A double-edged sword role of IFN-?-producing iNKT cells in sepsis: Persistent suppression of Treg cell formation in an Nr4a1-dependent manner

Sepsis, a leading cause of mortality in intensive care units worldwide, lacks effective treatments for advanced-stage sepsis. Therefore, understanding the underlying mechanisms of this disease is crucial. This study reveals that iNKT cells have an opposing role in the progression of sepsis by suppressing Treg differentiation and function. The activation of iNKT cells by a-Galcer enhances IFN-? production. Blocking antibodies or transferring IFN-?-deficient iNKT cells demonstrates that iNKT cells inhibit Treg differentiation through IFN-? production. Additionally, iNKT cell-mediated Treg inhibition prevents secondary infection caused by Listeria monocytogenes during the post-septic phase. The transcriptomic analysis of Tregs further reveals that the suppressive function of Tregs is impaired by iNKT cells. Finally, we demonstrate that iNKT cells inhibit Treg differetiation in a Nr4a1-dependent manner. Our data uncover the dual function of iNKT cells in sepsis progression and provide a potential treatment target for this adverse long-term outcome induced by sepsis. Overall design: The CD4+ T cells were isolated from splenocytes of septic mice treated with a-galcer or PBS (two days after CLP performance) using the negative CD4+T cell isolation kit, following the manufacturer's instructions. Subsequently, the Treg cell population, characterized by CD25highCD127low markers, was sorted using BD FACSAria™ Fusion Flow Cytometers. To ensure an adequate number of enriched Treg cells, Treg cells were collected from three mice. The isolated Treg cells with a purity about 90% were subjected to TRIzol treatment, and total RNA was extracted according to the manufacturer's protocol.

败血症（Sepsis）是全球重症监护病房中致死率最高的病因之一，目前针对晚期败血症尚无有效的治疗方案。因此，阐明该疾病的潜在发病机制至关重要。本研究揭示，恒定自然杀伤T细胞（invariant natural killer T cells, iNKT cells）可通过抑制调节性T细胞（regulatory T cells, Treg）的分化与功能，在败血症进展中发挥调控作用。经α-半乳糖苷神经酰胺（a-Galcer）激活iNKT细胞后，可促进干扰素-γ（IFN-γ）的产生。通过使用阻断性抗体或过继转输干扰素-γ缺陷型iNKT细胞的实验证实，iNKT细胞可通过分泌干扰素-γ抑制Treg细胞的分化。此外，在败血症后期阶段，iNKT细胞介导的Treg细胞抑制作用可抵御由单核细胞增生李斯特菌（Listeria monocytogenes）引发的继发感染。对Treg细胞的转录组分析进一步证实，iNKT细胞会削弱Treg细胞的免疫抑制功能。最后，本研究证实iNKT细胞可通过依赖于核受体Nr4a1的方式抑制Treg细胞的分化。本研究数据揭示了iNKT细胞在败血症进展中的双重功能，并为败血症诱发的不良远期结局提供了潜在治疗靶点。整体实验设计：采用阴性分选CD4+T细胞分离试剂盒，依照试剂盒说明书的操作步骤，从经α-半乳糖苷神经酰胺或磷酸盐缓冲液（PBS）处理的败血症小鼠（盲肠结扎穿刺术（cecal ligation and puncture, CLP）造模后2天）的脾脏细胞中分离CD4+T细胞。随后，以CD25highCD127low为表面标志物的Treg细胞群，通过BD FACSAria™ Fusion流式细胞仪进行分选。为确保获得足够数量的富集Treg细胞，本实验从3只小鼠体内收集Treg细胞。纯度约为90%的分离得到的Treg细胞经TRIzol试剂处理后，依照试剂盒说明书的操作流程提取总RNA。