NGF-TRKA Facilitates the Activation of Lung Fibroblasts and Modulates the Immune Response in Pulmonary Fibrosis Via the FAK/ERK Signaling Pathway

A growing body of evidence suggests that immune dysregulation plays a pivotal role in the pathogenesis of idiopathic pulmonary fibrosis (IPF); however, the precise regulatory mechanisms remain unclear. We aim to identify key inflammatory factors and immune cells that significantly contribute to the onset and progression of IPF, and to elucidate their specific mechanisms of action. The potential causal relationships between 731 immunocyte phenotype, 41 inflammatory cytokines and IPF were conducted using bidirectional two-sample Mendelian randomization (TwoSampleMR) analysis. The candidate targets were further screened in the peripheral blood of patients with IPF, as well as in both in vivo and in vitro models of pulmonary fibrosis (PF). In vitro experiments were performed by treating MRC5 cells with nerve growth factor (NGF) and the tropomyosin receptor kinase A (TRKA) inhibitor K-252a, or by transfecting the cells with siRNA targeting TRKA. Fibroblast activation was assessed using CCK-8 assays, wound healing assays, transwell migration assays, reverse transcription quantitative PCR (RT-qPCR), and western blot analysis. Additionally, co-immunoprecipitation (CoIP) was employed to verify the interaction between NGF and TRKA in MRC5 cells. In vivo studies examined the impact of NGF on immune responses and PF, as well as the antifibrotic effects of K-252a on these parameters. These evaluations were performed through histopathological examination, immunohistochemical analysis, immunofluorescence analysis, western blotting, and enzyme-linked immunosorbent assay (ELISA). The genetically predicted onset of IPF has been correlated with the expression of NGF, which is significantly upregulated in the peripheral blood of individuals diagnosed with IPF. Furthermore, genetic analyses have implicated the involvement of two distinct immune cell populations in the initiation of IPF, with a particularly strong association observed between CD4 on CD39 + regulatory T cells (Tregs) and the risk of developing IPF (odds ratio [OR] = 1.13, 95% confidence interval [CI] = 1.00 – 1.28, false discovery rate-adjusted p-value [PFDR] = 0.0476). The cellular markers CD4, CD39 and Forkhead box P3 (FOXP3) exhibit elevated expression in murine models of PF. A positive correlation between NGF and the expression levels of CD4 and FOXP3 was also identified in patients with IPF. Mechanistically, NGF binds to its receptor TRKA, thereby activating the focal adhesion kinase / extracellular signal-regulated kinase (FAK / ERK) signaling pathway, which induces endoplasmic reticulum stress (ER stress) in pulmonary fibroblasts and promotes their activation. Additionally, NGF exacerbates bleomycin (BLM)-induced PF and enhances the expression of cellular markers characteristic of CD4 + Foxp3 + CD39 + activated Tregs, including CD4, CD39, FOXP3, interleukin-10 (IL-10), granzyme B (GZMB), and interleukin-2 receptor alpha (IL-2RA). Conversely, treatment with K-252a significantly inhibits the progression of PF and attenuates associated immunological responses.In murine models of pulmonary fibrosis, there is an increased co-localization of NGF with CD4, CD39, and FOXP3, whereas treatment with K-252a reduces this co-localization. Our findings indicate that the NGF-TRKA signaling pathway contributes to fibroblast activation and the cellular immune response in IPF. Consequently, targeting NGF-TRKA may constitute a promising therapeutic strategy for the treatment of pulmonary fibrosis.