BMSCs-Derived Extracellular VesiclemiR-29a-3p Improved the Stability of Rat Myasthenia Gravis by Regulating Treg/Th17 Cells.

INTRODUCTION: Myasthenia gravis (MG) is an autoimmune disorder. Microvesicle-derived miRNAs have been implicated in autoimmune diseases. However, the role of microvesicle-derived miR-29a-3p in MG remains poorly understood. This study aimed to investigate the therapeutic effect and mechanism of miR-29a-3p derived from stem cell microvesicles (MVs) on experimental autoimmune myasthenia gravis (EAMG) rats. METHODS: EAMG was induced in rats by injection of the subunit of the rat nicotinic anti-acetylcholine receptor (AChR) R97-116 peptide.Besides the control group, EAMG rats were randomly allocated into the EAMG model group, MV group, MV-NC-agomir group, and MV- miR-29a-3p-agomir group. RESULTS: Our results found that BMSCs-MV promoted miR-29a-3p expression in gastrocnemius of EAMG rats. Bone marrow mesenchymal stem cells (BMSCs) derived microvesicle miR-29a-3p improved the hanging ability and swimming time of EMGA rats and weakened the degree of muscle fiber atrophy. Furthermore, microvesicles from miR-29a-3p overexpressing BMSCs reduced the content of AchR-Ab in the serum of EAMG rats. BMSC-derived microvesicle miR-29a-3p further suppressed the expression of IFN-γ and enhanced the IL-4 and IL-10 in the serum of EAMG rats by restoring the Th17/Treg cells balance. DISCUSSION: BMSCs-derived microvesicle miR-29a-3p improved the stability of rat myasthenia gravis by regulating Treg/Th17 cells. It may be an effective treatment for MG.

Randomized trial of autologous bone marrow mesenchymal stem cells transplantation for hepatitis B virus cirrhosis: regulation of Treg/Th17 cells.

BACKGROUND AND AIM: Liver cirrhosis is one of the major consequences of hepatitis B virus (HBV) infection, and transplantation of autologous bone marrow mesenchymal stem cells (ABMSCs) is one of promising therapies for patients with HBV-related liver cirrhosis (HBV-LC). However, the mechanism is unclear. The aim of the current study was to explore the role of Treg/Th17 cells in ABMSCs transplantation in patients with HBV-LC. METHODS: In this prospective study, 56 patients were enrolled and randomly assigned to transplantation group and control group. After 24-week follow-up, 39 patients completed the study (20 cases in transplantation group and 19 cases in control group). The Model for End-Stage Liver Disease scores, liver function, changes of Treg/Th17 cells, as well as related transcription factors and serum cytokines, were determined. RESULTS: Although patients in both groups showed significant improvement after Entecavir treatment, ABMSC transplantation further improved patients' liver function. Moreover, there was a significant increase in Treg cells and a marked decrease in Th17 cells in the transplantation group compared with control, leading to an increased Treg/Th17 ratio. Furthermore, mRNA levels of Treg-related transcription factor (Foxp3) and Th17-related transcription factor (RORγt) were increased and decreased, respectively. In addition, serum transforming growth factor-ß levels were significantly higher at early weeks of transplantation, while serum levels of interleukin-17, tumor necrosis factor-α, and interleukin-6 were significantly lower in patients in the transplantation group compared with control. CONCLUSION: ABMSCs transplantation was effective in improving liver function in patients with HBV-LC, which was mediated, at least in part, through the regulation of Treg/Th17 cell balance.

Ento-A alleviates DSS-induced experimental colitis in mice by remolding intestinal microbiota to regulate SCFAs metabolism and the Th17 signaling pathway.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by changes in the metabolism of short chain fatty acids (SCFAs), dysregulation of gut microbiota, and an imbalance of Treg/Th17. Herein, we explore the effects of the Ento-A (an alcohol extract of Periplaneta americana L.) on a mouse model of UC. First, a chronic and recurrent UC model was constructed in BALB/c mice by 2.2% DSS administration. UC mice were continuously treated for 14 days with Ento-A (50, 100, 200 mg/kg, i.g.) or a negative control. Ento-A alleviated many of the pathological changes observed in UC mice, such as body weight loss, disease activity index, changes in colon length, and colonic mucosal damage index. Ento-A also decreased levels of proinflammatory cytokines (IL-1ß, IL-6, IL-17A, and TNF-α), increased levels of anti-inflammatory cytokines (IL-10 and TGF-ß1) and repaired the intestinal mucosal barrier. Additionally, Ento-A regulated the proportions of Th17 cells, and Treg cells in mesenteric lymph nodes harvested from treated mice (as assessed by Flow cytometry), and the expression levels of IL-17A and Foxp3 in colon (as assessed by immunohistochemistry). 16 S rRNA gene sequencing revealed that Ento-A regulated gut microbiota. GC-MS analysis demonstrated that Ento-A also restored SCFAs content in the intestinal tract. Finally, transcriptomic analysis revealed that Ento-A regulated the IL-17 signaling pathway. In summary, Ento-A regulates the diversity and abundance of intestinal flora in UC mice, enhancing the secretion of SCFAs, subsequently regulating the IL-17 signaling pathway, and ultimately repairing the intestinal mucosal barrier.

YTE-17 inhibits colonic carcinogenesis by resetting antitumor immune response via Wnt5a/JNK mediated metabolic signaling.

The density and composition of lymphocytes infiltrating colon tumors serve as predictive factors for the clinical outcome of colon cancer. Our previous studies highlighted the potent anti-cancer properties of the principal compounds found in Garcinia yunnanensis (YTE-17), attributing these effects to the regulation of multiple signaling pathways. However, knowledge regarding the mechanism and effect of YTE-17 in the prevention of colorectal cancer is limited. In this study, we conducted isobaric tags for relative and absolute quantification (iTRAQ) analysis on intestinal epithelial cells (IECs) exposed YTE-17, both in vitro and invivo, revealing a significant inhibition of the Wnt family member 5a (Wnt5a)/c-Jun N-terminal kinase (JNK) signaling pathway. Subsequently, we elucidated the influence and mechanism of YTE-17 on the tumor microenvironment (TME), specifically focusing on macrophage-mediated T helper 17 (Th17) cell induction in a colitis-associated cancer (CAC) model with Wnt5a deletion. Additionally, we performed the single-cell RNA sequencing (scRNA-seq) on the colonic tissue from the Wnt5a-deleted CAC model to characterize the composition, lineage, and functional status of immune mesenchymal cells during different stages of colorectal cancer (CRC) progression. Remarkably, our findings demonstrate a significant reduction in M2 macrophage polarization and Th17 cell phenotype upon treatment with YTE-17, leading to the restoration of regulatory T (Treg)/Th17 cell balance in azoxymethane (AOM)/dextran sodium sulfate (DSS) model. Furthermore, we also confirmed that YTE-17 effectively inhibited the glycolysis of Th17 cells in both direct and indirect co-culture systems with M2 macrophages. Notably, our study shed light on potential mechanisms linking the non-canonical Wnt5a/JNK signaling pathway and well-established canonical ß-catenin oncogenic pathway in vivo. Specifically, we proposed that Wnt5a/JNK signaling activity in IECs promotes the development of cancer stem cells with ß-catenin activity within the TME, involving macrophages and T cells. In summary, our study undergoes the potential of YTE-17 as a preventive strategy against CRC development by addressing the imbalance with the immune microenvironment, thereby mitigating the risk of malignancies.

Formononetin reverses Treg/Th17 imbalance in immune-mediated bone marrow failure mice by regulating the PI3K/Akt signaling pathway.

BACKGROUND: Severe aplastic anemia (SAA) is a syndrome of bone marrow failure which is life-threatening. Recent studies have demonstrated that CD4 + T cell subsets, including T regulatory (Treg) and T helper 17 (Th17) cells, play a pivotal role in the pathogenesis of SAA. Formononetin (FMN) is a natural compound extracted from the traditional Chinese medicine Huangqi, which has the ability to regulate the imbalance of Treg/Th17 cells in some inflammatory diseases. Nevertheless, the therapeutic effect of FMN in SAA has yet to be definitively established. Therefore, the objective of this research was to investigate the effect of FMN on SAA and elucidate its underlying mechanism. METHODS: In vivo experiments, the mice were divided into the following five groups: control, model, low-dose FMN, high-dose FMN, and positive control cyclosporine A group. The immune-mediated bone marrow failure (BMF) mouse model was established by the total body X-ray radiation and lymphocyte infusion. After 10 days of continuous administration of FMN, the numbers of Treg/Th17 cells in the bone marrow and spleen were assessed by flow cytometry. The protein expressions of PI3K/Akt pathway in the bone marrow and spleen was assessed by immunohistochemistry and western blotting. In vitro, the impact of FMN on the differentiation of naive CD4 + T cells into Treg cells was investigated by flow cytometry and ELISA. RESULTS: In comparison with the control group, the model group showed a reduction in bone marrow nucleated cells, a significant decrease in peripheral blood cells, and an altered CD8 + /CD4 + T cell ratio. These findings indicate the successful establishment of a mouse model of immune-mediated BMF. After FMN treatment, there were the increased levels of red blood cells and hemoglobin. In addition, FMN mitigated the bone marrow destruction and restored the CD8 + /CD4 + T cell ratio. Furthermore, in comparison with the control group, the model group showed the decreased levels of Treg cells and the increased levels of Th17 cells. After FMN treatment, there was a significantly increased number of Treg cells and a decreased number of Th17 cells. Additionally, FMN remarkably down-regulated the expression levels of PI3K and Akt proteins in immune-mediated BMF mice. CONCLUSIONS: FMN alleviates immune-mediated BMF by modulating the balance of Treg/Th17 cells through the PI3K/Akt signaling pathway.

Synergism Effect of Dendrobine on Cisplatin in Treatment of H1299 by Modulating the Balance of Treg/Th17.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for about 80% of lung cancers. Cisplatin is a broad-spectrum anti-cancer drug and is often used in combination with other drugs. Research suggests that dendrobine, a pyrrolizidine derivative alkaloid, exhibits antitumor activity in various cancers. This study explores the effect of dendrobine combined with cisplatin on NSCLC and its underlying molecular mechanism. METHODS: The effects of dendrobine combined with cisplatin on tumor progression were evaluated by xenograft model (in vivo) and clonogenic survival assay (in vitro) using H1299 cell line. Annexin V staining was used for detecting apoptotic cells. The population of T cells, B cells and other subpopulations in the peripheral blood was determined by flow cytometry. RESULTS: Dendrobine combined with cisplatin prolonged the survival of mice implanted with H1299 cells and reduced tumor volume compared with single drug application. However, dendrobine exhibited no effect on H1299 cells in clonal survival assays with or without cisplatin treatment and did not promote cisplatin-induced apoptosis in vitro. Importantly, dendrobine suppressed the regulatory T cells (Treg cells) and enhanced the T helper 17 cells (Th17 cells). Treatment of dendrobine significantly reduced Foxp3, and increased the level of IL-17 in serum. CONCLUSION: Dendrobine displayed a synergistic effect with cisplatin to exert anti-tumor effect in vivo, which might be achieved by modulating the balance of Treg/Th17 cells rather than regulating cell apoptosis.

Genetic variation in FOXP3 and ROR-γ genes in pediatric acute lymphocytic leukemia (ALL) patients: correlation with associated cytokines.

BACKGROUND: FOXP3 and ROR-γ genes are master regulators of the Treg and Th17 differentiation, respectively. This work was planned to investigate the impact of FOXP3 (rs3761548C/A and rs3761549C/T) and ROR-γ (rs9017A/G & rs9826A/G) gene polymorphism on the vulnerability of pediatric Egyptians to acute lymphoblastic leukemia (ALL). Furthermore, we evaluated the impact of these genetic variations on Treg/Th17-related cytokines. METHODS: FOXP3 SNPs were genotyped using PCR-based restriction fragment length polymorphism (PCR-RFLP), while ROR-γ SNPs polymorphism were performed by PCR-sequence-specific primer (PCR-SSP). An Enzyme-linked immunosorbent assay (ELISA) was used to assess the levels of Treg/Th17 associated cytokines on 128 ALL children and 124 healthy donors. RESULTS: Compared to controls, patients had a significant increase (p < 0.01/p < 0.05) in FOXP3rs3761548CC genotype and a significant decrease (p < 0.001/p < 0.01) inrs3761548CA genotype. A significant elevation (p < 0.001/p < 0.01) in ROR-γ rs9017AA genotype and a significant reduction (p < 0.01/p < 0.05) in rs9017AG genotype were detected in ALL patients versus controls. An insignificant change in FOXP3 (rs3761549C/T) and ROR-γ (rs9826A/G) genotypes was demonstrated between both groups. ROR-γ GG and GA haplotypes were significantly decreased (p < 0.05/p < 0.05; p < 0.05/p < 0.05) in ALL subjects compared to healthy ones. Relapsed patients had a significantly higher (p < 0.05/P < 0.05) frequency of FOXP3 rs3761548CA genotype than non-relapsed subjects. ROR-γ rs9017AG and rs9826GG genotypes might be associated with the increase in IL-23 plasma level. CONCLUSIONS: Our preliminary data provided evidence for the impact ofFOXP3 (rs3761548C/A) and ROR-γ (rs9017A/G) gene polymorphisms and the occurrence of ALL in Egyptian children. Another large-scale prospective study should be conducted to validate these findings.

Nuciferine Regulates Immune Function and Gut Microbiota in DSS-Induced Ulcerative Colitis.

Nuciferine, a major aporphine alkaloid obtained from the leaves of Nelumbo nucifera, exhibits anti-cancer and anti-inflammatory properties; however, its protective effects against inflammatory bowel diseases (IBD) has never been explored. In this study, an ulcerative colitis (UC) model was established in BALb/c mice by the continuous administration of 5% dextran sulfate sodium (DSS) in drinking water for 1 week. From day 8 to day 14, the DSS-treated mice were divided into a high-dose and a low-dose nuciferine treatment group and were intraperitoneally injected with the corresponding dose of the drug. Body weight loss, disease activity index (DAI), and colon length were measured. Histological changes were observed using hematoxylin and eosin staining. T lymphocyte proliferation was assessed by MTT assay. The ratio of CD3+, CD4+, CD8+, Th1, Th2, Th17, and Treg cells were estimated by flow cytometry. Finally, 16S rRNA sequencing was performed to compare the composition and relative abundance of the gut microbiota among the different treatment groups. The results showed that nuciferine treatment led to a significant improvement in symptoms, such as histological injury and colon shortening in mice with DSS-induced UC. Nuciferine treatment improved the Th1/Th2 and Treg/Th17 balance in the DSS-induced IBD model, as well as the composition of the intestinal microflora. At the phylum level, compared with the control group, the abundance of Firmicutes and Actinobacteriota was decreased in the model group, whereas that of Bacteroidetes increased. Meanwhile, at the genus level, compared with the control group, the numbers of the genera Lachnospiraceae_Clostridium, Bilophila and Halomonas reduced in the model group, while those of Bacteroides, Parabacteroides, and Paraprevotella increased. Notably, nuciferine administration reversed this DSS-induced gut dysbiosis. These results indicated that nuciferine modulates gut microbiota homeostasis and immune function in mice with DSS-induced UC.

Fractalkine mediates lymphocyte inflammation and tubulointerstitial lesions by modifying the Treg/Th17 balance in lupus-prone MRL/lpr mice.

In this study, we first analyzed the expression level of fractalkine (FKN) in the serum of patients with lupus nephritis (LN) and the distribution of peripheral blood Treg cells, and explored FKN and Treg cells, systemic lupus erythematosus disease activity index 2000 (SLEDAI-2K) correlation. Subsequently, we explored the specific role of FKN in tubule interstitial lesions (TILs) and regulatory T (Treg) cells/T helper (Th) 17 cell balance in lupus model mice. Treated with an anti-FKN antibody (aFKN), recombinant FKN (rFKN), or an isotype antibody (IgG) in MRL/MpJ-Faslpr/J and C57BL/6 mice, and then detected TIL level and forkhead box p3 (Foxp3), IL-10, IL-17 and IL-6 expression levels in the kidney and spleen in the proportion of Treg and Th17 cells. Finally, then use aFKN, rFKN, or IgG to intervene in polarized Tregs with IL-6, TGF-ß, IL-23, anti-interferon, and Th17 cells with anti-IL-4 after transforming to transform growth factor (TGF)-ß and interleukin (IL)-2 in isolated mouse spleen lymphocytes. The results showed that the expression level of FKN was positively correlated with SLEDAI-2K and negatively correlated with the distribution of Treg cells. After treatment with aFKN in lupus model mice, kidney damage was delayed, TIL formation was reduced, Foxp3 and IL-10 levels were up-regulated, IL-17 and IL-6 levels were down-regulated in renal tissues, Th17 cell subsets and Treg cell subsets were reduced The increase is in the spleen, and rFKN treatment has the opposite effect in mouse. In addition, after interfering with polarized cells by aFKN, it was found that IL-17 and IL-6 expression levels were down-regulated in Th17 cells, Foxp3 and IL-10 levels in Tregs were up-regulated, and rFKN treatment had the opposite effect in vitro. These results indicate that FKN participates in and promotes SLE target organ damage including: secretion of inflammatory factors and renal TIL, and most importantly, these effects might have been due to modification of the Treg/Th17 cell balance.

1,25(OH)2D3 induces regulatory T cell differentiation by influencing the VDR/PLC-γ1/TGF-ß1/pathway.

Vitamin D has been recommended as an immune modulator in recent years, in addition to regulating calcium-phosphorous-bone metabolism. Clinical studies on organ transplantation found that vitamin D sufficiency patients were less likely to develop acute cellular rejection within one year after transplantation compared to those with vitamin D deficiency. Thus, a high percentage of regulatory T cells might play a key role in preventing acute cellular rejection (ACR). In this report, we studied the specific effects of 1,25(OH)2D3 on human T cell diff ;erentiation, and determined the potential molecule mechanism behind. Results showed that 1,25(OH)2D3 induced the differentiation of T-regulatory cells (Treg cells), while inhibiting Th17 cell proliferation. In addition, 1,25(OH)2D3 promoted secretion of the anti-inflammatory cytokine, transforming Growth Factor beta1 (TGF-ß1) but suppressed pro-inflammatory cytokines such as interleukin-17 (IL-17). Phospholipase C gamma 1 (PLC-γ1) is an indispensable signaling protein downstream of the classical TCR signaling pathway and was shown to play a crucial role in T cell activation, while Naive T cells expressed less PLC-γ1. Here we showed that Vitamin D could significantly upregulate PLC-γ1 expression, which then induced expression of TGF-ß1. In summary, 1,25(OH)2D3 indirectly modulates the differentiation of Treg/Th17 cells by aff ;ecting the VDR/PLC-γ1/TGF-ß1pathway. These results indicate that administration 1,25(OH)2D3 supplements may be a beneficial treatment for organ transplantation recipients.

Decrease of Markers Related to Bone Erosion in Serum of Patients with Musculoskeletal Disorders after Serial Low-Dose Radon Spa Therapy.

Musculoskeletal disorders (MSDs) are the most frequent cause of disability in Europe. Reduced mobility and quality of life of the patients are often associated with pain due to chronic inflammation. The inflammatory process, accompanied by a destruction of the cartilage and bone tissue, is discussed as a result of (A) the infiltration of immune cells into the joints, (B) an altered homeostasis of the joint cavity (synovium) with a critical role of bone remodeling cells, and (C) release of inflammatory factors including adipokines in the arthritic joint. In addition to the classical medication, low-dose radiation therapy using photons or radon spa treatments has shown to reduce pain and improve the mobility of the patients. However, the cellular and molecular mechanisms of anti-inflammatory effects of radon are yet poorly understood. We analyzed blood and serum samples from 32 patients, suffering from MSDs, who had been treated in the radon spa in Bad Steben (Germany). Before and after therapy, we measured the levels of markers related to bone metabolism (collagen fragments type-1, cartilage oligomeric matrix protein, receptor activator of NFκB ligand, and osteoprotegerin) in the serum of patients. In addition, adipokines related to inflammation (visfatin, leptin, resistin, and adiponectin) were analyzed. Some of these factors are known to correlate with disease activity. Since T cells play an important role in the progression of the disease, we further analyzed in blood samples the frequency of pro- and anti-inflammatory T cell subpopulations (CD4+IL17+ T cells and CD4+FoxP3+ regulatory T cells). Overall, we found a decrease of collagen fragments (CTX-I), indicating decreased bone resorption, presumably by osteoclasts, in the serum of MSD patients. We also observed reduced levels of visfatin and a consistent trend toward an increase of regulatory T cells in the peripheral blood, both indicating attenuation of inflammation. However, key proteins of bone metabolism were unchanged on a systemic level, suggesting that these factors act locally after radon spa therapy of patients with MSDs.

Role of alteration in Treg/Th17 cells' balance in nephropathic patients with Type 2 diabetes mellitus.

INTRODUCTION: In type 2 diabetes mellitus, the adaptive immune system drives systemic inflammation, promoting insulin resistance and related complications, such as diabetic nephropathy. Increased infiltration of activated T lymphocytes has been found in patients with diabetic nephropathy. T-cell influx and accumulation are the factors that aggravate diabetic nephropathy and link with glomerular filtration surface and albumin excretion. An appropriate balance between pro-inflammatory (T helper 17: Th17, and T helper 1: Th1) and anti-inflammatory (regulatory T cells: Tregs) subsets of T cells is critical to maintain homeostasis and avoid inflammatory disease. The aim of this study was to determine the balance between T helper 17 and regulatory T cells in type 2 diabetic patients who have diabetic nephropathy. METHODS: This case control study was conducted between December 2013 and June 2014 in Theodor Bilharz Research Institute in Egypt. Forty patients and 20 healthy volunteers were recruited in the study, and three groups were formed, i.e. two groups of cases with 20 patients in each group and one group of 20 controls) The groups were 1) 20 type 2 diabetic patients with nephropathy (group A); 2) 20 type 2 diabetic patients without nephropathy (group B); and 3) 20 healthy individuals (control group). Evaluation of T cells was done by standard 2-color flow cytometry. RESULTS: The study found higher mean of Th17 counts and Th17/Treg ratio among type 2 diabetic nephropathy patients compared to other groups; but a lower mean of Treg count was identified among type 2 diabetic nephropathy patients than in the other groups (p-value = 0.001). CONCLUSION: The important role for regulatory T cells in the protection against nephropathy in type 2 diabetic patients was demonstrated, and also it was observed that T helper 17 cells were associated with renal affection.

Women with recurrent spontaneous abortion have decreased 25(OH) vitamin D and VDR at the fetal-maternal interface

Immunological mechanisms have been proposed to underlie the pathogenesis of recurrent spontaneous abortion (RSA). Vitamin D has a potent immunomodulatory effect, which may affect pregnancy outcome. The objective of this study was to investigate 25-hydroxyvitamin D [25(OH) D] concentration and vitamin D receptor (VDR) expression in the decidual tissues of RSA patients. Thirty women with RSA (RSA group) and thirty women undergoing elective abortion (control group) were recruited during 2016 from gynecology outpatient clinics. We measured 25(OH) D, interleukin (IL)-17, IL-23, transforming growth factor β (TGF-β), VDR and 1-α-hydroxylase (CYP27B1) in decidual tissues collected during the abortion procedure. In the RSA group, 25(OH) D and TGF-β were significantly decreased while IL-17 and IL-23 were significantly increased compared with the control group. VDR expression was significantly decreased in the RSA group compared with the control group. Logistic regression analysis showed a significant negative correlation between 25(OH) D in decidual tissues and RSA. These results indicated that vitamin D concentrations in the decidua are associated with inflammatory cytokine production, suggesting that vitamin D and VDR may play a role in the etiology of RSA.