Treg Cells In Peripheral Blood Research Articles

Anti-pruritic and anti-inflammatory effects of oxymatrine in a mouse model of allergic contact dermatitis

BackgroundAllergic contact dermatitis (ACD) is a highly prevalent inflammatory disease of the skin. As a result of the complex etiology in ACD, therapeutic compounds targeting refractory pruritus in ACD lack efficacy and lead to numerous side effects. ObjectiveIn this study, we investigated the anti-pruritic effects of oxymatrine (OMT) and explored its mechanism of action in a mouse model of ACD. Method72 male SPF C57BL/6 mice were randomly divided into control group, ACD model group, dexamethasone positive control group (0.08 mg kg−1) and 3 OMT groups (80, 40, 20 mg kg−1). OMT was administrated by intraperitoneal injection 1 h before video recording on day 10, 24 h after 2nd challenge with SADBE. Cheek skin fold thickness was measured before treatment and after recording. H&E staining was used for pathological observation. RT-qPCR, Immunohistochemistry and LEGENDplexTM assay were used to detect cytokines levels. The population of Treg cells in peripheral blood were detected via flow cytometry. ResultsOMT treatment significantly decreases the skin inflammation and scratching bouts. It rescues defects in epidermal keratinization and inflammatory cell infiltration in ACD mice. Administration of OMT significantly reduced levels of IFN-γ, IL-13, IL-17A, TNF-α, IL-22 and mRNA expression of TNF-α and IL-1β. Furthermore, it increased the percentage of Treg cells in peripheral blood of ACD mice. ConclusionWe have demonstrated that OMT exhibits anti-pruritic and anti-inflammatory effects in ACD mice by regulating inflammatory mediators. OMT might emerge as a potential drug for the treatment of pruritus and skin inflammation in the setting of ACD.

Expression of PD-1 on peripheral blood Treg cells is related to the diagnosis, prognosis and treatment of T cell non-Hodgkin lymphoma

PurposeThe aim of study was to explore the PD-1 expression on Treg cells and its association with T-NHL. Methods137 patients newly diagnosed with T-NHL and 115 healthy controls were enrolled. The expression level of PD-1 was measured by flow cytometry at the time of diagnose and 3–8 course of treatment. ResultsMedian fluorescence intensity (MFI) of PD-1 on Treg cells in T-NHL patients was significantly higher than that in healthy controls (P < 0.001). MFI of PD-1 in medium/high-risk T-NHL patients were higher than that in low-risk patients (P < 0.05). After treatment with Chidamide combined with chemotherapy, MFI of PD-1 significantly decreased (P < 0.05). In patients with high PD-1 expression (percentage>19.6% and MFI > 580), EFS was significantly lower than patients with low PD-1 expression (percentage<19.6% and MFI < 580). ConclusionsThe PD-1expression on peripheral blood Treg cells of T-NHL patients is related to the diagnosis, prognosis and treatment of disease.

Expression of GRIM-19 in unexplained recurrent spontaneous abortion and possible pathogenesis.

Is aberrant expression of gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) associated with unexplained recurrent spontaneous abortion (URSA)? GRIM-19 deficiency may regulate regulatory T cell/T helper 17 cell (Treg/Th17) balance partly through reactive oxygen species (ROS)-mammalian target of rapamycin (mTOR) signaling axis in URSA. Immunological disorders may cause impaired maternal immune tolerance to the fetus and result in fetal rejection. The differentiation of Treg and Th17 cells is controlled by phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling pathway. GRIM-19 participates in the immune response, but its role in URSA is largely unknown. The current study included 28 URSA patients and 30 non-pregnant healthy women. The proportion of Treg and Th17 cells in peripheral blood of URSA patients and control subjects were assessed with flow cytometry. The expression of GRIM-19 in peripheral blood lymphocytes (PBLs) was measured with quantitative real-time PCR and western blot analysis. Furthermore, the ROS level in the PBLs of URSA patients and control subjects were assessed by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Then, Akt/mTOR expression in the PBLs was measured. Downregulation of GRIM-19 in Jurkat cells was performed by specific siRNA. Then, intracellular ROS production and the expression of p-mTOR, which is known to enhance Th17 differentiation and decrease Treg cell differentiation, were detected. Finally, N-acetylcysteine (NAC) was used to decrease the intracellular ROS level, and the expression of p-mTOR was measured. The proportion of Treg cells was reduced in URSA patients, whereas the proportion of Th17 cells was increased. The expression of GRIM-19 was significantly lower in PBLs of URSA patients. Furthermore, there is a considerable increase in intracellular ROS production and a high level of p-Akt and p-mTOR expression in the PBLs of URSA patients compared with the control subjects. In parallel to this, downregulation of GRIM-19 in the Jurkat cells by siRNA results in an increased ROS production and an increased expression of p-mTOR. Importantly, the upregulation of p-mTOR resulting from GRIM-19 loss was significantly reversed in the cells treatment with ROS inhibitor N-acetyl-l-cysteine (NAC), indicating that ROS was indeed required for GRIM-19 depletion induced p-mTOR expression. None. A large number of researches have confirmed that the differentiation of Treg and Th17 cells is controlled by PI3K/Akt/mTOR signaling pathway. We have not shown the regulatory role of ROS and PI3K/Akt/mTOR in Treg and Th17 differentiation in this study. Our study has demonstrated that GRIM-19 deficiency may play a role in regulating Treg/Th17 balance partly through ROS-mTOR signaling axis in URSA. The present study offers a new perspective to the roles of GRIM-19 in immunoregulation. This work was supported by the National Natural Science Foundation of China (Grant numbers 81571511, 81701528, 81370711 and 30901603), the Shandong Provincial Natural Science Foundation (Grant numbers ZR2017PH052 and ZR2013HM090) and the Science Foundation of Qilu Hospital of Shandong University, Fundamental Research Funds of Shandong University (Grant numbers 2015QLQN50 and 2015QLMS24). The authors declare that there is no conflict of interest that could prejudice the impartiality of the present research.

Human GMSCs Treat Murine Model with Aplastic Anemia Through Regulating the Balance between Treg and Th17 Cells

Severe aplastic anemia (AA) is a rare autoimmune disease characterized by severe pancytopenia and bone marrow failure. The recent finding of an abnormal quantity and function of T helper (Th) and regulatory T (Treg) cells in peripheral blood of patients has emerged as the major cause of the condition. Accumulating evidence has revealed that human gingiva-derived mesenchymal stem cells (GMSCs) are emerging as a new line of mesenchymal stem cells may have the potential to control or even treat autoimmune diseases through correcting the balance between Th and Treg cells. Given that GMSCs have a robust immune regulatory function and regenerative ability, we investigated the effect of GMSCs on preventing T cell-mediated bone marrow failure (BMF) in an AA mouse model. We observed that infusion of GMSCs markedly attenuated histological BM damage, controlled immunologically-mediated disorders in AA mice, significantly improved AA mice survival by significantly reducing cell infiltration of both CD8 cells and CD4 T cells (including Th1 and Th17 cells) in lymph nodes and decreased pro-inflammatory cytokines levels in lymph nodes and plasma. Moreover, GMSCs up-regulated the levels of Tregs and increased Foxp3 expression in lymph nodes. Our results demonstrate that the predominant Th1 and Th17 cell infiltration in the AA model could be significantly reduced while Treg cells are increased by GMSCs treatment. Collectively, our data demonstrate that GMSCs can attenuate T cell-mediated bone marrow failure through targeting the balance of Th17 and Treg cells, implicating that the use of GMSCs many have potential clinical application in prevention and treatment of patients with AA. Funding Statement: This study was supported by the National Key R&D Program of China (2017YFA0105800), the National Natural Science Foundation of China (No. 81671611), the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2016ZT06S252), the National Natural Science Foundation of Guangdong Province (No. 2014A030313131) and NIH R61 AR073409. Declaration of Interests: All authors have no conflict of interest. Ethics Approval Statement: This study was carried out in accordance with the recommendations of the animal use protocol, which was approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University (Approve number: [2018]02-195-01). Human gingiva samples were collected following routine dental procedures at the Division of Dentistry in the Third Hospital at the Sun Yat-sen University, which were approved by the medical ethics committees of Institutional Review Boards (IRB) at the Third Hospital at the Sun Yat-sen University.

Percentages of peripheral blood γδ T cells and regulatory T cells and expression of associated cytokines in infants with human cytomegalovirus infection

To investigate the percentages of peripheral blood γδ T cells and regulatory T cells (Treg) and the expression of associated cytokines, interleukin 17 (IL-17) and transforming growth factor-β1 (TGF-β1), in infants with human cytomegalovirus (HCMV) infection. Twenty-two infants with HCMV infection (HCMV group) and 22 healthy infants who underwent physical examination (control group) were enrolled in this study. The percentages of peripheral blood γδ T cells and Treg cells were determined by flow cytometry. The levels of IL-17 and TGF-β1 in plasma were measured using ELISA. Compared with the control group, the HCMV group had significantly higher percentage of γδ T cells and IL-17 level (P<0.01) and significantly lower percentage of Treg cells and TGF-β1 level (P<0.01). In the HCMV group, the percentage of γδ T cells was negatively correlated with the percentage of Treg cells and TGF-β1 level (P<0.05), but positively correlated with IL-17 level (P<0.05); the percentage of Treg cells was positively correlated with TGF-β1 level (P<0.05), but negatively correlated with IL-17 level (P<0.05); there was no correlation between IL-17 level and TGF-β1 level (P>0.05). There is an imbalance between γδ T cells and Treg cells in the peripheral blood of infants with HCMV infection, and γδ T cells may be involved in the secretion of IL-17.

CD4+ CD25+ FOXP3+ T cell frequency in the peripheral blood is a biomarker that distinguishes intestinal tuberculosis from Crohn's disease.

BackgroundDistinguishing between Crohn’s Disease (CD) and Intestinal Tuberculosis (ITB) has been a challenging task for clinicians due to their similar presentation. CD4+FOXP3+ T regulatory cells (Tregs) have been reported to be increased in patients with pulmonary tuberculosis. However, there is no such data available in ITB. The aim of this study was to investigate the differential expression of FOXP3+ T cells in patients with ITB and CD and its utility as a biomarker.MethodsThe study prospectively recruited 124 patients with CD, ITB and controls: ulcerative colitis (UC) and patients with only haemorrhoidal bleed. Frequency of CD4+CD25+FOXP3+ Tregs in peripheral blood (flow cytometry), FOXP3 mRNA expression in blood and colonic mucosa (qPCR) and FOXP3+ T cells in colonic mucosa (immunohistochemistry) were compared between controls, CD and ITB patients.ResultsFrequency of CD4+CD25+FOXP3+ Treg cells in peripheral blood was significantly increased in ITB as compared to CD. Similarly, significant increase in FOXP3+ T cells and FOXP3 mRNA expression was observed in colonic mucosa of ITB as compared to CD. ROC curve showed that a value of >32.5% for FOXP3+ cells in peripheral blood could differentiate between CD and ITB with a sensitivity of 75% and a specificity of 90.6%.ConclusionPhenotypic enumeration of peripheral CD4+CD25+FOXP3+ Treg cells can be used as a non-invasive biomarker in clinics with a high diagnostic accuracy to differentiate between ITB and CD in regions where TB is endemic.

Effects of Thalidomide on the Ratio of Th17 to Treg Cells in Peripheral Blood and Expression of IL-17 and IL-35 in Patients with Multiple Myeloma

To explore the effects of thalidomide on the ratio of Th17 to Treg cells in peripheral blood and expression of IL-17 and IL-35 in patients with multiple myeloma(MM), so as to provide reference for the clinical treatment of patients with MM. A total of 82 MM patients treated with thalidomide from January 2014 to December 2016 were enrolled in MM group, 30 healthy subjects were selected as control (control group). The ratio of T cell subsets and Treg cells accounted for CD4+T cell were detected by flow cytometer. The levels of IL-17 and IL-35 in serum were measured by ELISA, and the differences of various indexes were compared between 2 groups. Compared with the control group, the ratio of Th17 cells in peripheral blood and serum levels of IL-17 of MM patients were significantly increased, the ratio of Treg cells and the level of IL-35 were significantly decreased and the ratio of Th17/Treg cells was significantly increased in the patients with multiple myeloma before treatment (P<0.05). The ratio of Th17 cells in peripheral blood and serum levels of IL-17 in patients with multiple myeloma after treatment with thalidomide were significantly lower than those before treatment, and the ratio of Treg cells and levels of IL-35 were significantly higher than those before treatment, and the ratio of Th17 / Treg cells was higher than that before treatment (P<0.05). The indexes in ineffective treatment were not significantly changed (P> 0.05). The unbanlace of Th17/Treg cell ratio and abnormality of IL-17, IL-35 levels play an important role in the progression of multiple myeloma. The anti-MM mechanism of thalidomide may relate with the regulation of Th17 / Treg cell ratio and expression levels of IL-17 and IL-35.

Change of Peripheral Blood Treg/Thl7 in Cognitive Impairment with Chronic Renal Failure Patients

Background/Aims: To investigate the changes in peripheral blood Treg/Th17 cell balance and its significance in patients with chronic renal failure (CRF) and cognitive impairment. Methods: A total of 71 patients with CRF were enrolled as a study group. The patients were divided into a cognitive impairment group and a normal cognitive function group according to the Mini-Mental State Examination (MMSE). Peripheral blood Treg and Th17 cells were analyzed by flow cytometry and their relevant cytokines (IL-17, IL-10 and TGF-β) and other biochemical indicators, including C-reactive protein (CRP) and IL-6, were determined by ELISA. Results: Thepatients with both CRF and cognitive impairment were older than the cognitive normal groups. Peripheral blood Treg cells by Flow cytometry (the CRF cognitive impairment group 5.57±1.3%, CRF group with normal cognitive function 7.5 ± 0.9% and normal control group 9.7 ± 1.7%,P<0.05) and its related cytokines (IL-10 and TGF-β) by ELISA detection were lower in the group with cognitive impairment than in the group without cognitive impairment ( IL-10, 7.4±4.2 pg/mL, 13.8±3.9 pg/mL, 18.3±3.2 pg/mL; TGF-β 335.6±175.3 pg/mL, 512.7 ± 114.6 pg/mL, 953.8±373.4 pg/mL P < 0.05, respectively).However, Th17 cell numbers (the CRF cognitive impairment group 3.3 ± 0.7%, CRF group with normal cognitive function2.2 ± 0.5% and normal control group 1.5 ± 0.3%),and cytokine levels (IL-17, IL-6 and CRP) were higher in the group with cognitive impairment IL-6 (21.3 ± 5.1 pg/mL), IL-17 (18.5 ± 4.2 pg/mL) and CRP (20.3 ± 5.9 mg/L) in the CRF group with cognitive impairment when compared with the CRF group and normal cognitive function (12.2 ± 4.5 pg/mL, 12.1 ± 3.7 pg/mL and 13.5 ± 4.6 mg/L, respectively) or the normal control group (9.2 ± 5.8 pg/mL, 7.4 ± 2.6 pg/mL and 3.2 ± 1.3 mg/L, respectively, P<0.05). The frequencies of Treg in patients with CRF were positively correlated with the MMSE scores ((r = 0.518, P < 0.05), but the Th17 numbers were negatively correlated (r = -0.435, P < 0.05). Conclusion: An imbalance of peripheral blood Treg/Th17 cells is associated with cognitive impairment in patients with CRF.

Low-dose lymphocyte immunotherapy rebalances the peripheral blood Th1/Th2/Treg paradigm in patients with unexplained recurrent miscarriage

BackgroundThe published results regarding lymphocytes immunotherapy for unexplained recurrent miscarriage (uRM) patients are conflicting due to different screening criteria and therapeutic protocols. The objective of the present study is to evaluate the effectiveness of immunotherapy using low-dose lymphocytes in patients with uRM and Th1/Th2/Treg paradigm disorders.MethodsSixty-four uRM patients who received low-dose lymphocytes immunotherapy served as the immunotherapy group, while the other 35 women who did not receive the treatment served as the control group. The proportions of peripheral blood Th1 cells, Th2 cells and Treg cells; and the concentration of TGF-β1 in serum were detected by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively, before and after the immunotherapy.ResultsThe proportion of Th1 cells was significantly decreased while the proportions of Th2 cells and Treg cells were significantly increased in immunotherapy group patients after treatment. In addition, the concentration of TGF-β1 in serum was significantly higher after immunotherapy than before. Forty-three uRM patients achieved pregnancy after receiving immunotherapy and 5 patients underwent miscarriages in the immunotherapy group (11.6%, 5/43), while 8 of the 23 pregnant patients experienced a miscarriage in the control group (34.8%, 8/23; p < 0.05).ConclusionsLow-dose lymphocyte immunotherapy is beneficial for restoring balance in the Th1/Th2/Treg paradigm and improving pregnancy outcome in uRM patients.Trial registrationNCT03081325. ClinicalTrials.gov. Retrospectively registered July 2015.

Short-term cytokine stimulation reveals regulatory Tcells with down-regulated Foxp3 expression in human peripheral blood.

The identification of regulatory Tcells (Treg cells) in human peripheral blood is an important tool in diagnosis, research, and therapeutic intervention. As compared to lymphoid tissues, the frequencies of circulating Treg cells identified as CD4+ CD25+ Foxp3+ are, however, low. We here show that many of these cells remain undetected due to transient down regulation of Foxp3, which rapidly decays in the absence of cytokine-mediated STAT5 signals. Short-term incubation of PBMCs or isolated CD4+ Tcells, but not of lymph node cells, with IL-2, -7, or -15 more than doubles the frequency of Foxp3+ CD25+ among CD4+ Tcells detectable by flow cytometry. This increase is not due to cell division but to upregulation of both proteins. At the same time, the uncovered Treg cells up-regulate CD25 and down-regulate CD127, making them accessible to viable cell sorting. "Latent" Treg cells have a demethylated FOXP3 TSDR sequence, are enriched in naïve, non-cycling cells, and are functional. The confirmation of our findings in RA and SLE patients shows the feasibility of uncovering latent Treg cells for immune monitoring in clinical settings. Finally, our results suggest that unmasking of latent Treg cells contributes to the increase in circulating CD4+ CD25+ Foxp3+ cells reported in IL-2 treated patients.

Quantification of Treg cells in peripheral blood and lymph nodes of dogs with multicentric lymphoma

ABSTRACT Lymphoma is a malignant tumor characterized by cell proliferation of lymphoid origin and corresponds to 90% of all hematopoietic neoplasms of dogs. Regulatory T cells (Tregs) have been the target of many investigations in oncology due to their potential of down-regulating immune responses, as well as ensuring the maintenance of active mechanisms of tumor suppression. The aims of the present study were to compare the percentage of Tregs in peripheral blood between dogs with multicentric lymphoma and healthy animals, together with the percentage of Tregs in peripheral blood and lymph nodes of dogs with multicentric lymphoma. Twenty-six animals were enrolled in the study: 10 healthy dogs comprised the control group (CG) and 16 dogs with multicentric lymphoma comprised the Lymphoma Group (LG). We observed that dogs in the LG showed a significantly higher Tregs expression in peripheral blood compared to the CG. No significant difference was observed between Tregs expression in lymph nodes and peripheral blood of the LG, however. With these results, it is possible to conclude that multicentric lymphoma is a neoplasm with high Tregs expression, which poses this as a condition of interest when investigating treatments that can suppress Regulatory T cells.

Relationship between HBeAg from HBsAg positive mothers and regulatory T cells in neonates and its influence on HBV intrauterine transmission

Objective: To explore the relationship between HBeAg in HBsAg positive mothers and CD(4)(+)CD(25)(+)Foxp3(+)regulatory T cells (Treg) in newborns, as well as how they would influence the increasing risk on HBV intrauterine transmission. Methods: We collected information on general demographic characteristics and delivery on 270 HBsAg positive mothers and their newborns from the Third People's Hospital of Taiyuan. Fluorescence quantitative polymerase chain reaction (FQ-PCR) and chemiluminescence immunoassay (CLIA) were used to detect HBV DNA and HBV serological markers in peripheral blood from both mothers and neonates. The expression of Treg and other immune cells in peripheral blood of neonates were detected with flow cytometry (FCM). Results: Maternal HBeAg positive rates were associated with an increased risk of intrauterine transmission (OR=4.08, 95%CI: 1.89-8.82). Rates of Treg in newborns born to HBsAg-positive mothers were higher than that of the negative group (Z=2.29, P=0.022). Each pair of the subjects was assigned to five different groups according to the HBeAg titers of mothers. Frequencies of both Treg and HBeAg in newborns and HBV DNA in mothers between the above said 5 groups showed similar trends of changing patterns and the differences between groups were statistically significant(χ(2)=18.73, P<0.001; χ(2)=181.60, P<0.001; χ(2)=183.09, P<0.001). Results from partial correlation analysis showed that after adjusting for neonatal HBeAg and maternal HBV DNA, mother's HBeAg titers were positively related to the percentage of Treg in their newborns (r(s)=0.19, P=0.039). In addition, the frequencies of Treg were negatively correlated with pDC and CD(4)(+) T cell in their newborns (r(s)=-0.21, P=0.017; r(s)=-0.23, P=0.009). Conclusion: HBeAg from HBsAg positive mothers might have inhibited the function of neonatal DC cells and T cells to reduce the immune response to HBV by up-regulating the proportion of Treg and finally increased the risk of HBV intrauterine transmission.

Mesenchymal Stem Cells in Rat Hepatic Fibrosis MModel: Benefits? Mechanism of Action?: 2017 Presidential Poster Award

Introduction: Hepatic fibrosis may evolve to liver cirrhosis which may ultimately develop into hepatocellular carcinoma. Mesenchymal stem cells (MSCs) have potential to trans-differentiate into hepatic cells and thus may be a curative option in liver cirrhosis. Natural killer (NK) cells are a component of the innate immune system that destroy compromised host cells and have critical antifibrotic role. Aim: To show the effects of bone marrow MSCs transferred via the tail vein to the rats having hepatic fibrosis produced by common bile duct ligation (CBDL) with a special focus on the changes of the NK population distribution. Methods: Rats were divided into three groups; 1- CBDL rats that were injected with phosphate-buffered saline (PBS), 2- CBDL rats injected with MSCs, 3- healthy rats that were sham operated and given MSCs. All rats were sacrificed at the end of the experiment. We analyzed the in vivo functional outcomes by measuring the serum glucose, bilirubin and ALT levels. Splenocytes isolated from the collected spleens were cultured and the supernatants were analyzed for IL (interleukin)-1α, TNF (tumor necrosis factor)-α, IFN (interferon)-γ and IL-10 using flow cytometry. Results: Liver fibrosis developed in CBDL rats while the healthy rats did not show any alteration in liver architecture. Although healthy group had significantly lower serum bilirubin, ALT and glucose levels than CBDL rats, the difference regarding those parameters did not differ between groups 1 and 2. T-lymphocyte proliferation was significantly suppressed in group 2 compared to group 1. IL-1α, TNF-α and IFN-γ levels in supernatants of cultured lymphocytes in group 2 were significantly higher than group 3 while they tended to be lower than group 1. NK and Treg cells in peripheral blood significantly increased in group 2 compared to group 1. Histologically, hepatic fibrosis scores were significantly alleviated in group 2 compared to group 1 (p=0.039). NK cells in the general hepatic parenchyma and in the portal areas were both increased significantly in group 2 compared to group 1. Conclusion: Our findings suggest bone marrow derived MSCs may be beneficial in alleviating the hepatic fibrosis by suppressing the T cell proliferation and the proinflammatory cytokines and inducing NK cell recruitment to liver parenchyma in rats. Thus, MSC treatment may appear promising in liver injury and future clinical therapies are warranted. Immunophenotyping of leukocytes. CD3+CD4+ T lymphocyte, CD3+CD8+ Cytotoxic T cell, CD4+CD25+ T regulatory cell and CD161a NK cell in all groups by flow cytometry. (B) CD3+CD4+ % T lymphocyte, CD3+CD8+ % Cytotoxic T cell, CD4+CD25+ %T regulatory cell and CD161a NK cells were displayed statistically in all groups..*P < 0.05, **P < 0.01.Figure

Changes in myeloid-derived suppressor cells (MDSC), Treg and Th17 cells in peripheral blood of patients with cervical human papillomavirus (HPV) infection

Objective To investigate the changes in myeloid-derived suppressor cells (MDSC), regulatory T cells (Treg) and T helper 17 (Th17) cells in peripheral blood of patients with HPV infection and cervical diseases including ectopic cervical columnar epithelium (CE), cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CC), and to analyze the roles of MDSC, Treg and Th17 cells in the development of cervical diseases. Methods This study enrolled 120 HPV-positive patients with cervical diseases (18 cases of CE, 50 cases of CIN, 52 cases of CC), 20 HPV-negative patients with cervical diseases (10 cases of CE and 10 cases of CIN) and 20 healthy subjects from March 2011 to December 2016. Changes in MDSC, Treg and Th17 cells in peripheral blood of all patients were detected by flow cytometry. Results Percentages of MDSC, Treg and Th17 cells in peripheral blood of patients who were positive for HPV were significantly higher than those in HPV-negative patients (P<0.05). Besides, percentages of these cells gradually increased with the exacerbation of cervical disease (CE-CINⅠ-CINⅡ-CINⅢ-CC) (P<0.05). Patients who were infected with high-risk HPV had significantly higher percentages of MDSC, Treg and Th17 cells in peripheral blood than those infected with low-risk HPV (P<0.05). Changes of MDSC, Treg and Th17 cells in peripheral blood of HPV-positive patients with cervical cancer were related to International Federation of Gynecology and Obstetrics (FIGO) stage, tumor differentiation, lymph node metastasis and vascular invasion (P<0.05). MDSC, Treg and Th17 cells in peripheral blood of all patients were significantly reduced after treatment (P<0.05). Patients whose status changed from HPV-positive to HPV-negative following treatment also showed significantly decreased MDSC, Treg and Th17 cells in peripheral blood as compared with those who remained HPV-positive (P<0.05). MDSC, Treg and Th17 cells in peripheral blood of HPV-positive patients with cervical diseases were positively correlated (r=0.599, 0.677, 0.648; P<0.05). Conclusion MDSC, Treg and Th17 cells in peripheral blood of patients with HPV infection are associated with the pathological process, severity and clinical pathological features of cervical diseases as well as therapeutic effects. Therefore, they can be used as biomarkers to detect the occurrence and development of HPV-positive cervical diseases. Key words: Human papillomavirus; Cervical cancer; Myeloid-derived suppressor cell; Regulatory T cell; T helper 17 cell

Enrichment of Human CCR6+ Regulatory T Cells with Superior Suppressive Activity in Oral Cancer.

Human oral squamous cell carcinoma (OSCC) constitutes an inflammatory microenvironment enriched with chemokines such as CCL20, which promote cancer cell invasion and tumor progression. We found that in OSCC there is a correlation between the expression of CCL20 and FOXP3 mRNA. Therefore, we hypothesized that OSCC may favor the recruitment and retention of regulatory T (Treg) cells that express the CCL20 receptor, CCR6. Interestingly, most (∼60%) peripheral blood Treg cells express CCR6, and CCR6+ Treg cells exhibit an activated effector/memory phenotype. In contrast, a significant portion (>30%) of CCR6- Treg cells were found to be CD45RA+ naive Treg cells. Compared to CCR6- naive or memory Treg cells, CCR6+ Treg cells exhibit stronger suppressive activity and display higher FOXP3 expression along with lower methylation at the Treg-specific demethylated region of the FOXP3 gene. This predominance of CCR6+ Treg cells was also found in the draining lymph nodes and tumor-infiltrating lymphocytes of OSCC patients with early or late clinical staging. Moreover, CCR6+ Treg cells isolated from tumor-infiltrating lymphocytes or draining lymph nodes maintained similar phenotypic and suppressive characteristics ex vivo as did their counterparts isolated from peripheral blood. These results suggest that CCR6 marks activated effector or memory Treg phenotypes with superior suppressive activity in humans.

Th17/regulatory T cell imbalance in sepsis patients with multiple organ dysfunction syndrome: attenuated by high-volume hemofiltration.

We assessed the Th17 (T-helper cell)/Treg (Regulatory T cell) imbalance in sepsis patients with multiple organ dysfunction syndrome (MODS) and the clinical benefits of continuous high-volume hemofiltration (HVHF). 48 sepsis patients, including 22 patients with MODS (MODS group, n = 22) and 26 without (non-MODS group, n = 26), and 20 healthy volunteer controls were enrolled. The patients in MODS group were randomly divided into the continuous blood purification (CBP) group (n = 11) receiving conventional comprehensive treatment plus high-volume hemofiltration and the non-CBP group (n = 11) receiving conventional comprehensive treatment only. At day 28, all 48 patients were divided into the survival group (n = 36) and the non-survival group (n = 12). Venous blood samples, collected at 0, 6, 12 and 24 hours during hemofiltration (or equivalent times in the non-CBP group), were assessed by flow cytometry to detect the Th17 and Treg cells in peripheral blood. Serum cytokines (such as IL-6, IL-17, IL-23, IL-10 and TGF-β1) were detected by enzyme-linked immune sorbent assay (ELISA). Th17%, Treg%, Th17/Treg, IL-6, IL-17, IL-23, IL-10 and TGF-β1 were significantly higher in MODS and non-MODS group than in the health control (p<0.05), while Th17%, Treg%, Th17/Treg and measured cytokines were significantly higher in the MODS group compared to the non-MODS group (p<0.05). After HVHF treatment, Th17%, Treg%, Th17/Treg, IL-6, IL-17, and IL-10 were significantly reduced (p<0.05), while there were no significant changes in the non-CBP group (p>0.05). In addition, APACHE II and SOFA scores decreased markedly after HVHF treatment. Baseline Th17%, Treg%, Th17/Treg, IL-6, IL-17, IL-23, IL-10 and TGF-β1 were significantly higher in the non-survival group compared to the survival group. Both Th17% and Th17/Treg were positivity correlated with concentration of IL-6 and APACHE II score (p<0.01). The level of Th17/Treg imbalance in sepsis is related to the occurrence and prognosis of MODS. High-volume hemofiltration can attenuate the Th17/Treg imbalance in sepsis patients, possibly by removing inflammatory mediators.

Role of PIM2 in allergic asthma.

T cell-associated inflammation, particularly type 2 inflammation, has an important role in asthma pathogenesis, which is suppressed by regulatory T cells (Tregs). Proviral integration site for Moloney murine leukemia virus 2 (PIM2), a member off the serine/threonine kinase family, promotes the growth and survival of T cells and influences the function of Treg cells. However, whether PIM2 affects asthma pathogenesis remains unclear. Peripheral blood mononuclear cells and Treg cells from asthmatic and healthy subjects were obtained, and the expression level of PIM2 was measured by reverse transcription-quantitative polymerase chain reaction and immunocytochemistry. In addition, BALB/c female mice sensitized and challenged by ovalbumin were used as an asthma model, and PIM2 inhibitor was injected during the challenge period to observe the effect of PIM2 on asthma. The asthma symptoms were recorded, and airway hyper-responsiveness (AHR), expression levels of cytokines in the serum or bronchoalveolar lavage fluid (BALF), and the number of BALF leukocytes were evaluated. In addition, hematoxylin and eosin staining and immunohistochemistry of lung tissues was performed. The results demonstrated that PIM2 was overexpressed in patients with asthma in natural Treg cells. Inhibition of PIM2 attenuated asthma symptoms, and improved AHR and airway inflammation compared with asthmatic mice without inhibition of PIM2. In addition, expression levels of interleukin (IL)-10 and forkhead box protein 3 (FOXP3) in BALF were increased following PIM2 inhibition (IL-10, 470.3±21.78 vs. 533.7±25.55 pg/ml, P<0.05; FOXP3, 259±4.68 vs. 279.3±3.68 pg/ml; asthma and PIM2 inhibition groups, respectively; P<0.05). In conclusion, PIM2 may exhibit an important role in asthma pathogenesis and exacerbate AHR, airway inflammation and asthma symptoms. These effects of PIM2 may be dependent on Treg cells and the secretion of IL-10 by Tregs. The results of the present study suggest that PIM2 may be a potential target molecule for asthma treatment.

Decreased frequency of peripheral blood CD8+CD25+FoxP3+regulatory T cells correlates with IL-33 levels in pre-eclampsia

ABSTRACTObjective: We aimed to investigate the role of CD8+CD25+Foxp3+regulatory T (Treg) cells in pre-eclampsia (PE).Methods: This was a cross-sectional study of 46 patients with PE and 24 normotensive women within the third trimester of gestation. We analyzed the percentages of CD8+CD25+Foxp3+Treg cells in peripheral blood using flow cytometry and the serum levels of interleukin (IL)-6, IL-17A, IL-10, TGF-β1, IL-1β, and IL-33 by Luminex 200.Results: We found that patients with PE had lower percentages of CD8+CD25+Foxp3+Treg cells than normotensive pregnant women. In addition, the percentage of CD8+CD25+Foxp3+Treg cells was positively correlated with IL-33 concentration and negatively correlated with IL-17A concentration in patients with PE. We also found that IL-33 treatment can induce proliferation of CD8+CD25+Foxp3+Treg cells in vitro.Conclusions: These findings suggest that the reduced CD8+CD25+Foxp3+Treg cells may play a role in the pathogenesis of PE.AbbreviationsPE: pre-eclampsia; PBMCs: peripheral blood mononuclear cells; CTLA-4: cytotoxic T-lymphocyteantigen-4; APCs: antigen presenting cells; TGF-β: transforming growth factor-β; IL: interleukin; Treg: cells regulatory T cells; PBS: phosphate-buffered saline; Foxp3: forkhead Box protein 3; HELLPs: hemolysis, elevated liver enzyme and low platelet syndrome

Relationship between peripheral Treg/Th17 cells and pregnancy outcomes in the first trimester of patients with unexplained recurrent spontaneous abortion

Objective To investigate Treg and Th17 cell numbers in the peripheral blood of patients with unexplained recurrent spontaneous abortion (URSA), with an aim to elevate their potential for predicting pregnancy outcomes. Methods Peripheral blood was collected from 60 URSA pregnant women and 56 healthy pregnant women during their first trimester in the Department of Reproductive Endocrinology of the Second Affiliated Hospital of Soochow University from January 2014 to June 2015. The counts of regulatory T cell (Treg), T helper cell 17 (Th17), CD4 positive T cell (CD4+ T cell) and total lymphocytes in the peripheral blood were determined by flow cytometry. Calculated the ratios of Treg/CD4+ T cells, Treg/total lymphocytes, Th17/CD4+ T cells, Th17/total lymphocytes and Treg/Th17, then the pregnancy outcomes were followed up. According to the different pregnancy outcomes, the patients were divided into three groups: URSA successful delivery group (23 cases), URSA re-miscarriage group (37 cases) and normal successful delivery group (44 cases). Non-parametric test were used to compare the differences between groups. Results There were no difference between the URSA re-miscarriage group and URSA successful delivery group in the CD4+ T cell counts[(1 570.16±519.56) vs (1 571.83±450.37), t=0.01, P=0.99]and total lymphocytes[(4 497.11±975.48) vs (4 558.52±1 032.54), t=0.23, P=0.82]. However, the ratios of Treg/CD4+ T cells[(1.09±0.62) vs (3.32±2.30), t=4.55, P=0.000 1], Treg/total lymphocytes[(0.42±0.24) vs (1.31±1.14), t=3.67, P=0.001 3]and Treg/Th17[(0.56±0.41) vs (2.51±2.38), t=3.89, P=0.000 8]in the URSA miscarriage again group were significantly lower whereas the ratios of Th17/CD4+ T cells[(1.85±0.92) vs (0.93±0.36), t=5.40, P<0.001]and Th17/total lymphocytes[(0.64±0.31) vs (0.31±0.14), t=5.52, P<0.001]were significantly higher. Similarly, There were no difference between the URSA re-miscarriage group and normal successful delivery group in the CD4+ T cell counts[(1 570.16±519.56) vs (1 566.86±651.36), t=0.03, P=0.98]and total lymphocytes[(4 497.11±975.48) vs (4 720.91±1 572.92), t=0.78, P=0.44]. However, the ratios of Treg/CD4+ T cells[(1.09±0.62) vs (3.17±1.81), t=7.16, P<0.001], Treg/total lymphocytes[(0.42±0.24) vs (1.03±0.51), t=7.08, P<0.001]and Treg/Th17[(0.56±0.41) vs (1.87±1.68), t=4.98, P<0.001]in the URSA re-miscarriage group were significantly lower whereas the ratios of Th17/CD4+ T cells[(1.85±0.92) vs (1.12±0.50), t=4.29, P<0.001]and Th17/total lymphocytes[(0.64±0.31) vs (0.36±0.15), t=5.01, P<0.001]were significantly higher. Conclusions Treg and Th17 cell counts in the peripheral blood of patients with URSA might be useful in predicting their pregnancy outcomes. The decrease of Treg cell counts and the increase of Th17 cell counts might imply another abortion.(Chin J Lab Med, 2017, 40: 174-179) Key words: Abortion, habitual; Pregnancy trimester, first; T-lymphocytes, regulatory; Th17 cells

Detection of Th17/Treg cells and related factors in gingival tissues and peripheral blood of rats with experimental periodontitis.

Objective(s):This study aimed to investigate the role and the possible mechanisms involved in the immunoregulation of experimental periodontitis by Th17/Treg.Materials and Methods:Experimental periodontitis was established by silk thread ligation with Porphyromonas gingivalis daubing in the bilateral maxillary second molar of Male Sprague-Dawley (SD) rats. Alveolar bones were scanned by Micro-CT. Histological examination was stained with H&E. The proportions of Th17 and Treg cells in peripheral blood were detected by flow cytometry. RT-PCR was used to measure the expression of RORγt, Foxp3 mRNA in the gingival tissues. The concentrations of IL-17, IL-10, and TGF-β in peripheral blood and gingival crevicular fluid were measured by ELISA.Results:Experimental rats showed profound bone resorption and inflammatory cell infiltration. The percentages of Th17 significantly increased in the peripheral blood, which was consistent with gingival tissues study that Th17 cells related transcription factor RORγt mRNA and IL-17 increased in the course of periodontitis. The percentages of CD25+Foxp3+ Treg significantly increased in the peripheral blood, which was consistent with gingival tissues study that Treg cells related transcription factor Foxp3 mRNA and cytokines IL-10 and TGF-β increased in the course of periodontitis. The ratio of Th17/Treg cells was significantly increased in the peripheral circulation, however, the Th17/Treg balance is in wave motion in inflamed gingival tissues in the different stages of periodontitis.Conclusion:Th17/Treg balance may be associated with the progression of periodontitis and pathological tissue destruction. Moreover, local inflammation would result in the up-regulation ratio of Th17/Treg in peripheral blood, which may influence some periodontally involved systemic diseases.