key: cord-0312042-6xxrxcb9
authors: Horkowitz, Alexander P.; Schwartz, Ashley V.; Alvarez, Carlos A.; Herrera, Edgar B.; Thoman, Marilyn L.; Chatfield, Dale A.; Osborn, Kent G.; Feuer, Ralph; George, Uduak Z.; Phillips, Joy A.
title: Acetylcholine regulates pulmonary inflammation and facilitates the transition from active immunity to tissue repair during respiratory viral infection
date: 2020-07-02
journal: bioRxiv
DOI: 10.1101/2020.07.02.184226
sha: 5bb6f1e15784d61cac0346310aed4c43e6b8509b
doc_id: 312042
cord_uid: 6xxrxcb9

Inflammatory control is critical to recovery from respiratory viral infection. Acetylcholine (ACh) secreted from non-neuronal sources, including lymphocytes, plays an important, albeit underappreciated, role in regulating immune-mediated inflammation. This study was designed to explore the role of ACh in acute viral infection and recovery. Using the murine model of influenza A, cholinergic status in the lungs and airway was examined over the course of infection and recovery. The results showed that airway ACh remained constant through the early stage of infection and increased during the peak of the acquired immune response. As the concentration of ACh increased, cholinergic lymphocytes appeared in the airway and lungs. Cholinergic capacity was found primarily in CD4 T cells, but also in B cells and CD8 T cells. The cholinergic CD4+ T cells bound to influenza-specific tetramers at the same frequency as their conventional (i.e., non-cholinergic) counterparts. In addition, they were retained in the lungs throughout the recovery phase and could still be detected in the resident memory regions of the lung up to two months after infection. Histologically, cholinergic lymphocytes were found in direct physical contact with activated macrophages throughout the lung. When ACh production was inhibited, mice exhibited increased tissue inflammation, altered lung architecture, and delayed recovery. Together, these findings point to a previously unrecognized role for ACh in the transition from active immunity to recovery and pulmonary repair following respiratory viral infection.

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

Sections 35 were treated with biotin and streptavidin blocking solutions (Vector Laboratories

(Iba1): polyclonal rabbit anti-Iba1 antibody; Wako Pure 38 Chemicals Industries, Ltd, Osaka Japan] at 1:500 at 4C overnight. A goat secondary antibody

After staining with secondary antibodies, all sections 41 were washed three times with phosphate buffered saline and incubated with a streptavidin-AlexaFluor 594

1:500 diluted in 2% normal 43 goat serum. For tri-color stained sections, sections were incubated for 30 minutes in a light free environment 44 with an anti-GFP AlexaFluor-488 conjugate [anti-GFP, rabbit polyclonal antibody

OR] diluted 1:5 in 2% normal goat serum. Specificity controls for immunostaining included

Sections were overlaid with Vectashield anti-fade mounting 48 medium (Vector Laboratories

Sections were observed by fluorescence microscopy 50 (Ziess Axio Observer D1 Inverted Phase Contrast Fluorescent Microscope). Green, red, and blue channel 51 images were merged using AxioVision software

Automated Image Segmentation and Quantification of Immunofluorescence 55 For regional comparison of the lungs between treatment groups, images were categorized into three 56 categories based on anatomical region: open alveolar space (open), bronchus associated lymphoid tissue 57 (BALT), and area peripheral to large airways (peri-bronchial)

Exposure times were held constant in red, green, and blue 60 channels for each image captured, although only the signal in the red channel was quantified, as Iba1 was 61 marked with the AlexaFluor 594 fluorochrome

The same exposure time was utilized for both the red and green channels for comparison of auto fluorescent 63 tissues within each section, such as red blood cells and fibrin deposits, commonly seen as a result of vascular 64 leakage in inflamed tissues. For Iba1 immunofluorescence quantification

While there are a variety of thresholding methods present in the literature, Otsu's 77 method is the most accurate and most widely used(31-34). In the program, Otsu's method determines a 78 threshold that distinguishes the background from the region of interest. The determined threshold is then used 79 to segment each image, removing background noise and displaying the red stain. The final step is the 80 calculation of the total area of red stain present in the image as well as the intensity of the stains. Total pixel while total image intensity is calculated by summing the intensity levels of all elements remaining in the 83 segmented image

Statistical analysis was computed using R and R Studio. One-way ANOVA and two-87 tailed paired t-tests were performed *, p<0.05 was considered statistically significant

the alveolar space and peri-bronchial regions, as well as the full lung when all 54 measurements were compiled into a single data set. Greater total fluorescent intensity was also observed in the BALT 55 region of infected HC3 treated groups compared to infected vehicle control groups

As shown in Fig 8, infected 61 vehicle control lung tissue was characterized by having mild/moderate multifocal perivascular mixed infiltrate 62 and alveolar/interstitial mixed infiltrate of lymphocytes and neutrophils. However, infected HC3 treated lung 63 tissue exhibited histological abnormalities not seen in the vehicle control lungs. The lungs from infected 64 animals treated with HC3 were characterized as having moderate perivascular as well as alveolar/interstitial 65 mixed infiltrate of lymphocytes and neutrophils (Fig 8A). In addition, pathological anomalies including as 66 multifocal type II pneumocyte proliferation (Fig 8B), mild multifocal squamous cell metaplasia (Fig 8C), and 67 mild fibroplasia

Representative images of H&E stained sections of lung tissue at 10x (scale bar 100um) and 40x (scale bar 72 10um) magnification from healthy animals, infected vehicle control animals, and infected HC3 treated animals 73 from left to right. Flu infected vehicle control lung tissue with mild/moderate multifocal perivascular mixed 74 infiltrate (yellow arrow) and alveolar/interstitial mixed infiltrate of lymphocytes and neutrophils

Flu infected HC3 treated lung tissue with moderate perivascular (yellow arrows) as well as alveolar/interstitial 76 mixed infiltrate of lymphocytes and neutrophils (green arrows). B-D. Infected HC3 treated lung tissue shows 77 multifocal type 2 pneumocyte proliferation (yellow arrows), mild multifocal squamous cell metaplasia (green 78 arrows), and mild fibroplasia

More cells expressed Iba1 in the infected, HC3 treated 73 animals, resulting in larger areas of immunofluorescence. These animals also displayed higher mean staining 74 intensity, indicating that overall Iba1 expression was increased following disruption of ACh synthesis during 75 infection. Although Iba1 has historically been used as an activated macrophage marker(53), the secreted Iba1 76 protein also acts as an independent inflammatory stimulus

If altered cholinergic capacity plays a role in the 88 diminished immune response or delayed recovery to respiratory infection shown by the elderly, then improving 89 cholinergic function could result in enhanced immune function during aging. Since the elderly are at the 90 greatest risk of death not just from influenza infection but also from the ongoing COVID-19 pandemic, these 91 are questions of the utmost importance. One key feature of age-related immunodeficiency is in the increased 92 basal inflammatory status known as inflammaging(61, 62)

IL1b, and IL6 were significantly decreased following six months of donepezil therapy(65)

Furthermore, donepezil treatment is associated with decreased overall mortality, including pneumonia-98 associated mortality (66, 67)

Cholinergic lymphocytes appear in the lungs and airways during the recovery 07 phase of influenza, and are found in direct physical contact with activated macrophages. Artificially decreasing 08 the airway ACh concentration results in extended morbidity

Plasma concentration of acetylcholine in young women

Extraneuronal localization of acetylcholine and its 16 release upon nicotinic stimulation in rabbits

Species differences in the 18 concentration of acetylcholine, a neurotransmitter, in whole blood and plasma

Maintenance of constant blood acetylcholine 21 content before and after feeding in young chimpanzees

Non-neuronal cholinergic system in 23 regulation of immune function with a focus on alpha7 nAChRs

Expression and Function of the

Cholinergic System in Immune Cells

Anti-high mobility group box-1 27 monoclonal antibody treatment provides protection against influenza A virus (H1N1)-induced pneumonia in 28 mice

Allograft inflammatory 30 factor-1 in the pathogenesis of bleomycin-induced acute lung injury

Immunochemical and immunocytochemical 32 characterization of cholinergic markers in human peripheral blood lymphocytes

Beyond 35 neurotransmission: acetylcholine in immunity and inflammation

Acetylcholine-37 synthesizing T cells relay neural signals in a vagus nerve circuit

Lymphocyte-derived ACh 39 regulates local innate but not adaptive immunity

Choline acetyltransferase-41 expressing T cells are required to control chronic viral infection

Nicotinic acetylcholine receptor 43 alpha7 subunit is an essential regulator of inflammation

Acute lung injury 45 is reduced by the α7nAChR agonist PNU-282987 through changes in the macrophage profile

Expression and function of genes encoding 48 cholinergic components in murine immune cells

A review of inflammatory mechanism in airway diseases

Targeting the "cytokine storm" for 52 therapeutic benefit

Viral and host factors related to the clinical outcome of 54 COVID-19

Observations on excess mortality associated with epidemic 56 influenza

The immune response to 58 influenza in older humans: beyond immune senescence

The Non-neuronal cholinergic system: an emerging drug target in the 60 airways

The non-neuronal cholinergic system in the airways: an 62 unappreciated regulatory role in pulmonary inflammation?

The non-neuronal cholinergic system: 64 basic science, therapeutic implications and new perspectives

Innate immune induction and 66 influenza protection elicited by a response-selective agonist of human C5a

Fast sample preparation and 70 liquid chromatography-tandem mass spectrometry method for assaying cell lysate acetylcholine

Simultaneous analysis of multiple neurotransmitters by 73 hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry

Viral persistence and 76 chronic immunopathology in the adult central nervous system following Coxsackievirus infection during the 77 neonatal period

A Threshold Selection Method from Gray-Level Histograms

A survey of thresholding techniques. Computer Vision, Graphics, and 81 Image Processing

A review on Otsu image segmentation algorithm

A new approach for detecting abnormalities in 85 mammograms using a computer-aided windowing system based on Otsu's method

Gesture image segmentation with Otsu's method 88 based on noise adaptive angle threshold. Multimedia Tools and Applications

Cutting edge: Tissue-retentive lung 90 memory CD4 T cells mediate optimal protection to respiratory virus infection

Allograft inflammatory factor-1 93 augments production of interleukin-6, -10 and -12 by a mouse macrophage line

Signaling pathways and genes that 96 inhibit pathogen-induced macrophage apoptosis--CREB and NF-kappaB as key regulators

LPS induces the interaction of a transcription factor, LPS-99 induced TNF-alpha factor, and STAT6(B) with effects on multiple cytokines

Vagus nerve stimulation 02 attenuates the systemic inflammatory response to endotoxin

Cholinergic agonists inhibit HMGB1 release 04 and improve survival in experimental sepsis

Requisite role of the cholinergic alpha7 nicotinic acetylcholine receptor 09 pathway in suppressing Gram-negative sepsis-induced acute lung inflammatory injury

A novel regulator of lung inflammation and immunity: pulmonary 12 parasympathetic inflammatory reflex

Acetylcholine-producing NK cells attenuate CNS 14 inflammation via modulation of infiltrating monocytes/macrophages

Lung niches for the generation 17 and maintenance of tissue-resident memory T cells

Cholinergic systems in non-nervous tissues

Splenic nerve is required 21 for cholinergic antiinflammatory pathway control of TNF in endotoxemia

acetylcholine, a signalling molecule synthezised by surface cells of rat and man

Pulmonary 27 inflammation is regulated by the levels of the vesicular acetylcholine transporter

Activation of the alpha7 nAChR reduces 30 acid-induced acute lung injury in mice and rats. American journal of respiratory cell and molecular biology

Physiology and immunology of the cholinergic antiinflammatory pathway

Deficiency of α7 Nicotinic Acetylcholine Receptor Attenuates 35 Bleomycin-Induced Lung Fibrosis in Mice

Inflammatory Factor 1 as an Immunohistochemical Marker for Macrophages in Multiple Tissues and 38 Laboratory Animal Species

Functional characterization of the rat 40 chemokine KC and its importance in neutrophil recruitment in a rat model of pulmonary inflammation

Impaired immune responses in the lungs of aged mice following influenza 43 infection

The paradox of nicotinic acetylcholine receptor upregulation by nicotine

Nicotinic acetylcholine receptors: upregulation, age-related effects, 47 and associations with drug use

Differential effects of nicotine and aging on 49 splenocyte proliferation and the production of Th1-versus Th2-type cytokines

Diurnal rhythms in ornithine decarboxylase activity and 52 norepinephrine and acetylcholine synthesis in submaxillary lymph nodes and spleen of young and aged rats 53 during Freund's adjuvant-induced arthritis

Effect of melatonin on 24-hour rhythms of 55 ornithine decarboxylase activity and norepinephrine and acetylcholine synthesis in submaxillary lymph nodes 56 and spleen of young and aged rats

Inflammaging as a major characteristic of old people: can it be prevented or cured? Nutr 58 Rev

Metabolic 60 Alterations in Aging Macrophages: Ingredients for Inflammaging?

Treatment with an acetylcholinesterase 62 inhibitor in Alzheimer patients modulates the expression and production of the pro-inflammatory and anti-63 inflammatory cytokines

Acetylcholinesterase inhibitors effects on 65 oncostatin-M, interleukin-1β and interleukin-6 release from lymphocytes of Alzheimer's disease patients

Cholinesterase Inhibitors and Cell-Derived Peripheral Inflammatory Cytokines in Early Stages of Alzheimer's 69 Disease

Alzheimer's 71 disease medication and risk of all-cause mortality and all-cause hospitalization: A retrospective cohort study

Donepezil is associated with decreased in-hospital mortality as a result 74 of pneumonia among older patients with dementia: A retrospective cohort study