Giri Prashant S, Bharti Ankit H, Begum Rasheedunnisa, Dwivedi Mitesh
C. G. Bhakta Institute of Biotechnology, Faculty of Science, Uka Tarsadia University, Surat, Gujarat, India.
Aura Skin Care, Laxmi Icon 2nd Floor, Vyara, Gujarat, India.
Immunology. 2022 Nov;167(3):314-327. doi: 10.1111/imm.13538. Epub 2022 Jul 12.
NFATs and FOXP3 are linked with impaired regulatory T-cells (Tregs) in generalized vitiligo (GV). To elucidate calcium mediated NFATc1 signalling pathway and its effect on Treg suppressive capacity in GV. Calcium levels, calcineurin, NFATc1 and GSK-3β activity and cell proliferation were assessed in 52 GV patients and 50 controls by calcium assay kit, calcineurin phosphatase assay kit, TransAM NFATc1 kit, GSK-3β ELISA and BrdU cell proliferation assay. Transcripts (CNB, CAM, GSK3B, DYRK1A and calcium channel genes) and protein (IFN-γ, IL-10 and TGF-β) expressions were assessed by qPCR and ELISA, respectively. Reduced plasma and intracellular Tregs calcium levels and ORAI1 transcripts suggested altered calcium homeostasis in GV Tregs (p = 0.00387, p = 0.0048, p < 0.0001), which led to decreased calcineurin and NFATc1 activity in GV Tregs (p = 0.0299, p < 0.0001). CNB and CAM transcripts were reduced in GV Tregs (p < 0.0001, p = 0.0004). GSK-3β activity, GSK3B and DYRK1A transcripts significantly increased in GV Tregs (p = 0.0134, p < 0.0001 and p < 0.0001). Plasma (p = 0.0225, p = 0.032) and intracellular Treg (p = 0.0035, p = 0.005) calcium levels, calcineurin (p = 0.001) and NFATc1 (p = 0.001, p < 0.0001) activity and ORAI1 (p = 0.0093, p < 0.0001), CAM and CNB (p = 0.0214) transcripts significantly decreased in active vitiligo (AV) and severe GV (sGV) Tregs. Calcium treatment significantly increased intracellular calcium and ORAI1 transcripts in GV Tregs (p = 0.0042, p = 0.0035). Moreover, calcium treatment enhanced calcineurin and NFATc1 activity in GV Tregs (p = 0.0128, p < 0.0001). Remarkably, calcium treatment increased Treg mediated suppression of CD4 and CD8 T-cells (p = 0.015, p = 0.006) in GV and increased Tregs associated cytokines: IL-10 (p = 0.0323, p = 0.009), TGF-β (p = 0.0321, p = 0.01) and decreased IFN-γ production (p = 0.001, p = 0.016) by CD4 and CD8 T-cells. Intracellular calcium levels positively correlated with calcineurin (r = 0.83; p < 0.0001) and NFATc1 (r = 0.61; p < 0.0001) activity, suggesting the enhanced Treg immunosuppressive capacity after calcium treatment. Our study for the first time suggests that reduced plasma calcium and ORAI1 transcripts are linked to calcium uptake defects in Tregs, which leads to reduced calcineurin and NFATc1 activation, thereby contributing to decreased Tregs immunosuppressive capacity in GV. Elevated GSK-3β activity and GSKB and DYRK1A transcripts are involved in reduced NFATc1 activity in GV Tregs. Overall, the study suggests that calcium-NFATc1-signalling pathway is likely to be involved in defective Tregs function and can be implicated for development of effective Treg mediated therapeutics for GV.
核因子活化T细胞（NFATs）和叉头框蛋白P3（FOXP3）与泛发性白癜风（GV）中调节性T细胞（Tregs）功能受损有关。为阐明钙介导的NFATc1信号通路及其对GV中Treg抑制能力的影响，采用钙检测试剂盒、钙调神经磷酸酶磷酸酶检测试剂盒、TransAM NFATc1试剂盒、GSK-3β酶联免疫吸附测定（ELISA）和BrdU细胞增殖检测法，对52例GV患者和50例对照者的钙水平、钙调神经磷酸酶、NFATc1和GSK-3β活性以及细胞增殖情况进行了评估。分别通过定量聚合酶链反应（qPCR）和ELISA检测转录本（CNB、CAM、GSK3B、双重特异性酪氨酸磷酸化调节激酶1A（DYRK1A）和钙通道基因）和蛋白（干扰素-γ（IFN-γ）、白细胞介素-10（IL-10）和转化生长因子-β（TGF-β））的表达。GV患者血浆和细胞内Tregs钙水平以及ORAI1转录本降低，提示GV Tregs中钙稳态改变（p = 0.00387，p = 0.0048，p < 0.0001），这导致GV Tregs中钙调神经磷酸酶和NFATc1活性降低（p = 0.0299，p < 0.0001）。GV Tregs中CNB和CAM转录本减少（p < 0.0001，p = 0.0004）。GV Tregs中GSK-3β活性、GSK3B和DYRK1A转录本显著增加（p = 0.0134，p < 0.0001和p < 0.0001）。活动期白癜风（AV）和重度GV（sGV）Tregs中，血浆（p = 0.0225，p = 0.032）和细胞内Treg钙水平（p = 0.0035，p = 0.005）、钙调神经磷酸酶（p = 0.001）和NFATc1活性（p = 0.001，p < 0.0001）以及ORAI1（p = 0.0093，p < 0.0001）、CAM和CNB（p = 0.0214）转录本显著降低。钙处理显著增加了GV Tregs中的细胞内钙和ORAI1转录本（p = 0.0042，p = 0.0035）。此外，钙处理增强了GV Tregs中钙调神经磷酸酶和NFATc1活性（p = 0.0128，p < 0.0001）。值得注意的是，钙处理增加了GV中Treg介导的对CD4和CD8 T细胞的抑制作用（p = 0.015，p = 0.006），并增加了与Tregs相关的细胞因子：IL-10（p = 0.0323，p = 0.009）、TGF-β（p = 0.0321，p = 0.01），并减少了CD4和CD8 T细胞产生的IFN-γ（p = 0.001，p = 0.016）。细胞内钙水平与钙调神经磷酸酶（r = 0.83；p < 0.0001）和NFATc1活性（r = 0.61；p < 0.0001）呈正相关，提示钙处理后Treg免疫抑制能力增强。我们的研究首次表明，血浆钙降低和ORAI1转录本与Tregs中钙摄取缺陷有关，这导致钙调神经磷酸酶和NFATc1激活减少，从而导致GV中Tregs免疫抑制能力下降。GV Tregs中GSK-3β活性升高以及GSKB和DYRK1A转录本与NFATc1活性降低有关。总体而言，该研究表明钙-NFATc1信号通路可能参与Tregs功能缺陷，并且可能与开发有效的Treg介导的GV治疗方法有关。
