scholarly journals Therapeutic Effect of Exogenous Treg Cells on Collagen-Induced Arthritis and Rheumatoid Arthritis

2019 ◽  
Author(s):  
Shutong Li ◽  
Hongxing Wang ◽  
Hui Wu ◽  
Guoqing Zhang ◽  
Xiaotian Chang
Keyword(s):  
Rheumatoid Arthritis ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Synovial Fibroblast ◽  
Treg Cells ◽  
Cell Counting ◽  
Fibroblast Cells ◽  
Collagen Induced Arthritis ◽  
Gm Csf

Abstract Background Regulatory T (Treg) cells have anti-inflammatory and anti-autoimmune functions. The proportion and functions of Treg cells are perturbed in rheumatoid arthritis (RA) patients. Methods Human Treg cells were induced to amplify in vitro and cocultured with RA synovial fibroblast cells (RASFs). The proliferation and apoptosis of RASFs were determined by the cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Human Treg cells were also injected to collagen-induced arthritis (CIA) rats via the tail vein. Changes in lymphocyte subtypes and cytokines in the peripheral blood and spleen were observed by flow cytometry. Results After coculture with the Treg cells, the proliferation of RA synovial fibroblast cells decreased (p<0.01), and the rate of apoptosis increased (p=0.037). The human Treg cells were injected into the tail veins of collagen-induced arthritis (CIA) rats. The severity of the CIA was reduced (p<0.01) following the injection, the percentages of rat endogenous Treg cells in the peripheral blood and spleen increased significantly (p=0.007 and p<0.01, respectively), and the proportion of B cells decreased (p=0.031). The levels of interleukin IL-5 and IL-6 and the Th1/Th2 ratio in the peripheral blood were significantly decreased (p=0.013, 0.009 and 0.012, respectively). The number of NK cells and the levels of IL-4, IL-13, TNF-α, IFN-γ and GM-CSF in the peripheral blood and spleen did not change significantly. Conclusion These results suggest that exogenous Treg cells play a therapeutic role in RA and CIA. Treg cell treatment could serve as a therapy for RA.

scholarly journals AB0094 FUNCTIONAL TREG CELLS MAY BE CONVERTED INTO T EFFECTOR PHENOTYPE ON EXPOSURE TO INFLAMMATORY MILIEU IN RHEUMATOID ARTHRITIS (RA) SYNOVIAL FLUID: IN-VITRO STUDY

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1346.1-1347
Author(s):  
V. Kommoju ◽  
C. Kavadichanda ◽  
V. Negi
Keyword(s):  
Rheumatoid Arthritis ◽  
Flow Cytometry ◽  
Synovial Fluid ◽  
Cell Surface ◽  
Inflammatory Cytokines ◽  
Peripheral Blood ◽  
Treg Cells ◽  
Cell Surface Expression ◽  
Inflammatory Milieu

Background:The debate on functional versus numerical difference in T regulatory cell population among patients with Rheumatoid arthritis (RA) is not clear. Tregs expressing Inflammatory subset phenotype markers, such as Th1(cxcr3, Tbet) and Th17(ccr6,Rorϒ) are reported. (1) Though the reported numbers of synovial fluid tregs are higher in RA, (2) the fate of Tregs on entering the synovial inflammatory milieu from peripheral blood (PB) has not yet been investigated.Objectives:To compare Treg frequencies in PB and synovial fluid between osteoarthritis (OA) and RATo compare cytokine levels in PB and SF between OA and RATo study the effect of autologous synovial fluid on RA and OA Treg isolated form peripheral bloodMethods:The Peripheral Blood (PB) and synovial fluid (SF) of RA (n=80) and OA (n=30) patients were analyzed for CD4+T-cell subset frequencies and phenotypes by flow cytometry. Cytokine concentrations in plasma and SF were measured by cytometric bead array. Tregs from 5 RA-PB and 5 OA-PB were isolated and cultured in autologous synovial fluid for 24 hrs. Phenotypic expression of Th1 and Th17 chemokines on the cell surface were analyzed by flow cytometry and expression levels of T-bet, RORγ and FOXP3 in those Treg cells were measured with quantitative real-time PCR (RT-qPCR).Results:The PB and SF frequencies of Th1, Th17 and Tregs are shown in Table 1. The pro-inflammatory cytokines were high in the plasma and SF of RA but the anti-Inflammatory cytokines were similar (Fig 1.A&B). Treg cells were isolated from RA and OA PB and cultured in autologous SF for 24 hrs. RA Treg showed increased cell surface expression of CXCR3+ and CCR6+ (Fig 1C) and there was no difference in OA Treg. Gene expression studies showed an increased expression of T-bet, RORγ and decreased expression of Foxp3 in RA Tregs while there was no difference in OA Tregs before and after in-vitro culture (Fig 1D).Table 1.Tcell subsets in Rheumatoid Arthritis and Osteoarthritis.CD4 subtypeRAOAPBSFPBSFN=80N=30N=30N=30Th126.65 ± 5.5934.99 ± 1.3025.97 ± 7.2426.45 ± 1.87*Th25.19 ± 1.916.36 ± 1.735.24 ± 2.151.44 ± 0.10*Th1714.05 ± 3.2921.18 ± 2.0410.81 ± 2.78*2.45 ± 0.23*Treg10.68 ± 2.4712.53 ± 2.1011.162.9513.04 ± 2.57*P<0.05Conclusion:Tregs in RA may be converted to Th1 and Th17 phenotype on exposure to inflammatory cytokine in the synovial fluid, thus losing their regulatory functions. Understanding factors influencing stability of Treg cells may help improve future therapeutics.References:[1]Jinlin Miao & Ping Zhu. Functional Defects of Treg Cells: New Targets in Rheumatic Diseases, Including Ankylosing Spondylitis. Current Rheumatology Reports 2018; 20: 30[2]Penatti et al. Differences in serum and synovial CD4+ T cells and cytokine profiles to stratify patients with inflammatory osteoarthritis and rheumatoid arthritis. Arthritis Research & Therapy.2017; 19:103Acknowledgments:Department of Science and technology, India for the research grant.Disclosure of Interests:None declared

scholarly journals Therapeutic Effect of Exogenous Regulatory T Cells on Collagen-induced Arthritis and Rheumatoid Arthritis

2020 ◽  
Vol 29 ◽  
pp. 096368972095413
Author(s):  
Shutong Li ◽  
Hongxing Wang ◽  
Hui Wu ◽  
Xiaotian Chang
Keyword(s):  
Rheumatoid Arthritis ◽  
B Cells ◽  
Cell Transplantation ◽  
Treg Cell ◽  
Therapeutic Effect ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Treg Cells ◽  
Therapeutic Effects ◽  
Collagen Induced Arthritis

Regulatory T (Treg) cells have anti-inflammatory functions and heighten immune tolerance. The proportion and functions of Treg cells are perturbed in rheumatoid arthritis (RA), contributing to the excessive immune activation associated with this disease. We therefore hypothesized that supplementation with foreign Treg cells could be used to treat RA. To investigate the therapeutic effects of exogenous Treg cells on RA and its mechanism, we used human Treg cells to treat collagen-induced arthritis (CIA) in a rat model to observe whether exogenous Treg cells can treat the disease across species. Successful treatment would indicate that Treg cell transplantation in humans is more likely to affect RA. In the present study, human Treg cells were collected from healthy human peripheral blood and culture-expanded in vitro. Induced human Treg cells were injected into CIA rats via the tail vein. The rats’ lymphocyte subtypes, cytokines, and Th1/Th2 ratios were measured using flow cytometry. In the rats, following injection of the human Treg cells, the severity of CIA was significantly reduced ( P < 0.01), the proportion of endogenous Treg cells increased in the peripheral blood and spleen ( P = 0.007 and P < 0.01, respectively), and the proportion of B cells decreased ( P = 0.031). The IL-5 level, IL-6 level, and Th1/Th2 ratio in the peripheral blood were decreased ( P = 0.013, 0.009, and 0.012, respectively). The culture-expanded human Treg cells were also cultured with synovial fibroblast cells from RA patients (RASFs). After coculture with Treg cells, RASFs showed reduced proliferation ( P < 0.01) and increased apoptosis ( P = 0.037). These results suggest that exogenous and induced Treg cells can produce a therapeutic effect in RA and CIA by increasing endogenous Treg cells and RASF apoptosis and reducing B cells, the Th1/Th2 ratio, and secretion levels of IL-5 and IL-6. Treg cell transplantation could serve as a therapy for RA that does not cause immune rejection.

Identification and characterisation of citrullinated antigen-specific B cells in peripheral blood of patients with rheumatoid arthritis

2015 ◽  
Vol 75 (6) ◽  
pp. 1170-1176 ◽  
Author(s):  
Priscilla F Kerkman ◽  
Emeline Fabre ◽  
Ellen I H van der Voort ◽  
Arnaud Zaldumbide ◽  
Yoann Rombouts ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Flow Cytometry ◽  
B Cells ◽  
Peripheral Blood ◽  
Memory B Cells ◽  
Protein Antigens ◽  
High Frequencies ◽  
Culture Supernatants ◽  
Citrullinated Antigen

ObjectivesImmunity to citrullinated antigens is a hallmark of rheumatoid arthritis (RA). We set out to elucidate its biology by identifying and characterising citrullinated antigen-specific B cells in peripheral blood of patients with RA.MethodsDifferentially labelled streptavidin and extravidin tetramers were conjugated to biotinylated CCP2 or control antigens and used in flow cytometry to identify citrullinated antigen-specific B cells in peripheral blood. Tetramer-positive and tetramer-negative B cells were isolated by fluorescence activated cell sorting (FACS) followed by in vitro culture and analysis of culture supernatants for the presence of antibodies against citrullinated protein antigens (ACPA) by ELISA. Cells were phenotypically characterised by flow cytometry.ResultsBy combining differentially labelled CCP2 tetramers, we successfully separated citrullinated antigen-specific B cells from non-specific background signals. Isolated tetramer-positive B cells, but not tetramer-negative cells, produced large amounts of ACPA upon in vitro stimulation. Phenotypic analyses revealed that citrullinated antigen-specific B cells displayed markers of class-switched memory B cells and plasmablasts, whereas only few cells displayed a naïve phenotype. The frequency of tetramer-positive cells was high (up to 1/500 memory B cells with a median of 1/12 500 total B cells) and correlated with ACPA serum titres and spontaneous ACPA production in culture.ConclusionsWe developed a technology to identify and isolate citrullinated antigen-specific B cells from peripheral blood of patients with RA. Most cells have a memory phenotype, express IgA or IgG and are present in relatively high frequencies. These data pave the path for a direct and detailed molecular characterisation of ACPA-expressing B cells and could lead to the identification of novel therapeutic targets.

In Vitro Evaluation of Chitosan Induced Cytotoxicity against Cancerous Cell lines: MCF-7, Saos-2 and HeLa

2019 ◽  
Vol 19 ◽  
Author(s):  
Zeinab Abedian ◽  
Niloofar Jenabian ◽  
Ali Akbar Moghadamnia ◽  
Ebrahim Zabihi ◽  
Roghayeh Pourbagher ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Anticancer Agents ◽  
Fibroblast Cells ◽  
Apoptosis Induction ◽  
Cytotoxic Effects ◽  
Cancerous Cell ◽  
Significant Concentration ◽  
Mcf 7

Objective/ Background: Cancer is still the most common cause of morbidity in world and new powerful anticancer agents without severe side effects from natural sources is important. Methods: The evaluation of cytotoxicity and apoptosis induction was carried out in MCF-7,HeLa and Saos-2 as cancerous cell lines with different histological origin and human fibroblast served as control normal cell. The cells were treated with different concentrations of chitosan and the cytotoxicity was determined using MTT assay after 24, 48 and 72 h .The mode of death was evaluated by flow cytometry . Results: While both types of chitosan showed significant concentration-dependently cytotoxic effects against the three cancerous cell lines, fibroblast cells showed somehow more compatibility with chitosan. On the other hand, there were no significant differences between LMWC and HMWC cytotoxicity in all cell lines. The flow cytometry results showed the apoptosis pattern of death more in Saos-2 and HeLa while necrosis was more observable with MCF7. Also higher viability with both types of chitosan was seen in fibroblast as normal cells Conclusion: Chitosan shows anticancerous effect against 3 cancerous cell lines, while it is compatible with normal diploid fibroblast cells. Furthermore, it seems that the molecular weight of chitosan does not affect its anticancerous property.

scholarly journals Regulatory T Cells Fail to Suppress Fast Homeostatic Proliferation In Vitro

Life ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 245
Author(s):  
Daniil Shevyrev ◽  
Valeriy Tereshchenko ◽  
Elena Blinova ◽  
Nadezda Knauer ◽  
Ekaterina Pashkina ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Autoimmune Diseases ◽  
Peripheral Blood ◽  
Treg Cells ◽  
Homeostatic Proliferation ◽  
Suppressive Activity ◽  
Healthy Donors

Homeostatic proliferation (HP) is a physiological process that reconstitutes the T cell pool after lymphopenia involving Interleukin-7 and 15 (IL-7 and IL-15), which are the key cytokines regulating the process. However, there is no evidence that these cytokines influence the function of regulatory T cells (Tregs). Since lymphopenia often accompanies autoimmune diseases, we decided to study the functional activity of Tregs stimulated by HP cytokines from patients with rheumatoid arthritis as compared with that of those from healthy donors. Since T cell receptor (TCR) signal strength determines the intensity of HP, we imitated slow HP using IL-7 or IL-15 and fast HP using a combination of IL-7 or IL-15 with anti-CD3 antibodies, cultivating Treg cells with peripheral blood mononuclear cells (PBMCs) at a 1:1 ratio. We used peripheral blood from 14 patients with rheumatoid arthritis and 18 healthy volunteers. We also used anti-CD3 and anti-CD3 + IL-2 stimulation as controls. The suppressive activity of Treg cells was evaluated in each case by the inhibition of the proliferation of CD4+ and CD8+ cells. The phenotype and proliferation of purified CD3+CD4+CD25+CD127lo cells were assessed by flow cytometry. The suppressive activity of the total pool of Tregs did not differ between the rheumatoid arthritis and healthy donors; however, it significantly decreased in conditions close to fast HP when the influence of HP cytokines was accompanied by anti-CD3 stimulation. The Treg proliferation caused by HP cytokines was lower in the rheumatoid arthritis (RA) patients than in the healthy individuals. The revealed decrease in Treg suppressive activity could impact the TCR landscape during lymphopenia and lead to the proliferation of potentially self-reactive T cell clones that are able to receive relatively strong TCR signals. This may be another explanation as to why lymphopenia is associated with the development of autoimmune diseases. The revealed decrease in Treg proliferation under IL-7 and IL-15 exposure can lead to a delay in Treg pool reconstitution in patients with rheumatoid arthritis in the case of lymphopenia.

mTOR pathway regulates the differentiation of peripheral blood Th2/Treg cell subsets in patients with pemphigus vulgaris

2021 ◽  
Author(s):  
Kuan Lai ◽  
Wenjing Zhang ◽  
Songshan Li ◽  
Zhiwen Zhang ◽  
Shuangde Xie ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Pemphigus Vulgaris ◽  
Treg Cells ◽  
Mtor Pathway ◽  
Cd4 T Cell ◽  
Cell Ratio ◽  
Loss Of Balance

Abstract Pemphigus vulgaris (PV) is a chronic and potentially life-threatening autoimmune blistering disease. Aberrant mTOR pathway activity is involved in many autoimmune diseases. This study investigated the correlation of mTOR pathway (PI3K/AKT/mTOR/p70S6K) activity with the loss of balance in T helper 2/regulatory T (Th2/Treg) cells in the peripheral blood of PV patients. CD4+ T cells were isolated from 15 PV patients and 15 healthy controls (HCs), the ratios of Th2/CD4+ T cells and Treg/CD4+ T cells, the activity of the mTOR pathway (PI3K/AKT/mTOR/p70S6K), the transcription factors and cytokines of Th2 and Treg cells were detected. Primary CD4+ T cells from PV patients were cultured under Th2- or Treg-polarizing conditions with or without rapamycin in vitro. We found that PV patients showed significantly elevated serum IL-4 when compared with HCs, and serum IL-4 level was positively correlated with the titer of anti-Dsg1/3 antibody and disease severity, while the serum TGF-β level was negatively correlated with the titer of anti-Dsg3 antibody and disease severity. Meanwhile, PV patients showed increased Th2/CD4+ T cell ratio; decreased Treg/CD4+ T cell ratio; elevated mRNA of PI3K, AKT, mTOR and protein of PI3K (P85), AKT, p-AKT (Ser473), mTOR, p-mTOR (Ser2448), p-p70S6K (Thr389), GATA3; reduced protein of forkhead box protein 3. Rapamycin inhibited Th2 cell differentiation and promoted Treg cell differentiation in vitro. These data suggest a close association between mTOR pathway activation and the loss of balance in Th2/Treg cells in peripheral blood of PV patients. Inhibiting mTORC1 can help restore the Th2/Treg balance.

scholarly journals Secreted KIAA1199 promotes the progression of rheumatoid arthritis by mediating hyaluronic acid degradation in an ANXA1-dependent manner

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wei Zhang ◽  
Guoyu Yin ◽  
Heping Zhao ◽  
Hanzhi Ling ◽  
Zhen Xie ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Hyaluronic Acid ◽  
Dependent Manner ◽  
Collagen Induced Arthritis ◽  
Intracellular Degradation ◽  
Main Pathway ◽  
First Time

AbstractIn inflamed joints, enhanced hyaluronic acid (HA) degradation is closely related to the pathogenesis of rheumatoid arthritis (RA). KIAA1199 has been identified as a hyaladherin that mediates the intracellular degradation of HA, but its extracellular function remains unclear. In this study, we found that the serum and synovial levels of secreted KIAA1199 (sKIAA1199) and low-molecular-weight HA (LMW-HA, MW < 100 kDa) in RA patients were significantly increased, and the positive correlation between them was shown for the first time. Of note, treatment with anti-KIAA1199 mAb effectively alleviated the severity of arthritis and reduced serum LMW-HA levels and cytokine secretion in collagen-induced arthritis (CIA) mice. In vitro, sKIAA1199 was shown to mediate exogenous HA degradation by attaching to the cell membrane of RA fibroblast-like synoviosytes (RA FLS). Furthermore, the HA-degrading activity of sKIAA1199 depended largely on its adhesion to the membrane, which was achieved by its G8 domain binding to ANXA1. In vivo, kiaa1199-KO mice exhibited greater resistance to collagen-induced arthritis. Interestingly, this resistance could be partially reversed by intra-articular injection of vectors encoding full-length KIAA1199 instead of G8-deleted KIAA119 mutant, which further confirmed the indispensable role of G8 domain in KIAA1199 involvement in RA pathological processes. Mechanically, the activation of NF-κB by interleukin-6 (IL-6) through PI3K/Akt signaling is suggested to be the main pathway to induce KIAA1199 expression in RA FLS. In conclusion, our study supported the contribution of sKIAA1199 to RA pathogenesis, providing a new therapeutic target for RA by blocking sKIAA1199-mediated HA degradation.

scholarly journals Tolerogenic Splenic IDO+Dendritic Cells from the Mice Treated with Induced-Treg Cells Suppress Collagen-Induced Arthritis

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Jie Yang ◽  
Yiming Yang ◽  
Huahua Fan ◽  
Hejian Zou
Keyword(s):  
Dendritic Cells ◽  
Immune Responses ◽  
Foxp3 Expression ◽  
Treated Group ◽  
Treg Cells ◽  
Therapeutic Effects ◽  
Dependent Manner ◽  
Collagen Induced Arthritis

TGF-β-induced regulatory T cells (iTregs) retain Foxp3 expression and immune-suppressive activity in collagen-induced arthritis (CIA). However, the mechanisms whereby transferred iTregs suppress immune responses, particularly the interplay between iTregs and dendritic cells (DCs)in vivo, remain incompletely understood. In this study, we found that after treatment with iTregs, splenic CD11c+DCs, termed “DCiTreg,” expressed tolerogenic phenotypes, secreted high levels of IL-10, TGF-β, and IDO, and showed potent immunosuppressive activityin vitro. After reinfusion with DCiTreg, marked antiarthritic activity improved clinical scores and histological end-points were observed. The serological levels of inflammatory cytokines and anti-CII antibodies were low and TGF-βproduction was high in the DCiTreg-treated group. DCiTregalso induced new iTregsin vivo. Moreover, the inhibitory activity of DCiTregon CIA was lost following pretreatment with the inhibitor of indoleamine 2,3-dioxygenase (IDO). Collectively, these findings suggest that transferred iTregs could induce tolerogenic characteristics in splenic DCs and these cells could effectively dampen CIA in an IDO-dependent manner. Thus, the potential therapeutic effects of iTregs in CIA are likely maintained through the generation of tolerogenic DCsin vivo.

Quantitative analysis of cadherin-11 and β-catenin signalling during proliferation of rheumatoid arthritis-derived synovial fibroblast cells

2015 ◽  
Vol 67 (8) ◽  
pp. 1075-1082 ◽  
Author(s):  
Ryosuke Yoshioka ◽  
Yasuhiro Kita ◽  
Asako Nagahira ◽  
Atsushi Manno ◽  
Naoyuki Makita ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Quantitative Analysis ◽  
Synovial Fibroblast ◽  
Fibroblast Cells
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