Measurement of canine Th17 cells by flow cytometry

Abstract Objective The diagnosis of RA patients remains a challenge, especially in ACPA-negative disease. Novel T-cell subsets, particularly Th17 may be useful, although data on Th17 frequency using flow cytometry in RA are conflicting. We investigated whether a novel epigenetic qPCR assay for the quantification of Th17 could differentiate patients with RA from those with symptoms evolving towards an alternative diagnosis. Methods We used a qPCR assay measuring the extent of the methylation at a key position in the IL-17 and CD4 genes. Assays were performed on whole blood from 49 healthy controls (HC) and 165 early arthritis clinic patients. Flow cytometry was further used to detect the expression of CXCR4 on Th17 cells. Results In 75 inflammatory arthritis patients who progressed to RA, the qPCR assays showed significantly fewer Th17 cells compared with 90 patients who did not (P<0.0001). Regression models demonstrated a high predictive value for RA development (75.8% correct prediction), and particularly for the ACPA-negative group (n = 125) where Th17 and swollen joint count (SJC) were the only predictors (73% correct prediction). The chemokine receptor CXCR4 had significantly higher expression on Th17 from early RA patients (n = 11) compared with HC (n = 15). Conclusion The results of the epigenetic qPCR assay showed that low levels of Th17 cells were predictive of developing RA, particularly in the ACPA-negative patients. This could have value for insights into pathogenesis and management. The results suggest the recruitment of Th17 to the inflammatory disease site, consistent with high CXCR4 expression.

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Elevated Th22 cells correlated with Th17 cells in patients with high liver stiffness in nonalcoholic fatty liver disease

European Journal of Inflammation ◽

10.1177/2058739218802678 ◽

2018 ◽

Vol 16 ◽

pp. 205873921880267 ◽

Cited By ~ 2

Author(s):

Si-Biao Su ◽

Wen Chen ◽

Fei-Fei Huang ◽

Jian-Feng Zhang

Keyword(s):

Flow Cytometry ◽

Liver Disease ◽

Plasma Level ◽

Fatty Liver ◽

Nonalcoholic Fatty Liver Disease ◽

Fatty Liver Disease ◽

Th17 Cells ◽

Liver Stiffness ◽

Nonalcoholic Fatty Liver ◽

Th22 Cells

The aims of this article are to investigate the effects of Th22 and Th17 cells and plasma cytokines in patients with nonalcoholic fatty liver disease (NAFLD) and to examine the correlation between Th22 and Th17 cells levels and disease progression. Blood samples from 70 patients with NAFLD and 26 healthy controls (HCs) were collected. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) analysis were performed. ELISA revealed interleukin (IL)-22 plasma level in FibroScan subgroup patients was 2.2-fold higher than that in the HCs ( P < 0.01), body mass index (BMI) subgroup patients were 2.3-fold higher than HCs ( P < 0.01), IL-17A plasma level was 5.6-fold higher in FibroScan subgroup patients than that in the HCs ( P < 0.01), and 5.8-fold higher in BMI subgroup than HCs ( P < 0.01). Flow cytometry revealed median proportions of Th22 cells in FibroScan group was greater than that in the HCs (3.85% vs 0.86%; P < 0.001). Similar results were obtained for Th17 cells (6.36% vs 0.9%; P < 0.001). The liver stiffness measurement value was highly correlated with the proportion of Th17 and Th22 cells and moderately correlated with the plasma level of IL-17A, IL-22, and the BMI value. In conclusion, the elevated proportions of Th17 and Th22 cells were significantly correlated with the degree of liver cirrhosis in NAFLD patients. They may play an inflammatory and immune response role in progression from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) or fibrosis.

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T Cell Subsets and Associated Cytokines in Chronic Graft-Versus-Host-Disease Using G-CSF Primed Bone Marrow in Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v128.22.4580.4580 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4580-4580

Author(s):

Monica M Rivera Franco ◽

Eucario Leon Rodriguez ◽

Diana Gomez Martin ◽

Javier Merayo Chalico ◽

Jorge Alcocer Varela

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Stem Cell ◽

Th17 Cells ◽

Mononuclear Cells ◽

Chronic Gvhd ◽

The Other ◽

Hematopoietic Stem ◽

Graft Versus Host ◽

Ifn Γ

Abstract Background Graft versus host disease (GVHD) is the major complication of allogeneic hematopoietic stem cell transplantation. It is characterized by an imbalance between the effector and regulatory arms of the immune system which results in the over production of inflammatory cytokines. Regulatory T (T regs) cells and T helper 17 (Th17) cells are two recently described lymphocyte subsets with opposing actions. Both can develop from naïve CD4+ T cell precursors under the influence of TGFβ1. Th17 lymphocytes, are key effector cells in rodent models of human diseases including GVHD. The other subset, T regs, is essential for dominant immunologic tolerance. At our institution, patients transplanted using G-CSF primed bone marrow (G-BM), have a lower incidence of acute and chronic GVHD when compared to those transplanted with peripheral blood and not primed bone marrow. Some microenvironment characteristics of this hematopoietic stem cells (HSC) source remain unknown, as well as the difference between Tregs, Th17 and cytokine levels in patients who develop GVHD and those who do not. Objective To analyze the characteristics of thirty-eight G-BM donor samples, identifying lymphocytes subsets and associated cytokines, and comparing patients who developed chronic GVHD (cGVHD) and those who did not. Materials and Methods A prospective analysis was performed in 38 G-BM samples from donors from 1999 to 2016. Mononuclear cells were defrosted, counted, and viability was evaluated. A 24 hour resting with RPMI, and posterior activation with PMA (50 ng/ml) for 48 hours was performed. Cells were harvested and cytokines were evaluated by flow cytometry (CBA assay). From each sample, one million mononuclear cells were permeabilized, fixed, and stained with CD4-FITC, IL17A-PE, IFN-γ APC, and IL-4 PECy7, for their posterior phenotipication by flow cytometry. The samples were obtained in a BD LSR Fortessa cytometry, and analyzed with the Flow-Jo software. Patients (recipients) information was analyzed using SPSS v.21. Results GVHD incidence was reported as following: Three (8%) patients developed acute GVHD (2 grade II, and 1 grade IV), 11 patients (29%) developed chronic GVHD (9% extensive, and 91% limited), and 24 patients did not present either. Mononuclear cells from G-BM from donors of patients who developed cGVHD showed a pro inflammatory response, characterized by an increased concentration of IL-17A (15.5 vs 0.71 pg/mL, p=0.013), TNF-α (80.27 vs 0.13 pg/mL, p=0.001), and IL-6 (4953.6 vs 11.75 pg/mL, p=0.025), after a mitogenic stimulation, compared to cells from donors of patients who did not developed GVHD. On the other hand, a decreased IL-10 production (2.62 vs 52.81 pg/mL, p=0.001) was documented in mononuclear cells from donors of patients who developed chronic GVHD, compared to donor cells of patients who did not. No significant difference in the production of IL-2, IL-4, and IFN-γ was observed. There was no difference in Th1 and Th2 between both groups, but mononuclear cells from donors of patients who developed chronic GVHD had a higher percentage of Th17 (1.02% vs 0.46%, p<0.001), and less Tregs (0.88% vs 1.95%, p<0.001), compared to those who did not developed GVHD. Conclusions Patients who develop cGVHD (29%) are characterized by a pro inflammatory response with an increased production of IL-17A, IL-6, and IFN-γ, and also a major percentage of Th17 cells. Also, a decreased suppressive response was documented with reduced IL-10 and Tregs levels. The low incidence of cGVHD show that G-CSF primed bone marrow is an excellent source for allogeneic HSC transplantations, and would be useful to compare these results with other HSC sources. Disclosures No relevant conflicts of interest to declare.

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Amino Terminal Recognition by a CCR6 Chemokine Receptor Antibody Blocks CCL20 Signaling and IL-17 Expression via β-arrestin

10.21203/rs.3.rs-183666/v1 ◽

2021 ◽

Author(s):

Sara Gómez-Melero ◽

Fé Isabel García-Maceira ◽

Tania García-Maceira ◽

Verónica Luna-Guerrero ◽

Gracia Montero-Peñalvo ◽

...

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Chemokine Receptor ◽

Th17 Cells ◽

Therapeutic Potential ◽

Inflammatory Diseases ◽

Specific Binding ◽

Isolation And Purification ◽

Amino Terminal ◽

Important Target

Abstract Background: CCR6 chemokine receptor is an important target in inflammatory diseases. Th17 cells express CCR6 and a number of inflammatory cytokines, including IL-17 and IL-22, which are involved in the propagation of inflammatory immune responses. CCR6 antagonist would be a potential treatment for inflammatory diseases such as psoriasis or rheumatoid arthritis. The aim of this study is to develop an antagonistic monoclonal antibody (mAb) against human CCR6 receptor (hCCR6).Results: We generate monoclonal antibodies against hCCR6 immunizing Balb/c mice with hCCR6 overexpressing cells. The antibodies were tested by flow cytometry for specific binding to hCCR6, cloned by limiting dilution and resulted in the isolation and purification monoclonal antibody 1C6. By ELISA and flow cytometry, was determined that the antibody obtained binds to hCCR6 N-terminal domain. The ability of 1C6 to neutralize hCCR6 signaling was tested and we determined that 1C6 antibody were able to block response in β-arrestin recruitment assay with IC50 10.23 nM, but did not inhibit calcium mobilization. In addition, we found in a chemotaxis assay that 1C6 reduces the migration of hCCR6 cells to their ligand CCL20. Finally, we determined by RT-qPCR that the expression of IL-17A in Th17 cells treated with 1C6 was inhibited.Conclusions: In the present study, we applied whole cell immunization for successfully obtain an antibody that is capable to neutralize hCCR6 signaling and to reduce hCCR6 cells migration and IL-17 expression. These results provide an efficient approach to obtain therapeutic potential antibodies in the treatment of CCR6-mediated inflammatory diseases.

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Profiles of Th1, Th2, Th17 and Treg Cells in Patients with Chronic Idiopathic Thrombocytopenic Purpura.

Blood ◽

10.1182/blood.v112.11.3404.3404 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3404-3404

Author(s):

Rong-Fu Zhou ◽

Jian Ou-yang ◽

Da-Yu Chang ◽

Jing-Yan Xu ◽

Bing Chen ◽

...

Keyword(s):

Flow Cytometry ◽

Idiopathic Thrombocytopenic Purpura ◽

Peripheral Blood ◽

Th17 Cells ◽

Treg Cells ◽

Chronic Idiopathic Thrombocytopenic Purpura ◽

Active Stage ◽

Thrombocytopenic Purpura ◽

Chronic Itp ◽

Ifn Γ

Abstract Objective: To explore the profiles of Th1,Th2, Th17 and Treg cells in patients with chronic idiopathic thrombocytopenic purpura. Methods: Samples of peripheral blood were collected from 30 chronic ITP patients ( 9 males and 21 females), aged 41, 21 being in active stage, and 9 in remission stage, and 9 healthy persons in control (3 males and 6 females), aged 36. Peripheral blood was cultured, and activated with PMA/ionomycin when Th1, Th2 and Th17 cells were detected. Flow cytometry was used to measure the intracellular cytokines interferon (IFN)-γ, interleukin (IL)-4 and interleukin (IL)-17 so as to identify the Th1 cells (CD3+ CD8− IFN-γ+ IL-4− cells), Th2 cells (CD3+ CD8− IFN-γ − IL-4+ cells) and IL-17 cells (CD3+ CD8− IL-17+ cells); Treg cells were identified to CD4+ CD25+ Foxp3+ cells and uncultured peripheral blood was used to measured the CD4+ CD25+ Foxp3+ cells by flow cytometry. The ratios of Th1/Th2 were calculated. Results: The Th1/Th2 ratio for patients in active stage was 15.04±9.67, significantly higher than those for patients in remission stage (7.17±5.38, P <0.05) and in control (8.47±3.78, P <0.05); the percentage of Treg cells of the patients in active stage was 0.89±0.58%, significantly decreased than those of patients in remission stage (6.41±1.86%, P <0.001) and in control (6.06±0.85%, P <0.001); the percentage of Th17 cells was 1.94±0.77% for patients in active stage, 2.16±0.52% for patients in remission stage and 1.82±0.58% for patients in control, respectively, and there was no statistic significance between them. Conclusion: Chronic ITP is a Th1 predominant disease; decreased number and function of Treg cells might be one of mechanisms that cause immune regulation dysfunction in chronic ITP; Th17 cells might not play a role in the development of chronic ITP.

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Abnormalities of Bone Marrow Immune Micro-Environment in Patients with Immune Thrombocytopenia

Blood ◽

10.1182/blood.v126.23.3464.3464 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3464-3464

Author(s):

Yang Song ◽

Yu-tong Wang ◽

Xiao-jun Huang ◽

Yuan Kong

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Endothelial Cells ◽

Th17 Cells ◽

Immune Thrombocytopenia ◽

Perivascular Cells ◽

Platelet Production ◽

Cd34 Cells ◽

Significant Difference

Abstract Background: Immune thrombocytopenia (ITP) is an immune-mediated disease that is characterized by excessive platelet destruction and decreased platelet production. Although antiplatelet antibodies are considered as the primary immunologic defect in ITP, dysfunctional cellular immunity is also important in the pathophysiology of ITP. The current publications have observed excessive activation and proliferation of platelet auto-antigen-reactive CTLs, production abnormal Th cells, abnormal numbers and function of Tregs in peripheral blood of ITP, but no one focus on the bone marrow (BM) micro-environment in ITP patients. Many cell types including osteoblastic, perivascular, endothelial cells, and various mature immune cells contribute to the BM micro-environment. We have recently reported that the impaired BM vascular micro-environment may affect the thrombopoiesis of CD34+ cells by disrupting the interaction between megakaryocytes and BM endothelial cells (BMECs), resulting in the delayed platelet engraftment in allotransplant patients with prolonged isolated thrombocytopenia (Kong Y, et al. Biol Blood Marrow Transplant. 2014; 20:1190-1197). In mice model, the cross-talk between megakaryocytes and BMECs in BM vascular micro-environment regulates the megakaryocyte maturation and thrombopoiesis. Therefore, we hypothesized that the abnormal BM vascular micro-environment and immune micro-environment may operate in the occurrence of ITP. Aims: To investigate whether abnormal BM vascular and immune micro-environment are involved in ITP patients. Methods: The compartments of BM immune micro-environment were analyzed by flow cytometry in 26 untreated ITP patients and 26 healthy donors (HD). The fractions of T cells, including Th1, Tc1,Th2, Tc2 ,Th17 and Treg were identified as CD3+ CD8- IFN-gama+, CD3+ CD8- IFN-gama+, CD3+ CD8+ IL4+, CD3+ CD8+ IL-4+, CD3+ CD8- IL17A+ and CD3+ CD4+ CD25+ Foxp3+, respectively. The BMECs and perivascular cells, acting as key elements of vascular micro-environment, were identified as CD45- CD34+ VEGFR2+ and CD45- CD34- CD146+, respectively. Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) using rabbit anti-human CD34 and CD146 primary antibodies were performed on each BM trephine biopsies (BMB) derived from the patients and controls. Results: The proportion of Th1 cells and Tc1 cells among the bone marrow mononuclear cells (BMMNCs) was significantly increased in ITP patients compared to HD (27.7% ± 11.6% vs. 16.3% ± 7.7%, P<0.001; 39.8%±17.7% vs. 24.1%±11.8%, P<0.005), whereas there was no significant difference in the percentages of Th2 and Tc2 cells. In addition, the proportion of Th17 cells in ITP patients was remarkable higher than HD (3.2%±0.51%1.5%vs 1.7%±1.0%, P<0.0001). We also found the significantly decreased percentage of Treg in ITP patients compared to HD (2.5%±2.0% vs 3.7%±2.6%, P<0.001). However, the frequency of CD34+ cells as well as BMECs and perivascular cells were similar in BM between the ITP patients and HD. Consistent with our flow cytometry data, histological analysis of the recipient BMBs in situ showed no significant differences in CD34-positive BMECs and CD146-positive perivascular cells between ITP patients and HD. Summary/Conclusion: The BM CD34+ cells and vascular micro-environment were normal in ITP patients. However, the abnormal BM immune micro-environment, including the excessive polarization of Th1, Tc1 and Th17 cells and a remarkable decrease of Treg cells were observed in ITP patients. Our data indicated that the desregulated T cells responses in BM may abrogate the thrombopoiesis through the impaired megakaryocytes maturation and decreased platelet production, and eventually contributing to the occurrence of ITP. Acknowledgment: Supported by the National Natural Science Foundation of China (grant nos. 81370638&81230013), and the Beijing Municipal Science and Technology Program (grant nos. Z141100000214011& Z151100004015164& Z151100001615020). Disclosures No relevant conflicts of interest to declare.

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JIA patient T cells differentiate into Th1, Th17 and Th1.17 effector cells under Th1 polarizing conditions

10.1101/2021.10.01.21264425 ◽

2021 ◽

Author(s):

Anna E Patrick ◽

Tashawna Esmond ◽

Kayla Shoaff ◽

David M Patrick ◽

David K Flaherty ◽

...

Keyword(s):

Flow Cytometry ◽

T Cell ◽

Cell Cultures ◽

Th17 Cells ◽

Mononuclear Cells ◽

Healthy Children ◽

Th1 Cell ◽

Peripheral Blood Mononuclear ◽

Ifn Gamma

Objective. T helper cells develop into discrete Th1, Th2 or Th17 lineages that selectively express IFN-gamma, IL-4/IL-5/IL-13, or IL-17, respectively and actively silence signature cytokines expressed by opposing lineages. Our objective was to compare Th1, Th2 and Th17 polarization in cell culture models using JIA patient samples. Methods. Peripheral blood mononuclear cells were isolated from JIA or healthy prepubescent children. T cell naive and memory phenotypes were assessed by flow cytometry. T cell proliferation was measured using a fluorescence-based assay. Th cell cultures were generated in vitro and IFN-gamma, IL-17, and TNF-alpha measured by ELISA and flow cytometry. Results. JIA Th1 cells produced increased IFN-gamma and inappropriately produced IL-17. JIA Th17 cells produced increased IL-17. JIA Th1 cell cultures develop dual producers of IFN-gamma and IL-17, which are Th1.17 cells. JIA Th1 cultures expressed elevated levels of both T-bet and ROR-gamma-T. RNA sequencing confirmed activation of immune responses and inappropriate activation of IL-17 signaling pathways in Th1 cultures. A subset of JIA patient samples was disproportionally responsible for the enhanced IFN-gamma and IL-17 phenotype and Th1.17 phenotype. Conclusions. This study reveals that JIA patient uncommitted T cell precursors, but not healthy children, inappropriately develop into inflammatory effector Th1.17 and Th17 cells under Th1 polarizing conditions.

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High Resolution Mass Cytometry (CyTOF) in Aplastic Anaemia (AA) Can Identify an Aberrant Treg Subset with Pro-Inflammatory Properties, Predicting Poor Response to Immunosuppressive Therapy

Blood ◽

10.1182/blood.v124.21.1600.1600 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1600-1600 ◽

Cited By ~ 2

Author(s):

Shahram Kordasti ◽

Thomas Seidl ◽

Richard J Ellis ◽

Austin Kulasekararaj ◽

Xingmin Feng ◽

...

Keyword(s):

Flow Cytometry ◽

High Resolution ◽

Immunosuppressive Therapy ◽

Peripheral Blood ◽

Th17 Cells ◽

Potential Role ◽

Mass Cytometry ◽

Intracellular Staining ◽

Treg Subset ◽

Pre Treatment

Abstract Introduction Immune mediated self-response is an established feature of aplastic anaemia (AA) and up to 70% of patients respond to immunosuppressive therapy (IST). Reduced number and function of Tregs and an increase in Th17 cells, similar to other autoimmune conditions, have been reported in AA. Nevertheless, the potential role of these cells in response to IST is not fully understood. Our understanding of human Tregs and Th17 cells and their plasticity has been greatly improved by discovery of phenotypically and functionally distinct subsets. However, due to low number of such cells and technical difficulties for multicolour staining, it is challenging to investigate these subsets in AA and their potential role in response to IST. Mass cytometry (as implemented in the Fluidigm CyTOF instrument) is a technology which allows functional phenotypying of currently up to 40 parameters on a single cell level. In contrast to conventional polychromatic flow cytometry, where the overlap of fluorochrome emission spectra limit the maximum number of 12-14 markers, mass cytometry uses metal isotope-conjugated antibodies to enable "deep immune profiling". We have designed mass cytometry panels for both cell surface and intracellular antigens and used the CyTOF instrument to acquire immune signatures of peripheral blood (PB) samples of AA patients. Patients and Methods Peripheral blood samples from 9 AA patients (7 responders to IST, 2 non-responsers) and 2 healthy donors were collected at diagnosis and following treatment with rabbit or horse ATG and ciclosporin. PBMCs were rested overnight and stained (surface and intracellular) without and with 4 hours stimulation with Phorbol 12-myristate 13-acetate (PMA)/ ionomycin. We designed and optimised 2 separate panels of antibodies based on 41 surface markers, transcription factors and cytokines (table 1). Each antibody is tagged with a unique lanthanide isotope. Acquired data were analysed as .fcs files by both conventional gating and multidimensional Spanning tree Progression of Density normalized Events (SPADE) clustering. Results Viable Lymphocytes were identified by gating on cell length, DNA content, Rh viability dye and CD45 expression. Tregs were then clustered based on CD3, CD4, CD25, CD27, CD127, CD45RA, CD45RO, CD62L and FOXP3. Treg subset II (as defined by Sakaguchi, et al) was identified within total Tregs, based on CD45RA_lo, CD127_lo and FOXP3_hi. The relative expression of all the other markers on Treg II cluster was then calculated based on median values and scale cofactor. AA Tregs at diagnosis expressed significantly higher CD45RA (p<0.001), CD127 (p<0.001), CD7 (p=0.04), CD69 (p=0.03), CD103 (p=0.02), T-bet (p=0.01), and IL-4 (p=0.008) compared to HD, whereas the expression of CD152 (CTLA-4) and HLA-DR were significantly lower in AA (p=0.04 and p=0.03). When patients were grouped as responders and non-responders to IST, Tregs from non-responders patients expressed significantly higher CD11b (p=0.04), CD45RA (p<0.001), CD127 (p<0.001), CCR4 (p=0.02), IL-2 (p=0.03), IL-4 (p=0.02), IL-6 (p=0.04), T-bet (p=0.008), IL-17 (p=0.01) and GATA3 (p<0.001) while the expression of FOXP3 (p=0.03), CD7 (p=0.01) and CD25 (p=0.03) was lower compared to responders. Following IST the expression of CD45RA, CD7 and IL-4 by Tregs were reduced (p=0.004, p<0.001 and p=0.02) whereas CD152 increased (p<0.01) compared to pre-treatment Tregs. When Treg cytokine profiles following response to IST were compared to pre-treatment Tregs from non responder patients, we have noticed a significantly lower secretion of other pro-inflammatory cytokines including IL-6 (p=0.01) and IL-17 (p=0.03) as well as IL-2 (p=0.03) by post IST Tregs. These Tregs were also less T-bet positive (p=0.02)(figures 1 and 2). Conclusions We demonstrate the ability of mass cytometry to define in high resolution the immunophenotypes of very specific subsets of Tregs in AA that is extremely challenging by conventional flow-cytometry. Our data indicate that a combination of pro-inflammatory and activation markers defines a subset of Tregs, which could potentially predict response to IST. Similar studies in a larger cohort of patients should delineate a more specific immune signature for the evaluation of response and follow up of AA patients following non-transplant treatments. Table1: Markers for surface and intracellular staining. Table1:. Markers for surface and intracellular staining. Disclosures No relevant conflicts of interest to declare.

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Melatonin, an Endogenous Hormone, Modulates Th17 Cells via the Reactive Oxygen Species/TXNIP/HIF-1α Axis to Alleviate Autoimmune Uveitis

10.21203/rs.3.rs-1192974/v1 ◽

2022 ◽

Author(s):

Dan Liang ◽

Jun Huang ◽

Zhuang Li ◽

Yunwei Hu ◽

Zuoyi Li ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Flow Cytometry ◽

T Cells ◽

Th17 Cells ◽

Reactive Oxygen ◽

Treg Cells ◽

Oxygen Species ◽

T Helper ◽

Autoimmune Uveitis

Abstract Background Melatonin, an indoleamine produced by the pineal gland, plays a pivotal role in maintaining circadian rhythm homeostasis. Recently, the strong antioxidant and anti-inflammatory properties of melatonin have attracted attention of researchers. We evaluated the therapeutic efficacy of melatonin in experimental autoimmune uveitis (EAU), which is a representative animal model of human autoimmune uveitis. Methods EAU was induced in mice via immunization with the peptide interphotoreceptor retinoid binding protein 1-20 (IRBP1−20). melatonin was then administered via intraperitoneal injection to induce protection against EAU. With EAU induction for 14 days, clinical and histopathological scores were employed to evaluate the disease progression. T lymphocytes accumulation, the expression of inflammatory cytokines in the retinas were assessed via flow cytometry and RT-PCR. In vivo and in vitro experiments, T helper 1 (Th1), T helper 17 (Th17) and regulatory T (Treg) cells were detected via flow cytometry, the level reactive oxygen species(ROS) from CD4+ cells were tested via flow cytometry, and the expression of thioredoxin-interacting protein (TXNIP) and hypoxia-inducible factor 1 alpha (HIF-1α)proteins were also quantified via western blot analysis, to elucidate the mechanism of melatonin inhibiting EAU. Results Melatonin treatment resulted in notable attenuation of ocular inflammation in EAU mice, evidenced by decreasing optic disc edema, few signs of retinal vasculitis, and minimal retinal and choroidal infiltrates. Mechanistic studies revealed that melatonin restricted the proliferation of peripheral Th1 and Th17 cells and potentiated Treg cells by suppressing their transcription factors. In vitro studies corroborated that melatonin restrains the polarization of retina-specific T cells towards Th17 and Th1 cells in addition to enhancing the proportion of Treg cells. Pretreatment of retina-specific T cells with melatonin failed to induce EAU in naïve recipients. Furthermore, the ROS/ TXNIP/ HIF-1α pathway was shown to mediate the therapeutic effect of melatonin in EAU. Conclusions Melatonin regulates autoimmune T cells by restraining effector T cells and facilitating Treg generation, indicating that melatonin could be a hopeful treatment alternative for autoimmune uveitis.

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Inhibition of CXCR4 by MicroRNA-1192 Reduces the Activation of Th17 Cells and Expression of Inflammation Factors in a Mouse Model of Vulvovaginal Candidiasis

Cellular Physiology and Biochemistry ◽

10.1159/000494475 ◽

2018 ◽

Vol 50 (3) ◽

pp. 893-910 ◽

Cited By ~ 2

Author(s):

Jing Liu ◽

Huai-Zhen Wang ◽

Yong Sun

Keyword(s):

Flow Cytometry ◽

Epithelial Cells ◽

Mouse Models ◽

Th17 Cell ◽

Th17 Cells ◽

Vulvovaginal Candidiasis ◽

Inflammatory Factors ◽

Cell Percentage ◽

Vaginal Epithelial Cells ◽

Protein Expressions

Background/Aims: Vulvovaginal candidiasis (VVC) is a disease commonly occurring in sexually active women. The involvement of microRNAs in several kinds of infectious diseases has been highlighted in a number of researches. Therefore, we conducted the present study in order to investigate whether microRNA-1192 (miR-1192) would significantly target CXCR4 in Th17 cells as well as inflammatory factors in mouse models suffering from VVC. Methods: Seventy-five mice were selected as test subjects for this study, of which twenty-five were used as the normal control, while the rest were treated with estradiol or oil-treated in order to establish VVC mouse models (each n = 25). Protein expressions of CXCR4, IL-6, IL-17, and IL-23 were all measured using both an immunohistochemistry and ELISA. The Th17 cell percentage in peripheral blood and the expression of RORγt in Th17 cells were detected using a flow cytometry. Mouse vaginal epithelial cells were isolated from normal mice, after which the mice were treated with estradiol to regulate their estrogen, followed by treatments involving the miR-1192 mimic, miR-1192 inhibitor, siRNA-CXCR4, and miR-1192 inhibitor + si-CXCR4. The cell cycle, apoptosis, and proliferation were all examined by using an additional flow cytometry as well as the employment of the MTT assay. The miR-1192, CXCR4, IL-6, IL-17, and IL-23 expressions in tissues and cells were both measured using both RT-qPCR and western blot assay techniques. Results: The mice treated with either estradiol or oil had presented to us lowered levels in miR-1192 expression as well as higher levels in both Th17 cell percentage and expression of RORγt in Th17 cells, along with mRNA and protein expressions of CXCR4, IL-6, IL-17, and IL-23. In cell experiments, the mouse vaginal epithelial cells that had been treated with miR-1192 inhibitor had shown us a decreased cell proliferation rate and contrarily increased expressions of CXCR4, IL-6, IL-17, and IL-23 mRNA, protein, and cell apoptosis rate; these results were opposite to the ones found in the mice treated with miR-1192 mimic. Conclusion: Our results provided significant evidence that miR-1192 could directly development and progression of VVC by restraining the CXCR4 gene in the VVC mice.

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