03 QVYSLIRPNENPAHK as described(26). Negative control CD4 tetramer was I-A(b) human CLIP 87-101 04 PVSKMRMATPLLMQA. Tetramers were obtained from the NIH Tetramer Core Facility. Samples were 05 incubated with tetramer for 60 minutes at room temperature. For all flow cytometric studies, data acquisition 06 and analysis were performed on an Accuri C6 (BD Biosciences, San Jose, CA) flow cytometer using CFlow 07 Plus Analysis software. 08 09 Mass Spectrometry: Choline and ACh were measured in cell-free BAL fluid by hydrophilic interaction liquid 10 chromatography coupled to tandem mass spectrometry (HILIC LC-MS/MS), using stable isotope-labeled 11 internal standards (choline-d4 and acetylcholine-d4) as described (27, 28) . Briefly, an aliquot of the cell-free 12 BAL fluid was spiked with a mixture of deuterated choline/deuterated ACh. The sample was adjusted to 50% 13 methanol and ice partitioned to remove proteins. The remaining sample was lyophilized, dissolved in 19 of a 4% paraformaldehyde solution. The trachea was clamped and all lungs were removed and fully 20 submerged in the 4% paraformaldehyde solution overnight fixation (~18 hour), with hemostats still attached 21 and fully restricting flow through the trachea. The following day, hemostats were removed, lung lobes were 22 dissected from one another and fully submerged in 70% Ethanol (EtOH), which was replaced 6-8 hours. One 23 day prior to processing, EtOH was discarded and replaced for a further 24 hours before being sent for 24 processing and paraffin embedding. Formalin fixed paraffin embedded (FFPE) lung lobes were serially 25 sectioned at 5um on a Leica RM2125 RTS Rotary Microtome, and mounted on Prism (Prism Research Glass, 26 Raleigh, NC) positively charged microscope slides. Formalin fixed paraffin-embedded (FFPE) sections were 27 stained with Hematoxylin and Eosin (H&E) for histological analysis using standard methods. Tissue sections of 28 uninfected, infected vehicle control, and infected HC3 treated lung tissue were analyzed by a veterinary 29 pathologist who was blinded to treatments and groups.
Animals were infected with a sublethal dose of influenza A/PR8 (H1N1) and weighed daily to monitor 92 morbidity (Fig 1A) . Separate cohorts were euthanized at specified time points and BAL fluid was isolated in 93 order to measure the airway ACh concentration. Airway choline concentration was also measured as a 94 biomarker for local ACh hydrolysis(23). There was no change in the airway ACh concentration over time; 95 however, the airway choline concentration changed over the course of infection. From a background 96 concentration of 1200ng/ml prior to infection, the airway choline concentration increased to 4800 ng/mL 8 dpi 97 and peaked at 6500 ng/mL 10 dpi. By 15 dpi, BAL choline concentration diminished to 1800 ng/mL, similar to 98 the starting concentration measured 0 dpi ( Fig 1B) . Comparing the weight change curve to the choline 99 concentration changes shows that ACh hydrolysis reaches a peak in the influenza-infected lungs shortly after 00 the point of peak weight loss.
To determine the source of airway ACh and examine a possible role for non-neuronal ACh production 02 during influenza infection, we examined the kinetics of lymphocyte populations infiltrating airways and lungs 03 during infection using ChAT (BAC) -eGFP transgenic mice (ChAT mice) with endogenous choline 04 acetyltransferase transcriptional regulatory elements directing eGFP expression, alongside C57BL/6 mice as 05 non-reporter controls. Wild-type, influenza-infected C57BL/6 mice were used as controls for green fluorescent 06 protein (GFP) fluorescence. Starting 8 dpi, GFP + cells were detected in the airway of the ChAT mice ( Fig 1C) .
07 The number of ChAT-GFP + lymphocytes present in BAL samples followed the same kinetic pattern as the total 08 lymphocyte population (Fig 1D) , increasing rapidly starting 7 dpi and reaching peak numbers between 8-10 dpi 09 ( Fig 1E) . However, the percentage of ChAT-GFP + lymphocytes remained above 7% of the total lymphocyte 10 population in the airways through 28 dpi ( Fig 1F) . 
Flow cytometry identified ChAT-GFP + subsets of both CD4 + and CD8 + T cell populations in the airways 26 during peak days of infection (8-10 dpi) (Fig 2) . The majority of these ChAT-GFP + lymphocytes were CD4 + 27 (69.7%) while 23.3% were CD8 + , indicating more cholinergic helper T cells present in BALF samples than 28 cholinergic cytotoxic T cells, respectively. When the analysis was extended to lung tissue, ChAT-GFP was 29 detected in B220 + B-1 lymphocytes as well as CD4 and CD8 T cells. In airway and lung tissue, the highest 30 percentage of GFP + cells were CD4 + T cells. The CD4 population also expressed the most GFP fluorescence 31 on a per-cell basis (Table 1) 
Surface staining indicated that the cholinergic CD4 T cells were uniformly CD44 hi CD62L lo (Fig 4) . This 61 matches the overall surface phenotype of cholinergic CD4 T cells from multiple reports, but it also corresponds 62 with a specific subset associated with long term memory known as the T resident memory population. To 63 explore the possibility that cholinergic CD4 T cells made up part of the TRM population, mice were infected 64 with influenza A and allowed to recover for two months with no manipulation, then they were given an 65 intravenous injection of fluorescent anti-CD45 antibody and sacrificed ten minutes later. As shown in Fig 4B, 66 CD4 positive cells in the lungs can be divided into two subsets, based on staining by the injected CD45. Those 67 cells not exposed to the circulation were left unstained following iv injection. These represent the T resident 
The co-localization of cholinergic lymphocytes and activated macrophages indicated a potential role for 00 targeted ACh delivery to activated macrophages during the later stages of influenza infection. To examine the 01 requirement for ACh during recovery from influenza infection, we used the choline reuptake inhibitor 02 Hemicholinium-3 (HC3) to disrupt ACh synthesis during the time period associated with the increased airway 03 ACh concentration (Fig 1) . Mice were infected with influenza A/PR8 (H1N1) as above. After seven days, mice 04 were stratified into treatment cohorts according to the amount of weight loss to ensure equivalent pre-05 established morbidity in each cohort. One cohort was treated with HC3 from days 7 through 12 (infected HC3 06 treated), while the infected vehicle control cohort was injected with saline to control for handling/injection 07 stress. Additional control cohorts were injected with HC3 or PBS without having been infected. One infection 08 cohort was sacrificed 10 dpi to measure the airway choline concentration. BAL samples from the HC3 treated 09 cohort exhibited increased airway choline concentration compared to infected vehicle control groups, indicating 10 that HC3 was inhibiting choline uptake in the pulmonary airways ( Fig 6A) . All influenza infected cohorts lost 11 weight as expected. The infected control cohort began to regain weight 9 dpi and had returned to 96% of their 12 starting weight by 15 dpi. In contrast, the infected HC3 treated cohort did not begin to regain weight until 11 dpi 13 and only returned to 91% of their starting weight by 15 dpi. The uninfected cohort treated with HC3 did not 14 display any treatment-associated weight change (Fig 6B) . Flow cytometric analysis showed an increase in the 15 number of neutrophils in HC3 treated animals compared to vehicle control animals ( Fig 6C) .
19 and Methods. A. Airway choline was measured ten days after infection as in Figure 1. B 
We used Iba1 as a marker of overall inflammation(8, 29, 36-38) to determine the effect of HC3 27 treatment during the later stage of infection (Fig 7) . Fluorescent signal from immunofluorescence stained 28 slides was quantified spatially to determine the degree of inflammation at set time points. A novel automated 29 algorithm was designed and implemented in MATLAB 2019b Image Processing Toolbox to accurately 
In addition to inducing epithelial proliferation, ACh mediates changes in macrophage gene expression.
48 The anti-inflammatory activity of ACh binding to theα7nAchR on macrophages, resulting in diminished NF-κB 49 nuclear translocation and decreased inflammatory cytokine production is well described(39, 51) and has been 50 documented in the lungs (22, 42, 50) . Recently, loss of α7nAChR signal transduction was shown to decrease 51 expression of the canonical M2 marker Arginase-1(52). In addition, use of an α7nAchR agonist decreased the 52 LPS-induced inflammatory response and reversed the inflammatory profile, particularly regarding M1 and M2 53 polarization, while also improving lung function and remodeling in a model of acute lung injury(15). Based on 54 these findings and those in the current study, we hypothesize that ACh produced by cholinergic lymphocytes 55 acts on macrophages to decrease pro-inflammatory cytokine release and initiate tissue repair 56 during the recovery phase of respiratory viral infection.
In these studies, we used a novel analysis tool to quantify Iba1 expression, relating to pulmonary 58 inflammation. An automated MATLAB algorithm was implemented to accurately isolate the red stain in the 59 Iba1 fluorescent images. The algorithm automatically reads images from a folder sequentially, segments the 60 red stains and computes the amount of red stain present in each of the images. To perform the image 61 segmentation to isolate the red stain, Otsu's method of thresholding was implemented in order to remove 62 background noise with minimal human bias. The algorithm was automated for computational efficiency and