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

Abstract

BackgroundIdiopathic 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.MethodsPrimary 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.ResultsThe 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.ConclusionWe suggest a possible mechanism in which IPF fibroblast secreted TNF-α modifies neighboring fibroblast cell behavior.

CCN1 expression by fibroblasts is required for bleomycin-induced skin fibrosis

“…2 C). Integrin alpha 5 is an integrin subunit that binds fibronectin, thrombospondin-1 promotes ECM homeostasis, and ATF3 enhances the profibrotic effects of TGFbeta and contributes to bleomycin-induced skin fibrosis [ [34] , [35] , [36] ]. Of note, neither CCN2 nor alpha-smooth muscle actin (αSMA, ACTA2) mRNA expression was reduced in CCN1-deficient skin ( Fig.…”

Fibroblast–matrix interplay: Nintedanib and pirfenidone modulate the effect of IPF fibroblast‐conditioned matrix on normal fibroblast phenotype

“…Primary human lung fibroblasts were isolated from at least six IPF (histologically confirmed) tissues and six control samples (histologically normal lung distant from the resected tumour), as described by Epstein Shochet et al . The tissue was obtained at the time of biopsy.…”

Galectin-3 levels are elevated following nintedanib treatment

“…The isolation was performed as described previously. 20 Following extraction, the HLFs were cultured in Dulbecco’s modified Eagle’s medium supplemented with 15% FCS, L-glutamine (2 mM), and antibiotics (Biological Industries, Beit-Haemek, Israel). Cells were maintained in 5% CO 2 at 37°C.…”