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

Abstract 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

Peripheral blood telomere alterations in ground glass opacity (GGO) lesions may suggest malignancy

A ground glass opacity (GGO) lung lesion may represent early stage adenocarcinoma, which has an excellent prognosis upon prompt surgical resection. However, GGO lesions have broad differential diagnoses, including both benign and malignant lesions. Our objective was to study telomere length and telomerase activity in patients with suspected lung cancer in which GGO was the predominant radiographic feature. Knowledge of telomere biology may help distinguish malignant from benign radiographic lesions and guide risk assessment of these lesions. Peripheral blood samples were taken from 22 patients with suspected adenocarcinoma with the GGO radiographic presentation. Multidisciplinary discussion confirmed the need for surgery in all cases. We used an age and gender‐matched group without known lung disease as a control. Telomere length and aggregates were assessed by quantitative fluorescence in situ hybridization (QFISH) and quantitative PCR. Cell senescence was evaluated by senescence‐associated heterochromatin foci. Subjects with GGO lesions had a higher percentage of lymphocytes with shorter telomeres (Q‐FISH, P = 0.003). Furthermore, relative telomere length was also reduced among the GGO cases (qPCR, P < 0.05). Increased senescence was observed in the GGO group compared to controls (P < 0.001), with significant correlation between the senescence‐associated heterochromatin foci and aggregate formation (r = −0.7 and r = −0.44 for cases and controls, respectively). In conclusion, patients with resectable early adenocarcinoma demonstrate abnormal telomere length and cell senescence in peripheral blood leukocytes compared to control subjects. Abnormal telomere biology in the peripheral blood may increase suspicion of early adenocarcinoma among patients with GGO lesions

Hypoxia Inducible Factor 1A Supports a Pro-Fibrotic Phenotype Loop in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. The IPF-conditioned matrix (IPF-CM) system enables the study of matrix–fibroblast interplay. While effective at slowing fibrosis, nintedanib has limitations and the mechanism is not fully elucidated. In the current work, we explored the underlying signaling pathways and characterized nintedanib involvement in the IPF-CM fibrotic process. Results were validated using IPF patient samples and bleomycin-treated animals with/without oral and inhaled nintedanib. IPF-derived primary human lung fibroblasts (HLFs) were cultured on Matrigel and then cleared using NH4OH, creating the IPF-CM. Normal HLF-CM served as control. RNA-sequencing, PCR and western-blots were performed. HIF1α targets were evaluated by immunohistochemistry in bleomycin-treated rats with/without nintedanib and in patient samples with IPF. HLFs cultured on IPF-CM showed over-expression of ‘HIF1α signaling pathway’ (KEGG, p &lt; 0.0001), with emphasis on SERPINE1 (PAI-1), VEGFA and TIMP1. IPF patient samples showed high HIF1α staining, especially in established fibrous tissue. PAI-1 was overexpressed, mainly in alveolar macrophages. Nintedanib completely reduced HIF1α upregulation in the IPF-CM and rat-bleomycin models. IPF-HLFs alter the extracellular matrix, thus creating a matrix that further propagates an IPF-like phenotype in normal HLFs. This pro-fibrotic loop includes the HIF1α pathway, which can be blocked by nintedanib