Nitrated Fatty Acids Reverse Cigarette Smoke-Induced Alveolar Macrophage Activation and Inhibit Protease Activity via Electrophilic S-Alkylation

Nitrated fatty acids (NFAs), endogenous products of nonenzymatic reactions of NO-derived reactive nitrogen species with unsaturated fatty acids, exhibit substantial anti-inflammatory activities. They are both reversible electrophiles and peroxisome proliferator-activated receptor γ (PPARγ) agonists, but the physiological implications of their electrophilic activity are poorly understood. We tested their effects on inflammatory and emphysema-related biomarkers in alveolar macrophages (AMs) of smoke-exposed mice. NFA (10-nitro-oleic acid or 12-nitrolinoleic acid) treatment downregulated expression and activity of the inflammatory transcription factor NF-κB while upregulating those of PPARγ. It also downregulated production of inflammatory cytokines and chemokines and of the protease cathepsin S (Cat S), a key mediator of emphysematous septal destruction. Cat S downregulation was accompanied by decreased AM elastolytic activity, a major mechanism of septal destruction. NFAs downregulated both Cat S expression and activity in AMs of wild-type mice, but only inhibited its activity in AMs of PPARγ knockout mice, pointing to a PPARγ-independent mechanism of enzyme inhibition. We hypothesized that this mechanism was electrophilic S-alkylation of target Cat S cysteines, and found that NFAs bind directly to Cat S following treatment of intact AMs and, as suggested by in silico modeling and calculation of relevant parameters, elicit S-alkylation of Cys25 when incubated with purified Cat S. These results demonstrate that NFAs’ electrophilic activity, in addition to their role as PPARγ agonists, underlies their protective effects in chronic obstructive pulmonary disease (COPD) and support their therapeutic potential in this disease.

硝基脂肪酸（Nitrated fatty acids, NFAs）是一氧化氮（NO）衍生的活性氮物种（reactive nitrogen species）与不饱和脂肪酸（unsaturated fatty acids）经非酶促反应生成的内源性产物，具备显著的抗炎活性。它们既是可逆亲电试剂（reversible electrophiles），同时也是过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptor γ, PPARγ）激动剂，但其亲电活性对应的生理意义尚不明确。本研究检测了硝基脂肪酸对烟雾暴露小鼠肺泡巨噬细胞（alveolar macrophages, AMs）中炎性及肺气肿相关生物标志物的影响。经10-硝基油酸（10-nitro-oleic acid）或12-硝基亚油酸（12-nitrolinoleic acid）处理后，炎性转录因子NF-κB（inflammatory transcription factor NF-κB）的表达与活性均被下调，而PPARγ的表达与活性则得到上调；同时，炎性细胞因子（inflammatory cytokines）、趋化因子（chemokines）以及蛋白酶组织蛋白酶S（cathepsin S, Cat S，肺气肿性间隔破坏（emphysematous septal destruction）的关键介导因子）的产生均受到抑制。组织蛋白酶S的下调伴随肺泡巨噬细胞弹性蛋白酶活性的降低，而弹性蛋白酶活性正是间隔破坏的核心机制。在野生型小鼠（wild-type mice）的肺泡巨噬细胞中，硝基脂肪酸可同时下调组织蛋白酶S的表达与活性，但在PPARγ基因敲除小鼠（PPARγ knockout mice）的肺泡巨噬细胞中仅能抑制其活性，这提示存在不依赖PPARγ的酶抑制机制。本研究推测该机制为靶标组织蛋白酶S半胱氨酸残基的亲电S-烷基化反应（electrophilic S-alkylation）；后续实验证实，硝基脂肪酸可在完整肺泡巨噬细胞（intact AMs）经处理后直接结合组织蛋白酶S，且经计算机模拟建模（in silico modeling）与相关参数计算验证，当与纯化的Cat S共孵育时，硝基脂肪酸可诱导25位半胱氨酸（Cys25）发生S-烷基化修饰。上述结果表明，硝基脂肪酸除了作为PPARγ激动剂发挥生物学功能外，其亲电活性也是其在慢性阻塞性肺疾病（chronic obstructive pulmonary disease, COPD）中发挥保护作用的核心基础，同时也进一步支持了其在该疾病中的治疗潜力。