Abstract The neurotransmitter Acetylcholine (ACh) is produced by the enzyme choline acetyltransferase (ChAT). Recently, ChAT expression was found in leukocytes, however, the function of leukocyte-derived ACh is incompletely understood. Flow cytometry showed that most ChAT-expressing cells in the spleen, lymph nodes, lung, and pleural cavity of ChAT-GFP reporter mice are B cells. B-1 cells contain the highest frequencies of ChAT-expressors. B cell-specific Mb1-Cre+ ChAT fl/flknockouts (B Chat-KO) showed comparable bone marrow B cell development and steady-state serum IgG and IgM production as wild-type littermate controls. Given earlier studies showing TLR-mediated induction of ChAT in B cells, we studied infection of ChAT-GFP mice with Influenza A/Puerto Rico/8/34. Infection resulted in an accumulation of ChAT-expressing B cells in the lungs by 1- and 3-days post infection (dpi). Moreover, B Chat-KOmice showed significant reductions in viral loads at 1 dpi, suggesting that B cell-derived ACh regulates early innate antiviral immunity. Indeed, B Chat-KOshowed increased TNFa-expressing and CD86 hilung macrophages at 1dpi. Consistent with this, acetylcholine reduced TNFa-expression in LPS-stimulated macrophages in vitro. In contrast, Lck-Cre+ ChAT fl/flthat lack ChAT in T cells showed no difference in lung viral loads at 1dpi compared to controls, and the numbers of ChAT-expressing T cells in the lungs and draining lymph nodes did not increase until 7dpi. Together these data suggest a new regulatory function for B cells in early virus-induced lung inflammation via production of ACh. - Chan Zuckerberg Initiative to Colin Reardon and Nicole Baumgarth - NIH/NIAID R01AI148652 Nicole Baumgarth