Center for Immunology and Inflammation, The Feinstein Institutes for Medical Research, 350 Community Dr, Manhasset, NY, 11030, USA.
Elmezzi Graduate School of Molecular Medicine, Manhasset, NY, 11030, USA.
Mol Med. 2021 May 31;27(1):55. doi: 10.1186/s10020-021-00318-y.
Sepsis is a life-threatening disease syndrome caused by a dysregulated host response to infection and injury. Extracellular cold-inducible RNA-binding protein (eCIRP) acts as a damage-associated molecular pattern. Peritoneal cavity (PerC) B-1a cells attenuate inflammation and tissue injury by spontaneous releasing natural IgM and IL-10. Sialic acid-binding immunoglobulin-type lectin-G (Siglec-G) is a CD33-related receptor highly expressed in B-1a cells to serve critical immunoregulatory functions. In sepsis, B-1a cell numbers in PerC are decreased. We hypothesized that eCIRP causes the reduction of PerC B-1a cells and alters their function during sepsis.
Sepsis was induced in WT and CIRP mice by cecal ligation and puncture (CLP). PerC washout cells were collected and B-1a cells and Siglec-G were assessed by flow cytometry. Mice were i.p. injected with recombinant murine (rm) CIRP and after 20 h, Siglec-G expression in PerC B-1a cells were assessed. PerC B-1a cells were treated with rmCIRP for 4 h and Siglec-G expression was assessed. PerC B-1a cells were pre-treated with anti-Siglec-G Ab and then after stimulated with rmCIRP for 24 h, IL-6 levels in the culture supernatants were assessed.
eCIRP levels in the PerC were elevated in septic mice. In WT mice, the frequencies and numbers of total and Siglec-G B-1a cells in the PerC were significantly decreased in the CLP group compared to sham group, whereas in CIRP mice, their frequencies and numbers in sepsis were significantly rescued compared to WT septic mice. Mice injected with rmCIRP showed decreased frequencies and numbers of total and Siglec-G PerC B-1a cells compared to PBS-injected mice. In vitro treatment of PerC B-1a cells with rmCIRP demonstrated significant reduction in Siglec-G mRNA and protein compared to PBS group. PerC B-1a cells treated with anti-Siglec-G Ab had significantly higher production of IL-6 in response to rmCIRP compared to IgG control. Anti-Siglec-G Ab treated B-1a cells co-cultured with macrophages produced significantly higher levels of IL-6, and TNF-α, and lower levels of IL-10 compared to IgG-treated B-1a cells and macrophage co-cultures stimulated with rmCIRP.
eCIRP reduces PerC B-1a cell pool and skews them to a pro-inflammatory phenotype by downregulating Siglec-G expression. Targeting eCIRP will retain Siglec-G expressing B-1a cells in the PerC and preserve their anti-inflammatory function in sepsis.
败血症是一种危及生命的疾病综合征，是由宿主对感染和损伤的失调反应引起的。细胞外冷诱导 RNA 结合蛋白 (eCIRP) 作为一种损伤相关分子模式。腹腔 (PerC) B-1a 细胞通过自发释放天然 IgM 和 IL-10 来减轻炎症和组织损伤。唾液酸结合免疫球蛋白型凝集素-G (Siglec-G) 是一种高度表达于 B-1a 细胞的 CD33 相关受体，具有重要的免疫调节功能。在败血症中，PerC 中的 B-1a 细胞数量减少。我们假设 eCIRP 导致 PerC B-1a 细胞减少，并在败血症期间改变其功能。
通过盲肠结扎和穿孔 (CLP) 在 WT 和 CIRP 小鼠中诱导败血症。收集腹腔冲洗细胞，通过流式细胞术评估 B-1a 细胞和 Siglec-G。用重组鼠 (rm) CIRP 对小鼠进行腹腔内注射，20 小时后，评估 PerC B-1a 细胞中的 Siglec-G 表达。用 rmCIRP 处理 PerC B-1a 细胞 4 小时，评估 Siglec-G 表达。用抗 Siglec-G Ab 预处理 PerC B-1a 细胞，然后用 rmCIRP 刺激 24 小时后，评估培养上清液中的 IL-6 水平。
败血症小鼠 PerC 中的 eCIRP 水平升高。在 WT 小鼠中，与假手术组相比，CLP 组 PerC 中的总 B-1a 细胞和 Siglec-G B-1a 细胞的频率和数量明显减少，而在 CIRP 小鼠中，与 WT 败血症小鼠相比，其频率和数量明显恢复。与 PBS 注射小鼠相比，rmCIRP 注射小鼠的总 B-1a 细胞和 Siglec-G PerC B-1a 细胞的频率和数量均减少。与 PBS 组相比，体外用 rmCIRP 处理 PerC B-1a 细胞后 Siglec-G mRNA 和蛋白的表达显著降低。与 IgG 对照相比，用抗 Siglec-G Ab 处理的 PerC B-1a 细胞在 rmCIRP 刺激下产生的 IL-6 明显更高。与 rmCIRP 刺激的 IgG 处理的 B-1a 细胞和巨噬细胞共培养物相比，用抗 Siglec-G Ab 处理的 B-1a 细胞共培养物产生的 IL-6、TNF-α水平更高，IL-10 水平更低。
eCIRP 通过下调 Siglec-G 表达来减少 PerC B-1a 细胞池并使其向促炎表型倾斜。靶向 eCIRP 将保留 PerC 中的 Siglec-G 表达 B-1a 细胞，并在败血症中保留其抗炎功能。
