Alveolar macrophages drive lung fibroblast function in cocultures of IPF and normal patient samples.

Idiopathic pulmonary fibrosis (IPF) is characterized by increased collagen accumulation that is progressive and nonresolving. Although fibrosis progression may be regulated by fibroblasts and alveolar macrophage (AM) interactions, this cellular interplay has not been fully elucidated. To study AM-fibroblast interactions, cells were isolated from IPF and normal human lung tissue and cultured independently or together in direct 2-D coculture, direct 3-D coculture, indirect transwell, and in 3-D hydrogels. AM influence on fibroblast function was assessed by gene expression, cytokine/chemokine secretion, and hydrogel contractility. Normal AMs cultured in direct contact with fibroblasts downregulated extracellular matrix (ECM) gene expression whereas IPF AMs had little to no effect. Fibroblast contractility was assessed by encapsulating cocultures in 3-D collagen hydrogels and monitoring gel diameter over time. Both normal and IPF AMs reduced baseline contractility of normal fibroblasts but had little to no effect on IPF fibroblasts. When stimulated with Toll-like receptor (TLR) agonists, IPF AMs increased production of pro-inflammatory cytokines TNFα and IL-1ß, compared with normal AMs. TLR ligand stimulation did not alter fibroblast contraction, but stimulation with exogenous TNFα and TGFß did alter contraction. To determine if the observed changes required cell-to-cell contact, AM-conditioned media and transwell systems were utilized. Transwell culture showed decreased ECM gene expression changes compared with direct coculture and conditioned media from AMs did not alter fibroblast contraction regardless of disease state. Taken together, these data indicate that normal fibroblasts are more responsive to AM crosstalk, and that AM influence on fibroblast behavior depends on cell proximity.

Mechanomemory of pulmonary fibroblasts demonstrates reversibility of transcriptomics and contraction phenotypes.

Fibroblasts are cells responsible for producing extracellular matrix (ECM) components, which provides physical support for organs. Although these mesenchymal cells are responsive to mechanical cues in their environment, the permanence of these mechanophenotypes is not well defined. We investigated the mechanomemory of lung fibroblasts and determined how switching culture conditions modulate cell responses and function. Primary murine lung fibroblasts were isolated and cultured on 2D tissue culture plates or within 3D collagen hydrogels and were then passaged within the same or opposite culture condition to assess changes in gene expression, protein production, fibroblast subpopulation, contractile behavior, and traction forces. Compared to fibroblasts isolated on 2D tissue culture plates, fibroblasts within 3D hydrogels exhibited a decreased activation phenotype including reduced contraction profiles, diminished cell traction forces and decreased αSMA gene expression. Cells initially isolated via 2D culture and then cultured in 3D hydrogels exhibited a reversal in activation phenotype as measured by gene expression and contraction profiles. Bulk RNAseq identified groups of genes that exhibit reversible and non-reversable expression patterns. Overall, these findings indicate that lung fibroblasts have a mechanical memory that is altered by culture condition and can be reversible through precondition of cells within a softer 3D microenvironment.