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

Abstract

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-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).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.

MiR-608 overexpression in idiopathic pulmonary fibrosis (IPF)

“…Both targets were found to be down-regulated in IPF patient samples. Supporting these results, our recent work, showed that the IL-6R protein level is also reduced in tissue samples taken from IPF patient biopsies [ 14 ]. A recent study by Wu et al [ 13 ], suggested that Cdc42 is an important post-transcriptional regulator that may play a significant role in the process of inflammation.…”

“…To date, limited targets of miR-608 have been confirmed in-vitro, and were mostly performed in tumor cell lines [ 32 ]. Our study focused on two targets that were previously implicated in IPF, IL-6 and Cdc42 [ 13 , 14 ]. Both targets were found to be down-regulated in IPF patient samples.…”

“…They found that AT2 cells that lack Cdc42 are not able to differentiate into AT1 cells and, thus, cannot regenerate new alveoli following lung injury. Although IPF is not considered to be an inflammatory disease per se, pro-inflammatory factors, such as IL-6, TNF-alpha and IFN-y were shown to contribute to disease progression [ 14 , 33 ].…”

“…the minor ‘A-allele’ and the major ‘C-allele’, respectively), indicating weakened A-allele AChE–miR-608 interaction [ 11 ]. The impaired interaction of the A-allele of AChE with miR-608 predicted weakened AChE suppression, resulting in elevated levels of free miR-608 molecules to suppress other targets with tight miR-608 binding sites, such as Cdc42 and IL6, which were already shown to be involved in IPF progression [ 13 , 14 ]. In this study, we tested miR-608 expression and its targets in human IPF patient samples.…”

Genetic Variation in CCL18 Gene Influences CCL18 Expression and Correlates with Survival in Idiopathic Pulmonary Fibrosis—Part B

“…Given that CCL18 is associated with the progression and mortality in both SSc and IPF [ 12 , 14 ], the association of M2 macrophage phenotype with stat3 signaling in patients suffering from various types of pulmonary fibrosis including IPF may provide a molecular mechanism for CCL18 in fibrotic lung diseases [ 31 ]. In a recent study, the effect of IL-6 expression on TFG-ß related signaling pathways (STAT3, Smad3) in human lung fibroblasts derived from IPF patients could be antagonized by tocilizumab [ 32 ]. It is thus conceivable that inhibition of TGF-ß related signaling may also downregulate M2-macrophage activation and CCL18 expression.…”