Pulmonary Medicine Department, Meir Medical Department, 59 Tchernichovsky St, 44281, Kfar Saba, Israel.
Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
Respir Res. 2020 Feb 18;21(1):56. doi: 10.1186/s12931-020-1319-0.
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.
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).
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.
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.
特发性肺纤维化（IPF）是一种慢性且最终致命的疾病，其特征是肺功能逐渐下降。纤维化疾病，如 IPF，其特征是成纤维细胞的失控激活。由于已知微环境会影响细胞行为，激活的成纤维细胞可以反过来激活健康的邻近细胞。因此，我们研究了源自 IPF 患者的人肺成纤维细胞（HLF）的 IPF 旁分泌信号。
从 IPF（IPF-HLF）和对照供体（N-HLF）肺组织中建立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。从 IPF/N-HLF 中提取基线时的蛋白质和 RNA。通过 Western blot 和 qPCR 评估白细胞介素-6（IL-6）和 TGF-β相关信号转导因子（例如 STAT3、Smad3）。通过 ELISA 测量 IL-6 水平。通过 Tocilizumab（TCZ）（10ng/ml）阻断 IL-6 信号。
与 N-HLF 相比，IPF-HLF 显著过表达 IL-6 受体（IL-6R）、细胞因子信号转导抑制因子 3（SOCS3）、磷酸化 STAT3-Y705 和磷酸化 Smad3（p<0.05）。此外，它们被发现增殖更快，分泌更多的 IL-6，并表达更高水平的可溶性 IL-6R。TCZ 抑制了 IPF-HLF 的增殖增加。此外，IPF-HLF 衍生的上清液诱导直接和间接的 STAT3 激活，导致 N-HLF 中的 Smad3 磷酸化和 Gremlin 水平升高。这些作用也被 TCZ 成功阻断。
IPF-HLF 的旁分泌信号导致 IL-6R 过表达，进而影响 N-HLF 的存活。IL-6/STAT3/Smad3 轴促进了可能促进纤维化疾病的细胞反应。这种相互作用被 TCZ 成功阻断。
