OBJECTIVE To investigate the role of the inflammatory lipid mediator leukotriene B4 (LTB4 ) and its receptor, BLT1, in the development and progression of systemic sclerosis (SSc). METHODS Serum levels of LTB4 were compared in 64 patients with SSc and 80 healthy controls. Skin and lung tissue sections from patients with SSc and healthy donors were immunostained for leukotriene A4 hydrolase (LTA4 H), the critical enzyme for LTB4 synthesis, and BLT1, in combination with different cell markers. In mouse models of SSc using bleomycin or angiotensin II challenge or immunization with the DNA topoisomerase I, genetic or pharmacologic interruption of the LTB4 -BLT1 axis in mice was carried out to assess its effects on systemic disease features and myofibroblast markers. Immunoblotting was performed to examine the signaling pathway in fibroblasts and endothelial cells following stimulation with LTB4 or with serum from SSc patients. RESULTS Serum LTB4 levels were 44.93% higher in patients with SSc than in matched healthy controls (mean ± SD 220.3 ± 74.75 pg/ml versus 152.0 ± 68.05 pg/ml; P < 0.0001), and this was associated with the patient subsets of SSc-associated interstitial lung disease and diffuse cutaneous SSc. Levels of LTA4 H and BLT1 were increased in lesional areas of the skin and lungs of SSc patients, and both were abundant in myofibroblasts and endothelial cells. Interruption of the LTB4 -BLT1 axis in mouse models of SSc significantly mitigated dermal and pulmonary fibrosis, with 54.00% and 52.65% fewer α-smooth muscle actin-positive myofibroblasts accumulating in the skin and lungs of mice, respectively, after bleomycin challenge. Immunoblotting of cultures with recombinant LTB4 -stimulated fibroblasts and endothelial cells or with serum from SSc patients showed that fibroblast-myofibroblast and endothelial-mesenchymal transitions were promoted via BLT1, and that this was dependent on activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway but independent of the release of transforming growth factor β (TGFβ) by fibroblasts or endothelial cells. CONCLUSION The LTB4 -BLT1 axis may contribute to fibrosis in SSc by directly promoting myofibroblast differentiation via the PI3K/Akt/mTOR pathway, and this appears to operate independently of autocrine secretion of TGFβ.

中文翻译：

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白三烯B4-白三烯B4受体轴在系统性硬化中促进成肌纤维细胞分化和组织纤维化。

目的探讨炎性脂质介质白三烯B4（LTB4）及其受体BLT1在系统性硬化症（SSc）的发生和发展中的作用。方法比较了64例SSc患者和80例健康对照者的血清LTB4水平。对SSc患者和健康捐献者的皮肤和肺组织切片进行免疫染色，以检测白三烯A4水解酶（LTA4 H），LTB4合成的关键酶和BLT1，并结合不同的细胞标记物。在使用博来霉素或血管紧张素II攻击或用DNA拓扑异构酶I免疫的SSc小鼠模型中，对小鼠LTB4-BLT1轴进行了遗传或药理学中断，以评估其对全身疾病特征和成肌纤维母细胞标志物的影响。用LTB4或SSc患者的血清刺激后，进行免疫印迹检查成纤维细胞和内皮细胞中的信号传导途径。结果SSc患者的血清LTB4水平比健康对照者高44.93％（平均值±SD 220.3±74.75 pg / ml与152.0±68.05 pg / ml； P <0.0001），这与SSc的患者亚型有关相关性间质性肺疾病和弥漫性皮肤SSc。SSc患者皮肤和肺部病变区域的LTA4 H和BLT1水平升高，并且在成肌纤维细胞和内皮细胞中含量均很高。在SSc小鼠模型中，LTB4-BLT1轴的中断显着减轻了皮肤和肺纤维化，在小鼠的皮肤和肺中积聚的α-平滑肌肌动蛋白阳性肌成纤维细胞减少了54.00％和52.65％，博来霉素攻击后分别进行。用重组LTB4刺激的成纤维细胞和内皮细胞或来自SSc患者血清的培养物进行的免疫印迹表明，通过BLT1促进了成纤维细胞-成肌纤维细胞和内皮-间质转化，这取决于磷脂酰肌醇3激酶（PI3K）/的激活雷帕霉素（mTOR）途径的Akt /机制靶标，但与成纤维细胞或内皮细胞释放转化生长因子β（TGFβ）无关。结论LTB4-BLT1轴可通过PI3K / Akt / mTOR途径直接促进成纤维细胞分化，从而促进SSc的纤维化，并且这似乎独立于TGFβ的自分泌分泌而起作用。用重组LTB4刺激的成纤维细胞和内皮细胞或来自SSc患者血清的培养物进行的免疫印迹表明，通过BLT1促进了成纤维细胞-成肌纤维细胞和内皮-间质转化，这取决于磷脂酰肌醇3激酶（PI3K）/的激活雷帕霉素（mTOR）途径的Akt /机制靶标，但与成纤维细胞或内皮细胞释放转化生长因子β（TGFβ）无关。结论LTB4-BLT1轴可通过PI3K / Akt / mTOR途径直接促进成纤维细胞分化，从而促进SSc的纤维化，并且这似乎独立于TGFβ的自分泌分泌而起作用。用重组LTB4刺激的成纤维细胞和内皮细胞或来自SSc患者血清的培养物进行的免疫印迹显示，成纤维细胞-成纤维细胞和内皮-间质转化是通过BLT1促进的，这取决于磷脂酰肌醇3-激酶（PI3K）/的激活雷帕霉素（mTOR）途径的Akt /机制靶标，但与成纤维细胞或内皮细胞释放转化生长因子β（TGFβ）无关。结论LTB4-BLT1轴可通过PI3K / Akt / mTOR途径直接促进成纤维细胞分化，从而促进SSc的纤维化，并且这似乎独立于TGFβ的自分泌分泌而起作用。并且这取决于磷脂酰肌醇3-激酶（PI3K）/ Akt /雷帕霉素（mTOR）途径的机械靶标的激活，但与成纤维细胞或内皮细胞释放转化生长因子β（TGFβ）无关。结论LTB4-BLT1轴可通过PI3K / Akt / mTOR途径直接促进成纤维细胞分化，从而促进SSc的纤维化，并且这似乎独立于TGFβ的自分泌分泌而起作用。并且这取决于磷脂酰肌醇3-激酶（PI3K）/ Akt /雷帕霉素（mTOR）途径的机制靶标的激活，但与成纤维细胞或内皮细胞释放转化生长因子β（TGFβ）无关。结论LTB4-BLT1轴可通过PI3K / Akt / mTOR途径直接促进成纤维细胞分化，从而促进SSc的纤维化，并且这似乎独立于TGFβ的自分泌分泌而起作用。