Impact of Matrine on Th17/Treg Cell Balance in Coronary Heart Disease Rats by Regulating the RhoA-ROCK Signaling Pathway / 中药新药与临床药理

Objective To investigate the impacts of matrine on the balance of helper T cell 17(Th17)/regulatory T cell(Treg)and the Ras homolog gene family member A(RhoA)-Rho-associated coiled-coil forming protein kinase(ROCK)signaling pathway in coronary heart disease(CHD)rats.Methods A model of coronary heart disease was established.Rats were grouped into control group,model group(CHD group),low-dose matrine(50 mg·kg-1,Matrine-L)group,high-dose matrine(200 mg·kg-1,Matrine-H)group,and Matrine-H+LPA(200 mg·kg-1 matrine+10 mg·kg-1 LPA)group.Echocardiography was applied to detect cardiac function.Enzyme linked immunosorbent assay(ELISA)method was used to detect interleukin-17(IL-17)and transforming growth factor(TGF-β).The quantity of Th17,Treg and Th17/Treg ratio were detected by flow cytometry.Immunohistochemistry was applied to detect the protein expressions of endothelial nitric oxide synthase(eNOS)and endothelin 1(ET-1).Masson staining was carried out to observe the pathological changes of myocardial tissue.The myocardial infarction in each group of rats was observed by TCC staining.TUNEL staining was performed to detect cell apoptosis in myocardial tissue.Additionally,RhoA activity was detected by assay kit.Western Blot method was applied to detect the protein expressions levels of B-cell lymphoma factor 2(Bcl-2),Bcl-2 associated X protein(Bax),cysteine aspartate proteinase-3(Caspase-3),RhoA,ROCK1 and ROCK2.Results Compared with the control group,a large amount of blue collagen fiber deposition was observed in the myocardial tissue of CHD group.The expression levels of left ventricular end-diastolic volume(LVEDV),left ventricular end-systolic volume(LVESV),IL-17,Th17,Th17/Treg,ET-1,infarct size,cell apoptosis rate,TUNEL positive rate,Bax,Caspase-3,RhoA activity,RhoA,ROCK1,ROCK2 were obviously increased.The expression levels of left ventricular ejection fraction(LVEF),left ventricular shortening fraction(LVFS),TGF-β,Treg,eNOS,and Bcl-2 were obviously reduced(P＜0.05).Compared with the CHD group,blue collagen fibers in myocardial tissue of Matrine-L and Matrine-H groups gradually decreased.The expression levels of LVEDV,LVESV,IL-17,Th17,Th17/Treg,ET-1,infarct size,cell apoptosis rate,TUNEL positive rate,Bax,Caspase-3,RhoA activity,RhoA,ROCK1 and ROCK2 were obviously reduced in sequence.The expression levels of LVEF,LVFS,TGF-β,Treg,eNOS,and Bcl-2 were also obviously increased in sequence(P＜0.05).Compared with the Matrine-H group,blue collagen fibers in myocardial tissue of Matrine-H+LPA group increased.The expression levels of LVEDV,LVESV,IL-17,Th17,Th17/Treg,ET-1,infarct size,cell apoptosis rate,TUNEL positive rate,Bax,Caspase-3,RhoA activity,RhoA,ROCK1,ROCK2 were obviously increased,while the expression levels of LVEF,LVFS,TGF-β,Treg,eNOS and Bcl-2 were obviously reduced(P＜0.05).Conclusion Matrine regulates Th17/Treg cell balance and improves myocardial injury in rats with CHD by inhibiting the RhoA-ROCK signaling pathway.

Th17/Treg balance and macrophage polarization ratio in lower extremity arteriosclerosis obliterans

@#Objective: To explore the balance of peripheral blood T helper 17 cells/regulatory T cell (Th17/Treg) ratio and the polarization ratio of M1 and M2 macrophages in lower extremity arteriosclerosis obliterans (ASO). Methods: A rat model of lower extremity ASO was established, and blood samples from patients with lower extremity ASO before and after surgery were obtained. ELISA was used to detect interleukin 6 (IL-6), IL-10, and IL-17. Real-time RCR and Western blot analyses were used to detect Foxp3, IL-6, IL-10, and IL-17 expression. Moreover, flow cytometry was applied to detect the Th17/Treg ratio and M1/M2 ratio. Results: Compared with the control group, the iliac artery wall of ASO rats showed significant hyperplasia, and the concentrations of cholesterol and triglyceride were significantly increased (P<0.01), indicating the successful establishment of ASO. Moreover, the levels of IL-6 and IL-17 in ASO rats were pronouncedly increased (P<0.05), while the IL-10 level was significantly decreased (P<0.05). In addition to increased IL-6 and IL-17 levels, the mRNA and protein levels of Foxp3 and IL-10 in ASO rats were significantly decreased compared with the control group. The Th17/Treg and M1/M2 ratios in the ASO group were markedly increased (P<0.05). These alternations were also observed in ASO patients. After endovascular surgery (such as percutaneous transluminal angioplasty and arterial stenting), all these changes were significantly improved (P<0.05). Conclusions: The Th17/Treg and M1/M2 ratios were significantly increased in ASO, and surgery can effectively improve the balance of Th17/Treg, and reduce the ratio of M1/M2, and the expression of inflammatory factors.

Effect of Icariin on Peripheral Blood Dendritic Cells and Th17/Treg Balance in Myocardial Remodeling Model of Dahl Salt-sensitive Rats / 中国实验方剂学杂志

ObjectiveTo investigate the effect of icariin （ICA）-mediated vitamin D system on peripheral blood dendritic cells （DCs） and helper T cells 17 （Th17）/regulatory T cells （Treg） balance in myocardial remodeling model of Dahl salt-sensitive rats. MethodFifty SPF Dahl salt-sensitive rats were divided into model group， vitamin D group （3×10-5 mg·kg-1·d-1）， and high-， medium-， and low-dose ICA groups （120， 60， 30 mg·kg-1·d-1）， and 10 Dahl salt-resistant rats were used as normal group. The myocardial remodeling model was established by feeding rats with a high-salt diet containing 8% NaCl. After six weeks of modeling， the normal group and the model group were given an equal volume of ultrapure water by gavage， and other groups were continuously administrated for six weeks. Cardiac echocardiography， hematoxylin-eosin （HE） staining， and Masson staining were used to observe the pathological changes in cardiac structure and fibrosis. The levels of serum 25（OH）D3， B-type N-terminal pro-brain natriuretic peptide （NT-ProBNP）， interleukin （IL）-17， transforming growth factor （TGF）-β1， IL-12， and IL-10 were detected by enzyme-linked immunosorbent assay （ELISA）. The phenotype of peripheral blood DCs and the ratio of Th17/Treg cells of rats were detected by flow cytometry. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to detect the mRNA and protein expressions of vitamin D receptor （VDR），1α-hydroxylase （CYP27B1）， and 24-hydroxylase （CYP24A1） in peripheral blood DCs of rats. ResultCompared with the control group， the rats in the model group had pathological changes such as disordered arrangement of myocardial cells and cytoplasmic hypertrophy and swelling. Myocardial collagen fibers proliferated significantly， and the arrangement of myocardial fibers was disordered. The levels of serum 25（OH）D3 and IL-10 were significantly decreased， and the levels of serum IL-17， TGF-β1， IL-6， IL-12， and NT-ProBNP were significantly increased （P<0.05）. The costimulatory molecules CD40， CD80， CD86， and MHC-Ⅱ were highly expressed in the peripheral blood DCs， and the expression of CD11 and CD11b was lower （P<0.05）. The proportion of Th17 cells in the peripheral blood was significantly increased， and the proportion of Treg cells was decreased. The ratio of Th17/Treg was increased （P<0.05）. The mRNA and protein expressions of CYP24A1 in peripheral blood DCs increased， and the mRNA and protein expressions of CYP27B1 and VDR decreased （P<0.05）. Compared with the model group， the arrangement of myocardial fibers in each drug administration group was relatively regular， and the swelling of myocardial cells was significantly reduced. The pathological morphology of myocardial tissue was improved to varying degrees. The pathological changes in myocardial tissue were improved and alleviated to varying degrees. The drug could reduce the serum levels of NT-ProBNP， IL-17， TGF-β1， IL-6， and IL-12 and increase the level of serum 25（OH）D3 and IL-10 （P<0.05）. The expression of costimulatory molecules CD40， CD80， CD86， and MHC-Ⅱ in the peripheral blood DCs of rats was decreased， and the expression of CD11 and CD11b molecules was increased （P<0.05）. The drug could reduce the proportion of Th17 cells in peripheral blood and the ratio of Th17/Treg cells and increase the proportion of Treg cells （P<0.05）. It could decrease the mRNA and protein expressions of CYP24A1 in peripheral blood DCs of rats and elevate the mRNA and protein expression of VDR and CYP27B1 （P<0.05）. ConclusionICA can regulate the phenotype of peripheral blood DCs and the ratio of Th17/Treg cells by regulating the vitamin D system and play a role in improving myocardial remodeling from the perspective of immune balance.

Exploring Mechanism of Different Processed Products of Baiyaojian and Its Formula in Treating Ulcerative Colitis Based on Serum Metabolomics and Flow Cytometry / 中国实验方剂学杂志

ObjectiveTo explore the potential mechanism of different processed products of Baiyaojian and its compound Xiangmei pills in rats with ulcerative colitis（UC） by comparing the pharmacodynamic and metabolomic differences. MethodEighty SD rats were acclimatized and kept for 3 d， and randomly divided into 8 groups［blank group， model group， mesalazine group（0.4 g·kg-1）， Baiyaojian group（1.89 g·kg-1）， stir-fried Baiyaojian group（1.89 g·kg-1）， carbonized Baiyaojian group（1.89 g·kg-1）， and Xiangmei pills low and high dose groups（1.89， 5.67 g·kg-1）］， with 10 rats in each group. Rats in the blank group were administered physiological saline by gavage， and rats in the remaining 7 groups were orally administered 5% dextran sodium sulfate（DSS） solution daily for 8 consecutive days to induce UC model. After successful modeling， each dosing group was given the corresponding dose of drug solution by gavage， and the blank and model groups were given equal amounts of saline by gavage， and the drug was administered continuously for 8 d. Then serum levels of tumor necrosis factor-α（TNF-α）， interleukin（IL）-6， IL-10 and IL-1β were measured by enzyme-linked immunosorbent assay（ELISA）， hematoxylin-eosin（HE） staining was used to observe the histopathological changes of colon tissue， the proportion of T helper 17 cells（Th17） and regulatory T cells（Treg） in the peripheral blood of rats in each group was detected by flow cytometry. The endogenous metabolites in serum of rats were detected by ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry（UPLC-Q-Exactive Orbitrap MS）， and the differential metabolites were characterized by combining principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA）， and were analyzed according to the variable importance in the projection（VIP） value>1.0 and P<0.05， and potential metabolic pathways were analyzed according to Human Metabolome Database（HMDB）. ResultCompared with the blank group， the colon tissue of the model group was congested and the mucosa was ulcerated， the colon length was significantly reduced（P<0.01） and the quality was significantly increased（P<0.05）， the proportion of Th17/Treg in the peripheral blood and the serum levels of TNF-α， IL-6 and IL-1β were significantly increased， while the IL-10 expression wassignificantly reduced（P<0.05， P<0.01）. Compared with the model group， the colon tissue of UC rats in each treatment group was improved with scattered ulcers， reduced inflammatory cell infiltration， significantly increased colon length， and significantly decreased mass（P<0.05）， the proportion of Th17/Treg in the peripheral blood decreased， the expression of TNF-α，IL-6 and IL-1β was significantly reduced（P<0.05， P<0.01）， while the IL-10 expression was significantly increased（P<0.01）. The therapeutic effect of different administration groups on UC was in the order of high dose group of Xiangmei pills>low dose group of Xiangmei pills>carbonized Baiyaojian group>stir-fried Baiyaojian group>Baiyaojian group. And a total of 26 differential metabolites were screened in the metabolomics results. Compared with the blank group， 14 differential metabolites were up-regulated and 5 metabolites were down-regulated in the model group， and 14， 9， 14， 12 and 17 metabolites could be recalled in the Baiyaojian group， stir-fried Baiyaojian group， carbonized Baiyaojian group， Xiangmei pills low and high dose groups. The main metabolic pathways involved were citrate cycle pathway， pantothenic acid and coenzyme A biosynthesis pathway， aromatic hydrocarbon receptor（AhR） signaling pathway， glycolysis/gluconeogenesis pathway. ConclusionThe therapeutic effect of Baiyaojian on UC is significantly improved after charcoal stir-frying， and the efficacy is more prominent when combined with Angelicae Dahuricae Radix and Mume Fructus Carbonisata， which can provide a basis for the development of Baiyaojian compound preparations.

Development and application of regulatory T cells in corneal transplant rejection / 国际眼科杂志(Guoji Yanke Zazhi)

Corneal transplantation is an effective treatment for corneal blindness, and it is the only hope for patients with corneal blindness. Cornea has no blood vessels and no lymphatic vessels, which is called immune privilege organ, so the success rate of corneal transplantation is significantly higher than that of other organ transplantation, but the rejection reaction after corneal transplantation is still the main reason for the failure of corneal transplantation. The directional movement of immune cells to lymphoid tissue and inflammatory sites is the mainly immune response after organ transplantation. And the regulatory T cells(Treg)play a key role in immune regulation, which can induce immune tolerance by regulating and inhibiting the activation of effector T cells and reduce the rejection reaction after corneal transplantation. In addition, this review also discussed the effectiveness of applying cordyceps sinensis extract FTY720 to enhance the function of Treg. Based on this, we briefly reviewed the sources, mechanism of action and treatment of Treg after corneal transplantation, so as to provide some reference for the subsequent clinical application transformation and basic research.

Regulation of Immune Balance by Traditional Chinese Medicine in Treatment of Cough Variant Asthma： A Review / 中国实验方剂学杂志

Cough variant asthma （CVA） is a chronic respiratory disease with cough as its main symptom. The occurrence of CVA is closely related to non-specific airway inflammation， and its pathogenesis involves environmental， genetic， immune， and other factors. In recent years， the advantages of traditional Chinese medicine （TCM） in the treatment of CVA have attracted the attention of experts and scholars in China and abroad， especially its prominent role in regulating immune balance， relieving cough symptoms in CVA patients， and reducing recurrence. T Helper cells 1 （Th1）， T helper cells 2 （Th2）， T helper cells 17 （Th17）， and regulatory T cells （Treg） are derived from CD4+ T cells. Immune imbalance of Th1/Th2 and Th17/Treg is a new hotspot in the pathogenesis of CVA and a potential key target in the treatment of CVA by TCM. Th cell subsets are in dynamic balance under physiological conditions， maintaining respiratory immune homeostasis in which pro-inflammatory cytokines and anti-inflammatory cytokines are balanced. Immature helper T cells （Th0） can be differentiated into Th1， Th2， Th17， Treg， and other cell subsets due to cytokine types in the microenvironment in the stage of CVA maturation. The proliferation of Th2 cells leads to eosinophilic airway inflammation. Excessive differentiation of Th17 cells induces neutrophil airway inflammation. Th1/Th2 and Th17/Treg cells are mutually restricted in number and function， and the immune imbalance of Th1/Th2 and Th17/Treg is easy to aggravate the generation of inflammatory response. Restoring immune balance is particularly important for the airway anti-inflammatory therapy of CVA. In this paper， the imbalance of Th1/Th2 and Th17/Treg and the pathogenesis of CVA were systematically expounded. Meanwhile， the latest research on the regulation of immune imbalance by TCM compound， single TCM， and its effective ingredients in the treatment of CVA was reviewed. It provides ideas and references for revealing the scientific connotation of TCM regulating immune balance therapy of CVA， as well as the development of clinical treatment and basic research of CVA.

Foxp3+CD39+CD73+ regulatory T-cells are decreased in the peripheral blood of women with deep infiltrating endometriosis

Abstract Endometriosis's pathophysiology remains incompletely understood, with evidence pointing towards a dysregulated immune response. Regulatory T (Treg) cells, pivotal in maintaining self-tolerance, may facilitate the survival of ectopic endometrial cells within the abdominal cavity, thereby contributing to endometriosis development. This study aimed to assess the prevalence of CD39+CD73+ suppressor Treg cell subsets in the peripheral blood of endometriosis patients. This research focuses on the pivotal role of regulatory T-cells (Tregs), which are essential for maintaining immune tolerance and preventing autoimmune diseases. A case-control study was conducted, including 32 women diagnosed with endometriosis and 22 control subjects. The frequency of peripheral blood CD39+CD73+ suppressor Treg cells was quantified using flow cytometry. No significant differences were observed in the frequency of CD3+CD4+CD25High cells (Median [M]: 10.1; Interquartile Range [IQR]: 6.32‒18.3 vs. M: 9.72; IQR: 6.22-19.8) or CD3+CD4+CD25HighCD39+Foxp3+ cells (M: 31.1; IQR: 19.7-44.0 vs. M: 30.55; IQR: 18.5-45.5) between controls and patients. However, a significantly lower frequency of CD3+CD4+CD25HighCD39+CD73+ cells was observed in the endometriosis group compared to controls (M: 1.98; IQR: 0.0377-3.17 vs. M: 2.25; IQR: 0.50-4.08; p = 0.0483), suggesting a reduction in systemic immune tolerance among these patients. This finding highlights the potential role of CD39 and CD73 expression on Treg cells as biomarkers for assessing disease severity and progression. Furthermore, elucidating the mechanisms driving these alterations may unveil new therapeutic strategies to restore immune equilibrium and mitigate endometriosis symptoms.

Natural regulatory T cells increase significantly in pediatric patients with parasitic infections: Flow cytometry study

Background: The most accepted definition of regulatory T cells (Tregs) relies on the expression of several biomarkers, including CD4, CD25, and transcription factor, Foxp3. The Tregs maintain tolerance to self-antigens and prevent autoimmune diseases. Aim: The purpose of this study was to determine the difference in natural Treg levels in Entamoeba histolytica, Schistosoma mansoni, Giardia lamblia, Enterobius vermicularis, and Hymenolepis nana infected patients. Setting and Design: Fifty-one pediatric subjects (29 males and 22 females) were recruited from a tertiary care hospital, and were divided into infected and non-infected (control) groups. The mean age of the subjects was 8.7 years. Materials and Methods: Blood samples were collected from infected and non-infected groups, and change in the level of Tregs in these subjects was investigated by flow cytometry. Statistical Analysis Used: The statistical analysis of data was performed by SPSS software. Quantitative data used in this study included mean and standard deviation. Data from the two groups were compared by the Student's t-test. The age of the patient and infection status were used for multivariate logistic regression analysis. Odds ratios (ORs) were estimated within a 95% confidence interval, and a P value of <0.05 was considered significant. Results and Conclusions: The levels of natural regulatory T cells, indicated by the biomarkers, CD4+, CD25+, and Foxp3+, increase significantly in patients infected by Entamoeba histolytica, Schistosoma mansoni, Giardia lamblia, Enterobius vermicularis, and Hymenolepis nana as compared to controls. They also increase in cases of mixed infection as compared to infection by a single parasite.

Research Progress of Regulatory T Cells in the Pathogenesis of Multiple Myeloma --Review / 中国实验血液学杂志

The multiple myeloma (MM), the second most common hematologic malignancy, is malignant proliferative disease of plasma cells. Although the application of many targeted drugs has significantly prolonged the survival time of MM patients, it is still an incurable disease. In recent years, the immunosuppression caused by interaction between tumor microenvironment(TME) and tumor cells has attracted people's attention gradually. As a kind of immunosuppressive cells in TME, regulatory T cells (Treg) play an important role in the progress of MM. Treg is related to the proliferation and metastasis of tumors, and can lead to the progress of MM by promoting the angiogenesis and generating immunosuppressive TME. In this review, we briefly summarized the latest research progress on the impact of Treg on the pathogenesis of MM.

Changes in percentage of GATA3+ regulatory T cells and their pathogenic roles in allergic rhinitis / 南方医科大学学报

OBJECTIVE@#To investigate the changes in percentage of GATA3+ regulatory T (Treg) cells in patients with allergic rhinitis (AR) and mouse models.@*METHODS@#The nasal mucosa specimens were obtained from 6 AR patients and 6 control patients for detection of nasal mucosal inflammation. Peripheral blood mononuclear cells (PBMC) were collected from 12 AP patients and 12 control patients to determine the percentages of Treg cells and GATA3+ Treg cells. In a C57BL/6 mouse model of AR, the AR symptom score, peripheral blood OVA-sIgE level, and nasal mucosal inflammation were assessed, and the spleen of mice was collected for detecting the percentages of Treg cells and GATA3+ Treg cells and the expressions of Th2 cytokines.@*RESULTS@#Compared with the control patients, AR patients showed significantly increased eosinophil infiltration and goblet cell proliferation in the nasal mucosa (P < 0.01) and decreased percentages of Treg cells and GATA3+ Treg cells (P < 0.05). The mouse models of AR also had more obvious allergic symptoms, significantly increased OVA-sIgE level in peripheral blood, eosinophil infiltration and goblet cell hyperplasia (P < 0.01), markedly lowered percentages of Treg cells and GATA3+ Treg cells in the spleen (P < 0.01), and increased expressions of IL-4, IL-6 and IL-10 (P < 0.05).@*CONCLUSION@#The percentage of GATA3+ Treg cells is decreased in AR patients and mouse models. GATA3+ Treg cells possibly participate in Th2 cell immune response, both of which are involved in the occurrence and progression of AR, suggesting the potential of GATA3+ Treg cells as a new therapeutic target for AR.

Correlation and clinical significance of VISTA expression and cervical squamous cell carcinoma / 中华肿瘤杂志

Objective: To explore the relationship between the expression of the T-cell activation suppressor-immunoglobulin variable region (VISTA) and the development of cervical squamous cell carcinoma (CSCC), and the impact on the prognosis of CSCC patients. Methods: Cervical tissue samples from 116 CSCC, including 23 cervical intraepithelial neoplasia (CIN) grade I, 23 CIN grade Ⅱ-Ⅲ, and 23 chronic cervicitis patients, were collected from the First Hospital of Soochow University between March 2014 and April 2019. The expression of VISTA in each group was detected by immunohistochemistry (IHC). Survival data of CSCC patients were obtained by follow-up. The survival analysis was performed by Kaplan-Meier method, and survival differences between groups were compared by Log rank test. Prognostic impact factors were analyzed using a multifactorial Cox proportional hazards model. Results: The positive rate of VISTA expression in CSCC group was 32.8% (38/116), and which of grade Ⅱ-Ⅲ was 17.4% (4/23). VISTA expression results showed no positive expression patients in the cervical intraepithelial neoplasia grade I and chronic cervicitis groups. The differences between the CSCC group and other groups were statistically significant (P<0.01). In 116 CSCC patients, VISTA expression was associated with International Federation of Gynecology and Obstetrics (FIGO) stage and lymph node metastasis (P<0.01). The mean survival time of patients in the VISTA positive expression group was 30.7 months, and the 3-year survival rate was 44.7% (17/38). However, the mean survival time of the patients in the VISTA negative expression group was 49.1 months, and the 3-year survival rate was 87.2% (68/78). The Cox regression model found that VISTA expression positivity (P=0.001) and FIGO stage (P=0.047) were prognostic factors for CSCC, and patients with VISTA-positive CSCC had a 4.130-fold risk of death higher than those with VISTA-negative expression. Conclusions: The VISTA protein is highly expressed in CSCC tissues, and its expression level is closely related to the occurrence and development of CSCC. The expression of VISTA can be used as an independent predictor of CSCC prognosis and can provide a strong basis for the treatment of CSCC with immune checkpoint inhibitors.

Detection of regulatory T cells in peripheral blood of keloid patientsa nd its significance / 中华医学美学美容杂志

Objective:To explore the abnormally low expression of regulatory T cells (Tregs) in the peripheral blood of patients with keloids, its correlation with the formation and evolution of keloids.Methods:A total of 50 peripheral blood samples of patients diagnosed with keloids were collected in the first diagnosis of Changzhi City People′s Hospital from January 2019 to January 2021 as keloid group, including 22 males and 28 females, with an age range of 18-55 years and an average age of 32.13 years; the control group was normal healthy beauty seekers in the outpatient department of Changzhi People′s Hospital during the same period, and finally 25 peripheral blood samples were also collected, including 15 males and 10 females, with an age range of 18-55 years and an average age of 32.96 years. All patients had 2 ml of venous blood drawn on an empty stomach early in the morning before admission for treatment, and placed in an ethylene diamine tetraacetic acid (EDTA) potassium anticoagulation tube. Fresh peripheral blood samples were generated and sent for examination immediately. Flow cytometry was used to detect the CD4 + , CD25 + , CD127 + cells and low Tregs ratio in peripheral blood of keloid patients ( n=50) and normal healthy people ( n=25); the fluorescence intensity was analyzed, the light scattering data were saved, and Cell Quest Plot was used on the computer after the test. The point diagram and the group square diagram were analyzed by SPSS 24.0 for statistical software analysis; peripheral blood Tregs ratio was expressed as the mean ± standard deviation ( ±s), and the mean comparison between the two groups was conducted by using independent sample t test; multiple groups One-way analysis of variance was used for the comparison of the averages, and the difference was statistically significant at P<0.05. The keloid questionnaire and clinical grading were utilized to deeply analyze the relationship between the Tregs ratio in peripheral blood of keloid patients and the severity of keloid. Results:Compared with the normal control group, the peripheral blood Tregs ratio of the keloid group was significantly reduced [(4.39±1.31)% vs. (6.64±1.83)%, P<0.001]; according to the Sawada score scale, keloids were classified as mild, moderate and severe degrees; the Tregs ratio in peripheral blood of the moderate keloid group was significantly lower than that of the mild keloid group [(4.43±1.23)% vs. (5.37±1.12)%, P<0.05], while in the severe keloid group it was also significantly lower than the moderate keloid group [(3.55±0.97)% vs. (4.43±1.23)%, P<0.05]. Conclusions:The Tregs ratio of peripheral blood in patients with keloids is significantly decreased, suggesting that Tregs cell is one of the biomarkers to reflect the severity of keloids.

Expression of interleukin-36 and its modulation on the balance between regulatory T cells and Th17 cells in patients with myasthenia gravis / 中华神经科杂志

Objective:To investigate interleukin (IL)-36 expression in patients with myasthenia gravis (MG), and to study the modulatory function of IL-36 on regulatory T cells (Tregs) and Th17 cells in MG patients.Methods:Fifty-one MG patients (MG group) and 25 healthy controls (control group) were enrolled in this study in Xinxiang Central Hospital between July 2016 and August 2021. Peripheral blood was collected. Plasma and peripheral blood mononuclear cells (PBMCs) were isolated. Plasma IL-36α, IL-36β, IL-36γ, IL-36RA, IL-35, and IL-17 levels were measured by enzyme-linked immunosorbent assay. The percentages of Tregs and Th17 cells were measured by flow cytometry. Forkhead box protein P3 (FoxP3) and retinoid-related orphan receptor gamma t (RORγt) mRNA expressions were measured by real-time polymerase chain reaction. PBMCs or purified Tregs from MG patients were stimulated with recombinant IL-36β (5 ng/ml). Changes of Tregs and Th17 cell percentages, IL-35 and IL-17 secretions, FoxP3 and RORγt mRNA expressions, as well as immunosuppressive activity of Tregs were analyzed.Results:There were no statistically significant differences of IL-36α, IL-36γ, or IL-36RA between the control group and the MG group (all P>0.05). IL-36β level was notably higher in the MG group compared with the control group [(73.43±13.91) pg/ml vs (60.91±12.65) pg/ml, t=3.79, P<0.001]. Treg percentage [(4.67±1.33)% vs (6.32±1.81)%, t=4.48, P<0.001], IL-35 [(50.06±7.93) pg/ml vs (65.37±8.90) pg/ml, t=7.59, P<0.001] and FoxP3 mRNA expression (1.03±0.14 vs 1.57±0.46, t=7.78, P<0.001) was lower, while Th17 cell percentage [(1.05±0.15)% vs (0.94±0.21)%, t=2.61, P=0.011], IL-17 [(40.61±13.13) pg/ml vs (33.09±11.48) pg/ml, t=2.44, P=0.017] and RORγt mRNA expression (1.26±0.16 vs 1.03±0.13, t=6.08, P<0.001) was higher in the MG group ( P<0.05). There were no statistically significant differences of above indices between different genders, onset ages, afflicting with thymoma, or different Osserman types (all P>0.05). There were no statistically significant correlations between above indices and quantitative myasthenia gravis (QMG) score (all P>0.05). Recombinant IL-36β stimulation did not affect PBMCs proliferation in MG patients ( P=0.248), and reduced Tregs percentage [(3.05±0.66)% vs (4.18±1.07)%, t=4.23, P<0.001], IL-35 secretion [(48.12±10.93) pg/ml vs (56.96±13.73) pg/ml, t=2.36, P=0.023] and FoxP3 mRNA expression (0.99±0.17 vs 1.18±0.13, t=4.01, P<0.001), but did not affect Th17 cell percentage, IL-17 secretion or RORγt mRNA expression (all P>0.05). Recombinant IL-36β stimulation inhibited immunosuppressive activity of Tregs, which presented as enhanced cellular proliferation [(0.83±0.12)×10 5vs (0.69±0.15)×10 5, t=3.02, P=0.005] and reduced IL-35 secretion [(28.71±10.08) pg/ml vs (37.12±10.47) pg/ml, t=2.39, P=0.023]. Conclusion:Increased IL-36β contributed to the regulation of Tregs/Th17 cell balance probably through inhibition of Tregs function in MG patients.

Bacteroides fragilis ATCC25285 relieves colitis by inducing Treg cells differentiation via the TGF-β/Smad3 signaling pathway / 中国药科大学学报

@#The changes in intestinal flora are usually associated with different gastrointestinal diseases, and intestinal flora homeostasis can enhance immune tolerance and regulate intestinal immune balance.Previous studies have found that the increase of the relative abundance of Bacteroides fragilis (B.fragilis) in Bacteroides intestinalis can significantly enhance the expression of intestinal regulatory T cells (Treg) and anti-inflammatory cytokines, thus alleviating intestinal inflammation.However, the mechanism of B.fragilis regulating intestinal immunity is still unclear.In this study, an acute colitis model was constructed by giving 3% DSS in drinking water solution to SPF-grade C57BL/6 mice for 7 days, and exogenous supplementation B.fragilis was given to mice by gastric gavage to study its regulatory effect on intestinal immunity and its mechanism of action.The results showed that B.fragilis could improve the intestinal flora disorder in mice with colitis and increase the content of short-chain fatty acids (SCFAs), the main metabolite of the intestinal flora.By extracting mouse tissue lymphocytes, naive CD4+ T cells, and liposome-modified siRNA knockdown mouse Smad3, it was further discovered by flow cytometry that B.fragilis induced the expression of intestinal Treg cells and related cytokines through the TGF-β/Smad3 signaling pathway, which enhanced intestinal regulatory immunity and alleviated colitis.It was also found that B.fragilis activated TGF-β by increasing the expression of reactive oxygen species (ROS), thus inducing Treg cell differentiation and playing an immunomodulatory role.

Short-chain fatty acids ameliorate spinal cord injury recovery by regulating the balance of regulatory T cells and effector IL-17+ γδ T cells / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Spinal cord injury (SCI) causes motor, sensory, and autonomic dysfunctions. The gut microbiome has an important role in SCI, while short-chain fatty acids (SCFAs) are one of the main bioactive mediators of microbiota. In the present study, we explored the effects of oral administration of exogenous SCFAs on the recovery of locomotor function and tissue repair in SCI. Allen's method was utilized to establish an SCI model in Sprague-Dawley (SD) rats. The animals received water containing a mixture of 150 mmol/L SCFAs after SCI. After 21 d of treatment, the Basso, Beattie, and Bresnahan (BBB) score increased, the regularity index improved, and the base of support (BOS) value declined. Spinal cord tissue inflammatory infiltration was alleviated, the spinal cord necrosis cavity was reduced, and the numbers of motor neurons and Nissl bodies were elevated. Enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (qPCR), and immunohistochemistry assay revealed that the expression of interleukin (IL)‍-10 increased and that of IL-17 decreased in the spinal cord. SCFAs promoted gut homeostasis, induced intestinal T cells to shift toward an anti-inflammatory phenotype, and promoted regulatory T (Treg) cells to secrete IL-10, affecting Treg cells and IL-17+ γδ T cells in the spinal cord. Furthermore, we observed that Treg cells migrated from the gut to the spinal cord region after SCI. The above findings confirm that SCFAs can regulate Treg cells in the gut and affect the balance of Treg and IL-17+ γδ T cells in the spinal cord, which inhibits the inflammatory response and promotes the motor function in SCI rats. Our findings suggest that there is a relationship among gut, spinal cord, and immune cells, and the "gut-spinal cord-immune" axis may be one of the mechanisms regulating neural repair after SCI.

Effect of Fugan Huaxian Decoction （扶肝化纤汤） on Th17/Treg Balance and Related Transcription Factors in Hepatic Fibrosis with Healthy Qi Deficiency and Toxin Accumulation Pattern Model Rats / 中医杂志

ObjectiveTo investigate the possible mechanism of Fugan Huaxian Decoction （扶肝化纤汤， FHD） against hepatic fibrosis （HF） from the perspective of immunity. MethodsForty-eight SD rats were randomly divided into blank group， model group， colchicine group， FHD high-， medium- and low-dose group， with eight rats in each group. Except for the blank group， the disease-syndrome combined model of HF with healthy qi deficiency and toxin accumulation pattern was established during six weeks in the other five groups. After successful modeling， the high-， medium- and low-dose FHD groups were respectively given 37.5， 18.75 and 9.38 g/（kg·d） of FHD granules by gavage， while the colchicine group received 2 mg/ （kg·d） of colchicine tablets by gavage， and the blank group and the model group were given 10 ml/（kg·d） of purified water， all for 3 weeks. The general condition of the rats was recorded. After the treatment， the histopathological morphology of the liver was observed by HE staining， and the levels of interleukin 10 （IL-10） and interleukin 17 （IL-17） in serum were determined by enzyme-linked immunosorbent assay （ELISA）. The expression of helper T cells 17 （Th17） and regulatory T cells （Treg） in peripheral blood were detected by flow cytometry， and the Th17/Treg value was calculated. The mRNA expression of retinoic acid-related nuclear orphan receptor γ （RORγt） and fork-head/wing-like helix transcription factor （FoxP3） in liver tissue were detected by qRT-PCR. ResultsCompared to the general condition of rats in the blank group， those in the model group were listless， less active， stretched and pushed， arched and prone， having no resistance to gavage， significantly reduced food intake， loose stools， dirty anus， slow weight gain， dry and dull hair， purple and darkening skin of the limbs with ecchymoses， purple and black spots with varying degrees of the skin of the tail； hepatic fibrosis and hyperplasia of rats in model group were more obvious； serum IL-17， peripheral blood Th17 expression and Th17/Treg value， RORγt mRNA expression in the liver tissue significantly increased in the model group， while expression of IL-10， Treg and FoxP3 mRNA significantly decreased （P＜0.05 or P＜0.01）. Compared to those in the model group， the general condition of the rats and the liver fibrosis of HE stained liver tissue were improved in all the medication groups； the expression of IL-17 and Th17， Th17/Treg， and RORγt mRNA expression significantly decreased， while expression of IL-10， Treg， and FoxP3 mRNA increased in the high- and medium-dose FHD groups and the colchicine group； the expression of IL-17， Th17， and RORγt mRNA decreased， while the expression of IL-10 and FoxP3 mRNA increased in the low-dose FHD group （P＜0.05 or P＜0.01）. And more improvements were found in the FHD high-dose group than FHD medium- and low-dose groups and colchicine group （P＜0.05 or P＜0.01）. ConclusionFHD can may regulate immune balance and act against fibrosis by regulating the expression of specific transcription factors FoxP3 and RORγt， affecting the differentiation of Th17 and Treg cells and Th17/Treg balance， and regulating the secretion of IL-10 and IL-17.

Interleukin-38 suppresses the trans-differentiation of regulatory T cells to Th17 cells in patients with primary biliary cholangitis / 中华风湿病学杂志

Objective:To investigate the IL-38 level in patients with primary biliary cholangitis (PBC), and to assess the modulatory function of IL-38 on the phenotype shift from regulatory T cells (Tregs) to Th17 cells in PBC.Methods:Forty-six PBC patients and 24 controls were included. Serum IL-38, IL-35 and IL-17 levels were measured by enzyme-linked immunosorbent assay. CD4 +CD25 +CD127 dim/-Tregs and CD4 +IL-17A +Th17 cell proportions in peripheral blood mononuclear cells (PBMC) were assessed by flow cytometry. Forkhead box P3 (FoxP3) and retinoid acid receptor related orphan receptor γt (RORγt) mRNA expressions were semi-quantified by real-time PCR. Purified Tregs were stimulated with recombinant IL-38, and were cocultured with autologous PBMC. Tregs function was assessed by measuring cellular proliferation and cytokines expression in the supernatants. Tregs were also polarized for Th17 culture, and recombinant IL-38 was added for stimulation. The influence of IL-38 on trans-differentiation of Tregs into Th17 phenotype was investigated by measuring CCR4/CCR6 expression, IL-17 secretion and RORγt mRNA expression. Student′s t test or Mann-Whitney U test were used for comparison between groups. Results:Serum IL-38 level was significantly lower in PBC group compared with control group [68.02(48.51, 96.74) pg/ml vs 89.6(58.0, 265.5)pg/ml, Z=3.25, P=0.037]. Proportion of Tregs [(6.9±1.3)% vs (11.3±3.5)%, t=7.64, P<0.001], FoxP3 mRNA expression [(1.0±0.3) vs (1.9± 0.7), t=7.74, P<0.001] and serum IL-35 level [25.87(16.08, 37.26)pg/ml vs 30.99(24.81, 52.29)pg/ml, Z=2.02, P=0.047] were notably lower in PBC group, while Th17 cell proportion [(5.5±1.4)% vs (3.8±1.0)%, t=5.43, P<0.001)], RORγt mRNA expression [(1.4±0.6) vs (1.0±0.4), t=2.72, P=0.008] and serum IL-17 level [202.0(121.6, 311.6)pg/ml vs 104.5(79.8, 155.5)pg/ml, Z=4.43, P<0.001] were remarkably higher in PBC group. Purified Tregs were co-cultured with autologous PBMC. Cellular proliferation was increased in PBC group compared with control group [(6.5±1.40)×10 5vs (5.3±1.1)×10 5, t=2.49, P=0.020]. IL-35 and IL-10 secretions in the supernatants were lower in PBC group ( P<0.05), but interferon- γ was higher in PBC group ( P=0.011). Tregs were also polarized for Th17 culture. CCR4 and CCR6 mean fluorescence intensity (MFI), RORγt mRNA and IL-17 secretion were higher in PBC group compared with control group ( P<0.05). IL-38 stimulation enhanced the inhibitory activity of Tregs from both PBC group and control group, which presented as reduced cellular proliferation as well as enhanced IL-35 and IL-10 secretion ( P<0.05). IL-38 stimulation only suppressed transdifferentiation of Tregs into Th17 phenotype in PBC group, which presented as down-regulation of CCR4 MFI, CCR6 MFI and IL-17 secretion ( P<0.05). However, IL-38 did not affect Tregs trans-differentiation into Th17 phenotype in control group. Conclusion:IL-38 played an immune-protective role and suppressed inflammatory response in the disease progression of PBC. IL-38 inhibited transdifferentiation of Tregs into Th17 phenotype in PBC patients.

CD4 T lymphocyte subsets displa heterogeneous susceptibility to apoptosis induced by serum from patients with systemic lupus erythematosus

Abstract Background Serum from systemic lupus erythematosus (SLE) patients has been shown to induce T-lymphocyte (TL) apoptosis. Given that different cells of the immune system display different sensitivity to apoptosis, we set to evaluate the in vitro effect of SLE serum on regulatory T-cells (Treg), Th17, Th1 and Th2 from SLE patients and healthy controls. Methods Peripheral blood mononuclear cells from SLE patients or normal controls were exposed to a pool of sera from SLE patients or normal controls. Annexin V was used to label cells in apoptosis or necrosis. Annexin V-labeled Treg, Th17, Th1 and Th2 cells were determined using flow cytometry. Results Total CD3 + and CD4+cells from SLE patients showed higher frequency of spontaneous apoptosis/necrosis, whereas Th1 cells from SLE patients presented reduced spontaneous apoptosis/necrosis rate as compared with cells from controls. Incubation with SLE serum induced increased frequency of apoptotic/necrotic CD3 +, CD4 + and Th2 cells from normal controls or from SLE patients as compared with cultures incubated with normal human serum (NHS) or without human serum at all. Incubation with SLE serum did not increase the apoptosis/necrosis rate in Th1 or Th17 cells. Treg cells from SLE patients were more prone to apoptosis/necrosis induced by SLE serum than Treg cells from normal individuals. Th1, Th2, and Th17 cells presented increased apoptosis rates in cultures without human serum. Conclusion Our findings indicate that the serum of patients with active SLE stimulates apoptosis of CD4+T cells in general and exhibit differentiated effects on CD4+T-cell subsets.

Immunomodulatory mechanism of interleukin-7 receptor α antibody attenuating renal injury in MRL/lpr lupus mice / 中华肾脏病杂志

Objective:To investigate the effect of interleukin (IL)-7 receptor α (IL-7Rα) antibody on the immune inflammation and renal injury in MRL/lpr lupus mice.Methods:Fifteen 3-4-week-old female MRL/lpr lupus mice (specific pathogen free) weighing 15-16 g were bred to 14-week-old and randomly divided into three groups: IL-7Rα antibody intervention group, isotype antibody (positive control) group and normal saline (negative control) group. The mice in the threc groups were intraperitoneally injected with IL-7Rα antibody, isotype antibody and normal saline respectively, with 100 μg three times a week for 4 weeks. At the age of 18-week old, the mice were sacrificed. Twenty-four-hour urinary protein was detected by Coomassie brilliant blue method, serum creatinine was detected by peroxidase method, and the expression of autoantibody (anti-double strand DNA antibody) and inflammatory factors such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-21 was detected by enzyme-linked immunosorbent assay method. Renal pathology was detected by PAS and Sirius red staining, and CD3 and F4/80 in renal tissues were detected by immunohistochemistry method. Regulatory T cells, follicullar helper T cells (Tfh) and follicular regulatory T cells (Tfr) were detected by flow cytometry.Results:The 24-hour urinary protein, serum creatinine, serum anti-double strand DNA antibody and serum IFN-γ and IL-21 in the IL-7Rα antibody intervention group were significantly lower than those in the control groups (all P<0.01). However, there was no significant difference in serum TNF-α among the three groups ( F=0.39, P>0.05). The positive infiltrating cells of CD3 and F4/F80, and the ratio of type Ⅰ/Ⅲ collagen fibers ( F=41.11, P<0.01) of renal tissues in the IL-7Rα antibody intervention group were lower than those in the other two groups. Compared with the control groups, the ratio of regulatory T cells (CD4 +CD25 +Foxp3 +)/effector T cells (CD4 +CD25 +) in blood of IL-7Rα antibody intervention group increased ( F=21.64, P<0.01), while the ratio of Tfr (CD4 +CXCR5 +Foxp3 +)/Tfh (CD4 +CXCR5 +) in peripheral blood and spleen increased ( F=38.95, P<0.01; F=12.90, P<0.01). Conclusion:IL-7Rα antibody can reduce the production of autoantibodies such as anti-double strand DNA antibody and inflammatory factors by increasing the ratio of regulatory T cells and Tfr/Tfh, thus alleviating immune inflammation and renal damage in MRL/lpr lupus mice.

Regulatory T cells and Kawasaki disease / 国际儿科学杂志

Kawasaki disease is an acute, systemic vasculitis that easily injures coronary arteries and is the leading cause of acquired heart disease in children.Although the cause of Kawasaki disease remains unknown, it is widely believed that the pathogenesis of Kawasaki disease is an inflammatory cascade caused by a combination of infection and genetic predisposition.Regulatory T cells, which express Foxp3 + , CD4 + and CD25 + , are a T-cell subpopulation specialized in immune suppression.There are some correlations between regulatory T cells and Kawasaki disease in pathophysiology, treatment and prognosis.The dysfunction of regulatory T cells may be involved with the pathogenesis of Kawasaki disease, but there are few researches on it.This article reviews the progress of regulatory T cells in Kawasaki disease in recent years and summarizes the mechanism of regulatory T cells in the occurrence and repair of Kawasaki disease, prospecting the research future of targeted regulatory T cells therapy in the prevention of coronary artery lesions in Kawasaki disease.