Acetylcholine production by group 2 innate lymphoid cells promotes mucosal immunity to helminths

TL;DR: In this paper, the cholinergic phenotype of pulmonary ILC2s was associated with their activation state, could be induced by in vivo exposure to extracts of Alternaria alternata or the alarmin cytokines interleukin-33 (IL-33) and IL-25, and was augmented by IL-2 in vitro.

Abstract: Innate lymphoid cells (ILCs) are critical mediators of immunological and physiological responses at mucosal barrier sites. Whereas neurotransmitters can stimulate ILCs, the synthesis of small-molecule neurotransmitters by these cells has only recently been appreciated. Group 2 ILCs (ILC2s) are shown here to synthesize and release acetylcholine (ACh) during parasitic nematode infection. The cholinergic phenotype of pulmonary ILC2s was associated with their activation state, could be induced by in vivo exposure to extracts of Alternaria alternata or the alarmin cytokines interleukin-33 (IL-33) and IL-25, and was augmented by IL-2 in vitro. Genetic disruption of ACh synthesis by murine ILC2s resulted in increased parasite burdens, lower numbers of ILC2s, and reduced lung and gut barrier responses to Nippostrongylus brasiliensis infection. These data demonstrate a functional role for ILC2-derived ACh in the expansion of ILC2s for maximal induction of type 2 immunity.

Summary

Introduction

• Acetylcholine (ACh) is best known as a small-molecule neurotransmitter, but its role in cholinergic signaling also regulates the immune system.
• T cell expression of ChAT induced by IL-21 is essential for tissue trafficking required for T cell-mediated control of viral infection (6) .
• ILC2s have been shown recently to be both positively and negatively regulated by neurotransmitters such as neuromedin U (NMU) (10) (11) (12) and noradrenaline (13) , whereas group 3 innate lymphoid cells (ILC3s) upregulate lipid mediator synthesis in response to vagally-derived ACh (14) .
• These data demonstrate that the production and release of ACh by ILC2s is an important factor in driving type 2 immunity.

ILC2s synthesize and release acetylcholine during type 2 immunity

• The cholinergic phenotype of immune cells was monitored across the time course of a primary infection with N. brasiliensis using ChAT-eGFP BAC mice (3) .
• In these experiments, cells were isolated from infected animals at D11 p.i. to maximize the number of ACh-producing ILC2s obtained.
• A similar analysis of marker expression of the very few ChAT-eGFP + cells in naïve ChAT-eGFP BAC lungs revealed that these cells clearly showed a nILC2-like profile, with no obvious differences in marker expression to that of total ChAT neg ILC2s, including ICOS and ST2 (Figure 2F , 2G, 2H).
• A different scenario was observed in infected mice however, with a disparate profile for total ChAT-eGFP + ILC2s relative to total ChAT neg ILC2s (Figure 2F , 2G, 2H), corroborating the findings of previous analyses (Figure 1G, 1H) .
• Challenge with Alternaria induced a small increase in ChAT-eGFP expression in some lymphocyte populations, including CD4 +.

IL-25 and IL-33 induce the cholinergic phenotype of pulmonary ILC2s

• The authors data suggested that ChAT expression was associated with cellular activation, leading us to investigate whether known activators of ILC2s could induce this phenotype.
• Ex vivo stimulation of CD45 + cells isolated from naïve ChAT BAC -eGFP reporter mice with IL-33, but not IL-7, enhanced ILC2 ChAT-eGFP expression, suggesting that activation through alarmin signaling pathways specifically drives the ILC2 cholinergic phenotype (Figure 4A ).
• Lung ILC2s predominantly express the IL-33 receptor in naïve animals at immunological baseline, whereas iILC2s expressing the IL-25 receptor are thought to migrate to the lung from sites such as the gut following tissue damage such as that caused by helminth infection (18) .
• To assess the capacity of lung-resident ILC2s to upregulate ChAT-eGFP, the authors isolated CD45 + cells from the lungs of naïve ChAT BAC -eGFP reporter mice and stimulated them in vitro with different combinations of recombinant IL-33, IL-25, and IL-2, which are known to function as alarmins or promote proliferation and cytokine production (20, 21) .
• The authors assayed ChAT-eGFP expression by ILC2s after 24 h, and stimulation with IL-25 and IL-33 enhanced ChAT-eGFP expression.

RoRα-driven disruption of ChAT expression impairs pulmonary type 2 immunity to N. brasiliensis

• To determine whether synthesis of ACh by ILC2s played a role in immunity to helminth infection, the authors generated Rora Cre+ Chat LoxP mice in which a portion of the coding domain of the Chat gene is floxed (22) and excised by Cre-recombinase expressed under the control of Rora regulatory elements ( 23) (Figure S2A-B ).
• Chat deletion in Rora Cre+ Chat LoxP ILC2s was confirmed by PCR analysis and sequencing (Figure S2C , S2D, S2E).
• Infection of Rora Cre+ Chat LoxP and Chat LoxP littermate controls with N. brasiliensis revealed that the number of larvae recovered from the lungs were not significantly different between genotypes at 2 dpi, but higher intestinal worm burdens were observed at day 6 p.i. in Rora Cre+.
• During the anti-helminth immune response, IL-13 drives goblet cell hyperplasia and mucin production at epithelial barrier sites including the lung, where the predominant gel-forming mucins secreted by goblet cells are Muc5b and Muc5ac (25) .
• Infected Rora Cre+ Chat LoxP at D6 p.i. demonstrated reduced expression of Muc5b and Muc5ac in total lung tissue (Figure 5D ).

ILC2-derived ACh promotes autocrine population expansion of ILC2s to facilitate optimal antihelminth type 2 immunity.

• ILC2s are the major innate source of IL-13 during helminth infection, and ILC2-derived IL-13 is critical for expulsion of N. brasiliensis and induction of mucin expression in response to helminth infections (8, 9, 26) .
• Slightly fewer ILC2s were also observed in Rora Cre+ Chat LoxP lungs compared with Chat LoxP lungs at baseline (Figure 7A ), and the fold change for infection-induced increases in ILC2 numbers at this timepoint was not significantly different between genotypes (Figure S3A ).
• Interestingly, there appeared to be a degree of differential expression between ChAT-eGFP + and ChAT-eGFP neg with regards to AChR subtypes (Figure 7H, 7I ).
• In order to determine whether ACh might act as an autocrine factor to influence proliferation and activation of the cells, the authors isolated WT ILC2s from the lungs of N. brasiliensis-infected C57BL/6J mice and cultured them in vitro with IL-7 and IL-2 alone or in the presence of the mAChR antagonist 1,1dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) or the nAChR antagonist mecamylamine.
• Addition of 4-DAMP restricted the proliferative capacity of the cells, whereas mecamylamine had no effect when compared with vehicle-treated control cultures (Figure 7J, 7K, 7L ).

Discussion

• ILC2s play a pivotal role in translating epithelial cell cytokine production into robust type 2 immune responses.
• IL-25 was also a major regulator of ChAT expression, demonstrating that MyD88-dependent signaling is not essential for this in ILC2s.
• In support of this, the authors demonstrated that ILC2s express a range of nicotinic and muscarinic ACh receptors, and expression of the α7 nAChR by ILC2s has also been previously described (27) .
• A surprising observation was that ChAT expression by ILC2s was maintained several weeks after helminth eradication from the host (D21 p.i.).
• It will be interesting to determine whether ACh production by ILC2s plays a role during later, pro-repair activities in addition to the acute inflammatory phase of infection.

Materials and Methods

• The aim of this study was to determine the role of cholinergic signalling in the immune response to infection with a helminth parasite.
• The authors utilised ChAT-eGFP BAC reporter mice and flow cytometry to determine which cells synthesized acetylcholine (ACh), and conducted a kinetic analysis on their cholinergic phenotype throughout infection with the nematode parasite Nippostrongylus brasiliensis.
• Real-time qPCR was used to verify alterations in expression of Chat, and mass spectrometry used to confirm cellular secretion of ACh.
• Parasite recoveries and histological scores were conducted in blinded conditions.
• Chat LoxP mice were generated as previously described (22) and were backcrossed to F6-F10 generations on a B6 background with Rora Cre+ (23) a kind gift from Andreas Zembrzycki (Salk Institute, La Jolla, CA) to generate the Rora Cre+ Chat Loxp mice used in this study.

Murine model of allergic airway inflammation.

• Extracts of Alternaria alternata were obtained as a gift from Henry McSorley (University of Edinburgh) or purchased as lyophilized protein extract from Greer Laboratories (USA).
• Mice were lightly dosed with isoflurane before intranasal administration with 50 μg A. alternata extract in a final volume of 50 µl PBS.
• For isolation of bronchoalveolar cells, lungs were lavaged twice in a total of 2 ml PBS with 0.2% BSA and 2 mM EDTA.
• Samples were passed through 100 μm cell strainers and erythrocytes lysed.
• For intracellular staining, cells were fixed for 30 min at room temperature, then permeabilized using the FoxP3/transcription factor staining buffer kit and stained with fluorochrome-conjugated antibodies.

Ki67 and intracellular cytokine staining.

• To assess proliferative capacity of cells directly ex vivo, samples were processed to single cell suspension as described and rested in cDMEM (DMEM + 10% FCS, + 2mM L-glutamine + 100U ml -1 Penicillin + 100ug ml -1 streptomycin) for 1 hour at 37 o C/5%CO2, before extracellular staining, fixing and permeabilization as detailed.
• For each experimental run, lungs from 5 mice were pooled and sorted as a single sample and isolated ILC2 were then split equally between experimental treatment conditions.
• Polymerase chain reaction (PCR) was carried out in a 20 μl reaction volume containing: 0.25 pmol forward and reverse primers, 1.25 mM dNTPs, 0.5 U Taq polymerase (New England Biolabs), 1x Thermopol reaction buffer (New England Biolabs), 2 µl cDNA and dH20.
• Sections were stained with periodic acid-Schiff's and Haematoxylin and Eosin reagents, photographed at 40x or 100x magnification using a Zeiss Primo Star microscope and analyzed using Image J to determine the Histological Mucus Index (HMI) by established methods (49) .
• Subsequently, tissue slides were heated and boiled in pH 6 citrate buffer for 15 min using a microwave.

Figures

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Full text

University of Birmingham
Acetylcholine production by group 2 innate
lymphoid cells promotes mucosal immunity to
helminths
Roberts, Luke B; Schnoeller, Corinna; Berkachy, Rita; Darby, Matthew; Pillaye, Jamie;
Oudhoff, Menno J; Parmar, Naveen; Mackowiak, Claire; Sedda, Delphine; Quesniaux,
Valerie; Ryffel, Bernhard; Vaux, Rachel; Gounaris, Kleoniki; Berrard, Sylvie; Withers, David
R; Horsnell, William G C; Selkirk, Murray E
DOI:
10.1126/sciimmunol.abd0359
License:
None: All rights reserved
Document Version
Peer reviewed version
Citation for published version (Harvard):
Roberts, LB, Schnoeller, C, Berkachy, R, Darby, M, Pillaye, J, Oudhoff, MJ, Parmar, N, Mackowiak, C, Sedda,
D, Quesniaux, V, Ryffel, B, Vaux, R, Gounaris, K, Berrard, S, Withers, DR, Horsnell, WGC & Selkirk, ME 2021,
'Acetylcholine production by group 2 innate lymphoid cells promotes mucosal immunity to helminths', Science
Immunology, vol. 6, no. 57, eabd0359. https://doi.org/10.1126/sciimmunol.abd0359
Link to publication on Research at Birmingham portal
Publisher Rights Statement:
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version was published in Science Immunology on 05 Mar 2021:
Vol. 6, Issue 57, eabd0359, DOI: 10.1126/sciimmunol.abd0359.
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Download date: 10. Aug. 2022

Submitted Manuscript: Confidential template updated: July 5 2016
Science Immunology Manuscript Template Page 1 of 54
Title
1
2
Acetylcholine production by type 2 innate lymphoid cells promotes mucosal immunity to helminths
3
4
Authors
5
6
Luke B. Roberts
1,2
, Corinna Schnoeller
1
, Rita Berkachy
1
, Matthew Darby
3
, Jamie Pillaye
3,4
, Menno J
7
Oudhoff
5
, Naveen Parmar
5
, Claire Mackowiak
3
, Delphine Sedda
6
, Valerie Quesniaux
6
, Bernhard Ryffel
6
,
8
Rachel Vaux
1
, Kleoniki Gounaris
1
, Sylvie Berrard
7
, David R. Withers
4
, William G. C. Horsnell
3,4,6,*
, and
9
Murray E. Selkirk
1,8,*
10
11
Affiliations
12
13
1
Department of Life Sciences, Imperial College London, London, UK.
14
2
School of Immunology and Microbial Sciences, King’s College London, Great Maze Pond, London SE1
15
9RT, UK.
16
3
Wellcome Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular
17
Medicine, University of Cape Town, Cape Town, South Africa.
18
4
College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
19
5
CEMIR – Centre of Molecular Inflammation Research, Department of Clinical and Molecular Medicine,
20
NTNU – Norwegian University of Science and Technology, 7491 Trondheim, Norway
21
6
Laboratory of Molecular and Experimental Immunology and Neurogenetics, UMR 7355, CNRS-University
22
of Orleans and Le Studium Institute for Advanced Studies, Rue Dupanloup, 45000 Orléans, France.
23
7
Université de Paris, NeuroDiderot, Inserm, 75019 Paris, France
24
8
Further information and requests for resources and reagents should be directed to and will be fulfilled by
25
the Lead Contact, Murray E. Selkirk (m.selkirk@imperial.ac.uk)
26
27
*Correspondence: ME Selkirk, m.selkirk@imperial.ac.uk; WGC Horsnell, wghorsnell@gmail.com
28
29
30

Science Immunology Manuscript Template Page 2 of 54
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Abstract
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Innate lymphoid cells (ILCs) are critical mediators of immunological and physiological responses at mucosal
35
barrier sites. Whereas neurotransmitters can stimulate ILCs, the synthesis of small-molecule
36
neurotransmitters by these cells has only recently been appreciated. Type 2 innate lymphoid cells (ILC2s)
37
are shown here to synthesize and release acetylcholine (ACh) during parasitic nematode infection. The
38
cholinergic phenotype of pulmonary ILC2s was associated with their activation state, could be induced by
39
in vivo exposure to extracts of Alternaria alternata or the alarmin cytokines interleukin (IL)-33 and IL-25,
40
and was augmented by IL-2 in vitro. Genetic disruption of ACh synthesis by murine ILC2s resulted in
41
increased parasite burdens, lower numbers of ILC2s, and reduced lung and gut barrier responses to
42
Nippostrongylus brasiliensis infection. These data demonstrate a functional role for ILC2-derived ACh in
43
the expansion of ILC2s for maximal induction of type 2 immunity.
44
45
One-sentence summary
46
Synthesis of acetylcholine by type 2 innate lymphoid cells is important for optimal immune responses to
47
helminth infection.
48
49
MAIN TEXT
50
51
52
Introduction
53
54
Acetylcholine (ACh) is best known as a small-molecule neurotransmitter, but its role in cholinergic signaling
55
also regulates the immune system. This is best described in the cholinergic anti-inflammatory pathway
56
(CAIP), in which sensory perception of inflammatory stimuli leads to a vagal reflex culminating in α7
57
nicotinic receptor (nAChR) subunit-dependent inhibition of TNF-α, IL-1β and IL-18 production by splenic
58
macrophages (1, 2) . The identification of cells that synthesize ACh has been facilitated by the use of reporter
59
mice to visualize expression of choline acetyltransferase (ChAT), the enzyme which synthesizes ACh (3).
60
CD4
+
T cells with an effector/memory (CD44
+
CD62L
lo
) phenotype were identified as the source of ACh in
61
the spleen responsible for signaling to macrophages in the CAIP (4), and B cell-derived ACh inhibited
62
neutrophil recruitment during sterile endotoxemia (5). Additionally, CD4
+
and CD8
+
T cell expression of
63

Science Immunology Manuscript Template Page 3 of 54
ChAT induced by IL-21 is essential for tissue trafficking required for T cell-mediated control of viral
64
infection (6). Adaptive immunity is also regulated by ACh, and optimal type 2 effector responses to the
65
nematode parasite Nippostrongylus brasiliensis require signaling through the M3 muscarinic receptor
66
(mAChR) (7).
67
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Group 2 innate lymphoid cells (ILC2s) play an important role in initiating type 2 immune responses,
69
producing cytokines such as IL-13 and IL-5, which drive allergic inflammation and immunity to helminth
70
infection (8, 9). ILC2s have been shown recently to be both positively and negatively regulated by
71
neurotransmitters such as neuromedin U (NMU) (10–12) and noradrenaline (13) , whereas group 3 innate
72
lymphoid cells (ILC3s) upregulate lipid mediator synthesis in response to vagally-derived ACh (14).
73
Interestingly, ILCs expressing receptors responsive to neurotransmitters colocalize with neurons in mucosal
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tissues, forming neuroimmune cell units (NICUs) (15). ILC2s also express the neuropeptide calcitonin gene-
75
related protein, CGRP (16). ILC2s have been shown to express tryptophan hydroxylase 1 (Tph1), which is
76
the rate-limiting enzyme for the synthesis of the small-molecule neurotransmitter serotonin and have also
77
been shown to produce serotonin (17).
78
79
In this study, we demonstrate that pulmonary ILC2s upregulate their capacity to synthesize and release ACh
80
during infection with N. brasiliensis, and we show that the cholinergic phenotype of ILC2s is induced by
81
the alarmin cytokines IL-33 and IL-25. Rora
Cre+
Chat
LoxP
transgenic mice, which have ILC2s that do not
82
synthesize ACh, have impaired immunity to N. brasiliensis, reduced expression of type 2 cytokines IL-5 and
83
IL-13 in the lung, the mucins Muc5b and Muc5ac in the lung, and altered intestinal barrier responses. These
84
data demonstrate that the production and release of ACh by ILC2s is an important factor in driving type 2
85
immunity.
86

Results
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88
ILC2s synthesize and release acetylcholine during type 2 immunity
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The cholinergic phenotype of immune cells was monitored across the time course of a primary infection
90
with N. brasiliensis using ChAT-eGFP
BAC
mice (3). From day 4 post infection (D4 p.i., immediately
91
following the pulmonary migratory phase of parasite larvae) until at least D21 p.i., (long past the peak
92
of the acute phase of infection-driven inflammation) the proportion and number of CD45
+
cells in lung
93
tissue that expressed ChAT (ChAT-eGFP
+
) was elevated compared with uninfected (naïve) controls
94
(Figure 1A). Analysis of ChAT-eGFP
+
leukocytes revealed that most of these cells were from lymphoid
95
rather than myeloid lineages, as previously reported in other models and tissues (5) (Figure 1B). Of the
96
populations screened, expression of ChAT-eGFP was dramatically upregulated only in ILC2s at an early
97
time point (D4) in infection (Figure 1B, Figure S1A).
98
99
ChAT-eGFP expression by ILC2s in lung and bronchoalveolar lavage (BAL) samples increased by D4
100
p.i., peaked at D7, and remained elevated in both sites at D21. The proportion of ILC2s that were ChAT-
101
eGFP
+
was consistently greater in BAL than in the lungs (Figure 1C, 1D). Real-time (RT)-qPCR
102
confirmed that Chat expression in pulmonary ChAT-eGFP
+
ILC2s from infected ChAT-eGFP
BAC
mice
103
was upregulated in comparison to ILC2 from uninfected ChAT-eGFP
BAC
animals, as well as to ChAT-
104
eGFP
neg
ILC2, validating our reporter system (Figure 1E). HPLC-mass spectrometry was used to verify
105
that WT ILC2s synthesize and release ACh and showed that this was greatly enhanced during parasite
106
infection (Figure 1F). In these experiments, cells were isolated from infected animals at D11 p.i. to
107
maximize the number of ACh-producing ILC2s obtained. We observed that ChAT-eGFP
+
ILC2s had an
108
increased mean fluorescence intensity (MFI) for the IL-33 receptor subunit ST2 compared with ChAT-
109
eGFP
-
cells at D4 and D7 p.i. (Figure 1G), and for inducible T cell co-stimulator (ICOS) at D7 p.i.
110
(Figure 1H), suggesting that ChAT expression is associated with ILC2 activation state.
111
112
A striking degree of heterogeneity exists amongst ILC2s, including subtypes such as tissue-resident
113
‘natural’ ILC2s (nILC2s) and tissue-infiltrating ‘inflammatory’ ILC2s (iILC2s), which have been
114
described and delineated on the basis of differential levels of phenotypic marker expression in the lung
115

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