B cells modulate lung antiviral inflammatory responses via the neurotransmitter acetylcholine

The rapid onset of innate immune defenses is critical for early control of viral replication in an infected host, yet it can also lead to irreversible tissue damage, especially in the respiratory tract. Intricate regulatory mechanisms must exist that modulate inflammation, while controlling the infection. Here, B cells expressing choline acetyl transferase (ChAT), an enzyme required for production of the metabolite and neurotransmitter acetylcholine (ACh) are identified as such regulators of the immediate early response to influenza A virus. Lung tissue ChAT + B cells are shown to interact with a7 nicotinic Ach receptor-expressing lung interstitial macrophages in mice within 24h of infection to control their production of TNFa, shifting the balance towards reduced inflammation at the cost of enhanced viral replication. Thus, innate-stimulated B cells are key participants of an immediate-early regulatory cascade that controls lung tissue damage after viral infection.

TNFa regulates viral loads and is a target of ACh During viral respiratory tract infections, interstitial (IMs) and alveolar macrophages (AMs) are critical cellular components of the innate line of immune defense 1 .Overshooting activation can cause a local and systemic cytokine storm with detrimental effects on host survival, while following insu cient activation of the innate response or infection with high viral loads may result in a failure to control virus replication, leading to enhanced CD8 T cell-mediated immunopathology potentially resulting in host death [61][62][63][64][65][66][67][68] .
To assess the effects of ACh on lung macrophage in ammatory responses, we applied ACh to either total lung leukocytes or enriched lung-derived IMs, which were also stimulated with LPS.Application of ACh to lung cell suspensions led to a more than 50% reduction in the frequencies of TNFa generating CD64 + F4/80 + total macrophages, as well as CD11b-CD11c + SiglecF + AMs and CD11b + CD11c-SiglecF-IMs (Fig. 1a).Consistent with a direct effect on macrophages, ACh reduced TNFa production by enriched lung IMs in a dose-dependent manner (Fig. 1b).Next, ACh and its stabilizer, the acetylcholinesterase (AChE) inhibitor pyridostigmine bromide (PB) were applied intranasally to mice 12h prior to, at the time of, and 24h after intranasal infection with in uenza A/Puerto Rico/8/34 (A/PR8) (Fig. 1c).In this sublethal viral infection model, virus replication and innate-driven in ammation peaks within the rst 2-3 days, and virus-dose dependent weight loss peaks around 7-9 days post-infection (dpi) (Extended Data Fig. 1a).Differences in macrophage responses following intranasal ACh application were observed consistently in AMs and IMs at 24h after infection in a ACh dose-dependent manner.Intranasally applied ACh affected macrophages in both, the airways and the lung parenchyma.Application of ACh enhanced AM numbers in the lung parenchyma (Fig. 1d), suggesting their migration from the airways into the tissue.Both, the frequencies (Fig. 1e) and expression levels (Fig. 1f) of costimulatory marker CD86 and of MHCII by total lung macrophages decreased in an ACh dose-dependent manner, while surface expression levels of the activation inhibitor CD206 + increased (Fig. 1e).IMs showed dose-dependent decreases in CD86, MHCII, and CD64 expression (Fig. 1g).Moreover, in vitro LPS stimulation of total lung single cell suspensions from ACh-treated mice showed decreased macrophage TNFa expression levels in an ACh dose-dependent manner (Fig. 1h).Consistent with decreased in ammation, qRT-PCR analysis of lung homogenates from ACh-treated mice showed signi cant decreased expression of pro-in ammatory genes tnfa, il6, il1b, and chemokines ccl2, cxcl1, ccl5 and ccl7 compared to controls, while Il10, ifng, ifna1 and ifnb were unaffected (Fig. 1i).Thus, ACh can modulate lung tissue AMs and IMs, reducing their levels of activation, as well as chemokine and cytokine expression within 24h of a respiratory tract viral infection.
TNFa and Nfkb signaling pathways are known regulators of early innate responses to in uenza infection 69 .Consistent with those ndings, lung IMs but interestingly not AMs, isolated from A/PR8 infected C57BL/6 mice at 1 dpi showed increased TNFa production after in vitro restimulation compared to those from non-infected mice.Increasing the dose of in uenza A/PR8 used for infection also increased TNFa expression by IMs but again not AMs (Extended Data Fig. 1b).Early elaborated TNFa signi cantly contributed to control of in uenza A/PR8 viral replication, as in vivo blockade with anti-TNFa mAb administered prior to and at the time of infection (Fig. 1j) signi cantly increased lung viral loads at 1 dpi compared to infected and sham treated C57BL/6 controls (Fig. 1k).

B cells are the dominant ChAT expressing leukocyte population in the lung controlling In uenza A virus infection and TNFa production by interstitial macrophages
Given previous ndings that T cell-derived ACh can modulate macrophage responses, we performed ow cytometry on pleural cavity lavage uid, lung parenchyma, mediastinal lymph nodes (MedLN) and spleen of ChAT-GFP reporter mice 31 to assess for the presence of leukocytes capable of generating ACh in the respiratory tract (Fig. 2a-d and Extended Data Fig. 2a-d).In all tissues analyzed, by frequency and total cell count, most ChAT + cells were CD19 + B cells (Fig. 2c, d and Extended Data Fig. 2c, d).In pleural cavity and lung parenchyma, nearly 50% and 10% of B cells, respectively, expressed ChAT (Fig. 2b).
Unsupervised clustering of ow cytometry data revealed distinct clusters of ChAT-expressing B cells in the lung and pleural cavity (Extended Data Fig. 2e-g).ChAT + B cells were predominantly CD5+/-, CD19+, CD43+, IgM hi and IgD lo , CD138-, thus mostly, albeit not exclusively, B-1 cells (Fig. 2e and Extended Data Fig. 2h-l), consistent with a previous study 31 .While B-1 cells dominated ChAT expression in the pleural cavity by frequency and total numbers, due to their larger total cell numbers, conventional mature B cells outnumbered B-1 cells in spleen, lung, and MedLN (Extended Data Fig. 2h-l).
In the bone marrow (BM), ChAT expression was observed only rarely among pro-and pre-B cells (Hardy fractions A-D) but was seen at increased frequencies at the immature B cell stage (B220 hi IgM + IgD −/lo CD93+; Hardy Fraction E) (Extended Data Fig. 3a, b).Similar to peripheral tissues, BM cells with a mature, B-1-like cell phenotype (CD45R+/lo CD93-, CD43+/-, IgDlo, IgM+) displayed the highest frequencies of ChAT expression in the BM (Extended Data Fig. 3a, b).
ChAT-GFP was induced in spleen FO B cells with LPS but only marginally after anti-IgM stimulation, consistent with previous data suggesting ChAT-expression was MyD88-dependent 31 (Fig. 2f and Extended Data Fig. 4a).This may explain the relative large frequencies of ChAT-GFP + B-1 cells, as they respond more strongly to TLR-mediated signaling compared to conventional B cells and require MyD88 expression for survival and differentiation 70 .Stimulation of splenic B cells under conditions driving B cell differentiation reduced ChAT-GFP expression.This was seen following stimulation with both, LPS plus IL-4 and IL-5, as well as stimulation with anti-IgM, CD40L, IL4 and IL5 (Extended Data Fig. 4b, c).Similar results were obtained with sorted ChAT neg B-1 B cells, ruling out preferential activation of ChAT negative B cells (Extended Data Fig. 4d).Consistent with these ndings, plasmablasts and plasma cells obtained from respiratory tract draining MedLN of ChAT-GFP mice infected for 7 days with in uenza A/PR8 lacked ChAT-GFP expression (Extended Data Fig. 4e).The data suggest an innate-like, immediate-early role for ChAT-expressing B cells, independent of further differentiation.
To determine whether B cell derived ACh regulates early respiratory tract responses to viral infections, we A/PR8 infected mice with a B cell-speci c deletion of ACh (mb-1Cre +/− ChAT / mice, ChatBKO).
ChatBKO mice showed 10-fold reductions in lung viral loads at 1 dpi with in uenza A/PR8 compared to mb-1Cre −/− ChAT / control mice (Control, Fig. 2g), consistent with relatively high constitutive expression of ChAT by B cells prior to infection.While leukocyte-derived ACh effects in the spleen have been shown previously to require T cell-mediated ACh production 55,56 , T cell-speci c deletion of ChAT (Chat / -Lck Cre+/− ) did not affect in uenza virus loads at 1 dpi (Fig. 2h), consistent with their low frequencies in all tissues prior to infection (Extended Data Fig. 2).B cells remained the predominant population of ChAT-GFP + cells at early infection timepoints (Extended Data Fig. 5a-e).In addition, signi cant increases in absolute, but not relative, numbers of ChAT-GFP + B cells were measured at 1 and 3 dpi in the lungs of ChAT-reporter mice (Extended Data Fig. 5a-c).No accumulation of ChAT-GFP + T cells in the lungs were observed until 7 dpi, a time when in uenza-speci c CD4 T cells are known to enter the lung in large numbers and virus is largely cleared from the lungs (Extended Data Fig. 5d, e).Together these data indicate that innate signal-induced ChAT expressing respiratory tract B cells rapidly respond to innate stimulation to affect in uenza A virus replication in the lung.If primed and activated ChAT + T cells affect immune responses to in uenza infection via ACh release, they would not do so until later timepoints 71,58 .
Given the decreased control of in uenza virus replication in mice in which TNFa signaling was blocked (Fig. 1j), the effects of B cells on control of macrophage function during in uenza infection were evaluated by measuring TNFa production in µMT −/− mice, which lack mature B cells in all tissues, including the lungs (Extended Data Fig. 6a).Cells isolated from the lungs of control and µMT-/-mice at 1 dpi with A/PR8 showed signi cant increases in TNFa production by IMs but not AMs from µMT mice following in vitro restimulation compared to controls, supporting a modulating effect of B cells on IMs but not AMs immediately early after infection (Extended Data Fig. 6b).µMT −/− mice also showed increased lung monocyte in ltration and decreased AM numbers, suggesting that the absence of B cells caused enhanced in ammation and perhaps increased AM apoptosis (Extended Data Fig. 6c) [72][73][74] .Furthermore, µMT −/− mice had reduced frequencies of macrophages expressing the inhibitory receptor CD206 and decreased CD206 expression levels, but higher surface expression of activation markers F4/80, CD11b and CD64 (Extended Data Fig. 6d) and in IMs (Extended Data Fig. 6e).Thus, B cells regulate the activation state of IMs, but not AMs, in the respiratory tract immediately early after in uenza virus infection.
To determine the extent to which these effects of B cells were facilitated by their secretion of ACh, A/PR8 infected ChATBKO mice were analyzed at 1 dpi.Consistent with a major role for B cell derived ACh, ChatBKO derived IMs showed increased TNFa production as well as increased expression of CD86 very similar to IMs in µMT −/− mice, while AMs were unaffected (Fig. 2i, Extended Data Fig. 6f).Moreover, qRT-PCR analysis of lung homogenates from ChatBKO revealed increased expression of proin ammatory cytokine genes like il6, tnfa, il1b, and csf2, while il10 showed minimal change or downregulation compared to controls.ifna1 expression was reduced in the absence of B cell derived ACh, consistent with reductions in lung viral loads in the ChatBKO mice compared to controls (Extended Data Fig. 6g and Fig. 2g).
Depletion of ChAT in B cells did not signi cantly affect neutrophil numbers at 1dpi with A/PR8 (Extended Data Fig. 6h), a population that was shown previously to be affected by B cell derived ACh in a sepsis model 31 .Slight increases in lung monocyte and neutrophil cell counts, however, were noted by 7 dpi in ChatBKO mice (Extended Data Fig. 7a).Histolopathological evaluation of the respiratory tract at 7 dpi suggested enhanced epithelial degeneration in the nasal cavities and poorer overall health of ChATBKO mice compared to the controls (Extended Data Fig. 7b, c).The analysis also showed heightened CD8 T cell in ltration into the lungs and increased NK cells in the spleen, indicating enhanced systemic in ammation (Extended Data Fig. 7d, e).This was despite similar lung viral loads of control and ChatBKO mice by 7 dpi (Extended Data Fig. 7f), suggesting insu cient control of lung in ammation as main causes of increased pathology and immune cell activation.Altogether, the data demonstrate that B cell derived ACh acts on IMs but not AMs to inhibit lung in ammatory responses to in uenza infection at 1 dpi, inhibiting early control of lung viral loads, but signi cantly modulating production of various proin ammatory cytokines and chemokines and reducing the impact of the respiratory tract infection both locally and systemically.

B cells regulate innate immune cells in the lung parenchyma via ACh production
Previous studies demonstrated that immediate early in uenza virus infection is controlled in part by natural IgM production, secreted mostly by B-1 cells 75,76 , which we show here to be a ready source for ACh.However, lack of ChAT expression by B cells did not signi cantly affect total or virus-binding IgM levels (Extended Data Fig. 8a, b), further supporting an antibody independent role for B cells in inhibiting IMs' ability to secrete TNFa and in promoting viral replication.Similarly, the lack of B cell-expressed ChAT had no effect on extrafollicular plasmablast development or the frequencies of germinal center B cells (Extended Data Fig. 8c), in uenza-speci c IgM or IgG antibody-secreting cells in the MedLN at 7 and 14 dpi (Extended Data Fig. 8d), nor on serum in uenza-speci c IgM or IgG subclasses over a 4-week timecourse (Extended Data Fig. 8e).Deletion of ChAT in B cells also did not affect total numbers of innate leukocytes, T or B cell subsets in the spleen, or bone marrow B cell development when compared to control mice 77 (Extended Data Fig. 9a-e).The exception was a slight but signi cant reduction in the number of CD5 + B-1 cells in ChATBKO mice compared to ChAT x/ x Cre-negative controls (Extended Data Fig. 9f).Studies with non-oxed and Cre-expressing mb-1 Cre−/− ChAT +/+ mice showed similar reductions, suggesting that this effect on B-1 cell numbers was driven by mb-1 haploinsu ciency rather than ChAT expression (Extended Data Fig. 9g) [78][79][80]81 .
To assess the impact of B cell-speci c ACh generation on respiratory tract leukocytes we conducted single-cell RNA sequencing (scRNA-Seq), comparing cells from lung parenchyma of Control and ChatBKO mice prior infection (n = 4/group) (Fig. 3a).Post sample integration analysis revealed 16 distinct cell clusters within the lung parenchyma (Fig. 3b).Clusters 7, 9, 11, and 13 were CD45 neg and classi ed as non-immune cells, and the remainder were CD45 + leukocytes (Extended Data Fig. 10a and Supplemental Data 1).Among CD45 + leukocytes, B cells were identi ed as clusters 0, 6, 8, and 12, while T/NKT cells were present in clusters 15, 5, 2, and 3 and NK cells in cluster 1 (Extended Data Fig. 10b-d and Supplemental Data 1).Clusters 10, 14, and 4 represented myeloid cell compartments (Extended Data Fig. 10e-h and Supplemental Data 1).Cluster 10 exhibited markers indicative of AMs, cluster 14 granulocytes, and cluster 4 monocyte/monocyte-derived macrophages or IMs (Extended Data Fig. 10eh).The cluster 4 IMs expressed markers of activation, including tnf, socs3, cd80, and cd86 (Supplemental data 1).No notable differences were observed in lung cell subset frequencies between Control and ChatBKO mice (Extended Data Fig. 10i).To identify potential targets of B cell derived ACh, we compared the transcriptional pro les of lung parenchyma cell clusters from Control and ChatBKO mice.Amongst CD45 neg clusters, only cluster 11 showed signi cant differences in gene expression, as assessed by GSEA.Those differences included pathways associated with an IFN-g responses, IFN-a response and the PI3K-AKT-mTOR signaling pathway, all of which showed increases in the absence of B cell derived ACh (Supplemental data 2).Amongst the CD45 pos clusters, most B cells (cluster 0, 6 and 8) and CD8 T cells lacked signi cant changes (Supplemental data 2).NK and NKT cells (clusters 1 and 3) showed differential expression of immune-related pathways, including lower TNFa signaling via Nfkb pathway (Supplemental data 2).Amongst the myeloid clusters, AMs (cluster 10) showed no differences, while both granulocytes (cluster 14) and the biggest myeloid cluster (cluster 4), consisting of monocytes and IMs, showed the strongest differences between Control and ChatBKO mice with statistically signi cant differences in expression levels of 700 genes (Fig. 3c, Extended Data Fig. 10j and Supplemental Data 2).GSEA demonstrated upregulation of several hallmark pathways in IMs of ChatBKO mice, including kras signaling, myc targets V1, and notably, the apoptosis and the tnfa signaling via nfkb pathways (p adj <0.05 and p adj <0.0001) (Fig. 3d, e).The apoptosis genes dap, pmaip1, anxa1, mcl1, and activation and immunerelated genes pnrc1, nfe2l2, ccl4, rel, tnf, il1b, and cd83 were genes driving the difference, consistent with the ow cytometric data, suggesting that inhibition of viral replication via TNFa may occur through increased apoptosis 82,83 .Also consistent with the functional data obtained after in uenza infection, transcriptional analysis revealed no signi cant differences in the subset classi ed as AMs (Fig. 3f, g and Extended Data Fig. 10k).Thus, the lack of B cell derived ACh affected IMs but not AMs, further demonstrating the speci c impact of ChAT + B cells on monocyte/monocyte-derived macrophages/IMs.Flow cytometry supported the gene expression differences, with IMs from non-infected ChatBKO mice displaying increased TNFa production upon short-term restimulation with LPS in vitro, both by frequency and MFI, compared to controls (Fig. 3h and Extended Data Fig. 11a-c).Notably, the scRNA-sequencing data con rmed that changes among lung macrophages were restricted to IMs, as no signi cant differences were observed in AMs from ChatBKO and control mice regarding frequencies or total cells of TNFa producers, albeit a slight difference in TNFa MFI was observed (Fig. 3h and Extended Data Fig. 11a-c).Further characterization revealed signi cant increases in F4/80 expression on IMs and subtle differences in surface expression of CD11b and CD206 (Extended Data Fig. 11b, c) in cells from ChatBKO mice.These ndings indicate that B cell derived ACh signi cantly alters the functionality of lung IMs, but not AMs, revealing a distinct regulatory pathway by which B cells regulate in ammatory responses in the respiratory tract.

B cell derived ACh directly inhibits IMs via the α7 nicotinic ACh receptor (a7nAchR)
Given the overall ability of AMs to respond to ACh (Fig. 1a), the data suggest that the distinct and selective effects of ChAT + B cells on some myeloid cell clusters was driven by the location of B cells in the respiratory tract, rather than their differential ability to respond to ACh.Indeed, B cells are readily found in the lung interstitium, in fact they constituted the largest cluster of leukocytes by scRNAseq (Fig. 3b), but they are not present in the airways of not previously infected mice 84,85 .Given the extremely short half-life of ACh, this suggests that ChAT + B cells may exert a direct effect on IMs.
To assess this, allotypically marked lung IMs from CD45.1 + C57BL/6 wildtype (WT) mice were enriched to > 75% by magnetic cell separation and adoptively transferred intranasally into either CD45.2 + C57BL/6 WT controls or CD45.2 + ChatBKO, followed by infection with in uenza A/PR8.24h later, lung single cell suspensions were stimulated in vitro for 4h in the presence of LPS and Brefeldin A to assess cytokine production by the adoptively transferred macrophages (Fig. 4a).Signi cantly greater TNFa responses were seen from the transferred IMs placed into ChatBKO compared to those placed into Control mice (Fig. 4b).Thus, ruling out differences in IMs development and/or epigenetic changes in ChatBKO mice as a reason for their enhanced in ammatory responses following in uenza infection.In further support of direct B cell -IMs interaction, confocal microscopy revealed the co-localization of B220 (CD45R) + B cells and F4/80 + macrophages in the lung interstitium of ChAT-GFP reporter mice at 1 dpi with in uenza (Fig. 4c and Supplemental Fig. 3).GFP + B cells were often seen among small clusters of GFP-B cells, and in close proximity to one or more F4/80 + macrophages (Fig. 4c and Supplemental Fig. 3).These small clusters do not represent bronchus associated lymphoid tissues, which are not typically observed this early after infection of naïve mice.
Cells respond to ACh via several nicotinic and/or muscarinic cholinergic receptors.The inhibitory effects of T cell derived ACh on splenic macrophages were shown to depend on the a7 nicotinic (n)ACh receptor (R) [86][87][88][89][90][91][92] .Revealing a role for a7nAChR also in controlling in ammatory responses of lung macrophages by ACh, increased TNFa generation was observed in both AMs and IMs from 1 day in uenza A/PR8 infected mice that lacked this receptor (acra7-/-) compared to controls (Fig. 4d).
Additional IM cell adoptive transfer experiments were conducted to probe further for a direct impact of B cell derived ACh on IMs (Fig. 4e-f).For that equal numbers enriched IMs from allotype disparate 45.2 + acra7-/-mice and CD45.1 + WT controls were transferred i.n.into WT CD45.1/2 double-positive C57BL/6 mice which were then infected with in uenza A/PR8 (Fig. 4e).24h later, lung cells were stimulated in vitro with LPS, demonstrating signi cantly enhanced TNFa production in the CD45.2 + acra7-/-IMs compared to the co-transferred WT CD45.1 + cells (Fig. 4e).In contrast, when cell tracker dye labeled WT (CTV) and α7nAChR-de cient (eF670) CD45.2 + IMs were co-transferred into ChatBKO mice, which were subsequently infected for 24h, no signi cant difference in TNFa production was seen between acra7-/and WT cells (Fig. 4f).We conclude that B cells directly modulate cytokine production by lung IMs via ACh production immediately early after a respiratory tract infection.

Discussion
Immediate early induction of cytokines and chemokines is needed to orchestrate a strong and protective innate immune response to viral infections.The quality and magnitude of this response not only directly affects virus replication it also signi cantly effects the long-term consequences of this immune activation, balancing the need for viral replication control with the requirement to avoid host tissue damage.Lung IMs are increasingly recognized as critical components of infection-induced in ammatory cytokine and chemokine responses as well as facilitators of tissue repair.Their controlled activation is thus critical in balancing host immune responses.Here we demonstrate a new regulatory axis by which a7AChR + lung macrophages are regulated by ChAT + lung tissue B cells, the latter found co-localized with macrophages in the lung parenchyma but not the airways (Extended Data Fig. 12).The B cells' ability to generate ACh appears to directly regulate IM activation and TNFa production, and with it both, the control of in uenza virus replication.Its absence resulted in enhanced elaboration of other proin ammatory cytokines in the lung, as well as later in infection increased local T cell in ltration and respiratory tract pathology, as well as increased systemic NK cell activation.
The study adds to the growing list of immunomodulatory functions attributable to ACh; it also adds another antibody independent, immune regulatory function to B cells.ChAT expression by B cells in vitro was promoted by TLR4 stimulation in by both B-1 and B-2 cells, consistent with the dependency of B cell ChAT expression on MyD88 expression 31 .However, it remains to be determined whether TLR stimulation is the only innate signal responsible for ChAT expression by B cells.When B cells underwent activation and plasma cell differentiation in vitro following ligation with anti-BCR or IL-4, IL-5, and CD40L, TLRinduced ChAT expression was inhibited, suggesting that ChAT-induction is promoted only in B cells that do not participate in the T-dependent, antigen-speci c B cell response.Thus, perhaps suggesting a division of labor, in which antigen-speci c B cells remain ChAT negative and differentiate instead into antibody-producing plasmablasts and plasmacells, while those activated by innate signals, but not stimulation via the BCR, may be recruited into early innate immune response regulation.This would be consistent with the ndings that many ChAT + cells belong to the B-1 cell subset, a cell population that does not respond to BCR signaling with clonal expansion 103 , and immature B cells, another cell population that does not respond to BCR signaling with activation and differentiation unless rescued by provision of T cell help or other costimulatory signals [104][105][106] .Intriguingly, IL-10 producing B cells (Bregs) 22,107,108 are also most prevalent among B-1 and Immature B cells, and only rarely FO B cells.
ChAT + B cells store ACh in cytoplasmic vesicles 77 from which they can be released rapidly via stimulation with the intestinal neuropeptide cholecystokinin (CCK) 31 .What regulates ACh release by B cells in the lung parenchyma remains to be de ned but may involve other neuropeptides, as B cells express receptors for neuropeptides that can be released by specialized sensory nociceptive neurons and pulmonary neuroendocrine cells in the respiratory tract 29,112 .Of note, we found differences between the transcriptional pro le of various myeloid cell populations in the lungs of ChATBKO mice compared to controls already prior to the in uenza virus infection, suggesting the continuous release of ACh from B cells at this site, perhaps triggered by continuous environmental stimuli.
Previous studies showed that ACh can inhibit the JAK2-STAT3 pathway, although this inhibition was not entirely dependent on SOCS3, which is known to dephosphorylate STAT3 92,[113][114][115] .Another study identi ed ACh's effect on the nAChR/pERK pathway and the promotion of IL-10 production 42 .However, ACh does not seem to affect IL-10 in splenic or cavity macrophages 90 .Similarly, we found no difference in IL-10 transcript levels in ACh-treated mice, suggesting that the nAChR/pERK pathway might be speci c to myeloid-derived suppressor cells, and in this case might work through JAK2-STAT3, or another unidenti ed pathway.Future targeted analysis and experimental approaches are needed to investigate the speci c mechanisms of ACh's regulation of TNFa production and apoptosis pathways in IMs and how this contributes to overall in ammation and viral control during respiratory viral infections.Such advances could increase our understanding of lung in ammatory response regulation and identify potential new therapeutic targets and biomarkers of such diseases.
The known cholinergic anti-in ammatory pathway involves acetylcholine receptor (AChR) agonists inhibiting cytokine expression in human macrophages and in septic rats and mice, leading to improved survival 86,[89][90][91][92]116,117 . Later sudies emphasized the crucial role of the α7nAChR in inhibiting in ammation and preventing sepsis 87,88,118,119 .These effects have shown effectiveness in mitigating conditions such as rheumatoid arthritis, in ammatory bowel disease, and colitis, in both mice and humans [39][40][41][42][43] .Given the known role of ACh derived from T lymphocytes in regulating splenic macrophage TNFa production, the lack of effect of T cell derived ACh on macrophage function in the lung after in uenza infection was somewhat surprising.However, this is consistent with the nding that most ChAT-expressing leukocytes are B cells in the respiratory tract, spleen, and MedLN both at steady state and at 24hours after in uenza infection.Moreover, all ChAT-expressing T cells had an activated CD44hi CD62Llo phenotype, a cell population that does not appear in the lungs of infected mice until around day 5-7 of infection. Thus, indicatng profound differences in the kinetics and regulation of ACh production by B and T cells.
In conclusion, our studies indicate that B cell derived ACh regulates IMs during respiratory tract viral infections, cells that are increasingly identi ed as critical sources of protective innate but also harmful in ammatory responses and misdirected tissue repair leading to increased lung brosis following infections with respiratory tract pathogens such as in uenza virus and COVID-19.Despite the broad presence of ACh in neuronal synapses in all tissues, and strong expression of the numerous cholinergic receptors on many cell types, its regulatory effect on lung IMs during early infection depends on secretion by B cells that reside in close proximity in the same tissue space, revealing a new, highly cell and location-speci c regulatory axis controlling lung in ammation.
All mice were housed in SPF conditions in ventilated ltertop cages with food and water ad libitum at the University of California, Davis and the Johns Hopkins Bloomberg School of Public Health.Euthanasia was done by overexposing mice to CO 2 .All studies involving mice were conducted in compliance with, and after approval of protocols by the UC Davis Institutional Animal Care and Use Committee (IACUC) and by the Johns Hopkins University Animal Care and Use Committee (ACUC).
10PFU/mouse were used, unless otherwise stated in the gure legend, in 40 µL PBS 120 which was established as a sublethal dose generating on average less than 20% body weight loss over the course of infection.

Tissue processing and ow cytometry staining
Lymph node and spleen cell suspensions were prepared as previously outlined 120 .Brie y, tissues were ground between the frosted parts of two microscope slides and then incubated in ACK lysis buffer for 1 minute on ice to eliminate erythrocytes.Subsequently, the cells were passed through a 50 µm nylon lter and diluted for staining.
For lung tissue collection, lungs were harvested after left ventricle perfusion of the heart and then mechanically and chemically digested.The lungs were placed in 3 mL of DMEM F12 1X with 10% NCS in gentleMACS™ M Tubes (Milenty, # 130-093-236) and processed using a gentleMACS dissociator m_Lung_02 twice (Milteny).Following this, the lungs were incubated with DNAse I (50 U/mL) (Worthington-Biochem # LS002139) and Collagenase, Type I (250 U/mL) (Worthington-Biochem # LS004196) for 25 minutes at 37°C at 220 rpm shaking.After incubation, the lungs underwent another round of processing using the m_lung_02 program.The resulting cells were passed through a 50 µm nylon lter and diluted for staining, similar to lymph node and spleen preparations.
Single-cell suspensions were incubated with Fc receptor block (anti-CD16/32, made in-house) and Live/Dead Fixable Aqua or Near IR (Thermo Fisher, L34967 or L34994) in PBS for 20 minutes on ice.Subsequently, the cells were stained with uorescently labeled anti-surface receptor antibodies according to the manufacturer's instructions regarding temperature and duration (see methods Table 1 for the list of antibodies used) in staining media 120 .All uorophore-conjugated antibodies were titrated before use to ensure maximal differential staining between the negative and positive fractions.For intracellular staining, the eBioscience™ Foxp3 Transcription Factor Staining Buffer Set (Thermo Fisher REF 00-5523-00) was used following the manufacturer's instructions.
Macrophage cultures and ex-vivo re-stimulation auto-MACS-puri ed lung interstitial macrophages (IMs) or pleural/peritoneal cavity macrophages were cultured at 1x10 6 cells/mL per well in 100 µL of culture media as described above in 15-mL conical tubes and stimulated for designated times at 37°C with 5% CO 2 .Cells were analyzed via ow cytometry with uorescently labeled antibodies after Fc blocking with anti-CD16/32 (in-house) and Live/Dead staining.
For the detection of TNFα expression, total lung suspension or puri ed designated macrophages were stimulated with 100 ng/mL of LPS (Sigma # L6511) in the presence of Brefeldin A (Sigma # B6542) for 5 hours at 37°C.

Single cell RNA-sequencing (scRNA-Seq)
Lung single cell suspension was prepared as above and submitted for FACS-sorting on live cells (FACSAria) using Propidium Iodide (Miltenyi Biotec # 130-093-233) and cells were concentrated at 1000 cell/uL before submitting for 10X single cell RNA Sequencing.Cell viability was ensured to be > 90% prior sequencing submission.For single cell analysis, automatically called cells were further ltered to ensure usage of high-quality droplets with captured cells.RNA barcodes were ltered on Total UMI Count (> 500 UMIs), feature count (> 250 features), and percentage of mitochondrial genes (< 25%).Signac v1.9.0 121 was used to determine nucleosome signaling and transcription start site enrichment scores.ATAC barcodes were ltered on number of fragments mapping to peak regions (> 3,000 and < 20,000), percentage of fragments mapping to peak regions (> 15%), nucleosome signal scores (< 4) and transcription start site scores (> 1).Seurat v4.3.0 122 and Signac v1.9.0 121 used for handling of normalization, identi cation of variable genes, scaling, principal component analysis, UMAP dimensional reduction, and SNN generation followed by Leiden clustering for RNAseq and ATACseq data respectively.Batch effect correction was performed using Harmony 123 .Clusters were identi ed using a combination of marker genes and differential expression comparing each cluster to all other cells in the data set.Differential expression analyses were performed using Mann-Whitney U test.fgsea v1.24.0 124 was used to run gene set enrichment analysis (GSEA) with gene sets obtained from the Molecular Signatures Database 125 .Features were ranked by -log(pvalue) * sign(foldchange).
Percentages of cells by cluster and sample are the percentage of cells in a given cluster compared to the total number of cells per sample.Statistical comparisons shown are from t-test comparing the percentages.
Intercellular signaling analyses are based Domino 126 .UCell 127 was used to generate transcription factor activation scores by cell using transcription factor target gene sets from the Molecular Signatures Database 125 .ComplexHeatmap v2.14.0 128 and circlize v0.4.15 129 were used for visualizations.

Adaptive transfer experiments
WT or acra7-de cient interstitial macrophages (IMs) were puri ed using auto-MACS as described above (with purity > 75%) and labeled or not with either eF670 (Thermo Fisher # 65-0840-85) or Cell-trace violet (CTV) (Thermo Fisher # C34571) following the manufacturer's instructions.After labeling, the cells were washed with PBS, pooled together, and then adoptively transferred into mice intranasally (i.n.) in 20 µL of PBS at the indicated concentrations.

Quantitative real-time PCR (qRT-PCR) and viral qRT-PCR
For cytokine and chemokine mRNA measurement experiments, 4 small pieces of lung tissue were cut from each lobe and RNA was extracted as per manufacturer instruction (Qiagen, # 69504).All samples were compared to a GAPDH internal control.

ELISA assay
Sandwich ELISA assays were conducted following previously established protocols 134 .In brief, ELISA plates were coated with unlabeled anti-isotype antibodies or whole killed in uenza A/PR8/34 (200-400 HAU/mL; in-house).To minimize non-speci c binding, the plates were blocked with a blocking buffer containing 1% NBCS, 0.1% dried milk powder, and 0.05% Tween 20 in PBS.Serum obtained from tail bleeds or standards were pre-diluted in PBS and then added to the plate in serial dilutions.Antibody detection was achieved using biotinylated anti-isotype antibodies, followed by streptavidin-horseradish peroxidase.The reaction was visualized by adding TMB diluted in 0.05M Citric Acid and H2O2 for 10-15 minutes before stopping with 1N sulfuric acid.Absorbance was measured at 450nm and 595nm, and antibody concentrations were compared to standards.Prior to use in experiments, all reagents were titrated to ensure optimal performance.

ELISPOT assay
Antibody-secreting cells (ASCs) were quanti ed using ELISPOT, following established methods 120,134 .Brie y, Multi-Screen HA Filtration plates were coated with unlabeled anti-isotype antibodies or whole killed in uenza A/PR8/34 (200-400 HAU/mL; prepared in-house).Following blocking and addition of cell suspensions, antibody secretion was detected using biotinylated anti-isotype antibodies, followed by streptavidin-horseradish peroxidase.The resulting reaction was visualized using a previously described chemical method 134 .Plates were then enumerated using a computer-based system, the AID EliSpot Reader System (Autoimmune Diagnostika).
Statistical analysis and reproducibility

Supplementary Files
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Figures
Figures

Figure 2 B
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Figure 3 B
Figure 3