differentiation from CD4+ T cells, HDAC6 activity and Th17 cell differentiation were significantly decreased when treated with ACY-1215 or TSA. HDAC6 could interact with PKM2, and HDAC6 overexpression promoted the phosphorylation of STAT3 and PKM2 nuclear translocation. Furthermore, the STAT3 activator treatment reversed the effects of ACY-1215 or TSA treatment. ConclusionHDAC6i suppresses Th17 cell differentiation and attenuates HT via PKM2/STAT3 axis.

Aryl hydrocarbon receptor activation ameliorates experimental colitis by modulating the tolerogenic dendritic and regulatory T cell formation

BackgroundIntestinal immune dysfunction is involved in the onset of Crohn’s disease (CD). Dendritic cells (DCs), antigen-presenting cells, play a key role in the maintenance of intestinal immune homeostasis. The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor widely expressed in various immune cells, including DCs. Although AhR plays an important role in immune tolerance, its role in the DCs is unclear. The purpose of this study was to investigate whether the activation of AhR can induce tolerogenic DCs (tolDCs) and the differentiation of regulatory T (Treg) cells, as well as ameliorate experimental colitis.ResultsAhR activation in the DCs resulted in a lower expression of surface markers such as CD80, CD83, CD86, and pro-inflammatory cytokine production, and higher anti-inflammatory production (IL-1β, IL-23, and IL-12) compared to the control DCs. The surface dendrites in DCs were significantly reduced following AhR activation by 6-formylindolo [3,2-b]carbazole (FICZ). Such DCs with FICZ-mediated activation of AhR, namely tolDCs, promoted Treg cell differentiation. Adoptive transfer of tolDCs to a TNBS-induced colitis mouse model significantly alleviated the severity of inflammation by improving the colon length and decreasing the disease activity index (DAI) and histopathological score. Moreover, the transferred tolDCs decreased the frequency of Th17 cells and increased the frequency of Treg cells in the spleen and mesenteric lymph nodes (MLNs) in murine colitis models.ConclusionsActivation of AhR in the DCs could induce tolDCs, and the transplantation of tolDCs may help in relieving intestinal inflammation and maintaining the Th17/Treg differentiation balance. Thus, our data suggest that AhR may be a potential therapeutic target for CD.

Exosomes Secreted by Mesenchymal Stem Cells Induce Immune Tolerance to Mouse Kidney Transplantation via Transporting LncRNA DANCR.

Mesenchymal stem cells induce kidney transplant tolerance by increasing regulatory T (Treg) cells. Bone marrow mesenchymal stem cell exosomes (BMMSC-Ex) promote Treg cell differentiation. Long non-coding RNA differentiation antagonizing non-protein coding RNA (DANCR) is expressed in BMMSCs and can be encapsulated in exosomes. We aimed to explore the role of DANCR in BMMSC-Ex in immune tolerance after kidney transplantation and related mechanism. The isogenic/allograft kidney transplantation mouse model was established, and levels of serum creatinine (SCr) were determined. Hematoxylin-eosin staining was conducted to detect the inflammation, and immunohistochemistry was performed to detect the infiltration of CD4+ T cells. Levels of IFN-γ, IL-17, and IL-2 were examined by ELISA. Flow cytometry was conducted to determine Treg cells. In the allograft group, the inflammatory response was severe, CD4+ T cell infiltration, SCr levels, and plasma rejection-related factors were up-regulated, while injection of BMMSC-Ex reversed the results. BMMSC-Ex increased Treg cells in kidney transplantation mice. Interference with DANCR reversed the promoting effect of BMMSC-Ex on Treg cell differentiation. DANCR bound to SIRT1, promoted ubiquitination and accelerated its degradation. The injection of BMMSC-Ex (after interference with DANCR) promoted SIRT1 levels, inflammatory response, CD4+ T cell infiltration, SCr levels, and plasma rejection related factors' expression, while Treg cells were decreased. LncRNA DANCR in BMMSC-Ex promoted Treg cell differentiation and induced immune tolerance of kidney transplantation by down-regulating SIRT1 expression in CD4+ T cells.

Augmented Expansion of Treg Cells From Healthy and Autoimmune Subjects via Adult Progenitor Cell Co-Culture.

Recent clinical experience has demonstrated that adoptive regulatory T (Treg) cell therapy is a safe and feasible strategy to suppress immunopathology via induction of host tolerance to allo- and autoantigens. However, clinical trials continue to be compromised due to an inability to manufacture a sufficient Treg cell dose. Multipotent adult progenitor cells (MAPCⓇ) promote Treg cell differentiation in vitro, suggesting they may be repurposed to enhance ex vivo expansion of Tregs for adoptive cellular therapy. Here, we use a Good Manufacturing Practice (GMP) compatible Treg expansion platform to demonstrate that MAPC cell-co-cultured Tregs (MulTreg) exhibit a log-fold increase in yield across two independent cohorts, reducing time to target dose by an average of 30%. Enhanced expansion is coupled to a distinct Treg cell-intrinsic transcriptional program characterized by elevated expression of replication-related genes (CDK1, PLK1, CDC20), downregulation of progenitor and lymph node-homing molecules (LEF1 CCR7, SELL) and induction of intestinal and inflammatory tissue migratory markers (ITGA4, CXCR1) consistent with expression of a gut homing (CCR7lo β7hi) phenotype. Importantly, we find that MulTreg are more readily expanded from patients with autoimmune disease compared to matched Treg lines, suggesting clinical utility in gut and/or T helper type1 (Th1)-driven pathology associated with autoimmunity or transplantation. Relative to expanded Tregs, MulTreg retain equivalent and robust purity, FoxP3 Treg-Specific Demethylated Region (TSDR) demethylation, nominal effector cytokine production and potent suppression of Th1-driven antigen specific and polyclonal responses in vitro and xeno Graft vs Host Disease (xGvHD) in vivo. These data support the use of MAPC cell co-culture in adoptive Treg therapy platforms as a means to rescue expansion failure and reduce the time required to manufacture a stable, potently suppressive product.

Effects of Recombinant IL-35-BCG on Treg/Th17 Cell Imbalance and Inflammatory Response in Asthmatic Newborn Mice Induced by RSV.

Treg/Th17 cell imbalance and inflammatory response may occur in neonatal asthma. IL-35 and BCG have inhibitory effects on inflammatory responses in diseases. However, studies on neonatal asthma after combination of the two have not been reported so far. A respiratory syncytial virus (RSV)-induced neonatal asthma model was first developed in newborn mice. Pathological sections of lung tissue of asthmatic mice were observed by HE staining. Masson staining was used to observe the lung tissue and to compare the deposition of collagen fibers under bronchial epithelium in model mice. The expression of cytokines in serum was detected by ELISA. Giemsa staining analyzed each cell in bronchoalveolar lavage fluid (BALF). Flow cytometry was used to detect the differentiation and development of Treg and Th17 subgroups in BALF. The expression levels of inflammation-related factors were detected by RT-qPCR. Western blot was used to detect the expression of JNK pathway-related proteins. Recombinant IL-35-BCG improved the pathological response of asthmatic mice; inhibited the expression of IgE in serum, neutrophils, macrophages, and eosinophils in BALF; and increased the expression of lymphocytes. In addition, recombinant IL-35-BCG significantly inhibited Th17 differentiation, promoted Treg cell differentiation, and inhibited the expression of inflammatory factors in lung tissue homogenates, thereby reducing allergic airway inflammation. This process might be achieved by inhibiting the JNK signaling pathway. Recombinant IL-35-BCG can regulate Treg/Th17 cell imbalance and inflammatory response in asthmatic newborn mice induced by RSV through JNK signaling pathway, suggesting a new path to neonatal asthma treatment.

MTOR pathway regulates the differentiation of peripheral blood Th2/Treg cell subsets in patients with pemphigus vulgaris.

Pemphigus vulgaris (PV) is a chronic and potentially life-threatening autoimmune blistering disease. Aberrant mTOR pathway activity is involved in many autoimmune diseases. This study investigated the correlation of mTOR pathway (PI3K/AKT/mTOR/p70S6K) activity with the loss of balance in T helper 2/regulatory T (Th2/Treg) cells in the peripheral blood of PV patients. CD4+ T cells were isolated from 15 PV patients and 15 healthy controls (HCs), the ratios of Th2/CD4+ T cells and Treg/CD4+ T cells, the activity of the mTOR pathway (PI3K/AKT/mTOR/p70S6K), the transcription factors and cytokines of Th2 and Treg cells were detected. Primary CD4+ T cells from PV patients were cultured under Th2- or Treg-polarizing conditions with or without rapamycin in vitro. We found that PV patients showed significantly elevated serum IL-4 when compared with HCs, and serum IL-4 level was positively correlated with the titer of anti-Dsg1/3 antibody and disease severity, while the serum TGF-β level was negatively correlated with the titer of anti-Dsg3 antibody and disease severity. Meanwhile, PV patients showed increased Th2/CD4+ T cell ratio; decreased Treg/CD4+ T cell ratio; elevated mRNA of PI3K, AKT, mTOR and protein of PI3K (P85), AKT, p-AKT (Ser473), mTOR, p-mTOR (Ser2448), p-p70S6K (Thr389), GATA3; reduced protein of forkhead box protein 3. Rapamycin inhibited Th2 cell differentiation and promoted Treg cell differentiation in vitro. These data suggest a close association between mTOR pathway activation and the loss of balance in Th2/Treg cells in peripheral blood of PV patients. Inhibiting mTORC1 can help restore the Th2/Treg balance.

Baicalein Restores the Balance of Th17/Treg Cells via Aryl Hydrocarbon Receptor to Attenuate Colitis.

As one of the ligands of aryl hydrocarbon receptor (AhR), baicalein, isolated from Scutellaria baicalensis Georgi, has been proved to exert potential therapeutic effects on ulcerative colitis (UC), but its therapeutic mechanism remains obscure. Authentically, ulcerative colitis can be alleviated by regulating the differentiation of naïve CD4+ T cells via AhR activation. So, our study planned to prove the hypothesis that baicalein protected mice against UC by regulating the balance of Th17/Treg cells via AhR activation. Immunofluorescence and western blot results showed that baicalein could promote AhR activation and induce it to transfer to the nucleus. We further determined the effect of baicalein on naïve CD4+ T cell differentiation in vitro by magnetic cell separation and drug intervention. The results showed that baicalein could promote Treg cell differentiation by activating AhR. In vivo study, UC mice were established by free drinking of dextran sulfate sodium (DSS) for 7 days and then were orally administrated by baicalein (10, 20, and 40 mg/kg), TCDD (AhR agonist), and CH223191 (antagonist). The results demonstrated that baicalein improved the symptoms of UC mice, regulated the balance of Th17/Treg cells, and restored the balance of proinflammatory cytokines such as IL-17, IL-6, and TNF-α; anti-inflammatory cytokines such as IL-10 and TGF-β; and epithelial protective cytokine IL-22 in UC mice, and these effects were related to AhR. Taken together, our research found that baicalein might be a potential drug for UC via regulating Treg cell differentiation and maintaining immune homeostasis and attempted to shed a light on the pivotal role of AhR in these effects.

MiR-223-3p Inhibits Antigen Endocytosis and Presentation and Promotes the Tolerogenic Potential of Dendritic Cells through Targeting Mannose Receptor Signaling and Rhob.

Background The role of miR-223-3p in dendritic cells (DCs) is unknown. This study is aimed at investigating the effect of miR-223-3p on the antigen uptake and presentation capacities of DCs and the underlying molecular mechanism. Methods FITC-OVA antigen uptake and cell surface markers in bone marrow-derived DCs (BMDCs) were analyzed by flow cytometry. BMDCs were transfected with the miR-223-3p mimic or inhibitor. Cytokine levels were determined by ELISA. CD4+ T cell differentiation was determined by mixed lymphocyte culture assay. Results OVA treatment significantly downregulated miR-223-3p in BMDCs. The miR-223-3p mimic significantly inhibited OVA-induced antigen uptake and surface expression of MHC-II on BMDCs (P < 0.01). The miR-223-3p mimic increased TGF-β1 production in OVA-treated DCs (P < 0.01). Mixed lymphocyte reaction showed that the miR-223-3p mimic significantly promoted Treg cell differentiation. In addition, the miR-223-3p mimic significantly upregulated CD103 in DCs, indicating the promotion of tolerogenic DCs. The miR-223-3p mimic downregulated Rhob protein in OVA-induced DCs. Rhob knockdown significantly suppressed the ability of FITC-OVA endocytosis (P < 0.01) and surface MHC-II molecule expression (P < 0.01) in BMDCs, promoting promoted Treg cell differentiation. Mannose receptor (MR) knockdown significantly upregulated miR-223-3p, downregulated Rhob protein in OVA-treated DCs, inhibited the FITC-OVA endocytosis and surface MHC-II expression in BMDCs, and promoted Treg cell differentiation (all P < 0.01). Conclusion These data suggest that miR-223-3p has an inhibitory effect on the antigen uptake and presentation capacities of BMDCs and promotes Treg cell differentiation, which is, at least partially, through targeting MR signaling and Rhob.

065 CDK7 inhibitor suppresses psoriasis inflammation via inhibiting glycolysis to modulate Th17/Treg balance

Psoriasis is a chronic inflammatory disease characterized by hyper-activated Th17 and suppressive Treg cells, but the mechanism of Th17/Treg cells imbalance is still unclear. Cyclin-dependent kinase 7(CDK7) which is known as a cell cycle regulator has been reported an anti-inflammatory effect in immuno-cells. Here we firstly found CD4+T cells of psoriasis patients expressed higher levels of CDK7 along with an increased glycolysis levels than those in healthy controls. The chemical inhibitor of CDK7 called THZ1 restricted glycolytic metabolism in CD4+T cells of psoriasis patients as well as typical glycolysis related genes. More importantly, THZ1 could suppress Th17 cell differentiation and promote Treg cell differentiation even under Th17 polarizing condition in vitro. Intraperitoneal injection of THZ1 in mice exhibited an alleviated epidermal hyperplasia and alleviated inflammation caused by imiquimod(IMQ) treatment. THZ1-treated IMQ mice had significantly lower ratio of Th17 cells and higher ratio of Treg cells from the splenocytes, in comparison with vehicle-treated IMQ mice. Furthermore, we identified IL-23 as an upstream regulator that stimulated CDK7 expression and glycolysis through p-AKT-Hif-1α signalling pathway. Taken together, our results showed that abnormal CDK7 expression induced by IL-23 in CD4+ T cells in psoriasis patients contributed to the enhanced glycolysis levels which lead to the imbalance of Th17/Treg cells. CDK7 inhibitor THZ1 may serve as an immuno-modulator for psoriasis therapy in the future.

Regulating the balance of Th17/Treg cells in gut-lung axis contributed to the therapeutic effect of Houttuynia cordata polysaccharides on H1N1-induced acute lung injury

Our previous study had demonstrated that oral administration of Houttuynia cordata polysaccharides (HCP) without in vitro antiviral activity ameliorated gut and lung injuries induced by influenza A virus (IAV) in mice. However, as macromolecules, HCP was hard to be absorbed in gastrointestinal tract and had no effect on lung injury when administrated intravenously. The action mechanism of HCP was thus proposed as regulating the gut mucosal-associated lymphoid tissue (GALT). Actually, HCP treatment restored the balance of Th17/Treg cells firstly in GALT and finally in the lung. HCP reduced the expression of chemokine CCL20 in the lung and regulated the balance of Th17/Treg carrying CCR6+ (the CCL20 receptor), which was associated with specific migration of Th17/Treg cells from GALT to lung. In vitro, HCP inhibited Th17 cell differentiation through the downregulation of phospho-STAT3, whereas it promoted Treg cell differentiation by upregulating phospho-STAT5. Furthermore, its therapeutic effect was abolished in RORγt−/− or Foxp3−/− mice. These findings indicated that oral administration of macromolecular polysaccharides like HCP might ameliorate lung injury in IAV infected mice via directly regulating the balance of Th17/Treg cells in gut-lung axis. Our results provided a potential mechanism underlying the therapeutic effect of polysaccharides on pulmonary infection.

Tanshinone IIA Ameliorates CNS Autoimmunity by Promoting the Differentiation of Regulatory T Cells.

Tanshinone IIA (TSA), an important natural lipophilic diterpene compound from the traditional Chinese herb Salvia miltiorrhiza Bunge, has long been widely used for the prevention and treatment of various diseases because of its anti-inflammatory activities; however, the anti-inflammatory mechanism remains unknown. In the present work, we examined the effects of TSA on experimental autoimmune encephalomyelitis (EAE), a model of autoreactive T/B cell-mediated central nervous system (CNS) autoimmunity. The data showed that TSA significantly attenuates the severity of EAE when administered at the pre-onset and peak of clinical disease. In vivo, the protective effects of TSA on EAE mice are correlated with diminished inflammatory infiltration, demyelination, and GM-CSF-producing CD4+ T cells in the spinal cord and selectively increased regulatory T (Treg) cell frequencies in both the spinal cord and spleen. We further confirm that TSA can promote the polarization of naïve CD4+ T cells into Treg cells both by targeting dendritic cells (DCs) to drive transforming growth factor β1 (TGF-β1) upregulation and by directly targeting naïve CD4+ T cells in vitro. Most importantly, we showed that TSA-induced Treg cells display an effective suppressive activity at a level comparable to TGF-β1-polarized Treg Cells in vitro and in vivo. Taken together, our data provide evidence that TSA can promote Treg cell differentiation, and TSA may have a promising application as a therapeutic agent for the treatment of neuroinflammatory diseases.

Immature dendritic cells derived exosomes promotes immune tolerance by regulating T cell differentiation in renal transplantation.

Objective: To investigate the mechanism of immature dendritic cells-derived exosomes (imDECs) in the regulation of T cell differentiation and immune tolerance in renal allograft model mice.Results: imDECs significantly improved the percent of survival, relieved inflammatory response, and reduced CD4+T cell infiltration. In addition, imDECs reduced the rejection associated cytokines in allograft mice, and increased the percentage of Foxp3+CD4+T cells in spleen and kidney tissues. imDECs suppressed the IL17+CD4+T cells and promoted the Foxp3+CD4+T cells under Th17 polarization condition. Moreover, miR-682 was found to be highly expressed in imDECs which suppressed the IL17+CD4+T cells and promoted the Foxp3+CD4+T cells. Luciferase reporter assay showed ROCK2 was a target of miR-682, and ROCK mRNA level was negative correlated with miR-682 mRNA level.Conclusion: miR-682 was highly expressed in imDECs, and imDECs-secreted miR-682 promoted Treg cell differentiation by negatively regulating ROCK2 to promote immune tolerance in renal allograft model mice.Methods: Renal allograft model mice were established, and imDECs or mature dendritic cells-derived exosomes (mDECs) were injected into model mice. Rejection associated cytokines IFN-γ, IL-2, IL-17 levels in plasma were detected by ELISA. IL-17A, Foxp3, miR-682, ROCK2, p-STAT3, p-STAT5 expressions were measured by qRT-PCR or western blot.

Inhibition of ROCK activity regulates the balance of Th1, Th17 and Treg cells in myasthenia gravis

Aberrant ROCK activation has been found in patients with several autoimmune diseases, but the role of ROCK in myasthenia gravis (MG) has not yet been clearly investigated. Here, we demonstrated that ROCK activity was significantly higher in peripheral blood mononuclear cells (PBMCs) from MG patients. ROCK inhibitor Fasudil down-regulated the proportions of Th1 and Th17 cells in PBMCs of MG patients in vitro. Intraperitoneal injection of Fasudil ameliorated the severity of experimental autoimmune myasthenia gravis (EAMG) rats and restored the balance of Th1/Th2/Th17/Treg subsets. Furthermore, Fasudil inhibited the proliferation of antigen-specific Th1 and Th17 cells, and inhibited CD4 + T cells differentiated into Th1 and Th17 through decreasing phosphorylated Stat1 and Stat3, but promoted Treg cell differentiation through increasing phosphorylated Stat5. We conclude that dysregulated ROCK activity may be involved in the pathogenic immune response of MG and inhibition of ROCK activity might serve as a novel treatment strategy for MG.

Critical role of ROCK2 activity in facilitating mucosal CD4+ T cell activation in inflammatory bowel disease

Rho-associated kinase (ROCK) has been found to be involved in the pathogenesis of a variety of autoimmune diseases, but the role of ROCK in inflammatory bowel disease (IBD) is still elusive. In this study, we demonstrated that the levels of ROCK2, but not ROCK1, activity were significantly upregulated in peripheral blood mononuclear cells (PBMC) and inflamed mucosa from IBD patients using a ROCK activity assay, and that ROCK2 activity in intestinal mucosa was positively correlated with disease severity. Stimulation with TNF markedly upregulated ROCK2 activity in IBD CD4+ T cells through NF-κB signaling. Blockade of ROCK2 activity using Slx-2119 significantly suppressed proinflammatory cytokines in inflamed mucosa from IBD patients including IFX-unresponsive CD patients, and inhibited IBD CD4+ T cells to differentiate into Th1 and Th17 cells through downregulating phosphorylated Stat1 and Stat3, but promoted Treg cell differentiation through upregulating phosphorylated Stat5. Furthermore, oral administration of Slx-2119 markedly ameliorated intestinal mucosal inflammation in TNBS-induced colitis in mice and decreased proinflammatory cytokines productions in inflamed colon. Our data indicate that ROCK2 plays a critical role in inducing mucosal T cell activation and inflammatory responses in IBD and that inhibition of ROCK2 activity might serve as a novel therapeutic approach in the management of IBD.

Inhibition of Acute Graft-versus-Host Disease with Retention of Graft-versus-Tumor Effects by Dimethyl Fumarate.

Acute graft-versus-host disease (aGVHD) remains a clinical challenge and a major source of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Dimethyl fumarate (DMF), an activator of Nrf2, has been shown to have anti-inflammatory and immunomodulatory properties without significant immunosuppression. We therefore hypothesized that DMF could be potentially harnessed for the treatment of aGVHD with retention of graft-versus-tumor effect. In this study, we showed that DMF significantly inhibited alloreactive T cell responses in vitro in mixed lymphocyte reaction assay. Administration of DMF significantly alleviated the severity, histological damage, and the overall mortality of aGVHD in an MHC-mismatched aGVHD model. DMF administration reduced the activation and effector function of donor T cells in vitro and in vivo. In addition, DMF treatment upregulated antioxidant enzymes heme oxygenase-1 and glutathione S-transferase-α1 expressions. Furthermore, DMF treatment markedly increased the frequencies of Treg cells. Depletion of CD25+ cells in DMF recipients aggravated aGVHD mortality compared with IgG control recipients. DMF could promote Treg cell differentiation in a dose dependent manner by upregulating TGF-β expression in vitro. Most importantly, DMF administration preserved graft-versus-leukemia effect after bone marrow transplantation. In conclusion, our findings demonstrated DMF as a promising agent for the prevention of aGVHD after allo-HSCT.