Department of Rheumatology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China.
Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China.
J Transl Med. 2024 Apr 2;22(1):327. doi: 10.1186/s12967-024-05082-1.
Regulatory T cells (Tregs) are crucial in maintaining immune homeostasis and preventing autoimmunity and inflammation. A proportion of Treg cells can lose Foxp3 expression and become unstable under inflammation conditions. The precise mechanisms underlying this phenomenon remain unclear.
The PI16 gene knockout mice (PI16Foxp3) in Treg were constructed, and the genotypes were identified. The proportion and phenotypic differences of immune cells in 8-week-old mice were detected by cell counter and flow cytometry. Two groups of mouse Naïve CD4T cells were induced to differentiate into iTreg cells to observe the effect of PI16 on the differentiation and proliferation of iTreg cells, CD4CD25Treg and CD4CD25 effector T cells (Teff) were selected and co-cultured with antigen presenting cells (APC) to observe the effect of PI16 on the inhibitory ability of Treg cells in vitro. The effects of directed knockout of PI16 in Treg cells on inflammatory symptoms, histopathological changes and immune cell expression in mice with enteritis and autoimmune arthritis were observed by constructing the model of antigen-induced arthritis (AIA) and colitis induced by dextran sulfate sodium salt (DSS).
We identified peptidase inhibitor 16 (PI16) as a negative regulator of Treg cells. Our findings demonstrate that conditional knock-out of PI16 in Tregs significantly enhances their differentiation and suppressive functions. The conditional knockout of the PI16 gene resulted in a significantly higher abundance of Foxp3 expression (35.12 ± 5.71% vs. 20.00 ± 1.61%, p = 0.034) in iTreg cells induced in vitro compared to wild-type mice. Mice with Treg cell-specific PI16 ablation are protected from autoimmune arthritis (AIA) and dextran sulfate sodium (DSS)-induced colitis development. The AIA model of PI16 is characterized by the reduction of joint structure and the attenuation of synovial inflammation and in DSS-induced colitis model, conditional knockout of the PI16 reduce intestinal structural damage. Additionally, we found that the deletion of the PI16 gene in Treg can increase the proportion of Treg (1.46 ± 0.14% vs. 0.64 ± 0.07%, p < 0.0001) and decrease the proportion of Th17 (1.00 ± 0.12% vs. 3.84 ± 0.64%, p = 0.001). This change will enhance the shift of Th17/Treg toward Treg cells in AIA arthritis model (0.71 ± 0.06% vs. 8.07 ± 1.98%, p = 0.003). In DSS-induced colitis model of PI16, the proportion of Treg in spleen was significantly increased (1.40 ± 0.15% vs. 0.50 ± 0.11%, p = 0.003), Th17 (2.18 ± 0.55% vs. 6.42 ± 1.47%, p = 0.017), Th1 (3.42 ± 0.19% vs. 6.59 ± 1.28%, p = 0.028) and Th2 (1.52 ± 0.27% vs. 2.76 ± 0.38%, p = 0.018) in spleen was significantly decreased and the Th17/Treg balance swift toward Treg cells (1.44 ± 0.50% vs. 24.09 ± 7.18%, p = 0.012).
PI16 plays an essential role in inhibiting Treg cell differentiation and function. Conditional knock out PI16 gene in Treg can promote the Treg/Th17 balance towards Treg dominance, thereby alleviating the condition. Targeting PI16 may facilitate Treg cell-based therapies for preventing autoimmune diseases and inflammatory diseases. The research provides us with novel insights and future research avenues for the treatment of autoimmune diseases, particularly arthritis and colitis.
调节性 T 细胞（Tregs）在维持免疫稳态和预防自身免疫和炎症方面起着至关重要的作用。在炎症条件下，一部分 Treg 细胞会失去 Foxp3 表达并变得不稳定。其确切的机制尚不清楚。
构建了 Treg 细胞中 PI16 基因敲除小鼠（PI16Foxp3），并鉴定了其基因型。通过细胞计数器和流式细胞术检测 8 周龄小鼠免疫细胞的比例和表型差异。诱导两组小鼠 Naïve CD4T 细胞分化为诱导性调节性 T 细胞（iTreg），观察 PI16 对 iTreg 细胞分化和增殖的影响，选择 CD4CD25Treg 和 CD4CD25 效应 T 细胞（Teff），与抗原呈递细胞（APC）共培养，观察 PI16 对 Treg 细胞体外抑制能力的影响。通过构建抗原诱导关节炎（AIA）和葡聚糖硫酸钠盐（DSS）诱导的结肠炎模型，观察 Treg 细胞中 PI16 定向敲除对肠炎和自身免疫性关节炎小鼠炎症症状、组织病理学变化和免疫细胞表达的影响。
我们鉴定出肽酶抑制剂 16（PI16）是 Treg 细胞的负调控因子。我们的研究结果表明，Treg 细胞中条件性敲除 PI16 显著增强了其分化和抑制功能。与野生型小鼠相比，体外诱导的 iTreg 细胞中 Foxp3 表达（35.12±5.71%比 20.00±1.61%，p=0.034）显著增加。Treg 细胞特异性 PI16 消融的小鼠对自身免疫性关节炎（AIA）和葡聚糖硫酸钠盐（DSS）诱导的结肠炎发展具有保护作用。PI16 的 AIA 模型表现为关节结构减少，滑膜炎减轻，在 DSS 诱导的结肠炎模型中，PI16 的条件性敲除减少了肠道结构损伤。此外，我们发现 Treg 中 PI16 基因的缺失可以增加 Treg 的比例（1.46±0.14%比 0.64±0.07%，p<0.0001），减少 Th17 的比例（1.00±0.12%比 3.84±0.64%，p=0.001）。这种变化将增强 AIA 关节炎模型中 Th17/Treg 向 Treg 细胞的转移（0.71±0.06%比 8.07±1.98%，p=0.003）。在 PI16 的 DSS 诱导的结肠炎模型中，脾脏中 Treg 的比例显著增加（1.40±0.15%比 0.50±0.11%，p=0.003），Th17（2.18±0.55%比 6.42±1.47%，p=0.017）、Th1（3.42±0.19%比 6.59±1.28%，p=0.028）和 Th2（1.52±0.27%比 2.76±0.38%，p=0.018）明显减少，Th17/Treg 平衡向 Treg 细胞（1.44±0.50%比 24.09±7.18%，p=0.012）倾斜。
PI16 在抑制 Treg 细胞分化和功能方面起着重要作用。Treg 细胞中 PI16 基因的条件敲除可以促进 Treg/Th17 平衡向 Treg 优势倾斜，从而缓解病情。靶向 PI16 可能有助于基于 Treg 细胞的治疗方法，以预防自身免疫性疾病和炎症性疾病。本研究为治疗自身免疫性疾病，特别是关节炎和结肠炎提供了新的见解和未来的研究方向。
