Polymicrobial sepsis influences NK-cell-mediated immunity by diminishing NK-cell-intrinsic receptor-mediated effector responses to viral ligands or infections

The sepsis-induced cytokine storm leads to severe lymphopenia and reduced effector capacity of remaining/surviving cells. This results in a prolonged state of immunoparalysis, that contributes to enhanced morbidity/mortality of sepsis survivors upon secondary infection. The impact of sepsis on several lymphoid subsets has been characterized, yet its impact on NK-cells remains underappreciated–despite their critical role in controlling infection(s). Here, we observed numerical loss of NK-cells in multiple tissues after cecal-ligation-and-puncture (CLP)-induced sepsis. To elucidate the sepsis-induced lesions in surviving NK-cells, transcriptional profiles were evaluated and indicated changes consistent with impaired effector functionality. A corresponding deficit in NK-cell capacity to produce effector molecules following secondary infection and/or cytokine stimulation (IL-12,IL-18) further suggested a sepsis-induced NK-cell intrinsic impairment. To specifically probe NK-cell receptor-mediated function, the activating Ly49H receptor, that recognizes the murine cytomegalovirus (MCMV) m157 protein, served as a model receptor. Although relative expression of Ly49H receptor did not change, the number of Ly49H+ NK-cells in CLP hosts was reduced leading to impaired in vivo cytotoxicity and the capacity of NK-cells (on per-cell basis) to perform Ly49H-mediated degranulation, killing, and effector molecule production in vitro was also severely reduced. Mechanistically, Ly49H adaptor protein (DAP12) activation and clustering, assessed by TIRF microscopy, was compromised. This was further associated with diminished AKT phosphorylation and capacity to flux calcium following receptor stimulation. Importantly, DAP12 overexpression in NK-cells restored Ly49H/D receptors-mediated effector functions in CLP hosts. Finally, as a consequence of sepsis-dependent numerical and functional lesions in Ly49H+ NK-cells, host capacity to control MCMV infection was significantly impaired. Importantly, IL-2 complex (IL-2c) therapy after CLP improved numbers but not a function of NK-cells leading to enhanced immunity to MCMV challenge. Thus, the sepsis-induced immunoparalysis state includes numerical and NK-cell-intrinsic functional impairments, an instructive notion for future studies aimed in restoring NK-cell immunity in sepsis survivors.

脓毒症诱导的细胞因子风暴可引发严重的淋巴细胞减少症，并削弱存活/残留免疫细胞的效应功能，进而导致长期免疫麻痹状态，使脓毒症幸存者在继发感染时的发病率与死亡率进一步升高。目前已有研究阐明了脓毒症对多种淋巴亚群的影响，尽管自然杀伤细胞（NK-cells）在抗感染过程中发挥关键作用，但脓毒症对其的影响却未得到足够重视。本研究观察到，在盲肠结扎穿刺（cecal-ligation-and-puncture, CLP）诱导的脓毒症模型中，多组织内的NK细胞数量出现显著丢失。为阐明脓毒症对存活NK细胞造成的损伤，我们对其转录组谱进行了分析，结果显示其转录特征与效应功能受损的情况相符。后续实验发现，NK细胞在继发感染和/或细胞因子刺激（IL-12、IL-18）下产生效应分子的能力存在相应缺陷，这进一步表明脓毒症会诱导NK细胞的内在功能损伤。为专门探究NK细胞受体介导的功能，我们以识别鼠巨细胞病毒（murine cytomegalovirus, MCMV）m157蛋白的活化性Ly49H受体作为模型受体。尽管Ly49H受体的相对表达量未发生改变，但CLP模型小鼠体内Ly49H阳性NK细胞的数量减少，导致体内细胞毒性功能受损；同时，单个NK细胞介导Ly49H依赖的脱颗粒、杀伤以及效应分子产生的能力在体外也严重受损。机制层面的研究显示，通过全内反射荧光显微镜（total internal reflection fluorescence microscopy, TIRF microscopy）检测发现，Ly49H接头蛋白（DAP12）的活化与聚集过程受到了损伤，这一现象还与受体刺激后AKT磷酸化水平降低以及钙流能力受损相关。值得注意的是，在NK细胞中过表达DAP12可恢复CLP模型小鼠体内Ly49H/D受体介导的效应功能。最终，由于Ly49H阳性NK细胞在脓毒症下出现数量与功能损伤，宿主控制MCMV感染的能力显著受损。重要的是，CLP术后给予IL-2复合物（IL-2 complex, IL-2c）治疗可提升NK细胞的数量，但无法恢复其功能，不过该治疗仍可增强小鼠对抗MCMV攻击的免疫力。综上，脓毒症诱导的免疫麻痹状态同时包含NK细胞的数量减少与内在功能损伤，这一发现为未来旨在恢复脓毒症幸存者NK细胞免疫功能的研究提供了重要的理论启示。