Center for Immunology and Inflammation, The Feinstein Institutes for Medical Research, 350 Community Dr, 11030, Manhasset, NY, USA.
Departments of Surgery and Molecular Medicine, Zucker School of Medicine at Hofstra/Northwell, Manhasset, NY, USA.
Mol Med. 2024 Feb 1;30(1):17. doi: 10.1186/s10020-024-00790-2.
In sepsis, intestinal barrier dysfunction is often caused by the uncontrolled death of intestinal epithelial cells (IECs). CD4CD8αα intraepithelial lymphocytes (IELs), a subtype of CD4 T cells residing within the intestinal epithelium, exert cytotoxicity by producing granzyme B (GrB) and perforin (Prf). Extracellular cold-inducible RNA-binding protein (eCIRP) is a recently identified alarmin which stimulates TLR4 on immune cells to induce proinflammatory responses. Here, we hypothesized that eCIRP enhances CD4CD8αα IEL cytotoxicity and induces IEC death in sepsis.
We subjected wild-type (WT) and CIRP mice to sepsis by cecal ligation and puncture (CLP) and collected the small intestines to isolate IELs. The expression of GrB and Prf in CD4CD8αα IELs was assessed by flow cytometry. IELs isolated from WT and TLR4 mice were challenged with recombinant mouse CIRP (eCIRP) and assessed the expression of GrB and Prf in CD4CD8αα by flow cytometry. Organoid-derived IECs were co-cultured with eCIRP-treated CD4CD8αα cells in the presence/absence of GrB and Prf inhibitors and assessed IEC death by flow cytometry.
We found a significant increase in the expression of GrB and Prf in CD4CD8αα IELs of septic mice compared to sham mice. We found that GrB and Prf levels in CD4CD8αα IELs were increased in the small intestines of WT septic mice, while CD4CD8αα IELs of CIRP mice did not show an increase in those cytotoxic granules after sepsis. We found that eCIRP upregulated GrB and Prf in CD4CD8αα IELs isolated from WT mice but not from TLR4 mice. Furthermore, we also revealed that eCIRP-treated CD4CD8αα cells induced organoid-derived IEC death, which was mitigated by GrB and Prf inhibitors. Finally, histological analysis of septic mice revealed that CIRP mice were protected from tissue injury and cell death in the small intestines compared to WT mice.
In sepsis, the cytotoxicity initiated by the eCIRP/TLR4 axis in CD4CD8αα IELs is associated with intestinal epithelial cell (IEC) death, which could lead to gut injury.
在脓毒症中，肠屏障功能障碍通常是由肠上皮细胞（IECs）的失控性死亡引起的。CD4CD8αα 上皮内淋巴细胞（IELs）是一种定位于肠上皮内的 CD4 T 细胞亚群，通过产生颗粒酶 B（GrB）和穿孔素（Prf）发挥细胞毒性作用。细胞外冷诱导 RNA 结合蛋白（eCIRP）是一种新发现的警报素，可刺激免疫细胞上的 TLR4 诱导促炎反应。在这里，我们假设 eCIRP 增强 CD4CD8αα IEL 的细胞毒性，并在脓毒症中诱导 IEC 死亡。
我们通过盲肠结扎和穿孔（CLP）使野生型（WT）和 CIRP 小鼠发生脓毒症，并收集小肠分离 IELs。通过流式细胞术评估 CD4CD8αα IEL 中 GrB 和 Prf 的表达。用重组鼠 eCIRP（eCIRP）刺激 WT 和 TLR4 小鼠分离的 IELs，并通过流式细胞术评估 CD4CD8αα 中 GrB 和 Prf 的表达。在存在/不存在 GrB 和 Prf 抑制剂的情况下，将类器官衍生的 IEC 与 eCIRP 处理的 CD4CD8αα 细胞共培养，并通过流式细胞术评估 IEC 死亡。
与假手术组相比，脓毒症小鼠的 CD4CD8αα IEL 中 GrB 和 Prf 的表达明显增加。我们发现，WT 脓毒症小鼠的小肠中 GrB 和 Prf 水平升高，而 CIRP 小鼠的 CD4CD8αα IEL 在后未增加这些细胞毒性颗粒。我们发现，eCIRP 上调 WT 小鼠分离的 CD4CD8αα IEL 中的 GrB 和 Prf，但对 TLR4 小鼠的 CD4CD8αα IEL 则没有。此外，我们还揭示，eCIRP 处理的 CD4CD8αα 细胞诱导类器官衍生的 IEC 死亡，GrB 和 Prf 抑制剂可减轻这种细胞死亡。最后，脓毒症小鼠的组织学分析显示，与 WT 小鼠相比，CIRP 小鼠在小肠组织损伤和细胞死亡方面受到保护。
在脓毒症中，eCIRP/TLR4 轴在 CD4CD8αα IEL 中引发的细胞毒性与肠上皮细胞（IEC）死亡有关，这可能导致肠道损伤。
