Th17 Cells In Blood Research Articles

Heat shock protein 70 promotes the progression of type 2 diabetic nephropathy by inhibiting T-cell immunoglobulin and mucin domain-3 and thereby promoting Th17/Treg imbalance.

Diabetic nephropathy (DN) is the most common complication of diabetes mellitus. We aimed to investigate the role of regulatory T cells (Tregs) and helper T cells 17 (Th17) in the development and progression of DN. A mouse type 2 diabetic nephropathy (T2DN) model was established. Immunohistochemistry was used to detect the expression of HSP70 and Tim-3 in mouse kidney tissues, and western blotting was used to detect the expression levels of HSP70 and Tim-3. PAS staining and Masson's trichrome staining were used to detect the degree of kidney injury. Flow cytometry was used to detect the number of Th17 and Treg cells in blood and kidney tissues. The expression levels of interleukin 17 (IL-17) and interleukin 10 (IL-10) in the serum were measured via ELISA. The expression of HSP70 was significantly increased while the expression of Tim-3 was significantly decreased in the kidneys of mice in the T2DN group compared with those in the control (NC) group. Additionally, the inhibition of HSP70 upregulated the expression of Tim-3 in T2DN mice. The Th17/Treg ratio was significantly greater in the blood and kidneys of the mice in the T2DN group than in those of the NC group, the expression of serum IL-17 was increased, and the expression of IL-10 was decreased. Increased HSP70 inhibits Tim-3 expression in T2DN mouse kidney tissues, and subsequently causes a Th17/Treg imbalance and an inflammatory response, ultimately leading to kidney injury. The inhibition of HSP70 may alleviate the progression of T2DN.

Blocking CTLA-4 promotes pressure overload-induced heart failure via activating Th17 cells.

Targeting cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) with specific antibody offers long-term benefits for cancer immunotherapy but can cause severe adverse effects in the heart. This study aimed to investigate the role of anti-CTLA-4 antibody in pressure overload-induced cardiac remodeling and dysfunction. Transverse aortic constriction (TAC) was used to induce cardiac hypertrophy and heart failure in mice. Two weeks after the TAC treatment, mice received anti-CTLA-4 antibody injection twice a week at a dose of 10 mg/kg body weight. The administration of anti-CTLA-4 antibody exacerbated TAC-induced decline in cardiac function, intensifying myocardial hypertrophy and fibrosis. Further investigation revealed that anti-CTLA-4 antibody significantly elevated systemic inflammatory factors levels and facilitated the differentiation of T helper 17 (Th17) cells in the peripheral blood of TAC-treated mice. Importantly, anti-CTLA-4 mediated differentiation of Th17 cells and hypertrophic phenotype in TAC mice were dramatically alleviated by the inhibition of interleukin-17A (IL-17A) by an anti-IL-17A antibody. Furthermore, the C-X-C motif chemokine receptor 4 (CXCR4) antagonist AMD3100, also reversed anti-CTLA-4-mediated cardiotoxicity in TAC mice. Overall, these results suggest that the administration of anti-CTLA-4 antibody exacerbates pressure overload-induced heart failure by activating and promoting the differentiation of Th17 cells. Targeting the CXCR4/Th17/IL-17A axis could be a potential therapeutic strategy for mitigating immune checkpoint inhibitors-induced cardiotoxicity.

Soluble Fibrinogen–Like Protein 2 Downregulation and Th17/Treg Imbalance in a Taurocholate‐Induced Murine Experimental Model of Severe Acute Pancreatitis

ABSTRACTBackgroundSevere acute pancreatitis (SAP) is associated with tremendous systemic inflammation, T‐helper 17 (Th17) cells, and regulatory T (Treg) cells play an essential role in the inflammatory responses. Meanwhile, soluble fibrinogen–like protein 2 (Sfgl2) is a critical immunosuppressive effector cytokine of Treg cells and modulates immune responses. However, the impact of SAP induction on Sfgl2 expression and the role of Sfgl2 in immunomodulation under SAP conditions are largely unknown.MethodsA taurocholate‐induced mouse SAP model was established. The ratios of CD4+CD25+Foxp3+ Treg cells or CD4+IL‐17+ Th17 cells in blood and pancreatic tissues as well as surface expression of CD80, CD86, and major histocompatibility complex class II (MHC‐II) were determined by flow cytometry. Gene mRNA expression was determined by qPCR. Serum amylase and soluble factors were quantitated by commercial kits. Bone marrow–derived dendritic cells (DCs) were generated, and NF‐κB/p65 translocation was measured by immunofluorescence staining.ResultsSAP induction in mice decreased the Th17/Treg ratio in the pancreatic tissue and increased the Th17/Treg ratio in the peripheral blood. In addition, SAP was associated with a reduced level of Sfgl2 in the pancreatic tissue and blood: higher levels of serum IL‐17, IL‐2, IFN‐α, and TNF‐α, and lower levels of serum IL‐4 and IL‐10. Furthermore, the SAP‐induced reduction in Sfgl2 expression was accompanied by dysregulated maturation of bone marrow–derived DCs.ConclusionsSAP causes reduced Sfgl2 expression and Th17/Treg imbalance, thus providing critical insights for the development of Sfgl2‐ and Th17/Treg balance‐targeted immunotherapies for patients with SAP.

Quercetin through miR-147–5p/Clip3 axis reducing Th17 cell differentiation to alleviate periodontitis

BackgroundQuercetin (QU) plays an important role in treating periodontitis; however, the mechanism through which microRNAs regulate Th17 cell differentiation has not been determined. MethodsQU was administered intragastrically to periodontitis rats once a day for one month. The morphology of alveolar bone was observed by micro-CT, gingival tissue structure was observed by HE staining, IL-6, TNF-α, IL-17A, RORγt, FOXP3 and IL-10 were detected by immunohistochemical staining, and Th17 and Treg cells in the peripheral blood were detected by flow cytometry. CD4+T cells were induced to differentiate into Th17 cells in vitro. Cell viability was determined by CCK8, and IL-17A and RORγt were detected by qPCR. Th17 cells were detected by flow cytometry, microRNA sequencing and bioinformatics analysis were used to screen key microRNAs, the phenotypic changes of Th17 cells were observed after overexpressed microRNAs via mimics. TargetScan database, in situ hybridization, and dual-luciferase reporter experiment were used to predict and prove target genes of microRNAs. The phenotype of Th17 cells was observed after overexpression of microRNA and target gene. ResultsCompared with periodontitis group, the distance from cementoenamel junction(CEJ) to alveolar bone(AB) was decreased, the structure of gingival papilla was improved, IL-6, TNF-α, IL-17, and RORγt were downregulated, FOXP3 and IL-10 were upregulated, the proportion of Th17 decreased and Treg increased in peripheral blood after QU treatment. Compared with Th17 cell group, mRNA levels of IL-17A and RORγt were decreased, and proportion of Th17 cells was significantly lower in the coculture group. MiR-147–5p was low in control group, upregulated in Th17 cell group, and downregulated after QU intervention, it's eight bases were inversely related to 3′UTR of Clip3, miR-147–5p with Clip3 were co-located in cells of periodontal tissue. Compared with those in Th17-mimicsNC + QU cells, the mRNA levels of RORγt and IL-17A upregulated, and proportion of Th17 cells increased in Th17-miR-147–5p + QU cells. The miR-147–5p mimics inhibited the luciferase activity of the WT Clip3 3′UTR but had no effect on the Mut Clip3 3′UTR. Clip3 was significantly downregulated after the overexpression of miR-147–5p. Mimics transfected with miR-147–5p reversed the decrease in the proportion of Th17 cells induced by QU, while the overexpression of Clip3 antagonized the effect of miR-147–5p and further reduced the proportion of Th17 cells. Moreover, the overexpression of miR-147–5p reversed the decreases in the mRNA levels of IL-17 and RORγt induced by QU treatment, while pcDNA3.1 Clip3 treatment further decreased the mRNA levels of IL-17 and RORγt. ConclusionQU reducing inflammatory response and promoting alveolar bone injury and repair, which closely relative to inhibit the differentiation of CD4+T cells into Th17 cells by downregulating miR-147–5p to promote the activation of Clip3.

Increased Th17 and Treg levels in peripheral blood positively correlate with minimal residual disease in acute myeloid leukaemia.

Immune dysregulation plays a key role in acute myeloid leukemia (AML). We aimed to explore the correlation between T helper cell 17 (Th17) and the regulatory cells (Tregs) in the peripheral blood of patients with newly diagnosed (ND) AML and bone marrow blast cells, as well as minimal residual disease (MRD) before and after treatment. Changes in Th17 and Treg cells in the peripheral blood of 32 patients with ND AML were observed before and after induction chemotherapy with cytarabine for seven days and anthracycline for three days. The levels of inflammatory cytokines were measured using an enzyme-linked immunosorbent assay. Correlation analysis between bone marrow blast cells and Th17 and Treg cell frequencies was performed using the Pearson's correlation test. Frequencies of Th17 and Treg cells and MRD were assessed using flow cytometry. IL-6, IL-10, IL-17A, and GM-CSF levels gradually increased in patients with ND AML and CR and NR patients. The percentages of Th17 and Treg cells positively correlated with those of blast cells. In addition, the frequencies of Th17 and Treg cells in MRD-positive patients were higher than those in MRD-negative patients at the initial induction and after three months of chemotherapy. The frequencies of Tregs and Th17 cells positively correlated with MRD onset. Increased Th17 and Treg cell levels were positively correlated with onset of AML, poor remission, and MRD.

Allergens induce upregulated IL-18 and IL-18Rα expression in blood Th2 and Th17 cells of patients with allergic asthma.

Allergic asthma (AA) is closely associated with the polarization of T helper (Th)2 and Th17 cells. Interleukin (IL)-18 acts as an inducer of Th2 and Th17 cell responses. However, expressions of IL-18 and IL-18 receptor alpha (IL-18Rα) in blood Th2 and Th17 cells of patients with AA remain unclear. We therefore investigated their expressions in Th2 and Th17 cells using flow cytometric analysis, quantitative real-time PCR (qPCR), and murine AA model. We observed increased proportions of Th2, Th17, IL-18+, IL-18+ Th2, and IL-18+ Th17 cells in blood CD4+ T cells of patients with AA. Additionally, house dust mite seemed to upregulate further IL-18 expression in Th2 and Th17, and upregulate IL-18Rα expression in CD4+ T, Th2, and Th17 cells of AA patients. It was also found that the plasma levels of IL-4, IL-17A, and IL-18 in AA patients were elevated, and they were correlated between each other. In ovalbumin (OVA)-induced asthma mouse (AM), we observed that the percentages of blood CD4+ T, Th2, and Th17 cells were increased. Moreover, OVA-induced AM expressed higher level of IL-18Rα in blood Th2 cells, which was downregulated by IL-18. Increased IL-18Rα expression was also observed in blood Th2 cells of OVA-induced FcεRIα-/- mice. Collectively, our findings suggest the involvement of Th2 cells in AA by expressing excessive IL-18 and IL-18Rα in response to allergen, and that IL-18 and IL-18Rα expressing Th2 cells are likely to be the potential targets for AA therapy.

Fluvoxamine inhibits Th1 and Th17 polarization and function by repressing glycolysis to attenuate autoimmune progression in type 1 diabetes.

Fluvoxamine is one of the selective serotonin reuptake inhibitors (SSRIs) that are regarded as the first-line drugs to manage mental disorders. It has been also recognized with the potential to treat inflammatory diseases and viral infection. However, the effect of fluvoxamine on autoimmune diseases, particularly type 1 diabetes (T1D) and the related cellular and molecular mechanisms, are yet to be addressed. Herein in this report, we treated NOD mice with fluvoxamine for 2weeks starting from 10-week of age to dissect the impact of fluvoxamine on the prevention of type 1 diabetes. We compared the differences of immune cells between 12-week-old control and fluvoxamine-treated mice by flow cytometry analysis. To study the mechanism involved, we extensively examined the characteristics of CD4+ T cells with fluvoxamine stimulation using RNA-seq analysis, real-time PCR, Western blot, and seahorse assay. Furthermore, we investigated the relevance of our data to human autoimmune diabetes. Fluvoxamine not only delayed T1D onset, but also decreased T1D incidence. Moreover, fluvoxamine-treated NOD mice showed significantly attenuated insulitis coupled with well-preserved β cell function, and decreased Th1 and Th17 cells in the peripheral blood, pancreatic lymph nodes (PLNs), and spleen. Mechanistic studies revealed that fluvoxamine downregulated glycolytic process by inhibiting phosphatidylinositol 3-kinase (PI3K)-AKT signaling, by which it restrained effector T (Teff) cell differentiation and production of proinflammatory cytokines. Collectively, our study supports that fluvoxamine could be a viable therapeutic drug against autoimmunity in T1D setting.

Proportion and Clinical Significance of γδT17/Th17/Tc17 Cells in Peripheral Blood of Uygur Patients with Chronic Lymphoblastic Leukemia

To explore the distribution of γδT17/Th17/Tc17 cells in the peripheral blood of Uygur patients with chronic lymphoblastic leukemia (CLL) and clinical significance. ELISA method was used to detect the levels of IL-17, IL-23, IL-6, and IFN-γ in the peripheral blood serum of 53 newly diagnosed Uygur patients with CLL and 30 healthy controls. Flow cytometry was used to determine the proportion of γδT/γδT17/Th17/Tc17 cells in the peripheral blood of Uygur CLL patients and controls, and the changes of the abover indexes in CLL Binet staging were observed. Compared with the control group, the proportion of γδT cells, γδT17 cells, and Th17 cells in the peripheral blood of Uygur CLL patients increased significantly （P <0.05）. γδT17 cell proportion in total lymphocytes was significantly higher than Th17 and Tc17 cell proportions（P <0.05）, and proportions of γδT, γδT17 cells increased gradually as the disease progressed. The levels of cytokines IL-17, IL-23, and IL-6 in peripheral blood of Uygur patients with CLL were significantly higher than those in the control group（P <0.05）, while the level of cytokine IFN-γ was significantly lower（P <0.05）. The level of IL-17 in peripheral blood decreased gradually as the disease progressed（P <0.05）. γδT and γδT17 are abnormally highly expressed in Uygur CLL , which are related to the stage of disease and participate in the occurrence and development of CLL.

Regulatory T Cell and T Helper 17 Cell Imbalance in Patients with Unexplained Infertility.

Female infertility is a global health concern. The aim of this study was to investigate the relationship between regulatory T (Treg) cells and helper T cells 17 (Th17) in peripheral blood and unexplained infertility (UI). In addition, we explored potential valuable diagnostic biomarkers for patients with UI and ascertained whether Treg and Th17 cells are associated with primary and secondary UI. The patients underwent standard fertility evaluation test, including blood tests, ultrasound examination, fallopian tube tests, ovulation assessment, and male partner's semen analysis. According to the inclusion and exclusion criteria, this study enrolled 37 patients with UI (30 with primary UI and 7 with secondary UI) and 26 age-matched healthy volunteers as the control group. Flow cytometry was used to detect the frequency of Treg and Th17 cells. The area under the receiver operating characteristic curve (AUC) with a 95% confidence interval (CI) was used to assess the diagnostic performance. An AUC > 0.800 indicated good diagnostic performance. The percentage of Treg decreased significantly, whereas the percentage and absolute count of Th17 cells increased. Moreover, the Th17/Treg ratio in patients with UI increased significantly. As a diagnostic biomarker for UI, the Th17/Treg ratio exhibited remarkable diagnostic performance (AUC: 0.813 (95% CI = 0.709-0.917)). However, the percentages and absolute counts of Treg and Th17 cells in the peripheral blood of women with primary and secondary UI, as well as their Th17/Treg ratios, did not differ significantly. The distribution of Treg and Th17 cells is imbalanced in patients with UI. Therefore, the Th17/Treg ratio may be a promising indicator of UI. However, there were no significant differences in the distribution of Treg and Th17 cells between women with primary and secondary UI.

Effect of QingreHuoxue formula on Th17 cells and Tregs in mice with idiopathic membranous nephropathy.

This study aimed to evaluate the therapeutic effect of the QingreHuoxue formula on mice with Idiopathic Membranous Nephropathy (IMN) and its impact on Th17 cells and Tregs. A mouse model of IMN was established, and the mice were treated with traditional Chinese medicine, western medicine, or a combination of both. The efficacy and immunomodulatory effects of the QingreHuoxue formula were evaluated by examining renal pathology, urinary protein levels, peripheral blood Th17 and Treg cell counts, and comparing the expression levels of IL-17 and transforming growth factor-β1 in renal tissues. Compared to the untreated IMN model group, the IMN mice treated with TCM, western medicine, or the combination showed significant improvements in proteinuria, renal pathology, peripheral T lymphocyte counts, and IL-17 expression in renal tissues. Notably, the group treated with a combination of Chinese and western medicine exhibited better outcomes than the group treated with western medicine alone. The QingreHuoxue formula was effective in reducing proteinuria, modulating T cell immune function, and protecting renal tissue in mice with IMN.

Circulating JKAP levels may correlate with postpartum anxiety and depression through its interaction with T helper 17 cells.

Jun N-terminal kinase pathway-associated phosphatase (JKAP) regulates CD4+ T-cell differentiation and immunity, which are linked to mental disorders. This study aimed to explore the relationships between JKAP and T helper 17 (Th17)/regulatory T (Treg) ratio, as well as their associations with anxiety and depression in postpartum women. Serum JKAP were measured by enzyme-linked immunosorbent assay and blood Th17 and Treg cells were measured by flow cytometry in 250 postpartum women. Anxiety and depression were evaluated by the 6-item State-Trait Anxiety Inventory (STAI6) and Edinburgh Postnatal Depression Scale (EPDS). Anxiety and depression rates were 22.0 and 28.4%, respectively, among postpartum women. Notably, JKAP was negatively associated with the STAI6 (P=0.002) and EPDS scores (P<0.001) in postpartum women and was lower in postpartum women with anxiety (P=0.023) or depression (P=0.002) than in those without. Moreover, JKAP was inversely related to Th17 cells and Th17/Treg ratio but positively correlated with Treg cells in postpartum women (all P<0.001). Interestingly, Th17 cells and Th17/Treg ratio were both positively associated with STAI6 and EPDS scores in postpartum women (all P<0.001). Furthermore, Th17 cells and Th17/Treg ratio were lower in postpartum women with anxiety or depression than in those without (all P<0.01). Nevertheless, Treg cells were not linked to anxiety or depression in postpartum women. JKAP was negatively associated with Th17 cells and Th17/Treg ratio; moreover, they all related to anxiety and depression in postpartum women, indicating that JKAP may be involved in postpartum anxiety and depression via interactions with Th17 cells.

SLAMF3 promotes Th17 differentiation and is reversed by iguratimod through JAK1/STAT3 pathway in primary Sjögren's syndrome

ObjectiveThe signaling lymphocytic activation molecule family of receptors (SLAMF) is involved in the activation of T cells and plays important roles in the pathogenesis of autoimmune diseases. The purpose of this study is to observe the expression of SLAMF3 on CD4 + T cells and its effect on the differentiation of T helper 17 (Th17) in primary Sjögren's syndrome (pSS). Furthermore, we found iguratimod (IGU) could effectively reverse the aberrant Th17 differentiation through JAK1/STAT3 signaling. MethodsPeripheral blood mononuclear cells from 40 pSS and 40 healthy control subjects were enrolled for analysis of expression of SLAMF3 on CD4 + T and Th17 cells by flow cytometry. Serum IL-17 and SLAMF3 were detected by ELISA assay. Labial biopsies from 20 pSS patients and 20 non-pSS controls were performed immunohistochemical for staining expression of CD4, IL-17, and SLAMF3. Under the priming conditions with anti-CD3/CD28 or CD3/SLAMF3 antibodies on CD4 + T cells extracted from pSS and controls, the proportion of Th17 cells in CD4 + T cells and the amount of soluble IL-17A were assessed by flow cytometry and ELISA. Furthermore, RNA sequencing was performed for the transcriptomics study. Additionally, RNA level of RORγt and IL-17A and the protein level of RORγt, p-JAK1 and p-STAT3, were detected by real-time PCR and western blot. ResultsThe expression levels of SLAMF3 on CD4 + T and Th17 cells in the peripheral blood and salivary glands in pSS patients were significantly elevated than that in control groups. The serum IL-17A and SLAMF3 in pSS patients were much higher compared with the control group. Although co-stimulation of CD3/SLAMF3 could promote CD4 + T cells differentiate into Th17 cells both in pSS and controls, the CD4 + T cells from pSS have a more sensitive response in Th17 differentiation with the SLAMF3 stimulation. Transcriptomics results showed the CD3/SLAMF3 stimulation caused the activation of Th17 signaling and JAK1/STAT3 pathway. Quantitative PCR and western blotting confirmed the IGU (iguratimod), which is a safe clinical drug in treatment of autoimmune diseases, effectively reversed the increased Th17 proportion, the expression levels of RORγt, pJAK1, and pSTAT3 caused by CD3/SLAMF3 stimulation. ConclusionSLAMF3 upregulates Th17 cell differentiation of CD4 + T cells and IL-17A secretion through enriching RORγt and activating the transcriptomics participating in the pathogenesis of primary Sjögren's syndrome. IGU could inhibit the process through this therapeutic target in pSS.

FRI131 Abrogation Of Transglutaminase 2 In Myeloid Cells Ameliorates Angiotensin II Induced Vascular Stiffness In Female Mice

Abstract Disclosure: H. Naz: None. A.R. Aroor: None. A.T. Whaley-Connell: None. J. Guanghong: None. J.L. Hulse: None. C.M. Manrique Acevedo: None. G. Lastra: None. Introduction: Chronic low-grade inflammation and vascular stiffening contribute to cardiovascular disease (CVD) in obese and insulin-resistant women. Under these conditions, inappropriate activation of the renin angiotensin aldosterone system (RAAS) stimulates Transglutaminase 2 (TG2), a ubiquitously expressed enzyme that promotes vascular stiffening and remodeling. Additionally, Th17 cells contribute to CVD development in the setting of RAAS activation. However, it is unclear how TG2 activation mediates maladaptive immune responses associated with vascular stiffening in women. In this study, we hypothesize that TG2 abrogation inhibits Ang II-induced vascular stiffness in females by modulating Th17 immunity. Methods: 12-week-old female mice lacking TG2 expression (MyTG2KO) in myeloid cells, and control littermates (LM) were infused with Ang-II (1.4 mg/kg/day) for 2 weeks (n=4-8 per group). Prior to sacrifice, systolic blood pressure (SBP) was measured by tail-cuff method. Aortic stiffness was assessed by atomic force microscopy of thoracic aorta explants, and TG2 activity was examined by Alexa FluorTM 488 cadaverine. Analysis of Th17 cells in blood, spleen and epididymal fat (epi-fat) was done by flow cytometry. Cytokine bead array was used to measure cytokine levels in serum, RT-PCR was used to analyze cytokines in aortic tissue and TG2 knockdown efficiency in myeloid cells. Immunohistochemistry was used to examine fibrosis in aorta. Results: SBP was decreased in MyTG2KO-Ang-II as compared to LM-Ang-II cohort (129.6±5.6 vs 152.5 ±2 mmHg; p=0.04). TG2 mRNA expression in macrophages was lower in MyTG2KO mice as compared to LM (p=0.002). TG2 activity in aortic tissue increased in LM-Ang-II as compared to LM-saline (p=0.004) but was significantly lower in MyTG2KO-Ang-II mice (p=0.02). Aortic stiffness was lower in MyTG2KO-Ang-II mice relative to LM-Ang-II (4.0±0.6 vs 10.3±2.4 kPa; p=0.02). Compared to LM-Ang-II, MyTG2KO-Ang-II mice also had decreased Th17 lymphocytes (CD4+IL-17A+) in blood (3.5±0.2 vs 5.8 ±0.4 %; p=&amp;lt;0.0001), spleen (4.7±0.4 vs 7.1 ±0.5 %; p=0.004) and epi-fat (4.7±0.6 vs 6.8 ±0.5 %; p=0.02). MyTG2KO-Ang II mice also had significant lower levels of proinflammatory cytokines (TNF-a, IL6, IL17A, INF-g) in serum and mRNA expression of proinflammatory cytokines compared to LM-Ang-II mice and decreased total macrophage infiltration (CD68+) in the aorta. Ang-II treated MyTG2KO mice group also exhibited reduced fibrosis in the aorta relative to LM-Ang-II (p=0.003). Conclusion: The results demonstrate that abrogating TG2 in myeloid cells in female mice substantially reduces blood pressure, proinflammatory cytokines, and Th17 immune responses induced by Ang-II. Our findings have potential clinical implications for the prevention and management of CVD in women. Presentation: Friday, June 16, 2023

Role of Peripheral Blood T Helper 17 Cells in Non-Small Cell Lung Cancer of Different Clinical Stages, Pathological Types and Differentiation Degrees.

We aimed to explore the roles of T helper 17 (Th17) cells and cytokines interleukin 17 (IL-17), IL-6, IL-4, transforming growth factor β (TGF-β) and γ-interferon (IFN-γ) in the peripheral blood of patients with non-small cell lung cancer (NSCLC) of different clinical stages, pathological types, and differentiation degrees. A total of 120 NSCLC patients admitted and 80 healthy people receiving physical examinations from June 2019 to October 2021 were enrolled as the case group (TNM stage: I, II, III and IV; pathological type: adenocarcinoma and squamous cell carcinoma; differentiation degree: low and moderate-high) and the control group, respectively. The serum levels of IL-17, IL-6, IL-4, TGF-β and IFN-γ were measured using an enzyme-linked immunosorbent assay. Th17 cells were counted by flow cytometry. The case group had higher levels of Th17 cells, IL-17, IL-6, IL-4, and TGF-β and lower IFN-γ level in the peripheral blood than those of the control group (p < 0.05). NSCLC patients with different TNM stages had significantly different levels of Th17 cell, IL-17, IL-6, TGF-β, and IFN-γ (p < 0.05). The expression levels of Th17 and IL-6 in patients with lowly differentiated NSCLC were lower than those of patients with moderately and highly differentiated NSCLC, while the IFN-γ expression level followed an opposite trend (p < 0.05). The count of Th17 cells in the peripheral blood of NSCLC patients had significantly positive correlations with IL-17, IL-6, IL-4, and TGF-β levels, but negative correlation with IFN-γ level (p < 0.05). The count of Th17 cells increases significantly in NSCLC patients and has correlations with TNM stage and differentiation degree.

The expression of Th17/Treg in oral submucosal fibrosis carcinogenesis and the significance in the development of mucosal lesions.

It targets to explore the expression of Th17/Treg in oral submucous fibrosis (OSF) carcinogenesis and its significance in the development of mucosal lesions. In this research, 100 patients with OSF who visited our hospital for surgical treatment from March 2020 to April 2022 were selected. Based on pathological examination results, the patients were divided into 27 patients with oral leukoplakia (OK) group, 14 patients with oral lichen planus (OLP) group, 9 patients with oral squamous cell carcinoma (OSCC) group, and 50 patients with OSF group. It adopted flow cytometry (FC) to calculate the ratio of peripheral blood Th17 cells and Treg cells in four groups, and the Th17/Treg ratio was calculated; The area of oral mucosal lesions (OML) from patients was collected. It needs to compare the differences in Th17/Treg ratio and OML area among four groups and determine the correlation between indicators. ROC curve was used to analyze the diagnostic threshold of the Th17/Treg ratio for carcinogenesis. Except for the OK and OLP, it had statistical significance differences in Th17, Treg cells, and Th17/Treg ratio (P<0.001); The area of OML in the OK, OLP, and OSCC was higher than that in the simple OSF, with statistical significance (P<0.001); Th17 (%), Treg (%), and Th17/Treg all had direct ratio with the area of OML; The area of OML has directed ratio with the development of mucosal lesions (r>0, P<0.05); The areas under the ROC curve for patients with OSF combined with OK, OLP, or OSCC with Th17 (%), Treg (%), Th17/Treg, and OML area were 0.560, 0.986, 0.936, and 0.466, respectively. The expression of Th17/Treg is elevated in oral submucosal fibrosis and carcinogenesis. When mucosal lesions progress or become cancerous, the Th17/Treg ratio increases accordingly, and it has more clinical value than the increase in the OML area.

Circulating Th17/Treg as a promising biomarker for patients with rheumatoid arthritis in indicating comorbidity with atherosclerotic cardiovascular disease.

Immune and inflammatory responses have a pivotal role in the pathogenesis of rheumatoid arthritis (RA) and atherosclerotic cardiovascular disease (ASCVD). This study aims to explore the change of peripheral lymphocytes, especially the absolute and relative changes in peripheral T cells in RA patients with and without ASCVD. The changes in the lymphocyte subsets were assessed to provide a novel insight in diagnosing and preventing ASCVD in patients with RA. A propensity score matching system (1:1) was conducted to perform a matched case-control study with 169 pairs RA-ASCVD and RA participants. Univariate and multivariate analyses were performed to determine the association between peripheral lymphocytes and RA-ASCVD. Multivariate logistic regression analysis demonstrated that Th17 cell absolute, Th17 cell Ratio, Th17/Treg were associated with a significantly higher risk of ASCVD after model adjustment. Then we focused on Th17/Treg, multivariate logistic analyses in tri-sectional Th17/Treg groups showed that the odds of ASCVD is gradually increasing with Th17/Treg rank's rising after model adjustment. Finally, the restricted cubic spline of Th17/Treg and odds ratio of RA-ASCVD was conducted. Interestingly, we found a critical point of Th17/Treg (critical point = 0.2399). Th17/Treg shows a protective role in the odds of ASCVD when Th17/Treg < 0.2399. With smaller Th17/Treg, the protective efficiency is more obvious when Th17/Treg < 0.2399. Our study suggested that increasing absolute and percentage of Th17 cells in the peripheral blood of patients with RA was associated with the development of ASCVD. And Th17/Treg may be a promising biomarker for patients with RA in indicating comorbidity with ASCVD.

Cathepsin S inhibition in dendritic cells prevents Th17 cell differentiation in perivascular adipose tissues following vascular injury in diabetic rats.

In the context of diabetes mellitus (DM), the circulating cathepsin S (CTSS) level is significantly higher in the cardiovascular disease group. Therefore, this study was designed to investigate the role of CTSS in restenosis following carotid injury in diabetic rats. To induce DM, 60 mg/kg of streptozotocin (STZ) in citrate buffer was injected intraperitoneally into Sprague-Dawley rats. After successful modeling of DM, wire injury of the rat carotid artery was performed, followed by adenovirus transduction. Levels of blood glucose and Th17 cell surface antigens including ROR-γt, IL-17A, IL-17F, IL-22, and IL-23 in perivascular adipose tissues (PVAT) were evaluated. For in vitro analysis, human dendritic cells (DCs) were treated with 5.6-25 mM glucose for 24 h. The morphology of DCs was observed using an optical microscope. CD4+ T cells derived from human peripheral blood mononuclear cells were cocultured with DCs for 5 days. Levels of IL-6, CTSS, ROR-γt, IL-17A, IL-17F, IL-22 and IL-23 were measured. Flow cytometry was conducted to detect DC surface biomarkers (CD1a, CD83, and CD86) and Th17 cell differentiation. The collected DCs presented a treelike shape and were positive for CD1a, CD83, and CD86. Glucose impaired DC viability at the dose of 35 mM. Glucose treatment led to an increase in CTSS and IL-6 expression in DCs. Glucose-treated DCs promoted the differentiation of Th17 cells. CTSS depletion downregulated IL-6 expression and inhibited Th17 cell differentiation in vitro and in vivo. CTSS inhibition in DCs inhibits Th17 cell differentiation in PVAT tissues from diabetic rats following vascular injury.

203-OR: Features of Human Th-Like Regulatory T Cells in the Pathogenesis of Type 1 Diabetes

Background: The imbalance between Tregs, Bregs and proinflammatory Th cells plays a vital role in the pathogenesis and development of T1D. Accumulating evidence has revealed that Treg has phenotypic plastic changes into other subsets under pathological conditions. However, whether Tregs convert into other immune cell types in T1D and the potential influencing factors remain unknown. This study aimed to examine the changes of Th-like Tregs in T1D patients and determine whether Th cells and IL-10+ Bregs play roles in Treg polarization. Methods: A total of 49 T1D patients and 20 healthy controls (HC) were recruited in this study. Flow cytometry was used to measure Treg plasticity (including CD4+Foxp3+IFN-γ+ Th1-like, CD4+Foxp3+IL-4+ Th2-like and CD4+Foxp3+IL-17+ Th17-like Tregs), the proportion of CD27+CD39hi Bregs, and Th cell subsets (including Th1, Th2 and Th17 cells) in the peripheral blood. Cytokines were detected by ELISA. Results: The frequencies of Th1-like Tregs and Th17-like Tregs in T1D were lower than those in HC, while the proportion of Th17 cells was significantly increased in T1D. The frequency of Bregs was markedly higher in T1D relative to HC, but IL-10 expression of Bregs was reduced. Interestingly, compared with lean and overweight patients, Th1-like Tregs were higher in patients with normal weight, but Treg plasticity was not affected by disease status (age at diagnosis, islet autoantibodies and β-cell function) or T1D risk loci (rs3118470 or rs2104286) in IL2RA. Additionally, positive correlations between Th1-like Tregs and Th17-like Tregs and between Bregs and Th2-like Tregs were observed in T1D. Meanwhile, the frequency of Th1-like Tregs was negatively related to IL-6 and the frequency of Th2-like Tregs was negatively associated with TNF-α. Conclusion: Our results firstly explored the features of human Th-like Tregs in the pathogenesis in T1D, which might contribute to improve our understanding of the pathogenic mechanisms of T1D. Disclosure Y.Qin: None. Y.Li: None. L.Dai: None. Q.Wei: None. M.Huang: None. M.Zhang: None. Funding National Natural Science Foundation of China (81974103, 82000747)

Acupuncture regulates the Th17/Treg balance and improves cognitive deficits in a rat model of vascular dementia

ObjectiveThe present study was developed to explore the impact of acupuncture on the Th17/Treg balance in the brain and the periphery and associated changes in cognitive deficits in a rat model of vascular dementia (VD). MethodsMale Wistar rats (8 weeks old) were randomly assigned to sham-operated (Gs, n = 10), and operation (n = 30) groups. A VD model was established for all rats in the operation group via the permanent bilateral occlusion of the common carotid artery. Behavioral screening of these rats was conducted via a hidden platform trial at 2 months post-operation. These operation group rats were then further subdivided into impaired (Gi) and acupuncture (Ga) groups (n = 10/group). Acupuncture was performed over a 21-day period for rats in the Ga group. A Morris water maze (MWM) test was used to assess cognitive function for rats in all groups. Flow cytometry and fluorescent staining were used to detect Th17 and Treg cells in samples from these animals based on IL-17/FoxP3 or CD4+FoxP3+/CD4+RORγt+ staining profiles. ResultsRelative to the Gs group, escape latency values for rats in the Gi group were significantly increased. Following treatment, rats in the Ga group exhibited significant reductions in escape latency values as compared to rats in the Gi group (P < 0.05). The relative Treg proportion in the peripheral blood and spleen additionally trended upwards in these Ga rats as compared to those in the Gi group (P > 0.05), whereas the frequency of Th17 cells in the peripheral blood and spleen of Ga group rats trended downward relative to the Gi group (P > 0.05). Significantly fewer CD4+RORγt+ and RORγt+ cells were detected in the Ga group relative to the Gi group, whereas CD4+FoxP3+ and FoxP3+ cell counts were increased (P < 0.01). ConclusionIn summary, VD model rats exhibited dysregulated Th17/Treg homeostasis. Acupuncture treatment was sufficient to reduce the frequency and numbers of Th17 cells in these animals while increasing Treg cell levels, thereby alleviating cognitive deficits with respect to both spatial learning and memory impairment. Consequently, the therapeutic benefits of such acupuncture treatment may be attributable to the regulation of the Th17/Treg balance and associated improvements in cognitive function.

ERK/MEK Pathway Regulates Th17 Cell Differentiation in Patients with Pemphigus Vulgaris.

T helper (Th) cells are involved in the pathogenesis of pemphigus vulgaris (PV). However, the mechanism still needs more exploration. This study aimed to evaluate the molecular mechanism of the dysregulation of Th17 cells in the peripheral blood of patients with PV. Serum levels of IL-17 and anti-Dsg3 titres in patients with PV were analysed using ELISA. The mRNA expression of retinoic acid orphan receptor γt (RORγt) in CD4+ T cells was detected using reverse transcription-quantitative PCR (qPCR). The number of Th17 cells was examined using flow cytometry. Western blot analysis and immunofluorescent staining were also performed to investigate the expression levels of ERK/MAPK signalling proteins and Th17 lineage-associated proteins. The proportion of Th17 cells and Th17 spectrum-associated proteins (p-STAT3, RORγt and IL-17) were upregulated in CD4+ cells in PV patients. The increased transcriptional levels of RORγt and IL-17 correlated positively with the severity of PV. Elevated phosphorylation of the ERK signalling factors was found in the collected CD4+ T cells in PV patients. The inhibition of the ERK signalling pathway significantly reduced the differentiation of Th17 cells in PV patients in vitro. Th17 cells are essential in the dysregulation of PV, and ERK signalling is involved in Th17-type immunity and promotes the development of PV. The study here provides us with a potential therapeutic target for PV.