A74-37 Macrophage Piezo1 Drives Myofibroblast Differentiation in Co-culture

Abstract Rationale Idiopathic pulmonary fibrosis (IPF) is a severe, progressive interstitial lung disease in which epithelial injury triggers aberrant wound repair. At the leading edge of fibrosis, immune cells such as macrophages accumulate and drive the fibroblast to myofibroblast transition. In response to soluble and mechanical signals myofibroblasts secrete extracellular matrix proteins leading to a feed forward fibrotic focus. The impact of the macrophage mechanical signal on myofibroblast differentiation remains unknown. Our lab has shown that macrophage mechanosensitive ion channels such as TRPV4 and Piezo1 are required for lung inflammation. Thus, we hypothesized that macrophage Piezo1 drives myofibroblast differentiation and fibrosis. Methods Bone marrow derived macrophages (BMDMs) and mouse lung fibroblasts (MLFs) were isolated from wild-type, Piezo1fl/fl or Piezo1LysMcre mice. To determine the role of Piezo1 on macrophage-fibroblast crosstalk, control MLFs were plated with either Piezo1fl/fl or Piezo1LysMcre BMDM conditioned media (CM) or directly co-cultured. CD45 positivity distinguished macrophages from fibroblasts. Myofibroblasts were discerned by prominent transcellular stress fibers staining for alpha smooth muscle actin (α-SMA), phalloidin by immunofluorescence and cell area, shape, and elongation. Active and total TGF-β were measured in the co-culture supernatant by mink lung epithelial cells (MLEC) and ELISA respectively. To start to identify the mechanism of macrophage-fibroblast crosstalk, gene expression differences were compared in Piezo1 sufficient and depleted macrophages by bulk RNA-sequencing and Qiagen IPA pathway analysis. Results No significant difference in myofibroblast differentiation was seen upon CM transfer from Piezo1fl/fl or Piezo1LysMcre BMDMs, suggesting macrophage Piezo1 does not mediate secretion of a pro-fibrotic soluble factor. Surprisingly, Piezo1 depleted macrophages decreased myofibroblast differentiation by 50% upon co-culture compared with BMDM controls (Piezo1fl/fl). As TGF-β is common pro-fibrotic secretary factor, we measured and found no difference in active and total TGF-β as a consequence of Piezo1 depletion. In order to find critical pro-fibrotic pathways in macrophage-fibroblast crosstalk, we detected 597 significantly different genes changed upon downregulation of macrophage Piezo1 using bulk RNA sequencing. Pathway analysis revealed changes in cell adhesion and anti-fibrotic gene expression pathways suggesting a potential macrophage Piezo1 mechanism of action. Conclusion These data show that macrophage Piezo1 drives myofibroblast differentiation in a cell-cell contact dependent manner. Mechanisms by which macrophage Piezo1 drives macrophage-fibroblast crosstalk are a necessary prerequisite to identify novel targetable pathways to ameliorate fibrosis in IPF. This abstract is funded by: NIH