Induction, fate and function of inflammatory fibroblasts

PROJECT SUMMARY By single cell RNA sequencing we have identified multiple molecular states of fibroblasts that emerge from normal alveolar fibroblasts in response to fibrogenic stimuli. Using lineage tracing with a new line of mice we generated that specifically marks normal alveolar fibroblasts (Scube2-CreER), we

found that a subset marked by responses to inflammatory mediators (inflammatory fibroblasts) emerges relatively early after injury in mice. ScRNA sequencing from lungs of patients with pulmonary fibrosis suggest that inflammatory fibroblasts persist, even in the setting of advanced fibrosis. Computational

pseudo time analysis suggests that inflammatory fibroblasts give rise to fibrotic fibroblasts in both mice and humans. We identified SAA3 as a highly expressed specific marker of murine inflammatory fibroblasts and have generated a new murine line that allows us to mark, delete or genetically manipulate inflammatory fibroblasts. We propose to use these mice, together with mice we have

generated to specifically target alveolar fibroblasts (Scube2-CreER) to determine the mechanisms that lead to the emergence of inflammatory fibroblasts from alveolar fibroblasts and how these cells modulate inflammatory responses and fibrosis. We will compare the timing of inflammatory fibroblastic emergence

and their persistence and fate in 2 different models of pulmonary fibrosis, induced by bleomycin or silica, and in an inflammatory model, LPS, that we have found also induces inflammatory fibroblasts but does not cause fibrosis (aim 1). We hypothesize that NF-kB signaling will be a critical driver of

inflammatory fibroblasts in all 3 models but that the upstream activators, specific inflammatory genes induced and persistence of their induction will differ between the fibrotic and non-fibrotic stimuli. We will then compare responses in all 3 models after we specifically delete inflammatory fibroblasts or modulate their ability to respond to upstream inflammatory activators to determine the

functional role of these fibroblasts in driving fibrosis and the mechanisms underlying these effects. We will delete TGFbR2 or the mechanical sensor, Piezo 2 from inflammatory fibroblasts to assess their role in fate transition to fibrotic fibroblasts (aim 2). We hypothesize that in response to bleomycin and

silica (but not LPS) a subset of inflammatory fibroblasts will transition into fibrotic fibroblasts through Piezo2-mediated responses to increased lung stiffness and TGF-b signaling, and that this transition will be critical for the development of pulmonary fibrosis. We will utilize spatial transcriptomics with single cell resolution (Xenium), immunohistochemistry and multi-color in situ

hybridization on tissue sections from normal and fibrotic human lungs to interrogate the relevance of these findings to human pulmonary fibrosis. We expect that the insights gained from these studies could lay the groundwork for developing novel therapeutic strategies for treatment of pulmonary fibrosis.