BACKGROUND Idiopathic pulmonary fibrosis (IPF) is a chronic and ultimately fatal disease characterized by a progressive decline in lung function. Fibrotic diseases, such as IPF, are characterized by uncontrolled activation of fibroblasts. Since the microenvironment is known to affect cell behavior, activated fibroblasts can in turn activate healthy neighboring cells. Thus, we investigated IPF paracrine signaling in human lung fibroblasts (HLFs) derived from patients with IPF. METHODS Primary human fibroblast cultures from IPF (IPF-HLF) and control donor (N-HLF) lung tissues were established and their supernatants were collected. These supernatants were then added to N-HLFs for further culture. Protein and RNA were extracted from IPF/ N-HLFs at baseline. Interleukin-6 (IL-6) and TGF-β-related signaling factors (e.g. STAT3, Smad3) were evaluated by western blot and qPCR. IL-6 levels were measured by ELISA. IL-6 signaling was blocked by Tocilizumab (TCZ) (10 ng/ml). RESULTS IPF-HLFs were found to significantly overexpress IL-6 receptor (IL-6R), suppressor of cytokine signaling 3 (SOCS3), phospho-STAT3-Y705 and phospho-Smad3 in comparison to N-HLFs (p < 0.05). In addition, they were found to proliferate faster, secrete more IL-6 and express higher levels of the soluble IL-6R. IPF-HLF increased proliferation was inhibited by TCZ. Moreover, IPF-HLF derived supernatants induced both direct and indirect STAT3 activation that resulted in Smad3 phosphorylation and elevated Gremlin levels in N-HLFs. These effects were also successfully blocked by TCZ. CONCLUSIONS IPF-HLF paracrine signaling leads to IL-6R overexpression, which in turn, affects N-HLF survival. The IL-6/STAT3/Smad3 axis facilitates cellular responses that could potentially promote fibrotic disease. This interplay was successfully blocked by TCZ.

中文翻译：

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IL-6反信号介导特发性肺纤维化（IPF）成纤维细胞衍生的可溶性因子激活TGF-β途径。

背景技术特发性肺纤维化（IPF）是一种慢性且最终致命的疾病，其特征在于肺功能的逐渐下降。纤维化疾病（例如IPF）的特征在于成纤维细胞的活化不受控制。由于已知微环境会影响细胞行为，因此活化的成纤维细胞可以依次活化健康的邻近细胞。因此，我们调查了源自IPF患者的人肺成纤维细胞（HLF）中的IPF旁分泌信号传导。方法建立IPF（IPF-HLF）和对照供体（N-HLF）肺组织的原代人成纤维细胞培养物，并收集其上清液。然后将这些上清液添加到N-HLF中进行进一步培养。在基线时从IPF / N-HLF中提取蛋白质和RNA。白介素6（IL-6）和TGF-β相关信号传导因子（例如STAT3，通过蛋白质印迹和qPCR评估Smad3）。通过ELISA测量IL-6水平。IL-6信号传导被Tocilizumab（TCZ）（10 ng / ml）阻断。结果与N-HLF相比，发现IPF-HLF显着过表达IL-6受体（IL-6R），细胞因子信号传导抑制因子3（SOCS3），磷酸STAT3-Y705和磷酸Smad3（p <0.05）。另外，发现它们增殖更快，分泌更多IL-6并表达更高水平的可溶性IL-6R。IPF-HLF增加的增殖被TCZ抑制。此外，IPF-HLF衍生的上清液诱导了STAT3的直接和间接活化，导致Smad3磷酸化和N-HLFs的Gremlin水平升高。这些效果也被TCZ成功阻止。结论IPF-HLF旁分泌信号传导导致IL-6R过度表达，进而影响N-HLF存活。IL-6 / STAT3 / Smad3轴促进细胞反应，可能促进纤维化疾病。TCZ已成功阻止此相互作用。