Role of M3 muscarinic receptor in regulation of immunity to infectious pathogens

During the last decade, cholinergic signaling via acetylcholine and its receptors has emerged as an important regulator of immunity. Acetylcholine binds to and signals through two types of receptors; nicotinic and muscarinic receptors. Studies have shown that signaling through nicotinic receptors, particularly the α7 subtype on macrophages has potent anti-inflammatory effects. However, the role for muscarinic receptor has not yet been conclusively characterized. In this study, we demonstrate that M3 muscarinic receptor subtype is required for optimal protective immunity to two pathogens; the nematode Nippostrongylus brasiliensis and the bacterium Salmonella enterica sp. Tyhpimurium. M3R deficient mice (M3R-/-) were susceptible to infection with N.brasiliensis with decreased production of the protective cytokine IL-13. Furthermore, stimulation of lymphocytes with muscarinic agonists enhanced TH2 cytokine production in an M3R dependent manner

The M3 muscarinic receptor Is required for optimal adaptive immunity to Helminth and bacterial infection

Innate immunity is regulated by cholinergic signalling through nicotinic acetylcholine receptors. We show here that signalling through the M3 muscarinic acetylcholine receptor (M3R) plays an important role in adaptive immunity to both Nippostrongylus brasiliensis and Salmonella enterica serovar Typhimurium, as M3R-/- mice were impaired in their ability to resolve infection with either pathogen. CD4 T cell activation and cytokine production were reduced in M3R-/- mice. Immunity to secondary infection with N. brasiliensis was severely impaired, with reduced cytokine responses in M3R-/- mice accompanied by lower numbers of mucus-producing goblet cells and alternatively activated macrophages in the lungs. Ex vivo lymphocyte stimulation of cells from intact BALB/c mice infected with N. brasiliensis and S. typhimurium with muscarinic agonists resulted in enhanced production of IL-13 and IFN-γ respectively, which was blocked by an M3R-selective antagonist. Our data therefore indicate that cholinergic signalling via the M3R is essential for optimal Th1 and Th2 adaptive immunity to infection

The M3 muscarinic receptor is required for optimal adaptive immunity to helminth and bacterial infection

Innate immunity is regulated by cholinergic signalling through nicotinic acetylcholine receptors. We show here that signalling through the M3 muscarinic acetylcholine receptor (M3R) plays an important role in adaptive immunity to both Nippostrongylus brasiliensis and Salmonella enterica serovar Typhimurium, as M3R-/- mice were impaired in their ability to resolve infection with either pathogen. CD4 T cell activation and cytokine production were reduced in M3R-/- mice. Immunity to secondary infection with N. brasiliensis was severely impaired, with reduced cytokine responses in M3R-/- mice accompanied by lower numbers of mucus-producing goblet cells and alternatively activated macrophages in the lungs. Ex vivo lymphocyte stimulation of cells from intact BALB/c mice infected with N. brasiliensis and S. typhimurium with muscarinic agonists resulted in enhanced production of IL-13 and IFN-γ respectively, which was blocked by an M3R-selective antagonist. Our data therefore indicate that cholinergic signalling via the M3R is essential for optimal Th1 and Th2 adaptive immunity to infection

Publisher Correction: Reporting guideline for the early-stage clinical evaluation of decision support systems driven by artificial intelligence: DECIDE-AI (Nature Medicine, (2022), 28, 5, (924-933), 10.1038/s41591-022-01772-9)

In the version of this article initially published, a list of the DECIDE-AI expert group members and their affiliations was omitted and has now been included in the HTML and PDF versions of the article. *A list of authors and their affiliations appears online

Natural and Vaccine-Mediated Immunity to Salmonella Typhimurium is Impaired by the Helminth Nippostrongylus brasiliensis

The impact of exposure to multiple pathogens concurrently or consecutively on immune function is unclear. Here, immune responses induced by combinations of the bacterium Salmonella Typhimurium (STm) and the helminth Nippostrongylus brasiliensis (Nb), which causes a murine hookworm infection and an experimental porin protein vaccine against STm, were examined. Mice infected with both STm and Nb induced similar numbers of Th1 and Th2 lymphocytes compared with singly infected mice, as determined by flow cytometry, although lower levels of secreted Th2, but not Th1 cytokines were detected by ELISA after re-stimulation of splenocytes. Furthermore, the density of FoxP3+ T cells in the T zone of co-infected mice was lower compared to mice that only received Nb, but was greater than those that received STm. This reflected the intermediate levels of IL-10 detected from splenocytes. Co-infection compromised clearance of both pathogens, with worms still detectable in mice weeks after they were cleared in the control group. Despite altered control of bacterial and helminth colonization in co-infected mice, robust extrafollicular Th1 and Th2-reflecting immunoglobulin-switching profiles were detected, with IgG2a, IgG1 and IgE plasma cells all detected in parallel. Whilst extrafollicular antibody responses were maintained in the first weeks after co-infection, the GC response was less than that in mice infected with Nb only. Nb infection resulted in some abrogation of the longer-term development of anti-STm IgG responses. This suggested that prior Nb infection may modulate the induction of protective antibody responses to vaccination. To assess this we immunized mice with porins, which confer protection in an antibody-dependent manner, before challenging with STm. Mice that had resolved a Nb infection prior to immunization induced less anti-porin IgG and had compromised protection against infection. These findings demonstrate that co-infection can radically alter the development of protective immunity during natural infection and in response to immunization

M3R deficient mice have impaired memory immune responses during secondary infection with <i>N. brasiliensis</i>.

<p>(A) Number of <i>N. brasiliensis</i> larvae recovered from the lungs at day 2 post-secondary infection. (B) Levels of IL-13 and IL-4 in total lung homogenates at day 2 p.i. (C) Intracellular IL-13 and IL-4 cytokine production in lung CD4 T cells at day 2 post secondary infection. (D) Goblet cells in the lungs visualised by Periodic Acid Schiff (PAS) staining and enumerated by Histological Mucus Index (HMI) at day 2 p.i. White bar = WT BALB/c mice, black bar = M3R^−/− mice. (E) Recovery of adult worms from the intestines of WT BALB/c mice, alveolar macrophage numbers and expression of YM1 and RELMα after adoptive transfer of 5 × 10⁵ CD4 cells from naïve WT mice, WT mice infected with <i>N. brasiliensis</i>, naïve M3R^−/− mice and infected M3R^−/− mice, and infection with <i>N. brasiliensis</i> thereafter. Data are shown as mean ± SEM, n = 6 and are representative of two independent experiments. Statistical significance was calculated using the Mann-Whitney two-tailed t test and denoted by *p<0.05, **p<0.01.</p

M3R deficient mice are more susceptible to <i>S. typhimurium</i> infection and exhibit impaired CD4 T cell responses.

<p>(A) Recovery of <i>S. typhimurium</i> from the spleens of infected mice at 7, 18, 27 and 35 days p.i. and weight loss at different days p.i. (B) IFN-γ secretion by antigen-restimulated splenocytes at different days p.i. (C) IFN-γ mRNA, proportion and number of IFN-γ+ CD4 splenic T cells assayed at day 18 p.i. (D) The proportion of activated CD4 T cells in spleens at day 18 p.i. (E) Secretion of IFN-γ by splenocytes stimulated with sub-optimal anti-CD3 after day 26 p.i. is enhanced by ACh and muscarinic agonists (10 μM). Enhancement of IFN-γ by ACh and muscarinic agonists is blocked by atropine (AT, 100 μM) and the M3R-selective antagonist J104129 (J, 40 nM). Data are shown as mean ± SEM, n = 5–6 and are representative of at least two independent experiments. Statistical significance was calculated using the Mann-Whitney two-tailed t test and denoted by *p<0.05, **p<0.01.</p

M3R deficient mice exhibit delayed clearance of a primary <i>N. brasiliensis</i> infection and impaired T cell-associated protective responses.

<p>(A) Adult worm numbers in the small intestine were elevated in M3R^−/− mice in comparison to WT BALB/c on day 7 and 9 p.i. (B) Hypercontractile responses of jejunum in response to varying doses of acetylcholine measured at day 9 p.i. were absent in M3R^−/− mice. (C) Production of IL-13 by CD4 T cells is impaired in M3R^−/− mice: IL-13 mRNA levels and proportion of IL-13⁺ CD4 T cells from the MLN at day 7 p.i. (D) Proportion of activated (CD44^hi CD62L^lo) CD4 T cells in MLN at day 7 p.i. (E) Ca²⁺ mobilisation in response to ionomycin in CD4 T cells in MLN at day 7 p.i. Data are shown as mean ± SEM, n = 4–6 and are representative of at least two independent experiments. Statistical significance was calculated using the Mann-Whitney two-tailed t test and denoted by * p<0.05, ** p<0.01. White bar: WT BALB/c mice; black bar: M3R^−/− mice.</p

Signalling via the M3R potentiates Th2 cytokine production during <i>N. brasiliensis</i> infection.

<p>(A) Detection of mRNA for M1-M5 muscarinic receptors and β-actin (βA) in CD4 T cells isolated from MLN. (B) Ox40 expression on effector memory (CD44^hiCD62L^lo) subsets of CD4 T cells day 7 p.i. (C) Cytokine secretion by MLN cells day 7 p.i. after stimulation with sub-optimal anti-CD3 and muscarinic receptor agonists (10 μM, 48 hrs) and dose response to muscarine. Asterisks indicate significant differences between control and cholinergic stimulations. (D) IL-13 production from <i>N. brasiliensis</i> infected (day 7) WT and M3R^−/− MLN cells after sub-optimal anti-CD3 stimulation plus agonists ACh and Oxotremorine M (Oxo M) at 10 μM and the muscarinic receptor antagonist atropine (AT, 100 μM) or the M3R-selective antagonist J104129 (J, 40 nM). Data are shown as mean ±SEM, n = 6 and are representative of at least two independent experiments. *p<0.05, **p<0.01 determined by the Mann-Whitney two-tailed t test.</p

Immunoglobulin-switching patterns are maintained during co-infection but serum immunoglobulin responses to STm are reduced.

<p><b>A</b>) Spleen sections were generated for immunohistology from mice which were infected as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003341#pntd-0003341-g001" target="_blank">Figure 1A</a>. Sections were double-stained for IgD with IgG1, IgG2a, IgE or CD3. These sections were used to quantify extrafollicular plasma cells per mm² and determine the proportion of the spleen occupied by germinal centres. Germinal centres were identified as areas of the follicle which were IgD^lo. <b>B</b>) Serum anti-STm IgM, IgG, IgG2a and anti-Nb IgM, IgG and IgG1 antibody titres were quantified by ELISA against a total outer membrane preparation from STm and homogenized L3 larvae, respectively. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Data is representative of 4–6 mice per group with experiments performed twice for each time point. (NS = Non-significant, ^*P<0.05 and ^**P<0.005).</p