Fermat's Library | Differential Regulation of Allergic Airway Inflammation by Acetylcholine annotated/explained version.

Differential Regulation of Allergic

Airway Inﬂammation by Acetylcholine

Luke B. Roberts

1,2

, Rita Berkachy

, Madina Wane

, Dhiren F. Patel

Corinna Schnoeller

, Graham M. Lord

2,4

, Kleoniki Gounaris

, Bernhard Ryffel

Valerie Quesniaux

, Matthew Darby

, William G. C. Horsnell

5,6,7

and Murray E. Selkirk

Department of Life Sciences, Imperial College London, London, United Kingdom,

School of Immunology and Microbial

Sciences, King’s College London, Great Maze Pond, London, United Kingdom,

National Heart and Lung Institute, Imperial College

London, London, United Kingdom,

Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom,

Laboratory of Molecular and Experimental Immunology and Neurogenetics, UMR 7355, CNRS-University of Orleans and Le Studium

Institute for Advanced Studies, Rue Dupanloup, Orle

ans, France,

Institute of Infectious Disease and Molecular Medicine, Univer sity of

Cape Town, Cape Town, South Africa,

College of Medical and Dental Sciences, University of Birmingham, Birmingh am, United Kingdom

Acetylcholine (ACh) from neuronal and non-neuronal sources plays an important role in the

regulation of immune responses and is associated with the development of several

disease pathologies. We have previously demonstrated that group 2 innate lymphoid cell

(ILC2)-derived ACh is required for optimal type 2 responses to parasitic infection and

therefore sought to determine whether this also plays a role in allergic inﬂammation.

Rora

Cre+

Chat

LoxP

mice (in which ILC2s cannot synthesize ACh) were exposed to an

allergenic extract of the fungus Alternaria alternata, and immune responses in the airways

and lung tissues were analyzed. Airway neutrophilia and expression of the neutrophil

chemoattractants CXCL1 and CXCL2 were enhanced 24 h after exposure, suggesting

that ILC2-derived ACh plays a role in limiting excessive pulmonary neutrophilic

inﬂammation. The effect of non-selective depletion of ACh was examined by intranasal

administration of a stable parasite-secreted acetylcholinesterase. Depletion of airway ACh

in this manner resulted in a more profound enhancement of neutrophilia and chemokine

expression, suggesting multiple cellular sources for the release of ACh. In contrast,

depletion of ACh inhibited Alternaria-induced activation of ILC2s, s uppressing the

expression of IL-5, IL-13, and subseque nt eosinophilia. Depletion of ACh reduced

macrophages with an alternatively activated M2 phenotype and an increase in M1

macrophage marker expression. These data suggest that ACh regulates allergic airway

inﬂammation in several ways, enhancing ILC2-driven eosinophilia but suppressing

neutrophilia through reduced chemokine expression.

Keywords: lung, alternaria, ILC2, neutrophil, eosinophil, inﬂammation, chemokine, acetylcholine

INTRODUCTION

Acetylcholine (ACh) is best known as a neurotransmitter, but in recent years, it has been increasingly

implicated as an important signaling molecule in the immune system. It was ﬁrst identiﬁed as a negative

regulator of inﬂammatory cytokine production by macrophages in what is termed the cholinergic anti-

inﬂammatory pathway (1), and was subsequently demonstrated to inﬂuence the trafﬁcking, activation

and effector functions of T cells during infection (2, 3). Our studies have indicated that ACh is necessary

Frontiers in Immunology | www.frontiersin.org May 2022 | Volume 13 | Article 8938441

Edited by:

Susetta Finotto,

University Hospital Erlangen, Germany

Reviewed by:

Fumio Takei,

University of British Columbia, Canada

Roma Sehmi,

McMaster University, Canada

*Correspondence:

Luke B. Roberts

luke.roberts@kcl.ac.uk

Murray E. Selkirk

m.selkirk@imperial.ac.uk

Specialty section:

This article was submitted to

Mucosal Immunity,

a section of the journal

Frontiers in Immunology

Received: 10 March 2022

Accepted: 03 May 2022

Published: 27 May 2022

Citation:

Roberts LB, Berkachy R, Wane M,

Patel DF, Schnoeller C, Lord GM,

Gounaris K, Ryffel B, Quesniaux V,

Darby M, Horsnell WGC and

Selkirk ME (2022) Differential

Regulation of Allergic Airway

Inﬂammation by Acetylcholine.

Front. Immunol. 13:893844.

doi: 10.3389/fimmu.2022.893844

ORIGINAL RESEARCH

published: 27 May 2022

doi: 10.3389/fimmu.2022.893844

for effective control of the parasitic nematode Nippostrongylus

brasiliensis, in part by regulating physiological responses such as

smooth muscle contraction, but also contributing to adaptive

immunity by signaling through the M3 muscarinic acetylcholine

receptor (mAChR)to promote cytokine productionby CD4

Tcells

(2). We and others recently determined that group 2 innate

lymphoid cells (ILC2s) are a major source of ACh during

nematode infection and that genetic disruption of ACh synthesis

by ILC2s renders mice more susceptible to infection, with reduced

barrier responses in the lungs and gut (4, 5).

ACh is the major parasympathetic neurotransmitter in the

airways, and excessive cholinergic signaling contributes to the

pathology of asthma and chronic obstructive pulmonary disease

(COPD) by promoting bronchoconstriction, mucus production,

and airway remodeling (6). ACh also appears to play a pro-

inﬂammatory role, which impacts on the path ology of lung

disease (7). Neurons are a major source of ACh in the lungs, but

it is also synthesized and released by non-neuronal cells, namely,

pulmonary epithelial and immune cells, including ILC2s (4, 8–10).

Asthmatic inﬂammation is characterized by increased type 2

cytokines and eosinophilia in the lungs. ILC2s can be activated by

release of epithelial alarmins including interleukin (IL)-33, and thus

act as an important link between the innate and the adaptive

immune system in the initiation of allergic inﬂammation (11).

Because we had identiﬁed ILC2-derivedACh as an important factor

in promoting barrier responses to nematode infection, we were

interested in examining whether thisplayed a role in the initiation of

allergicinﬂammation in thelungs. Intranasal dosing of mice withan

extract of the fungal mold Alternaria alternata,acommon

environmental aeroallergen, induces rapid release of interleukin

(IL)-33 intothe airway lumen, although the initial trigger appears to

be the release of adenosine triphosphate (ATP) from epithelial cells

(12). ILC2s produce large amounts of IL-5 and IL-13 in response to

activation with IL-33, resulting in eosinophilia (13) and this has

been adopted as a robust model for innate induction of airway

inﬂammation (14).

In this study, we demonstrate that Rora

Cre+

Chat

LoxP

mice, in

which ILC2s cannot synthesize ACh, show enhanced neutrophilia

in the airways following acute exposure to an allergenic extract of A.

alternata. More profound neutrophilia was observed in wild-type

mice following non-selective enzymatic depletion of ACh in the

airways, which was accompanied by increased expression of

neutrophil chemokines and a reduction of macrophages with an

alternatively activated M2 phenotype. Non-selective depletion of

ACh in the airways also strongly inhibited ILC2 activation, cytokine

expression, and associated eosinophilia.

RESULTS

ILC2-Derived Acetylcholine Inhibits

Neutrophilia Following Exposure to

Alternaria Allergen Extract

It is unclear how cholinergic signalin g regulates pulmonary

immune responses during type 2-dominated airway

inﬂammation. We have recently shown that ILC2s are

signiﬁcant producer s of ACh followi ng alarmin-medi ated

activation in the context of type 2 immunity against parasitic

nematode infection and in response to A. alternata extract (4).

During Alternaria-induced inﬂammation, serine protease

activity releases IL-33 from airway epithelial cells (15), which

activates many immune cell types, including ST2

ILC2s. ILC2s

are central mediators of type 2 inﬂammation, including

eosinophilia (16), and we therefore explo red whether ILC2-

derived ACh was required for the generation of this response.

Administration of Alternaria to Rora

Cre+

Chat

LoxP

animals

resulted in increased numbers of CD45

cells in the lungs 24 h

post-exposure (Figure 1A). The numbers of eosinophils and

neutrophils in the lung tissue at baseline were c omparable

between Rora

Cre+

Chat

LoxP

, Chat

LoxP

, and C57BL/6J (WT) mice

(Figures 1B, C). Exposure of Rora

Cre+

ChatLoxP and Chat

LoxP

mice to Alternaria resulted in a comparable eosinophil inﬂux to

lung tissue (Figure 1B). Although Rora

Cre+

Chat

LoxP

animals

showed a trend toward increased numbers of neutrophils in

the lungs, this was not signiﬁcantly different from control groups

at this time point (Figure 1C) . Histological ly, lung ti ssue

appeared more inﬂamed in Alternaria-treated Rora

Cre

Chat

LoxP

and Chat

LoxP

mice relative to untre ated controls

(Figure 1D). In the airways, alveolar macrophages (AM) were

the dominant cell type in WT control mice, whereas signiﬁcantly

increased eosinophils and neutrophils were observed in

Alternaria exposed Rora

Cre+

Chat

LoxP

and Chat

LoxP

genotypes

(Supplemental Figure 1 and Figures 1E, F) as expected (14, 17).

Airway eosinophil numbers were comparable between Alternaria-

exposed genotypes (Figure 1E), but more neutrophils were observed

in Rora

Cre+

Chat

LoxP

mice (Figure 1F), indicating a role for ILC2-

derived ACh in regulating neutrophil inﬂux following exposure

to Alternaria.

Analysis of transcript expression for chemokines involved in

granulocyte trafﬁcking revealed that eotaxin-1 (Ccl11)was

unaffected in Rora

Cre+

Chat

LoxP

and Chat

LoxP

genotypes relative

to WT mice and was also unaffected by exposure to Alternaria

(Figure 1G). Transcripts for the neutrophil-attractant

chemokines CXCL1/CXCL2 were also similar in untreated

Rora

Cre+

Chat

LoxP

and Chat

LoxP

mice but were signiﬁcantly

elevated in Rora

Cre+

Chat

LoxP

mice following exposure to

Alternaria (Figure 1G).

Proinﬂammatory macrophages with a classical ‘M1’ proﬁle are

key producers of neutrophil chemoattractants including CXCL1

and CXCL2, whereas alternatively activated or ‘M2’ macrophages

do not characteristically express these molecules (18, 19).

Expression of the M1 marker Nos2 (inducible nitric oxide

synthase, iNOS) was increased in the total lung tissue of Rora

Cre

Chat

LoxP

mice, while the M2 markers Mrc1 (mannose receptor C-

type 1, CD206) and Arg1(arginase 1) were downregulated following

Alternaria exposure (Figure 1H). These data suggest that increased

neutrophil inﬁltration following Alternaria exposure might result

from an M1 macrophage bias in the absence of ILC2-derived ACh.

Analysis of IL-5 and IL-13 in bronchoalveolar lavage (BAL)

ﬂuid conﬁrmed that dosing with Alternaria promoted a type 2

immune response. However, removal of the capacity of ILC2s to

Roberts et al. Cholinergic Regulation of Airway Inﬂammation

Frontiers in Immunology | www.frontiersin.org May 2022 | Volume 13 | Article 8938442

synthesize ACh did not impact the overall type 2 cytokine

environment of the airways (Figures 2A, B). Expression of IL-

5 and IL-13 (Supplemental Figure 2A, Figures 2C–H) and the

activation markers ICOS or ST2 were not further altered in

pulmonary ILC2s from Rora

Cre+

Chat

LoxP

mice (Figures 2I, J).

Lung CD4

T cells were also analysed as they are also a potential

source of type 2 cytokines, can express RORa and are known

producers of ACh (20). However, as expected given the acute

nature of the model, expression of IL-5 and IL-13 by total CD4

T cells (Supplemental Figures 2B–G) was extremely limited,

and no differences between the genotypes were observed. A small

proportion of total CD4

T cells displayed a Th2 phenotype

(Gata3

ST2

) which was unaltered in the lungs of Alternaria-

treated mice (Supplemental Figures 2B, H), but again there

were no signiﬁcant differences between Rora

Cre+

Chat

LoxP

and

Chat

LoxP

genotypes.

Enzymatic Depletion of Airway

Acetylcholine Prevents Eosinophilia But

Exacerbates Neutrophilia Following Acute

Exposure to Alternaria Allergen Extract

ILC2-derived ACh did not appear to greatly inﬂuence the onset of

the type 2 immune response in the brief 24-hour timeframe of this

model. However, ILC2s are only one cell type capable of ACh

synthesis in the lung. We thus examined the ef fect of broader,

non-selective deple tion of ACh using a stable secreto ry

acetylcholinesterase (AChE) from N. brasiliensi s (21)anda

catalytically inactive form of the enzyme generated by site-directed

mutagenesis (22). The enzymatic activity of the preparations was

conﬁrmed by in-gel staining (Supplemental Figure 3A) and

Ellman’s assay, which also conﬁrmed that no inhibitors of AChE

activity were present in the Alternaria extracts (Supplemental

Figure 3B). Given our observations in Rora

Cre+

Chat

LoxP

mice, we

G H

D FE

FIGURE 1 | Granulocyte responses in the lung and airways of Rora

Cre+

Chat

LoxP

mice following acute fungal allergen challenge. Lung and airway leucocytes were

analyzed from C57BL/6J, Chat

LoxP

, and Rora

Cre+

Chat

LoxP

mice at baseline, and following Alternaria alternata allergen extract (ALT) exposure in Chat

LoxP

and

Rora

Cre+

Chat

LoxP

mice, 24 h after intranasal dosing. (A) Number of CD45

leucocytes per lung. (B) Number of eosinophils in the lung. (C) Number of neutrophils

in the lung. (D) Representative H&E stained lung sections. b, bronchiole; v, blood vessel; a, alveolus. (E). Number of eosinophils in the airways. (F) Number of

neutrophils in the airways. (G) Fold change of Ccl11 (eotaxin-1), Cxcl1, and Cxcl2 transcript expression in total lung tissue, compared to baseline Chat

LoxP

. (H) Fold

change of M1 macrophage marker Nos2 (inducible nitric oxide synthase, iNOS) and M2 macrophage markers Mrc1 (mannose receptor c-type 1, CD206) and Arg1

(arginase 1) transcript expression in total lung tissue, compared to baseline Chat

LoxP

. Data points represent individual animals. Data show pooled data points from 2

independent experiments with n = 3 C57BL/6J mice per group and n = 4–5 Rora

Cre+

Chat

LoxP

and Chat

LoxP

mice per experiment. Samples from untreated Chat

LoxP

and Rora

Cre+

Chat

LoxP

were collected on different days to C57BL/6J mice and ALT treated Chat

LoxP

and Rora

Cre+

Chat

LoxP

, using animals from the same generations/

litters and parents for both data sets. *p <0.05, **p <0.01, ***p <0.001, ns, non-signiﬁcant difference (p > 0.05).

Roberts et al. Cholinergic Regulation of Airway Inﬂammation

Frontiers in Immunology | www.frontiersin.org May 2022 | Volume 13 | Article 8938443

were particularly interested in examining the neutrophilic response

following ACh depletion. Therefore, we used BALB/c mice, which

have a dominant early neutrophilic response to Alternaria exposure

compared with C57BL/6 mice, whic h are more eosinophil-

dominant (17). The enzymes were co-administered intranasally

with Alternaria,asshowninSupplemental Figure 4A.

Intranasal administration of Alternaria to mice resulted in a

moderate inﬂux of neutrophils (Figures 3A–D ) and pronounced

eosinophilia in the lungs and airways after 48 hours, as

anticipated (Figures 3E–G)(15, 16). When Alternaria was co-

administered with enzymatically active AChE, elevated numbers

of neutrophils were observed in the lungs (Figures 3A–D), but

eosinophilia was strikingly reduced in both sites (Figures 3E–G).

Inactive AChE had no effect o n eosinophil or neutrophil

numbers, indicating that the effects of the active enzyme were

due to hydrolysis of ACh (Figures 3B–G). Treatment with active

AChE alone also resulted in signiﬁcantly increased numbers of

neutrophils (Figures 3B–D) and a small increase in eosinophils

(Figures 3E–G) relative to PBS and inactive AChE baseline

controls. Of note, eosinophil numbers in active AChE-treated

mice at baseline were lower than those in Alternaria + PBS/

inactive AChE-exposed tissues.

Granulocytes in the lungs also showed some phenotypic

alterations following the depletion of ACh (Figures 3H, I and

Supplemental Figures 4B–G). Eosinophils in the lung tissue, but

not the airways, of Alternaria-treated mice demonstrated

enhanced expression of Siglec-F, typical of recruited

inﬂammatory cells (23), and this was further enhanced by co-

administration of active but not inactive AChE (Figures 3H, I).

Exposure to active AChE alone also slightly enhanced eosinophil

Siglec-F expression, but to a lesser extent than following

Alternaria exposure (Figure 3H). Depletion of ACh resulted in

DFE

GIJH

FIGURE 2 | ILC2 responses of Rora

Cre+

Chat

LoxP

mice following acute fungal allergen challenge. Pulmonary ILC2s were analysed from PBS-treated C57BL/6J mice

and Alternaria alternata allergen extract (ALT) treated-Chat

LoxP

and Rora

Cre+

Chat

LoxP

mice 24 h after intranasal dosing. (A) IL-5 and (B) IL-13 expression in the airways

(bronchoalveolar lavage) as determined by ELISA. (C) Representative ﬂow cytometry plots for IL-5 and IL-13 expression by pulmonary ILC2s from the indicated mouse

strains and treatment groups. Numbers on the plots indicate the proportion of the parent ILC2 population for each gate. (D) Proportion of IL-5

ILC2s. (E) Proportion

of IL-13

ILC2s. (F) Proportion of IL-5

IL-13

ILC2s.(G–J) Mean ﬂuorescence intensity (MFI) of (G) IL-5 staining for IL-5

ILC2s (H) IL-13 staining for IL-13

ILC2s.

(I) ST2 staining for ILC2s. (J) ICOS staining for ILC2s. MFI data (G–J) are normalized to the mean of PBS treated C57BL/6J control values. Data points represent

individual animals. Data show pooled data points from 2 independent experiments with n = 3 C57BL/6J mice per group and n = 4–5 Rora

Cre+

Chat

LoxP

and Chat

LoxP

mice per experiment. *p <0.05, **p <0.01, ***p < 0.001, ns, non-signiﬁcant difference (p>0.05).

Roberts et al. Cholinergic Regulation of Airway Inﬂammation

Frontiers in Immunology | www.frontiersin.org May 2022 | Volume 13 | Article 8938444

reduced CD11b (Integrin alpha M) expression on the surface of

eosinophils a nd neutrophils (Supplemental Figur es 4B–E),

wheareas expression of Gr-1 (Ly6C/Ly6G antigen) on airway

neutrophils was enhanced by either Alternaria or depletion of

ACh (Supplemental Figures 4F).

Given the striking alterations to inﬂammatory cell inﬂux

caused by the depletion of ACh, we investigated whether this

was accompanied by an altered chemokine environment. The

level of CXCL1 in lung extracts was slightly increased by

exposure to active AChE alone but was greatly enhanced by

Alternaria + active AChE (Figure 3J). CXCL2 was not affected

by active AChE alone but was considerably enhanced by co-

administration of Alternaria and active AChE (Figure 3K). In

contrast, although eotaxin-1 was only elevated following

exposure to Alternaria, its maximal level was unaffected by

ACh depletion (Figure 3L). In all cases, co-administration of

an inactive enzyme with Alternaria had no signiﬁcant effect on

chemokine levels in the pulmonary tissues.

As previous studies have in dicated that endothelial cell

adhesion molecules could b e downregulated in response to

cholinergic agonists during inﬂammation (24, 25), we also

assessed whether hydrolysis of airway ACh affected their

expression. Administration of Alternaria induced pronounced

upregulation of E-Selectin (Sele)(Figure 3M)andmodest

increases in Intercellular adhesion molecule 1 (Ic am1)

(Figure 3N) and Vascular cell adhesion molecule 1 (Vcam1)

(Figure 3O) transcripts in lung tissue, but these were not further

affected by co-administration of either active or inactive AChE. A

small increase in Sele was also observed when active AChE was

administered alone, in the absence of Alternaria (Figure 3M).

ILC2 Activation and Type 2 Cytokine

Production in Response to Alternaria

Allergen Extract is Profoundly Inhibited

Following Enzymatic Depletion of

Airway Acetylcholine

As anticipated, administration of Alternaria resulted in increased

production of IL-5 and IL-13 by total unseparated cells from lung

tissue, but this was signiﬁcantly reduced following non-selective

BCD

FGH

FIGURE 3 | Granulocyte responses in the lung and airways following acute fungal allergen challenge in the context of enzymatic depletion of airway acetylcholine.

Mice were dosed with Alternaria extract (ALT) or vehicle (PBS) and/or inactive AChE (Ai), active AChE (Aa) or vehicle (PBS) in the combinations indicated. For dosing

regimen, see related Supplemental Figure 4A. (A) Representative H&E-stained lung sections. b, bronchiole; v, blood vessel; a, alveolus. (B) Representative ﬂow

cytometry plots for airway neutrophils from the indicated mouse strains and treatment groups. (C) Number of neutrophils in the lung. (D) Number of neutrophils in

the airways as retrieved by bronchoalveolar lavage (BAL). (E) Representative ﬂow cytometry plots for airway eosinophils from the indicated mouse strains and

treatment groups. (F) Number of eosinophils in the lung. (G) Number of eosinophils in the airways, retrieved by BAL. Normalised mean ﬂuorescence intensity (MFI) of

(H) Siglec-F staining for lung eosinophils and (I) Siglec-F staining for airway eosinophils. Quantiﬁcation of protein expression from total lung protein lysate for

(J) CXCL1. (K) CXCL2. (L) eotaxin-1(CCL11). qRT-PCR analysis of (M) Sele (E-selectin), (N) Icam1 (intercellular cell adhesion molecule 1), (O) Vcam1 (Vascular cell

adhesion protein 1) from total lung tissue. Transcript levels were normalized to reference genes peptidylprolyl isomerase A (Ppia) and eukaryotic translation elongation

factor 2 (Eef2) and calculated as ratios of pooled PBS control treated group samples (relative transcript level = 1). MFI data are normalised to the mean of PBS

control values. Numbers on the cytometry plots indicate proportion of the parent Live/CD45

gate. Data points represent individual animals. Data are representative

of 2 independent experiments with n = 5 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-signiﬁcant difference (p >0.05).

Roberts et al. Cholinergic Regulation of Airway Inﬂammation

Frontiers in Immunology | www.frontiersin.org May 2022 | Volume 13 | Article 8938445

depletion of airway ACh (Figures 4A, B). Active AChE

treatment alone did not alter total IL-5 production, although

IL-13 was increased (Figure 4B). Although ILC2-derived ACh

did not appear to impact on th ese changes (Figure 2), we

reasoned that ILC2 activity may still be dependent directly or

indirectly on cholinergic signaling and therefore investigated this

in more detail. Expression of IL-5 and IL-13 was increased by

pulmonary ILC2s in response to Alternaria, with a signiﬁcantly

higher proportion of ILC2s expressing the cytokines and higher

per-cell expression levels (Figures 4C–H). This was strongly

suppressed by active but not inactive AChE (Figures 4C–H).

Conversely, treatment with active AChE at baseline had the

opposite effect, increasing IL-5 and IL-13 production by lung

ILC2s relative to PBS and inactive AChE-treated controls

(Figure 4C–H). Cytokine production by ILC2s reﬂected the

activation status of the cells, as measured by ST2 and ICOS

expression, which were enhanced in Alternaria-treated mice

alone or with inactive AChE, and in active AChE only treated

mice, but restricted follow ing depletion of ACh with active

enzyme in the context of Alternaria exposure (Figures 4I, J).

Alternative Activation of Pulmonary

Macrophages is Inhibited by Depletion

of Acetylcholine

Both non-selective depletion of ACh and loss of ILC2-speciﬁc

capacity to synthesize ACh enhanced neutrophil

chemoattractants and cellular inﬂux into the lungs following

exposure to Alternaria, indicating that neutrophil trafﬁcking is

sensitive to suppression by cholinergic signaling. We therefore

A CB

DEF

GHIJ

FIGURE 4 | ILC2 responses following acute fungal allergen challenge in the context of enzymatic depletion of airway acetylcholine. Mice were dosed with Alternaria

extract (ALT) or vehicle (PBS) and/or inactive AChE (Ai), active AChE (Aa) or vehicle (PBS) in the combinations indicated. For dosing regimen, see related Supplemental

Figure 4A. (A) IL-5 and (B) IL-13 expression in the supernatant of total lung cells cultured with PMA/Ionomycin, as determined by ELISA. (C) Representative ﬂow

cytometry plots for IL-5 and IL-13 expression by pulmonary ILC2s from the indicated mouse strains and treatment groups. Numbers on the plots indicate proportion of

theparentILC2populationforeachgate.(D) Proportion of IL-5

ILC2s. (E) Proportion of IL-13

ILC2s.(F)Proportion of IL-5

IL-13

ILC2s. (G–J) Mean ﬂuorescence

intensity (MFI) of (G) IL-5 staining for IL-5

ILC2s, (H) IL-13 staining for IL-13

ILC2s, (I) ST2 staining for ILC2s, (J) ICOS staining for ILC2s. MFI data (G–J) are normalized

to the mean of PBS control values. Data points represent individual animals. Data are representative of 2 independent experiments with n = 5 mice per group. *p < 0.05,

**p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-signiﬁcant difference (p >0.05).

Roberts et al. Cholinergic Regulation of Airway Inﬂammation

Frontiers in Immunology | www.frontiersin.org May 2022 | Volume 13 | Article 8938446

examined the M1/M2 phenotypes of pulmonary macrophages

following enzymatic depletion of ACh. We analyzed three

macrophage populations: monocyte-derived airway

macrophages (Mo-AMF), tissue resident alveolar macrophages

(TR-AMF), and interstitial macrophages (IMF) in lung tissue

(Supplemental Figures 5A–E).

Mice exposed to Alternaria alone or Alternaria +inactive

AChE showed enhanced frequencies and numbers of Mo-AMF

(Figures 5A–B), no alterations in overall numbers of TR-AMF,

but reduced frequencies of these cells due to greatly enhan ced

neutrophil and eosinophil inﬂux, (Figures 5C, D), and

increased frequencies and numbers of total IMF in the lung

(Figures 5E, F). Alternaria +activeAChEtreatmentdidnot

alter total Mo-AMF (Figures 5A, B ) but reduced total TR-

AMF (Figures 5C, D) and resulted in a non-signiﬁcant trend

toward increased lung IMF (Figures 5E, F). Treatment with

inactive AChE alone did not alter macrophage populations

relative to PBS o nly treated controls, however active AChE

alone resulted in signi ﬁcantly more Mo-AMF (Figure 5B), less

TR-AMF (Figure 5D)andmoreIMF (Figure 5E). Notably,

Alternaria +activeAChEtreatment resulted in signiﬁcantly less

TR-AMF (Figure 5D)butmoreIMF (Figure 5E ), compared

to the active AChE alone.

A characterization of macrophage polarization phenotypes

revealed that Alternaria exposure combined with ACh depletion

reduced the numbers expressing mannose receptor c-type 1

(CD206) (Figures 5G–

L), a speciﬁc indicator of the M2

phenotype (26). Conversely, active AChE treatment alone

showed a tendency towards increased numbers of M2 Mo-

AMF (Figure 5H) and IMF (Figure 5L) relative to PBS and

inactive AChE-treated controls. To investigate whether this was

correlated with enhancement of the M1 phenotype, we gated on

CD206

MHCII

macrophages (Supplemental Figure 5E).

Active AChE a lone, and Alternaria treatment + PBS or

inactive AChE increased numbers of M1 Mo-AMF ,while

active AChE treatment and Alternaria co-exposure decreased

M1 Mo-AM and TR-AMF relative to Alternaria exposed

controls (Figures 5M–P). However, M1-like IMF were

increased signiﬁcantly in Alternaria + active AChE treated

mice, relative to increases observed in Alternaria + PBS and

inactive AChE controls as well as active AChE only treated

animals (Figures 5Q, R).

Cumulatively, these data indicate that cholinergic signaling is

an important and complex regulator of immune responses, both

at baseline during homeostasis and during fungal allergen-

induced, allergic inﬂammation in the murine lung.

CBD FE

GIHJ LK

MONP

FIGURE 5 | Alveolar and interstitial macrophage responses and alternative activation following acute fungal allergen challenge in the context of enzymatic depletion

of airway acetylcholine. Mice were dosed with Alternaria extract (ALT) or vehicle (PBS) and/or inactive AChE (Ai), active AChE (Aa) or vehicle (PBS) in the

combinations indicated. For dosing regimen, see related Supplemental Figure 4A. Monocyte-derived alveolar macrophages (Mo-AMF), tissue-resident alveolar

macrophages (TR-AMF) and lung interstitial macrophages (IMF) were analysed for M1 (CD206

MHCII

) and M2 (CD206

) phenotypes. (A) Mo-AMF as the

proportion of the total live cell population in the airways. (B) Number of Mo-AMF in the airways. (C) TR-AMF as the proportion of the total live cell population in the

airways. (D) Number of TR-AMF in the airways (E) IMF as the proportion of the total live CD45

cell population in the lung. (F) Number of IMF in the lung. (G) M2

Mo-AMF as the proportion of total Mo-AMF in the airways. (H) Number of M2 Mo-AMF in the airways. (I) M2 TR-AMF as the proportion of total TR-AMF in the

airways. (J) Number of M2 TR-AMF in the airways. (K) M2 IMF as the proportion of total IMF in the lung. (L) Number of M2 IMF in the lung. (M) M1 Mo-AMF as

the proportion of total Mo-AMF in the airways. (N) Number of M1 Mo-AMF in the airways. (O) M1 TR-AMF as the proportion of total TR-AMF in the airways.

(P) Number of M1 TR-AMF in the airways. (Q) M1 IMF as the proportion of total IMF in the lung. (R) Number of M1 IMF in the lung. CD206 and MHCII gating was

carried out based on absence of expression in ﬂuorescence minus one (FMO) controls. Data points represent individual animals. Data are representative of 2

independent experiments with n = 5 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001, ns, non-signiﬁcant difference (p >0.05).

Roberts et al. Cholinergic Regulation of Airway Inﬂammation

Frontiers in Immunology | www.frontiersin.org May 2022 | Volume 13 | Article 8938447

DISCUSSION

As we recently demonstrated that ILC2-derived ACh is required

for optimal type 2 responses to parasitic infection, we sought to

determine whether this also plays a role in allergic inﬂammation.

Rora

Cre+

Chat

LoxP

mice showed enhanced airway neutrophilia

following fungal allergen exposure, indicating that neutrophil

inﬂux is regulated in part by ILC2-derived ACh. This was further

exacerbated by non-selective depletion of ACh, suggesting that

there are multiple cellular sources of ACh which contribute to

the suppression of neutrophil inﬂux into the airways. We have

not determined the precise sources of these signals, which could

be neuronal, epithelial (9), or lymphoid (4).

A recent study showed that blockade of ACh synthesis by

inhibition of choline uptake in the airways promoted

neutrophilic inﬂammation and delayed the recovery of mice

infected with inﬂ uenza A (8), consistent with our current data. B

cells, CD4

, and CD8

T cells were identiﬁed as potential sources

of ACh via the use of transgenic choline acetyltransferase

(ChAT) reporter mice (8), and we have also identiﬁed ChAT

expression in several lymphocyte lineages in the lungs (4). B cell-

derived ACh has previously been demonstrated to inhibit

neutrophil recruitment to the peritoneum in an LPS-induced

model of sterile sepsis, and it was concluded that this resulted

from suppression of expression of intercellular adhesion

molecule 1 (ICAM-1) and vascular cell adhesion molecule 1

(VCAM-1) on vascular endothelial cells, signaling through

mAChRs (24). This built upon other studies which indicated

that ACh reduced expression of adhesion molecules and

suppressed chemokine release from endothelial cells (25) and

moreover suppressed expression of CD11b by neutrophils (27),

in both cases signaling through the a7 nicotinic acetylcholine

receptor (nAChR).

In our current study, no alteration in the expression of the

adhesion molecules ICAM-1, VCAM-1, and E-selectin in lung

tissue was observed after the depletion of ACh in the context of

Alternaria induced airway inﬂammation, and CD11b expression

on both neutrophils and eosinophils was suppressed rather than

enhanced. However, depletion of ACh resulted in clearly enhanced

expression of the neutrophil-attractant chemokines CXCL1 and

CXCL2, and this was also evident in Rora

Cre+

Chat

LoxP

mice. We

therefore conclude that ACh deﬁciency leads to alterations in

chemokine expression, which are primarily responsible for the

inﬂux of neutrophils into the lungs in this model of allergic

inﬂammation. However, depletion of ACh without exposure to

Alternaria did enhance pulmonary E-Selectin (Sele) expression in

addition to CXCL1, concomitant with enhanced neutrophilia,

suggesting that ACh plays an important role in the restriction of

neutrophil inﬂux in the absence of an overt inﬂammatory

stimulus. Previously, we have identiﬁed lung B cells as a

signiﬁcant source of non-neuronal ChAT and thus ACh

synthesis in mice (4). Therefore, in line with previous studies

(24, 28), it is possible that B cell production of ACh regulates

neutrophil trafﬁcking at homeostasis in the lung.

While neutrophilia was promoted, depletion of ACh also

prevented the characteristic eosinophilia associated with

Alternaria exposure, most likely due to restricted ILC2

activation and release of IL-13 and IL-5. These cytokines

operate in concert with eotaxin to recruit eosinophils to the

lungs (29, 30) and are also important in the maintenance of

cellular viability (31, 32). Expression of Siglec-F is increased in

eosinophils following lung inﬂammation (23) and has been

proposed to regulate cell numbers via induction of apoptosis

(33). In our current study, depletion of ACh resulted in enhanced

expression of Siglec-F on lung eosinophils, and it is therefore

possible that this contributes to impaired cell survival.

Conversely, ACh depletion in the absence of Alternaria

exposure slightly increased eosinoph il numbers in the lung.

This is most likely due to the activating effect that ACh

depletion at baseline appeared to have on lung ILC2s.

Recent evidence demonstrated that synthesis of ACh was

required for resolution of inﬂammation following respiratory

viral infection, accompanied by increased numbers of ChAT

lymphocytes in lung tissues (8). These lymphocytes were found

in direct physical contact with pulmonary macrophages (8),

suggesting that lymphocyte-derived ACh might play a role in

driving macrophage polarization to an anti-inﬂammatory

phenotype required for tissue repair (19, 34 ). Physical contact

of cells is particularly relevant as the labile nature of ACh

necessitates that intercellular signaling functions over relatively

short distances. Tissue resident alveolar macrophages are the

predominant leucocyte population present in the airways of

healthy mice at homeostasis. Most of these cells are CD206

and co-express additional markers of the M2 phenotype (26). In

contrast, inﬂammation promotes the inﬂux and development of

monocyte-derived airway macrophages, characterized by

expression of CD11b and Siglec-F (35–37). Depletion of ACh

reduced the M2 macrophage pool by suppressing the proportion

of monocyte-derived and interstitial macrophages that displayed

an M2-like phenotype and decreasing the overall number of

tissue-resident airway macrophages. This could result in part

from the reduction of type-2 cytokines but also because ACh acts

directly on macrophages to promote differentiation to the M2

state (38–40). Supportive of this, an increase in M1 macrophage

marker expression was observed following both ILC2-speciﬁc

ACh depletion and pan-ACh depletion. The enhanced

expression of CXCL1 and CXCL2 following depletion of ACh

in our model may t here fore reﬂect a shift in macrophage

phen otype fro m M2 to M1, although pericytes, endothelial,

and mast cells are also impor tant sources of neutrophil

chemokines (18, 41, 42).

The role of speciﬁc muscarinic and nicotinic acetylcholine

receptors in regulating pulmonary inﬂammation is complex.

Muscarinic receptor subtype-deﬁcient mice have been used to

examine cigarette smoke-induced airway inﬂammation, with the

conclusion that signaling through the M3 receptor was broadly

pro-inﬂammatory, characterized by elevated neutrophils,

macrophages, and lymphocytes in the airways and a

corresponding increase in the expression of CXCL-1, CCL2,

and IL-6, whereas signaling through the M1 and M2 mAChRs

was generally anti-inﬂammatory, characterized by a reduction in

cellular inﬁltration and chemokine levels (43). Tiotropium, a

Roberts et al. Cholinergic Regulation of Airway Inﬂammation

Frontiers in Immunology | www.frontiersin.org May 2022 | Volume 13 | Article 8938448

mAChR antagonist, which is functionally selective for the M3

subtype, is widely used to treat asthma and chronic obstructive

pulmonary disease and to alleviate bronchoconstriction and

mucus production, has been documented to reduce airway

inﬂammation and remodeling (44, 45).

The effects of cholinergic signaling on ILC2s are similarly

complex. Several reports indicate that a7 subtype-selective

nAChR agonists reduce ILC2 effector function and airway

hyperreactivity in an Alternaria allergic inﬂammation model

(46, 47), whereas others indicate that broader, non-selective

inhibition of nicotinic or m uscarinic receptors can block

activation (4, 5). A recent study demonstrated that inhibition

of mAChR signaling with tiotropium attenuated ILC2

proliferation, type 2 cytokine production and eosinophilia in

papain and IL-33-driven models of airway inﬂammation (48).

Tiotropium did not inhibit cytokine pr oduc tion by isolated

ILC2s in vitro, but did suppress IL-4 production by basophils,

suggesting that ACh activates ILC2s indirectly via effects on

basophils (48). Cholinergic signaling via mAChRs has also been

demonstrated to stimulate ATP sec retio n in diverse tissues,

suggesting another indirect route for activation of ILC2s by

induction of IL-33 release (49, 50). Exemplifying the

complexity of the effects o f c holin ergic signaling on ILC2s,

enzymatic depletion of ACh had opposite effects on ILC2

activity in the lung dependent on exposure to Alternaria.

Previously, we and others have shown that pulmonary ILC2s

undergo signiﬁcant alterations to ACh receptor expression

following he lminth and alarmin-induced activation, such as

loss of the a7 inhibitory nAChR and upregulation of the

excitatory M3 mAChR (4, 5). It is therefore possible that

cholinergic signaling at homeostasis is required to restrict ILC2

activity, but supports effector responses following tissue-speciﬁc

activation of the cells. Tissue signals, including IL-33, are known

to regulate resident ILC2 at homeostasis and upon activation,

increasing “basal activation” of the cells (51, 52). Therefore,

inhibitory cholinergic signaling may serve an important role in

the restriction of basal type 2 effector responses before the need

for full activation of ILC2s in the context of exogenous challenge

such as parasite infection and/or tissue damage.

Our data suggest that ACh has distinct effects associated with

fungal allergen-induced pulmonary inﬂammation, on the one

hand, promoting ILC2 cytokine production and eosinophilia,

and on the other hand, inhibiting neutrophilia via suppression of

CXCL1 and CXCL2 expression. ILC2-derived ACh plays a role

in inhibition of neutrophil inﬁltration, potentially by directing

macrophages towards an M2 phenotype, thus limiting

expression of neutrophil chemoattractants.

MATERIALS AND METHODS

Animals

This study was approved by the Animal Welfare Ethical Review

Board at Imperial College London and was licensed by and

performed under the UK Home Ofﬁce Animals (Scientiﬁc

Procedures) Act Personal Project Licence number 70/8193:

‘Immunomodulation by helminth parasites’. C57BL/6J and

BALB/c mice, aged 6–8 weeks old, were purchased from

Charles River. The Rora

Cre+

Chat

Loxp

mice used in this study

were generated as previously described (4).

Murine Model of Acute Fungal

Allergen Exposure

Extracts of A. alternata were purchased as lyophilized protein

extracts from Greer Laboratories (USA). Mice were anesthetized

with aerosolized isoﬂuorane before intranasal administration

with 50 mgofAlternaria in a ﬁnalvolumeof50µlof

phosphate buffered saline without Ca

or Mg

(PBS, Sigma).

Mice were exposed to a single dose of Alternaria for 24 or 48 h as

indicated. Control animals were dosed with 50 ml of PBS on the

same schedule. For co-administration of active or inactive AChE,

20 mg of either enzyme was mixed with 50 mgofAlternaria in

PBS and the volume adjusted to 50 µl for the ﬁrst dose, and 20 mg

of the enzyme alone was administered in 50 ml of PBS for the

second dose.

Expression of Active and Inactive AChE

From N. brasiliensis

N. brasiliensis AChE B was expressed in Pichia pastoris as a

secreted protein and puriﬁed from culture supernatants as

previously described (21). An enzymatically inactive form of

theenzymewasgeneratedvia site-directed mutagenesis,

changing the active site serine residue Ser-193 (Ser-200 in

Torpedo AChE) to alanine (S193A), using the Quickchange

site-directed mutagenesis kit (Stratagene) as pre viously

described (22). The mutation was conﬁrmed by sequencing

before expression in P. pastoris, and puriﬁcation conﬁrmed

that the enzyme was catalytically inactive. Proteins were passed

through endotoxin removal columns (Pierce) and endotoxin

removal was conﬁrmed using a LAL Chromogenic Endotoxin

Quantitation Kit (Pierce). Prote in concentrations were

determined using the Pierce Coomassie Plus (Bradford) Assay

Kit (Thermo Scientiﬁc). AChE activity was determined by the

method of Ellman with 1 mM acetylthiocholine iodide as the

substrate in the presence of 1 mM 5,5’-dithiobis(2-nitrobenzoic

acid) (DTNB) in 100 mM sodium phosphate pH 7.0 at 20°C. The

reaction was monitored by measuring the absorbance at 412 nm,

and the hydrolysis of acetylthiocholine iodide was calculated

from the extinction coefﬁcient of DTNB (53). One unit of AChE

was deﬁned as 1 m mol of substrate hydrolyzed per min at 20°C.

Denaturing and

Non-Denaturing Electrophoresis

Puriﬁed recombinant active and inactive enzymes were resolved

by SDS-PAGE on 10% polyacrylamide gels followed by staining

with Coomassie brilliant blue . The same preparations w ere

resolved under non-denaturing conditions by electrophoresis

in 7.5% polyacrylamide gels in Tris-Borate-EDTA buffer pH

8.0, and enzyme activity was assayed using the method of

Karnovsky and Roots (54).

Roberts et al. Cholinergic Regulation of Airway Inﬂammation

Frontiers in Immunology | www.frontiersin.org May 2022 | Volume 13 | Article 8938449

Tissue Harvest and Preparation

For isolation of bronchoalveolar cells, the lungs were lavaged

twice via the trachea in 2 ml of PBS with 0.2% BSA and 2 mM

EDTA. Erythrocytes were lysed, leukocytes resuspended and

counted. For lung single cell suspensions, lungs were perfused

with PBS via injection into the heart before harvest, and lung

leukocytes were isolated by dicing lung tissue and digesting for 1

h at 37°C with 300 U ml

−1

of collagenase-II (Gibco) + 150 mg

−1

of DNAse I (Sigma) in PBS without Mg

or Ca

, followed

by sample mashing through a 70 µm cell strainer into single-cell

suspension, followed by red blood cell lysis.

Flow Cytometry

Single cell suspensions were stained with ﬁxable viability dyes

(Invitrogen), then treated with rat anti- mou se CD32/ CD16

(FcBlock, BD Biosciences), washed, and stained for

extracellular markers using ﬂuorophore conjugated monoclonal

antibodies (eBioscience , Milteny i Biotec, or Biolegend). For

intracellular staining, cells were ﬁxed for 30 min at room

temperature, then permeabilized using the FoxP3/transcription

factor sta ining bu ffer kit (eBiosc ience) an d stained with

ﬂuorochr ome- conjugated antibodies. Unstained samples and

ﬂuorescence minus one control were used as appropriate.

Samples were analysed on a BD LSR Fortessa

™

analyzer.

Immunophenotyping of

Leucocyte Populations

Unless otherwise stated, leucocyte populations were identiﬁed by

ﬂowcytometrybygatinglivecells,followedbysinglecell

and CD45

gating, and then using the following markers: ILC2:

CD45

Lineage

CD127

GATA3

ICOS

ST2

;CD4

Tcells:

CD3

CD4

;neutrophils:CD11b

Siglec-F

GR-1

CD11c

−/lo

;

eosinophils: CD11b

Siglec-F

Gr-1

−/lo

CD11c

−

; tissue resident

alveolar macrophages (TR-AMF): CD11b

−

F4/80

Siglec-

CD11c

Gr-1

−

; monocyte-derived alveolar macrophages (MO-

AMF): CD11b

F4/80

Siglec-F

CD11c

Gr-1

−

; interstitial lung

macrophages (IMF): CD11b

F4/80

Siglec-F

CD11c

−/+

Gr-1

−

CD206 was used as a marker of alternatively activated M2

macrophage subsets. Absence of CD206 expression (CD206

−

)

and positive expression of MHCII (MHCII

)wereusedto

identify M1 macrophage subsets. The lineage panel consisted of

antibodies against CD3, CD4, CD8, CD5, B220, CD19, TER119,

CD49b, FcϵRI, CD11c, and Gr-1 (Ly6C/Ly6G). Scatter proﬁling of

gated myeloid populations was also used to validate identity, as

shown in Supplemental Figure 1.

Intracellular Cytokine Capture

Single cell suspensions were diluted to 5× 10

cells ml

−1

cDMEM (DMEM + 10% FCS, + 2 mM L-glutamine + 100 U ml

−1

penicillin + 100 ug ml

−1

streptomycin) and either stimulated for

4 h at 37°C/5% CO2 with 1 ug ml

−1

PMA/100 ng ml

−1

ionomycin

with 1× brefeldin-A (GolgiPlug, BD Biosciences) + 1 mM

monensin ((Sigma) or left unstimulated (golgi inhibitors

alone). Samples were stained, ﬁxed, and permeabilized as

described, and intracellular staining for Fc receptor blocking

followed by ﬂuorophore conjugated mAbs against IL-5 and IL-13

was performed in permeabilization buffer.

RT-qPCR

Total lung tissue was homogenized using a Tissuelyser II (Qiagen).

Total RNA was extracted by TRIzol/chloroform phase-separation,

DNAse-1 treated, then 1 mg of RNA was reverse transcribed using

the iScript cDNA synthesis kit (Biorad). RT-qPCR reactions were

carried out using the PowerUp SYBR Green Mix (ThermoFisher) in

an ABI 7500 Fast Real-time PCR thermocycler (Applied

Biosystems). RT-qPCR reactions were run in triplicate, with no

template and no RT controls. Relative expression of Ccl11, Cxcl1,

and Cxcl2 was calculated by the comparative cycle threshold (Ct)

method (2

−DDCT

)usingActb, Hprt,andGapdh as reference genes.

Eef2and Ppia wereusedas the referencegenesfor calculatingIcam1,

Vcam1,andSele expression. Primer sequences used for RT-qPCR

were as follows:

Nos2:F:5’- CCGGCAAACCCAAGGTCTAC-3’, R: 5’ -

CTGCTCCTCGCTCAAGTTCA-3’.

Arg1:F:5’-AAAGGCCGATTCACCTGAGC -3’, R: 5 ’-

CTGAAAGGAGCCCTGTCTTGTA-3’.

Mrc1:F:5’-GGAGGGTGCGGTACACTAAC-3’, R: 5’-

TCAGTAGCAGGGATTTCGTCTG-3’.

Ccl11:>F:5’-TGGCTCACCCAGGCTCCATC-3’ , R: 5 ’-

TCTCTTTGCCCAACCTGGTCTT-3’.

Cxcl1: >F: 5’-ACCCAAACCGAAGTCATAGCCA -3’, R: 5’-

TCAGAAGCCAGCGTTCACCA-3’.

Cxcl2:>F:5’-TC CAAAAGATACTG AACAAAGGCAA-3’,

R: 5’-ATCAGGTACGATCCAGGCTTCC-3’ .

Icam1

:F:5’-AGCTCGGAGGATCACAAACG-3’, R: 5’-

TCCAGCCGAGGACCATACAG-3’.

Sele:F:5’ -CCCAGTGCTTCTGGACCTTT-3’ , R: 5’ -

TTCACAGCTGAACACGTGGG-3’.

Vcam1:F:5’-CGACCTTCATCCC CACCATT-3’, R: 5’-

GGGGGCAACGTTGACATAAAG-3’.

Eef2:F:5’-CCCCAACATTCTCACCGACA-3’ , R: 5’-

AGAGAGCGCCCTCCTTAGTA-3’

Ppia:F:5’- GCAT ACAGGTCCTGGCATCT-3’, R: 5’-

ATGCTTGCCATCCAGCCATT-3’

Gapdh:>F:5’-GTCATCCCAGAGCTGAACGG-3’, R: 5’-

TACTTGGCAGGTTTCTCCAGG-3’.

Actb: >F: 5’-TTCCTTCTTGGGTATGGAATCCT-3’, R: 5’ -

TTTACGGATGTCAACGTCACAC-3’.

Hprt:F:5’-ACAGGCCAGACTTTGTTGGA-3’

, R: 5’-

ACTTGCGCTCATCTTAGGCT-3’.

Enzyme-Linked Immunosorbent Assay

Protein lysates were made by snap freezing the small right lung

lobe directly after tissue harvest. Harvested lung samples were

stored at −80°C and homogenised in PBS with an electric

homogeniser. Samples were centrifuged and the supernatant

collected. Samples were diluted to a ﬁnal concentration of 50

mg of lung tissue ml

−1

for use. Total lung cells were cultured at a

density of 5 × 10

ml for 12 h with 12.5 ng/ml PMA and 125 ng/

ml Ionomycin, before collecting culture supernatants and

centrifuging to clear residual cells, followed by freezing at −20°C

until analysis. Proteins were quantiﬁed using commercial ELISA

Roberts et al. Cholinergic Regulation of Airway Inﬂammation

Frontiers in Immunology | www.frontiersin.org May 2022 | Volume 13 | Article 89384410

kits according to the instructions of the manufacturer (Duoset,

R&D). For CXCL1 and CXCL2 detection, results were obtained on

a Tecan Sunrise 96-well Microplate reader F039300 with Tecan

Magellan Anlaysis Software V7.2 software. For Eotaxin-1 (Ccl11),

IL-5 and IL-13, results were obtained on a FLUOStar Omega

microplate reader.

Histology

Lung tissue was harvested and ﬁxed in 4% paraformaldehyde

overnight at 4°C. Tissue was parafﬁn embedded, sectioned and

stained with hem atoxylin and eosin (H&E) according to

standard techniques.

Statistical Analysis

Flow cytometry data was analyzed using FlowJ o software

(Treestar). Graphs and statistical tests were carried out using

Graphpad prism software (Graphpad). The normality of data

distribution was analyzed by the Shapiro–Wilk test. Parametric

data were analyzed by Welch’s t-test, non-parametric data were

analyzed by Mann–Whitney-U test. Data represent mean ± SEM

unless otherwise stated. Statistical signiﬁcance between groups is

indicated as *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001, n.s =

non-signiﬁcant difference (p>0.05).

DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be

made available by the authors, without undue reservation.

ETHICS STATEMENT

The animal study was reviewed and approved by the Animal

Welfare Ethical Review Board at Imperial College London and

was licensed by and performed under the UK Home Ofﬁce

Animals (Scientiﬁc Procedures) Act Personal Project Licence

number 70/8193: ‘Immunomodulation by helminth parasites’.

AUTHOR CONTRIBUTIONS

Conception and design: LR, RB, CS, and MS. Experimental work:

LR, RB, MW, CS, DP, MD, and MS. Analysis and interpretation:

LR, RB, MW, CS, and MS. Provision of Resources: LR, GL, KG,

SB, BR, VQ, WH, and MS. Drafting the initial manuscript: LR

and MS. Drafting the revised manuscript: LR and MS. A ll

authors listed have made a substantial, direct, and intellectual

contribution to the work and approved it for publication.

FUNDING

This work was funded by a project grant to MS from the BBSRC

(BB/R015856/1) and a PhD studentship to LR from the

Wellcome Trust (097011).

ACKNOWLEDGMENTS

We thank Lorraine Lawrence (NHLI, Imperial College London)

for processing histology and Robert Snelgrove (NHLI, Imperial

College London) for advice on chemokine analysis.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at:

https://www.frontiersin.org/articles/10.3389/ﬁmmu.2022.893844/

full#supplementary-material.

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Conﬂict of Interest: The authors declare that the research was conducted in the

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Roberts et al. Cholinergic Regulation of Airway Inﬂammation

Frontiers in Immunology | www.frontiersin.org May 2022 | Volume 13 | Article 89384413

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