TNF signaling mediates lipopolysaccharide-induced lung epithelial progenitor cell responses in mouse lung organoids.

Abstract

Bacterial respiratory infections are a major global health concern, often leading to lung injury and triggering lung repair mechanisms. Endogenous epithelial progenitor cells are crucial in this repair, yet the mechanisms remain poorly understood. This study investigates the response of lung epithelial progenitor cells to injury induced by lipopolysaccharide (LPS), a component of gram-negative bacteria, focusing on their regulation during lung repair. Lung epithelial cells (CD31^-CD45^-Epcam⁺) from wild-type and tumor necrosis factor (TNF) receptor 1/2 knock-out mice were co-cultured with wild-type fibroblasts. Organoid numbers and size were measured after 14 days of exposure to 100 ng/mL LPS. Immunofluorescence was used to assess differentiation (after 14 days), RNA sequencing analyzed gene expression changes (after 72 hours), and MTS assay assessed proliferative effects of LPS on individual cell types (after 24 hours). LPS treatment increased the number and size of wild-type lung organoids and promoted alveolar differentiation, indicated by more SPC⁺ organoids. RNA sequencing revealed upregulation of inflammatory and fibrosis-related markers, including Cxcl3, Cxcl5, Ccl20, Mmp13, and Il33, and enrichment of TNF-α signaling and epithelial-mesenchymal transition pathways. TNF receptor 1 deficiency inhibited LPS-induced progenitor cell activation and organoid growth. In conclusion, LPS enhances lung epithelial progenitor cell proliferation and differentiation via TNF receptor 1 signaling, highlighting potential therapeutic targets for bacterial lung injury.