Immunosuppression by Myeloid Cells in Pneumonia - Project 3

摘要 导致脓毒症的原发侮辱可能会导致免疫抑制的严重状态，从而使 易受铜绿假单胞菌(PA)等细菌继发感染的个体。PA- 诱发性肺炎可导致急性呼吸窘迫综合征(ARDS)。我们已经确定了两支安打 小鼠首次外周暴露于细菌脂多糖(LPS)后的模型 PA引起的肺部感染，导致肺部免疫抑制。这些小鼠的肺部表现出高度的 抗炎细胞因子IL-10的表达水平，但几乎检测不到前- 炎性细胞因子IL-6。与只用PA单次攻击的小鼠相比，两次攻击的小鼠表现出 发病率、肺损伤和肺部细菌传播增加。假单胞菌分泌不同的毒力 因子和宿主进而表达称为对氧磷酶(PONS)的酶来防御这些因子。 PON_2可诱导巨噬细胞从促炎性M1表型向抑制性M2表型转变 (MφS)我们先前证实，类似于髓系细胞的调节性髓系细胞数量增加。 肺内抑制细胞(MDSCs)对产生高水平IL-2的内毒素或细菌感染的反应 10.对人类败血症的研究表明，外周血中的MDSCs与慢性免疫抑制和 重症ARDS患者血浆IL-10水平升高与预后不良相关。在细胞类型中 它可以防御细菌感染，先天类T细胞的一个亚群，MAIT细胞，控制肺部感染 包括PA在内的不同细菌。我们未能在危重病患者的外周血中检测到MAIT细胞 增加了脓毒症期间继发感染的易感性是由宿主下降引起的可能性 保护性细胞。人外周血单个核细胞的单细胞RNA-SEQ分析 患者表现出基因表达的动态变化，这可能有助于预后。总而言之，这些 观察结果使我们假设，细菌性肺炎期间肺部的免疫抑制包括 主要的抗炎机制，如PPAR，γ和mTOR，导致增加的产生 调节髓系细胞和抗炎介质IL-10和PON_2的表达增加，但 减少宿主保护性介质(IL-6、肿瘤坏死因子-α)和细胞类型(MAIT细胞)的表达。 为了证明这一假设，我们将： 目的1.测定危重病人外周血单核细胞的免疫细胞动力学和基因信号。 MTOR在免疫抑制中的作用。 目的2.确定PPARγ和PON2在持续性抗炎细胞因子基因中的作用 在二次打击模型小鼠肺组织中的表达。

ABSTRACT A primary insult that precipitates sepsis can induce a profound state of immunosuppression, which renders an individual highly susceptible to secondary infections by bacteria such as Pseudomonas aeruginosa (PA). PA- induced pneumonia can cause acute respiratory distress syndrome (ARDS). We have established a 2-hit model in mice involving an initial peripheral exposure to bacterial lipopolysaccharide (LPS) followed by pulmonary infection with PA that induces immune suppression in the lung. The lungs of these mice show high levels of expression of the anti-inflammatory cytokine IL-10 but barely detectable expression of the pro- inflammatory cytokine IL-6. Compared to mice subjected to a single hit with PA only, those with 2-hits show increased morbidity, lung injury and lung bacterial dissemination. Pseudomonas secretes different virulence factors and the host in turn expresses enzymes called paraoxonases (PONs) to defend against these factors. PON2 was shown to induce a switch from pro-inflammatory M1 to suppressive M2 phenotype in macrophages (Mφs). We previously demonstrated increased numbers of regulatory myeloid cells resembling myeloid-derived suppressor cells (MDSCs) in the lung in response to LPS or bacterial infection that produce high levels of IL- 10. Studies of human sepsis have implicated peripheral blood MDSCs in chronic immune suppression and high plasma IL-10 levels were associated with poor prognosis in severely ill ARDS patients. Among cell types that defend against bacterial infections, a subset of innate-like T cells, MAIT cells, control infections of the lung by different bacteria including PA. We failed to detect MAIT cells in the peripheral blood of critically ill patients raising the possibility that susceptibility to secondary infections during sepsis is caused by a decline in host protective cells. Single cell RNA-seq (scRNA-seq) analysis of peripheral blood mononuclear cells (PBMCs) of patients show dynamic changes in gene expression, which may aid in prognosis. Collectively, these observations lead us to hypothesize that immune suppression in the lung during bacterial pneumonia involves dominance of anti-inflammatory mechanisms such as PPARγ and mTOR that induce increased generation of regulatory myeloid cells and increased expression of the anti-inflammatory mediators IL-10 and PON2 but decreased expression of host-protective mediators (IL-6, TNF-α) and cell types (MAIT cells). To prove this hypothesis, we will: Aim 1. Determine immune cell dynamics and gene signatures in PBMCs from critically ill patients and the role of mTOR in immune suppression. Aim 2. Determine the role of PPARγ and PON2 in persistent anti-inflammatory cytokine gene expression in the lungs of mice subjected to the 2-hit model.