Pulmonary exposure to multi-walled carbon nanotubes (MWCNTs) has been reported to exert strong pro-inflammatory and pro-fibrotic adjuvant effects in mouse models of allergic lung disease. However, the molecular mechanisms through which MWCNTs exacerbate allergen-induced lung disease remain to be elucidated. We hypothesized that protease-activated receptor 2 (PAR2), a G-protein coupled receptor previously implicated in the pathogenesis of various diseases including pulmonary fibrosis and asthma, may play an important role in the exacerbation of house dust mite (HDM) allergen-induced lung disease by MWCNTs. Wildtype (WT) male C57BL6 mice and Par2 KO mice were exposed to vehicle, MWCNTs, HDM extract, or both via oropharyngeal aspiration 6 times over a period of 3 weeks and were sacrificed 3-days after the final exposure (day 22). Bronchoalveolar lavage fluid (BALF) was harvested to measure changes in inflammatory cells, total protein, and lactate dehydrogenase (LDH). Lung protein and RNA were assayed for pro-inflammatory or profibrotic mediators, and formalin-fixed lung sections were evaluated for histopathology. In both WT and Par2 KO mice, co-exposure to MWCNTs synergistically increased lung inflammation assessed by histopathology, and increased BALF cellularity, primarily eosinophils, as well as BALF total protein and LDH in the presence of relatively low doses of HDM extract that alone produced little, if any, lung inflammation. In addition, both WT and par2 KO mice displayed a similar increase in lung Cc1-11 mRNA, which encodes the eosinophil chemokine CCL-11, after co-exposure to MWCNTs and HDM extract. However, Par2 KO mice displayed significantly less airway fibrosis as determined by quantitative morphometry compared to WT mice after co-exposure to MWCNTs and HDM extract. Accordingly, at both protein and mRNA levels, the pro-fibrotic mediator arginase 1 (ARG-1), was downregulated in Par2 KO mice exposed to MWCNTs and HDM. In contrast, phosphorylation of the pro-inflammatory transcription factor NF-κB and the pro-inflammatory cytokine CXCL-1 was increased in Par2 KO mice exposed to MWCNTs and HDM. Our study indicates that PAR2 mediates airway fibrosis but not eosinophilic lung inflammation induced by co-exposure to MWCNTs and HDM allergens.

中文翻译：

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蛋白酶激活受体2（PAR2）在多壁碳纳米管加剧屋尘螨诱发的小鼠过敏性肺病中的作用

据报道，肺部暴露于多壁碳纳米管（MWCNT）可在过敏性肺病小鼠模型中产生强烈的促炎和促纤维化佐剂作用。然而，多壁碳纳米管加剧过敏原诱发的肺部疾病的分子机制仍有待阐明。我们假设蛋白酶激活受体 2 (PAR2) 是一种 G 蛋白偶联受体，之前与包括肺纤维化和哮喘在内的多种疾病的发病机制有关，可能在屋尘螨 (HDM) 过敏原诱发的恶化中发挥重要作用多壁碳纳米管引起的肺部疾病。将野生型 (WT) 雄性 C57BL6 小鼠和 Par2 KO 小鼠在 3 周内通过口咽抽吸 6 次暴露于媒介物、MWCNT、HDM 提取物或两者，并在最终暴露后 3 天（第 22 天）处死。收集支气管肺泡灌洗液 (BALF) 以测量炎症细胞、总蛋白和乳酸脱氢酶 (LDH) 的变化。检测肺蛋白和 RNA 的促炎或促纤维化介质，并对福尔马林固定的肺切片进行组织病理学评估。在 WT 和 Par2 KO 小鼠中，共同暴露于 MWCNT 会协同增加经组织病理学评估的肺部炎症，并增加 BALF 细胞结构（主要是嗜酸性粒细胞）以及 BALF 总蛋白和 LDH（在单独产生的相对低剂量的 HDM 提取物存在下）肺部炎症即使有，也很少。此外，在同时暴露于MWCNT和HDM提取物后，WT和par2 KO小鼠的肺Cc1-11 mRNA（编码嗜酸性粒细胞趋化因子CCL-11）均表现出类似的增加。然而，通过定量形态测定法测定，与 WT 小鼠相比，在同时暴露于 MWCNT 和 HDM 提取物后，Par2 KO 小鼠表现出显着较少的气道纤维化。因此，在暴露于 MWCNT 和 HDM 的 Par2 KO 小鼠中，在蛋白质和 mRNA 水平上，促纤维化介质精氨酸酶 1 (ARG-1) 均下调。相反，在暴露于MWCNT和HDM的Par2 KO小鼠中，促炎转录因子NF-κB和促炎细胞因子CXCL-1的磷酸化增加。我们的研究表明，PAR2 介导气道纤维化，但不介导因同时暴露于 MWCNT 和 HDM 过敏原而引起的嗜酸性肺部炎症。