Division of Allergy, Pulmonary, and Critical Care Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, United States.
Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, United States.
Am J Physiol Lung Cell Mol Physiol. 2024 Jan 1;326(1):L29-L38. doi: 10.1152/ajplung.00123.2023. Epub 2023 Nov 22.
Cell-free hemoglobin (CFH) is elevated in the airspace of patients with acute respiratory distress syndrome (ARDS) and is sufficient to cause acute lung injury in a murine model. However, the pathways through which CFH causes lung injury are not well understood. Toll-like receptor 4 (TLR4) is a mediator of inflammation after detection of damage- and pathogen-associated molecular patterns. We hypothesized that TLR4 signaling mediates the proinflammatory effects of CFH in the airspace. After intratracheal CFH, BALBc mice deficient in TLR4 had reduced inflammatory cell influx into the airspace [bronchoalveolar lavage (BAL) cell counts, median TLR4 knockout (KO): 0.8 × 10/mL [IQR 0.4-1.2 × 10/mL], wild-type (WT): 3.0 × 10/mL [2.2-4.0 × 10/mL], < 0.001] and attenuated lung permeability (BAL protein, TLR4KO: 289 µg/mL [236-320], WT: 488 µg/mL [422-536], < 0.001). These mice also had attenuated production of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the airspace. C57Bl/6 mice lacking TLR4 on myeloid cells only (LysM.CreTLR4) had reduced cytokine production in the airspace after CFH, without attenuation of lung permeability. In vitro studies confirm that WT primary murine alveolar macrophages exposed to CFH (0.01-1 mg/mL) had dose-dependent increases in IL-6, IL-1 β, CXC motif chemokine ligand 1 (CXCL-1), TNF-α, and IL-10 ( < 0.001). Murine MH-S alveolar-like macrophages show TLR4-dependent expression of IL-1β, IL-6, and CXCL-1 in response to CFH. Primary alveolar macrophages from mice lacking TLR4 adaptor proteins myeloid differentiation primary response 88 (MyD88) or TIR-domain-containing adapter-inducing interferon-β (TRIF) revealed that MyD88KO macrophages had 71-96% reduction in CFH-dependent proinflammatory cytokine production ( < 0.001), whereas macrophages from TRIFKO mice had variable changes in cytokine responses. These data demonstrate that myeloid TLR4 signaling through MyD88 is a key regulator of airspace inflammation in response to CFH. Cell-free hemoglobin (CFH) is elevated in the airspace of most patients with acute respiratory distress syndrome and causes severe inflammation. Here, we identify that CFH contributes to macrophage-induced cytokine production via Toll-like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) signaling. These data increase our knowledge of the mechanisms through which CFH contributes to lung injury and may inform development of targeted therapeutics to attenuate inflammation.
细胞游离血红蛋白 (CFH) 在急性呼吸窘迫综合征 (ARDS) 患者的肺泡空间中升高，并且足以在小鼠模型中引起急性肺损伤。然而，CFH 引起肺损伤的途径尚不清楚。Toll 样受体 4 (TLR4) 是在检测到损伤和病原体相关分子模式后炎症的介质。我们假设 TLR4 信号转导介导 CFH 在肺泡空间中的促炎作用。在气管内 CFH 后，TLR4 缺失的 BALBc 小鼠的炎症细胞流入肺泡空间减少[支气管肺泡灌洗 (BAL) 细胞计数，TLR4 缺失型 (KO) 中位数：0.8×10/mL [IQR 0.4-1.2×10/mL]，野生型 (WT)：3.0×10/mL [2.2-4.0×10/mL]， < 0.001]，肺通透性降低（BAL 蛋白，TLR4KO：289µg/mL [236-320]，WT：488µg/mL [422-536]， < 0.001）。这些小鼠在肺泡空间中也产生了更少的白细胞介素 (IL)-1β、IL-6 和肿瘤坏死因子 (TNF)-α。仅在髓样细胞上缺乏 TLR4 的 C57Bl/6 小鼠（LysM.CreTLR4）在 CFH 后在肺泡空间中产生的细胞因子减少，而肺通透性没有降低。体外研究证实，WT 原代小鼠肺泡巨噬细胞暴露于 CFH（0.01-1mg/mL）时，IL-6、IL-1β、CXC 基序趋化因子配体 1 (CXCL-1)、TNF-α 和 IL-10 的产生呈剂量依赖性增加（ < 0.001）。鼠 MH-S 肺泡样巨噬细胞在 CFH 作用下显示 TLR4 依赖性表达 IL-1β、IL-6 和 CXCL-1。缺乏 TLR4 衔接蛋白髓样分化初级反应 88 (MyD88) 或 TIR 结构域包含衔接诱导干扰素-β (TRIF) 的 TLR4 缺失型小鼠的原代肺泡巨噬细胞显示，MyD88KO 巨噬细胞 CFH 依赖性促炎细胞因子产生减少 71-96%（ < 0.001），而 TRIFKO 小鼠的巨噬细胞细胞因子反应发生变化。这些数据表明，髓样 TLR4 信号通过 MyD88 是 CFH 对肺泡空间炎症反应的关键调节因子。细胞游离血红蛋白 (CFH) 在大多数急性呼吸窘迫综合征患者的肺泡空间中升高，并导致严重的炎症。在这里，我们确定 CFH 通过 Toll 样受体 4 (TLR4) 和髓样分化初级反应 88 (MyD88) 信号转导，导致巨噬细胞诱导的细胞因子产生。这些数据增加了我们对 CFH 导致肺损伤的机制的了解，并可能为开发靶向治疗以减轻炎症提供信息。
