Lyn expression in macrophages promotes TLR activation and restricts proliferation in an isoform-independent manner

Toll-like receptor (TLR) signaling is vital to macrophage function, and dysregulation is associated with many disease states. The Src-family kinase (SFK) Lyn serves activating and inhibitory roles downstream of TLRs, yet distinct functions of the isoforms LynA and LynB in TLR signaling have not been investigated. We used isoform-specific knockout mice (LynAKO and LynBKO) to interrogate the contribution of each isoform to TLR signaling in bone marrow-derived macrophages (BMDMs). Bulk RNA sequencing and cytokine analyses revealed that complete Lyn deficiency (LynKO) dampens TLR4- and TLR7-induced inflammatory gene expression and TNF production, but enhances extracellular matrix (ECM) gene expression and promotes cell-cycle progression and macrophage proliferation. In contrast, expression of either LynA or LynB was sufficient to preserve wild-type (WT) transcriptional responses and TNF production, despite moderately reduced total Lyn levels. These data suggest that Lyn promotes macrophage activation downstream of TLRs and restrains aberrant proliferation and matrix deposition in a largely dose-dependent rather than isoform-specific manner. Our findings provide new insight into the roles of LynA and LynB in TLR signaling and can inform future studies of macrophage-driven pa-thology in autoimmune disease, fibrosis, and cancer. Overall design: RNA sequencing of bone marrow-derived macrophages (BMDMs) from wild-type (WT), LynAKO, LynBKO, and total LynKO mice under different cell-culture conditions. BMDMs were differentiated for 7-days in macrophage-colony stimulating factor (M-CSF)-containing media, rested overnight, and then treated for 2h with M-CSF-free media (NT), lipopolysaccharide (LPS), or resiquimod (R848). Samples are biological replicates, with cells derived from 4 mice of each genotype (2 male and 2 female), independently differentiated and treated.