TGF-β, but not IL-4, polarized macrophages induce fibrotic responses in lung fibroblasts

Macrophages are implicated in the initiation and regulation of pulmonary fibrosis, where anti-inflammatory macrophages promote fibrosis. Interleukin-4 (IL-4) and Transforming Growth Factor (TGF)-β stimulation of naïve macrophages results in the polarization to anti-inflammatory macrophages which are often used for mechanistic in vitro studies of fibrotic properties of these cells. To assess the effect of IL-4- and TGF-β-polarized macrophages on fibrotic responses of normal human lung fibroblasts (NHLFs) in order to identify the most appropriate anti-inflammatory macrophage subset for use in in vitro model systems. Peripheral blood mononuclear cells, human THP-1 monocytes and B-cell precursor leukemia cells were stimulated with IL-4 or TGF-β to induce anti-inflammatory macrophage polarization. Naïve macrophages were used as control. Whole-genome transcriptomes were analysed using RNA-sequencing. NHLFs were cocultured with polarized macrophages derived from THP-1, and conditioned medium transfer experiments were performed. Fibrotic responses were assessed by immunofluorescence. Size exclusion columns were used to separate the conditioned medium on molecular weight. Separated conditioned medium was used to stimulated NHLFs to explore the candidates in TGF-β polarized macrophages that promote activation of NHLFs. TGF-β receptor kinase inhibitor RepSox was used to block TGF-β pathway signaling in NHLFs. Coculture with TGF-β-, but not IL-4-polarized macrophages, induced α-smooth muscle actin (αSMA) levels in NHLFs. Conditioned medium from TGF-β-, but not IL-4-, polarized macrophages induced αSMA, collagen type I and fibronectin protein levels in NHLFs. Transcriptional differences between TGF-β and IL-4-polarized macrophages were analysed and showed differences in gene expression levels of secreted factors, extracellular matrix (ECM)-affiliated proteins, and ECM regulators and glycoproteins between TGF-β and IL-4-polarized macrophages. Size exclusion experiments revealed that the profibrotic effect remained intact in the conditioned medium fraction containing proteins larger than 100kD. Subsequent RNA-sequencing analysis showed increased expression of several potential high molecular weight profibrotic proteins related to TGF-β signaling pathway activation in TGF-β-polarized macrophages. Pretreatment of NHLFs with RepSox blocked the profibrotic response of NHLFs induced by conditioned medium from TGF-β-polarized macrophages. TGF-β-polarized macrophages promoted fibrotic responses of NHLFs, whereas IL-4-polarized macrophages did not; the profibrotic activity of TGF-β-polarized macrophages is mediated through activation of TGF-β signaling pathway in NHLFs.