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.
The ECM propagates processes in idiopathic pulmonary fibrosis (IPF), leading to progressive lung scarring. We established an IPF-conditioned matrix (IPF-CM) system as a platform for testing drug ...candidates. Here, we tested the involvement of a PGE2 and PDE4 inhibitor, Roflumilast, in the IPF-CM system. Primary normal/IPF tissue-derived human lung fibroblasts (N/IPF-HLFs) were cultured on Matrigel and then removed to create the IPF-CM. N-HLFs were exposed to the IPF-CM/N-CM with/without PGE2 (1 nM) and Roflumilast (1 µM) for 24 h. The effect of the IPF-CM on cell phenotype and pro-fibrotic gene expression was tested. In addition, electronic records of 107 patients with up to 15-year follow-up were retrospectively reviewed. Patients were defined as slow/rapid progressors using forced vital capacity (FVC) annual decline. Medication exposure was examined. N-HLFs cultured on IPF-CM were arranged in large aggregates as a result of increased proliferation, migration and differentiation. A PGE2 and Roflumilast combination blocked the large aggregate formation induced by the IPF-CM (
< 0.001) as well as cell migration, proliferation, and pro-fibrotic gene expression. A review of patient records showed that significantly more slow-progressing patients were exposed to NSAIDs (
= 0.003). PGE2/PDE4 signaling may be involved in IPF progression. These findings should be further studied.
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) (
< 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 (
< 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.
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.
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.
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.
We suggest a possible mechanism in which IPF fibroblast secreted TNF-α modifies neighboring fibroblast cell behavior.
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 NH
OH, 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,
< 0.0001), with emphasis on
(PAI-1),
and
. 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.
Levels of tumor markers in pleural effusions may help to establish the diagnosis of pleural malignancy, but the precise diagnostic value of each marker remains unclear. The aim of this study was to ...assess the diagnostic value of five common pleural fluid tumor markers, carcinoembryonic antigen (CEA), cytokeratin fragment (CYFRA) 21‐1, cancer antigen (CA) 15‐3, CA 19‐9, and CA 125, and to review the literature from the past 15 years. Pleural fluid samples were collected prospectively from 116 patients and assayed for CEA, CYFRA 21‐1, CA 15‐3, CA 19‐9, and CA 125 levels. A MEDLINE search of the English‐language literature from the past 15 years was also done.
Effusions were classified as benign or malignant on the basis of their definitive pathologic or cytologic diagnoses. The levels of all pleural tumor markers were statistically significantly higher in the malignant group than in the benign group. The marker with the highest accuracy was CEA (85.3%); CA 15‐3, CYFRA 21‐1, and CA 19‐9 had similar accuracies (75.2%, 72.4%, and 71.5%, respectively), and CA 125 had the lowest accuracy (40.5%). On univariate analysis, tumor‐marker combinations did not result in a greater accuracy than that of CEA alone. On multivariate logistic regression, CA 15‐3 and CYFRA 21‐1 were significant predictors of malignancy. Among the nine reports in the literature comparing 11 different tumor markers, CEA, CA 15‐3, and CYFRA 21‐1 yielded the best results. We conclude that pleural fluid analysis should include CEA for the diagnosis of malignancy. CA 15‐3 and CYFRA 21‐1 may serve as alternative options.
Summary Background Mycobacterium kansasii infection is one of the most common causes of nontuberculous mycobacterial lung disease in the world. However, it is not possible to differentiate completely ...between M. kansasii and other nontuberculous mycobacteria (NTM) because of a lack of direct comparative studies. This retrospective study sought to identify their clinical and radiological features systematically. Methods The sample included 98 consecutive patients with a culture-positive diagnosis of NTM infection, derived from the databases of the Laboratory of Microbiology of a tertiary medical center and two outpatient tuberculosis centers. Sixty-four patients had M. kansasii infection. All patients fulfilled disease criteria for treatment. Data on patient background and clinical features were collected, and chest radiographs were evaluated. Results In the M. kansasii group, n = 27 (42%) were native-born Israelis compared to 9.4% ( n = 3) of all other NTM groups ( p = 0.0001). Similar rates of co-morbid diseases, including diabetes mellitus, heart disease, lung diseases, and malignancy were noted in both groups. Old TB was less common in the M. kansasii group compared to the other NTM (3.1% vs. 23.5%, p = 0.003). Clinical symptoms were significantly more common in patients with M. kansasii infection. On radiological study, M. kansasii infection was associated with more cavitations and unilaterality. Patients with M. kansasii infection had a higher likelihood of right upper lobe disease ( p = 0.001). Pleural effusions and lymphadenopathy were found only in a few patients in each group. Conclusion Major differences in the epidemiologic and clinical features of M. kansasii infection and other NTM have important diagnostic and clinical implications.
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