Long-term survivors of murine sepsis exhibit enhanced LPS-induced lung injury and proinflammatory immune reprogramming

Sepsis is the most common cause of hospitalization worldwide. Millions of people survive sepsis each year and are at risk for rehospitalization and death. Pulmonary complications such as respiratory failure due to pneumonia and exacerbation of chronic respiratory disease are among the most common reasons for rehospitalization in sepsis survivors. In order to prevent additional morbidity and death in patients surviving sepsis, we must establish biomarkers to identify patients at risk for pulmonary complications and develop treatments. Late complications in sepsis survivors, particularly nosocomial infections, are proposed to occur through persistent immune reprogramming after sepsis known as immunoparalysis. However, pro-inflammatory immune reprogramming in the form of primed or enhanced responses to secondary stimuli has also been described and could directly contribute to tissue injury and death. Primed immune responses and their contribution to long-term sepsis complications remains understudied. We hypothesize that primed immune responses to inflammatory stimuli in the lung after sepsis are associated with pulmonary complications in survivors of sepsis. To this end, we developed a model of antibiotic treated sepsis induced by cecal ligation and puncture followed three weeks later by secondary challenge with intranasal lipopolysaccharide to induce inflammatory lung injury. We find that mice surviving sepsis have enhanced lung injury responses in the setting of an exaggerated proinflammatory immune response, including primed Ly6Chi monocyte Tnf expression. Using RNA sequencing, we identified derangements in lung gene expression after CLP prior to LPS administration which may mediate enhanced lung injury in this model. One potential mediator, S100A8/A9, was also found to be elevated in the circulation of human sepsis survivors for up to 180 days after sepsis. These findings validate our model and identify S100A8/A9 as one of many potential biomarkers and therapeutic targets for patients at risk for long-term pulmonary complications after sepsis. The role of S100A8/A9, monocyte priming, and other factors predisposing to enhanced lung injury responses and pulmonary complications after sepsis warrant further investigation in humans and mice. Overall design: 12 post-mortem perfused lung tissue samples from male mice (C57BL/6) with and without sepsis, 21 days post CLP infection. Cases are CLP sepsis survivors and controls are unopertated healthy controls.

脓毒症（Sepsis）是全球范围内住院治疗的最常见病因。每年有数百万脓毒症患者得以存活，但仍面临再住院与死亡的风险。肺炎所致呼吸衰竭、慢性呼吸系统疾病急性加重等肺部并发症，是脓毒症幸存者再住院的最常见诱因之一。为预防脓毒症幸存者出现额外的发病与死亡情况，我们亟需确立可识别肺部并发症高危患者的生物标志物，并开发相应治疗手段。
脓毒症幸存者的远期并发症（尤以医院获得性感染为甚）被认为源于脓毒症后持续存在的免疫重编程现象，即免疫麻痹（immunoparalysis）。不过，也有研究报道了以次级刺激应答致敏或增强为表现的促炎免疫重编程，这类重编程可能直接导致组织损伤与死亡。致敏免疫应答及其在脓毒症远期并发症中的作用，目前仍未得到充分研究。
我们提出假说：脓毒症后肺部对炎症刺激产生的致敏免疫应答，与脓毒症幸存者的肺部并发症存在关联。为此，我们构建了经抗生素处理的盲肠结扎穿刺（cecal ligation and puncture, CLP）诱导脓毒症模型，并于造模三周后通过鼻内给予脂多糖（lipopolysaccharide, LPS）进行二次攻击，以诱发炎症性肺损伤。
我们发现，脓毒症存活小鼠在出现过度促炎免疫应答时，肺损伤反应会增强，这其中包括Ly6C⁺单核细胞的肿瘤坏死因子（Tnf）表达致敏。通过RNA测序（RNA sequencing），我们在脂多糖给药前的盲肠结扎穿刺术后小鼠肺组织中，鉴定出了可能介导该模型中增强型肺损伤的基因表达紊乱。其中一种潜在介导因子——S100A8/A9，在脓毒症后长达180天的人类脓毒症幸存者循环系统中也呈升高状态。
上述研究结果验证了我们的模型，并将S100A8/A9确定为脓毒症后远期肺部并发症高危患者的潜在生物标志物与治疗靶点之一。S100A8/A9、单核细胞致敏以及其他易导致脓毒症后肺损伤反应增强与肺部并发症的相关因素，均有待在人类与小鼠模型中开展进一步研究。
总体实验设计：取雄性C57BL/6小鼠的12份死后灌注肺组织样本，涵盖脓毒症组与对照组，样本采集于盲肠结扎穿刺术后21天。其中病例组为盲肠结扎穿刺脓毒症存活小鼠，对照组为未接受手术的健康小鼠。