Khan Farhan Ullah, Khongorzul Puregmaa, Gris Denis, Amrani Abdelaziz
Department of Pediatrics, Immunology Division, Université de Sherbrooke Faculté de Médecine et des Sciences de la Santé, 3001, 12 th Avenue North, Sherbrooke, QC, J1H 5 N4, Canada.
Department of Phamacology-Physiology, Université de Sherbrooke Faculté de Médecine et des Sciences de la Santé, 3001, 12 th Avenue North, Sherbrooke, QC, J1H 5 N4, Canada.
Cell Mol Biol Lett. 2025 Apr 17;30(1):47. doi: 10.1186/s11658-025-00727-5.
Tolerogenic dendritic cells (toDCs) are critical for maintaining immune homeostasis and preventing autoimmune disease development, such as type 1 diabetes (T1D). We have previously shown that DCs of non-obese diabetic (NOD) mice expressing active Stat5b (Stat5b-CA.DCs) acquire toDCs signature and protect against diabetes. However, the mechanisms involved in reprogramming DCs to adopt tolerogenic or immunogenic signatures are not fully known. This study investigates for the first time the role of USP7 in DC-mediated immune regulation in T1D using a transgenic NOD mouse model expressing an active form of Stat5b (NOD.Stat5b-CA).
Splenic DCs were purified from diabetes-prone NOD mice and diabetes-resistant NOD.Stat5b-CA transgenic mice and their tolerogenic and immunogenic phenotypes were analyzed by FACS. Their pro-and anti-inflammatory cytokine patterns, IRF4, IRF8, de-ubiquitin ligase USP7, and methyltransferase Ezh2 expression were assessed by FACS and Western blot. Moreover, the impact of USP7 inhibition in DCs on Th1/Th2/Th17 and Treg and diabetes onset was assessed using an in vivo DC-based transfer model.
In this study, we found that splenic Stat5b-CA.DCs expressed high levels of USP7, Ezh2, and PD-L-1/2 and contained a higher proportion of tolerogenic conventional DC2 (cDC2) subsets than immunogenic cDC1 compared to NOD mice DCs. We also found that the USP7 blockade increased Stat5b-CA.DCs maturation and proinflammatory cytokines production while decreasing anti-inflammatory cytokines and PD-L1 and PD-L2 expressions. Mechanistically, USP7 blockade in Stat5-CA.DCs promoted cDC1 over cDC2 subsets by increasing IRF8 expression in an Ezh2-dependent manner and decreasing IRF4 expression independently of Ezh2. USP7 blockade also increased Stat5b-CA.DC capacity to promote Th17 and to restrain Th2 and Treg cells. Importantly, the capacity of Stat5b-CA.DCs to protect NOD mice from diabetes were lost when treated with USP7 inhibitor.
Our findings underscore the role of the USP7/Ezh2 axis in maintaining tolerogenic DC functions that are required to tailor adaptive immune response and diabetes protection in NOD mice.
耐受性树突状细胞（toDCs）对于维持免疫稳态和预防自身免疫性疾病的发展至关重要，例如1型糖尿病（T1D）。我们之前已经表明，表达活性Stat5b的非肥胖糖尿病（NOD）小鼠的树突状细胞（Stat5b-CA.DCs）获得了toDCs特征并能预防糖尿病。然而，使树突状细胞重编程以采用耐受性或免疫原性特征所涉及的机制尚不完全清楚。本研究首次使用表达活性形式Stat5b的转基因NOD小鼠模型（NOD.Stat5b-CA），研究泛素特异性蛋白酶7（USP7）在T1D中树突状细胞介导的免疫调节中的作用。
从易患糖尿病的NOD小鼠和抗糖尿病的NOD.Stat5b-CA转基因小鼠中纯化脾树突状细胞，并通过流式细胞术分析它们的耐受性和免疫原性表型。通过流式细胞术和蛋白质免疫印迹法评估它们的促炎和抗炎细胞因子模式、干扰素调节因子4（IRF4）、干扰素调节因子8（IRF8）、去泛素化连接酶USP7和甲基转移酶Ezh2的表达。此外，使用基于树突状细胞的体内转移模型评估树突状细胞中USP7抑制对Th1/Th2/Th17和调节性T细胞（Treg）以及糖尿病发病的影响。
在本研究中，我们发现与NOD小鼠的树突状细胞相比，脾Stat5b-CA.DCs表达高水平的USP7、Ezh2和程序性死亡配体1/2（PD-L-1/2），并且与免疫原性的浆细胞样树突状细胞（cDC1）相比，含有更高比例的耐受性传统树突状细胞2（cDC2）亚群。我们还发现，USP7阻断增加了Stat5b-CA.DCs的成熟和促炎细胞因子的产生，同时降低了抗炎细胞因子以及PD-L1和PD-L2的表达。从机制上讲，Stat5-CA.DCs中的USP7阻断通过以Ezh2依赖的方式增加IRF8表达并独立于Ezh2降低IRF4表达，促进了cDC1而非cDC2亚群的产生。USP7阻断还增加了Stat5b-CA.DC促进Th17细胞并抑制Th2和Treg细胞的能力。重要的是，用USP7抑制剂处理后，Stat5b-CA.DCs保护NOD小鼠免受糖尿病影响的能力丧失。
我们的研究结果强调了USP7/Ezh2轴在维持耐受性树突状细胞功能中的作用，这些功能是在NOD小鼠中调节适应性免疫反应和糖尿病保护所必需的。
