背景:重症监护病房获得性肌无力(ICU-AW)是一种综合征,其特征是在慢性期败血症存活患者中经常观察到的长期肌肉无力。尽管ICU-AW与死亡率增加独立相关,有效的治疗方法尚未建立。程序性死亡-1(PD-1)抑制剂作为逆转脓毒症免疫衰竭的潜在治疗方法引起了人们的关注;然而,其对ICU-AW的影响仍有待阐明。这里,我们在临床前脓毒症模型中研究PD-1缺乏如何影响脓毒症诱导的骨骼肌功能障碍.
方法:通过用盲肠浆液处理野生型(WT)和PD-1敲除(KO)小鼠建立慢性脓毒症模型,然后用抗生素和生理盐水复苏。在第15-17天将小鼠安乐死。身体重量,肌肉重量,并测量了肢体肌肉强度。流式细胞术检测白细胞介素13(IL-13)和PD-1的表达。通过逆转录和定量聚合酶链反应(RT-qPCR)测量慢抽搐肌肉的信使RNA(mRNA)表达。在一项体外研究中,用脂多糖(LPS)和重组IL-13处理C2C12肌管,随后进行基因表达测量。
结果:WT败血症小鼠表现出肌肉重量降低(股四头肌,P<0.01;腓肠肌,P<0.05;胫骨前肌,P<0.01)和长期肌无力(P<0.0001),而PD-1KO败血症小鼠的肌肉重量和强度没有任何降低。慢抽搐特异性mRNA,包括肌红蛋白(Mb),肌钙蛋白I型1(Tnni1),肌球蛋白重链7(Myh7)在WT骨骼肌中减少(Mb,P<0.0001;Tnni1,P<0.05;和Myh7,P<0.05)在脓毒症诱导后,但PD-1KO脓毒症小鼠Tnni1和Myh7的mRNA表达增加(Mb,不显著;Tnni1,P<0.0001;Myh7,P<0.05)。用LPS处理C2C12肌管细胞降低慢抽搐mRNA的表达,IL-13(Mb,P<0.0001;Tnni1,P<0.001;Myh7,P<0.05)。IL-13的产生在ILC2s中显著高于骨骼肌中的T细胞(P<0.05)。检测了骨骼肌中产生IL-13的ILC2s,发现PD-1KO败血症小鼠增加,与WT脓毒症小鼠相比(P<0.05)。PD-1KO败血症小鼠增加了ILC2衍生的IL-13,并认为可以保护肌肉免受实验性ICU-AW的影响。
结论:实验性ICU-AW中的长期肌无力在PD-1KO小鼠中得到改善。在PD-1KO小鼠中,骨骼肌中ILC2衍生的IL-13产生增加,从而表明IL-13减轻脓毒症期间的肌肉无力。这项研究证明了PD-1阻断通过增加ILC2衍生的IL-13在脓毒症期间保持肌肉力量的作用,并且可能是脓毒症诱导的ICU-AW的有吸引力的治疗靶标。
BACKGROUND: Intensive care unit-acquired weakness (ICU-AW) is a syndrome characterized by a long-term muscle weakness often observed in sepsis-surviving patients during the chronic phase. Although ICU-AW is independently associated with increased mortality, effective therapies have yet to be established. Programmed death-1 (PD-1) inhibitors have attracted attention as potential treatments for reversing immune exhaustion in sepsis; however, its impact on ICU-AW remains to be elucidated. Here, we study how PD-1 deficiency affects sepsis-induced skeletal muscle dysfunction in a preclinical sepsis model.
METHODS: Chronic sepsis model was developed by treating wild-type (WT) and PD-1 knockout (KO) mice with caecal slurry, followed by resuscitation with antibiotics and saline. Mice were euthanized on days 15-17. Body weights, muscle weights, and limb muscle strengths were measured. Interleukin 13 (IL-13) and PD-1 expressions were examined by flow cytometry. Messenger RNA (mRNA) expressions of slow-twitch muscles were measured by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). In an in vitro study, C2C12 myotubes were treated with lipopolysaccharide (LPS) and recombinant IL-13 followed by gene expression measurements.
RESULTS: WT septic mice exhibited decreased muscle weight (quadriceps, P < 0.01; gastrocnemius, P < 0.05; and tibialis anterior, P < 0.01) and long-term muscle weakness (P < 0.0001), whereas PD-1 KO septic mice did not exhibit any reduction in muscle weights and strengths. Slow-twitch specific mRNAs, including myoglobin (Mb), troponin I type 1 (Tnni1), and myosin heavy chain 7 (Myh7) were decreased in WT skeletal muscle (Mb, P < 0.0001; Tnni1, P < 0.05; and Myh7, P < 0.05) after sepsis induction, but mRNA expressions of Tnni1 and Myh7 were increased in PD-1 KO septic mice (Mb, not significant; Tnni1, P < 0.0001; and Myh7, P < 0.05). Treatment of C2C12 myotube cells with LPS decreased the expression of slow-twitch mRNAs, which was restored by IL-13 (Mb, P < 0.0001; Tnni1, P < 0.001; and Myh7, P < 0.05). IL-13 production was significantly higher in ILC2s compared to T cells in skeletal muscle (P < 0.05). IL-13-producing ILC2s in skeletal muscle were examined and found to increase in PD-1 KO septic mice, compared with WT septic mice (P < 0.05). ILC2-derived IL-13 was increased by PD-1 KO septic mice and thought to protect the muscles from experimental ICU-AW.
CONCLUSIONS: Long-term muscle weakness in experimental ICU-AW was ameliorated in PD-1 KO mice. ILC2-derived IL-13 production in skeletal muscles was increased in PD-1 KO mice, thereby suggesting that IL-13 alleviates muscle weakness during sepsis. This study demonstrates the effects of PD-1 blockade in preserving muscle strength during sepsis through an increase in ILC2-derived IL-13 and may be an attractive therapeutic target for sepsis-induced ICU-AW.