Single-cell RNA sequencing of digested whole lungs of mb1Cre-/-ChATfl/fl and mb1Cre+/-Chatfl/fl mice taken before and at 24h after infection with a sublethal dose of Influenza A/Puerto Rico/8/24

Respiratory viral infections can lead to dysregulated inflammatory responses in the lungs. Acetylcholine (ACh), synthesized by the enzyme choline acetyltransferase (ChAT), is a well-known neurotransmitter but has also been proposed as an immunomodulator and a potential mediator of inflammation. Our findings demonstrate that ACh treatment inhibits lung macrophage activation in vitro and intranasal ACh administration 12h prior to infection with influenza A/Puerto Rico/8/34 reduces lung inflammation in vivo in mice 24h following influenza virus infection. Furthermore, we identified B cells as the primary source of ChAT in mice, both under steady-state conditions and during the first 7 days post influenza infection. Based on these findings, we hypothesized that B cell-derived ACh regulates lung innate immunity. To investigate this, we performed single-cell RNA sequencing on 4 samples using the Standard 3' 10X Chromium platform on >85% viable lung single-cell suspensions from 1 sample each of control (mb1Cre-/-Chatfl/fl) and ChATBKO (mb1Cre+/-Chatfl/fl) mice, collected before infection and 24 hours post-infection. Each sample (n=4) contained cells pooled from 2 mouse lungs. Our analysis identified 15 distinct cell clusters of lung cells. Mice lacking B cell-derived ACh showed transcriptional changes among lung interstitial macrophages but not alveolar macrophages, specifically affecting TNF-α signaling via NF-κB and apoptosis pathways. Prior to and following intranasal influenza A/PR8 infection, live lung cell suspensions were isolated using fluorescently activated cell sorting (FACS) (>85%) with propidium iodide to distinguish live and dead cells. The isolated cells were then submitted for library preparation and Standard 3' Chromium single-cell RNA sequencing technology. Four different samples were compared: S1 WT (mb1Cre-/-Chatfl/fl) non-infected S3 ChATBKO (mb1Cre+/-Chatfl/fl) non-infected S5 WT (mb1Cre-/-Chatfl/fl) 24h post-infection S7 ChATBKO (mb1Cre+/-Chatfl/fl) 24h post-infection Single cell suspension of lung parenchyma from 2 female,12 wk-old mice were pooled together for each sample