Amer Johnny, Salhab Ahmad, Abuawad Mohammad
Department of Allied Sciences, Faculty of Medicine and Health Sciences, An-Najah National University, Nablus, Palestine.
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, An-Najah National University, Nablus, Palestine.
Front Immunol. 2025 Jan 7;15:1466802. doi: 10.3389/fimmu.2024.1466802. eCollection 2024.
NK cells and NK-cell-derived cytokines were shown to regulate neutrophil activation in acute lung injury (ALI). However, the extent to which ALI regulates lung tissue-resident NK (trNK) activity and their molecular phenotypic alterations are not well defined. We aimed to assess the impact of 1,25-hydroxy-vitamin-D3 [1,125(OH)D] on ALI clinical outcome in a mouse model and effects on lung trNK cell activations.
Oleic acid (OA)-induced ALI in C57BL/6J mice and 1,25(OH)D treatment 2×/2 weeks were performed. Lung tissue was harvested to assess alveolar I/II cell apoptosis and lung injury marker of Surfactant-Protein-D (SP-D). Pulmonary edema markers of epithelial sodium channel, cystic fibrosis transmembrane conductance regulator, and aquaporin 5 were assessed by RT-PCR. Lung trNK cells were assessed for activation markers of CD107a and NKp46, vitamin D receptor (VDR), and programmed cell death protein-1 (PD-1) via flow cytometry. The bronchoalveolar lavage fluid (BALF) obtained was investigated for soluble receptor for advanced glycation end products (sRAGE), inflammatory cytokines, soluble 1,25(OH)D, and PDL-1. Naïve mice treated with DMSO (vehicle) were used as a control.
Flow cytometry analysis displayed a high apoptotic rate in alveolar I/II cells of threefold in ALI mice as compared to naïve mice. These findings were accompanied by elevated markers of pulmonary edema as well as lung injury markers of SP-D. Isolated lung trNK cells of the ALI mice exhibited reduced CD107a and NKp46 markers and cytotoxicity potentials and were correlated through significantly 2.1-fold higher levels of PD-1 and diminished VDR expressions as compared to naïve mice. BALF samples of ALI mice displayed high soluble PDL-1 and reduced soluble 1,25(OH)D levels compared to naïve mice. 1,25(OH)D treatment alongside OA led to a significant fourfold increase in the CD107a and NKp46 expressions to levels higher than the mice treated with the vehicle. Furthermore, 1,25(OH)D ameliorates free radical scavengers of GSH, GPX, CAT, and GPx-1; decreased pro-inflammatory cytokines and soluble PDL-1; and increased soluble 1,25(OH)D with amelioration in pulmonary edema markers and alveolar I/II apoptosis.
Our results indicate 1,25(OH)D's potential therapeutic effect in preventing clinical outcomes associated with ALI via regulating NK cells through inhibiting inflammatory cytokines and alleviating levels of PDL-1 and 1,25(OH)D released by lung tissue.
自然杀伤（NK）细胞及NK细胞衍生的细胞因子可调节急性肺损伤（ALI）中的中性粒细胞活化。然而，ALI对肺组织驻留NK（trNK）细胞活性的调节程度及其分子表型改变尚不明确。我们旨在评估1,25 - 二羟维生素D3 [1,25(OH)D]对小鼠模型中ALI临床结局的影响以及对肺trNK细胞活化的作用。
在C57BL/6J小鼠中诱导油酸（OA）所致的ALI，并进行为期2周、每周2次的1,25(OH)D治疗。采集肺组织以评估肺泡I/II型细胞凋亡及表面活性蛋白D（SP - D）这一肺损伤标志物。通过逆转录聚合酶链反应（RT - PCR）评估上皮钠通道、囊性纤维化跨膜传导调节因子及水通道蛋白5的肺水肿标志物。通过流式细胞术评估肺trNK细胞的CD107a和NKp46活化标志物、维生素D受体（VDR）及程序性细胞死亡蛋白1（PD - 1）。对所获支气管肺泡灌洗液（BALF）进行晚期糖基化终末产物可溶性受体（sRAGE）、炎性细胞因子、可溶性1,25(OH)D及程序性死亡配体1（PDL - 1）的检测。用二甲基亚砜（DMSO，溶剂）处理的未致敏小鼠作为对照。
流式细胞术分析显示，与未致敏小鼠相比，ALI小鼠肺泡I/II型细胞凋亡率高三倍。这些结果伴有肺水肿标志物及SP - D肺损伤标志物升高。ALI小鼠分离出的肺trNK细胞表现出CD107a和NKp46标志物及细胞毒性潜能降低，与未致敏小鼠相比，PD - 1水平显著高2.1倍且VDR表达降低。与未致敏小鼠相比，ALI小鼠的BALF样本显示可溶性PDL - 1水平高且可溶性1,25(OH)D水平降低。与OA同时进行的1,25(OH)D治疗使CD107a和NKp46表达显著增加四倍，达到高于用溶剂处理小鼠的水平。此外，1,25(OH)D改善了谷胱甘肽（GSH）、谷胱甘肽过氧化物酶（GPX）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶1（GPx - 1）的自由基清除剂；降低了促炎细胞因子和可溶性PDL - 1；增加了可溶性1,25(OH)D，同时改善了肺水肿标志物及肺泡I/II型细胞凋亡。
我们的结果表明，1,25(OH)D通过抑制炎性细胞因子、减轻肺组织释放的PDL - 1和1,25(OH)D水平来调节NK细胞，从而在预防与ALI相关的临床结局方面具有潜在治疗作用。
