Integrin alpha-5 silencing leads to myofibroblastic differentiation in IPF-derived human lung fibroblasts.

BACKGROUND AND OBJECTIVE: The term 'fibroblast' covers a heterogeneous cell population in idiopathic pulmonary fibrosis (IPF). The fibroblasts are considered as main effector cells, because they promote disease progression by releasing exaggerated amounts of extracellular matrix proteins and modifying cell microenvironment. As IPF-derived human lung fibroblasts (IPF-HLFs) were shown to express higher levels of integrin alpha-5 (ITGA5) than normal derived HLFs (N-HLFs), we explored the importance of ITGA5 to IPF progression. METHODS: IPF-HLF and N-HLF primary cultures were established. ITGA5 was silenced by specific small interfering RNA (siRNA)s and its effects on cell phenotype (e.g. cell number, size, cell death, migration) and gene expression (e.g. RNA sequencing, quantitative polymerase chain reaction [qPCR], western blot and immunofluorescence) were tested. Specific integrin expression was evaluated in IPF patient formalin-fixed paraffin embedded sections by immunohistochemistry (IHC). RESULTS: ITGA5-silencing resulted in reduced IPF-HLF proliferation rates and cell migration (p < 0.05), as well as elevated cell death. transforming growth factor beta (TGF-ß) targets (e.g. Fibronectin (FN1), Matrix metalloproteinase 2 (MMP2), TGFB1) were surprisingly elevated following ITGA5 silencing (p < 0.05). N-HLFs, however, were only slightly affected. Interestingly, ITGA5-silenced cells differentiated into myofibroblasts (e.g. elevated alpha-smooth muscle actin [αSMA], collagen1a, large cell size). RNA-sequencing revealed that following differentiation on 3D-Matrigel for 24 h, ITGA5 levels are reduced while integrin alpha-8 (ITGA8) are elevated in IPF-HLFs. This was confirmed in IPF patients, in which ITGA5 was mainly found in fibroblastic foci, while ITGA8 was mostly observed in old fibrous tissue in the same patient. CONCLUSIONS: ITGA5 expression facilitates a more aggressive proliferative phenotype. Downregulation of this integrin results in myofibroblastic differentiation, which is accompanied by elevated ITGA8. Specific targeting could present a therapeutic benefit.

TGF-ß pathway activation by idiopathic pulmonary fibrosis (IPF) fibroblast derived soluble factors is mediated by IL-6 trans-signaling.

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-ß-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.

Epidermal growth factor receptor paracrine upregulation in idiopathic pulmonary fibrosis fibroblasts is blocked by nintedanib.

Although present in normal cells, epidermal growth factor receptor (EGFR) is overexpressed in a variety of tumors and has been associated with decreased survival. Because activated fibroblasts are considered key effectors in fibrosis and because metastatic and fibrotic processes were shown to share similar signaling pathways, we investigated the contribution of EGFR signaling to idiopathic pulmonary fibrosis (IPF) progression in lung fibroblasts derived from patients with IPF (IPF-HLF). EGFR expression and EGFR-related signaling were evaluated by Western blot and immunohistochemistry. Supernatants (SN) from cultured IPF-HLF and N-HLF were added to N-HLF, and their effect on cell phenotype was tested. Growth factor levels in the SN were measured by ELISA-based arrays. EGFR activity was blocked by erlotinib (Tarceva, 0.1-0.5 µM). Expression of EGFR, phosphorylated (p)EGFR-1068 and pAkt-473 was significantly higher in IPF-HLF compared with lung fibroblasts from control donors (N-HLF) (P < 0.05). Apparent expression of p/total EGFR and pAkt-473 was found in the myofibroblastic foci of IPF patients. Erlotinib significantly inhibited IPF-HLF but not N-HLF proliferation. IPF-HLF-SN elevated N-HLF cell number, viability, EGFR expression, and pAkt-473 and ERK1/2 phosphorylation (P < 0.05). Because high basic fibroblast growth factor levels were found in the IPF-HLF-SN, nintedanib (10-100 nM) was used to inhibit fibroblast growth factor receptor (FGFR) activation. Unlike erlotinib, nintedanib completely blocked IPF-HLF-SNs' effects on the N-HLF cells in a concentration-dependent manner. In summary, IPF-HLF paracrine signaling elevates EGFR expression, which in turn, affects N-HLF survival. The FGF-EGFR interplay facilitates cellular responses that could potentially promote fibrotic disease. This interplay was successfully blocked by nintedanib.

Fibroblast paracrine TNF-α signaling elevates integrin A5 expression in idiopathic pulmonary fibrosis (IPF).

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a poor prognosis. Inflammatory cytokines play a significant role in IPF pathology. However, the fibroblast itself is also believed to be the primary effector in IPF. We hypothesized that the fibroblasts themselves secrete pro-inflammatory cytokines that could propagate IPF by affecting normal neighboring cells. Thus, we explored the effects of IPF fibroblast derived media on normal fibroblast characteristics. METHODS: Primary IPF/normal tissue derived fibroblast cultures were established and their supernatants were collected (IPF/N-SN, respectively). These supernatants were added to normal fibroblasts. Cell death (caspase-3, western blot), proliferation, viability (WST-1), migration (scratch test) and cell detachment (crystal violet and fibronectin adhesion assays) were tested. 10 inflammatory cytokines were measured by ELISA-based quantitative array. Integrin α5 (ITGA5), pIκBα, p/total STAT3 levels were measured by western blot/IHC. TNF-α involvement was confirmed using Infliximab ®, anti-TNF-α mAb. RESULTS: The IPF-SN facilitated fibroblast cell detachment and reduced cell migration (p < 0.05). Nevertheless, these effects were reversed when cells were seeded on fibronectin. The exposure to the IPF-SN also elevated ITGA5 levels, the fibronectin receptor, in addition to NFκB pathway activation (pIκBα↑ 150%, p < 0.05). In accordance, IPF derived fibroblasts were found to express higher ITGA5 than the normal cells (44%↑, p < 0.05). ITGA5 was also expressed in the fibroblastic foci. The IPF-SN contained high TNF-α levels (3-fold, p < 0.05), and Infliximab pretreatment successfully reversed all the above observations. CONCLUSION: We suggest a possible mechanism in which IPF fibroblast secreted TNF-α modifies neighboring fibroblast cell behavior.