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. 2013 Jan 22;110(4):1410-5.
doi: 10.1073/pnas.1221655110. Epub 2013 Jan 7.

Lymphocyte-derived ACh regulates local innate but not adaptive immunity

Colin Reardon  1 , Gordon S Duncan, Anne Brüstle, Dirk Brenner, Michael W Tusche, Peder S Olofsson, Mauricio Rosas-Ballina, Kevin J Tracey, Tak W Mak
Affiliations

Lymphocyte-derived ACh regulates local innate but not adaptive immunity

Colin Reardon et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A.. Olofsson, Peder [corrected to Olofsson, Peder S]

Abstract

Appropriate control of immune responses is a critical determinant of health. Here, we show that choline acetyltransferase (ChAT) is expressed and ACh is produced by B cells and other immune cells that have an impact on innate immunity. ChAT expression occurs in mucosal-associated lymph tissue, subsequent to microbial colonization, and is reduced by antibiotic treatment. MyD88-dependent Toll-like receptor up-regulates ChAT in a transient manner. Unlike the previously described CD4(+) T-cell population that is stimulated by norepinephrine to release ACh, ChAT(+) B cells release ACh after stimulation with sulfated cholecystokinin but not norepinephrine. ACh-producing B-cells reduce peritoneal neutrophil recruitment during sterile endotoxemia independent of the vagus nerve, without affecting innate immune cell activation. Endothelial cells treated with ACh in vitro reduced endothelial cell adhesion molecule expression in a muscarinic receptor-dependent manner. Despite this ability, ChAT(+) B cells were unable to suppress effector T-cell function in vivo. Therefore, ACh produced by lymphocytes has specific functions, with ChAT(+) B cells controlling the local recruitment of neutrophils.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
ChAT-GFP is expressed in multiple immune cells. (A) Flow cytometry for GFP expression was conducted on splenocytes for CD4+ and CD8+ T cells (Left) and marginal zone (MZ) and follicular B (FOB) cells (Right). ChAT+ cells were also enumerated in the PP and MLN (B) and in peritoneal B1a, B1b, and B2 B cells (C). Splenic DCs (D) and macrophages (E) were assessed for ChAT-GFP. (F) ChAT and ACh production was assessed by qRT-PCR (Left) and by MS from splenic B220+CD23+ B cells (Right) (n = 3 mice repeated three times). #P <0.001 by ANOVA.
Fig. 2.
Fig. 2.
MyD88-dependent signaling regulates ChAT expression in immune cells. (A) Sorted GFP− peritoneal (PEC) B1a, B1b, or B2 cells or splenic (SPL) B2 cells were stimulated (16 h) with anti-IgM/CD40 (10 μg/mL and 5 μg/mL, respectively), polyinosinic:polycytidylic acid [Poly(I:C); 100 μg/mL], CpG (10 μM), LPS (10 μg/mL), Pam3cys (1 μM), or R848 (1 μg/mL) and assessed for GFP. (B) FACS-sorted GFP− splenic B2 cells from MyD88+/+ Chat-GFP−, MyD88+/+ ChAT-GFP+, and MyD88−/− ChAT-GFP+ mice were stimulated with TLR ligands. ChAT expression in MyD88−/− mice was assessed by flow cytometry on SPL follicular B cells (C, Left) and CD4+ T cells (C, Right), summarized from PEC, SPL, MLN (mLN), pLN, and PP cells (D) (n = 3 mice repeated three times). *P < 0.05 by ANOVA.
Fig. 3.
Fig. 3.
Intestinal microbiota contribute to ChAT+ lymphocyte development. (A) Antibiotics (Abx) were administered in the drinking water to deplete the microbiota. (B) Follicular B cells were assessed in control (white bars) and antibiotic-treated (black bars) mice in the PP, MLN, and spleen. (C) Effect of microbiota depletion was also assessed in peritoneal B1a, B1b, and B2 B cells. (D) Analysis of the CD4+ T-cell population was also conducted in the PP, MLN, and spleen. In separate experiments, mice were gavaged with vehicle (control, open bars) or FTY720 (solid bars). GFP expression was assessed on cells from the PP, MLN, pLN, and spleen in CD4+ T cells (E) and B220+ B cells (F) (n = 3 to 4 mice per group, two experiments). Abs. No., absolute number. *P < 0.05 and #P < 0.001 by ANOVA.
Fig. 4.
Fig. 4.
Lymphocyte ChAT expression is transient and reinducible. ChAT-GFP mice were crossed to ChAT.Cre+TomatoLSL mice to allow for fate mapping. Cells from ChAT-GFP− ChAT.Cre+TomatoLSL (Upper) and ChAT-GFP+ ChAT.Cre+TomatoLSL (Lower) were assessed by flow cytometry. Tomato+ lymphocytes were assessed for GFP in splenic CD4+ T cells (A), B220+ B cells (B), and peritoneal B1a and B1b B cells (C). (D) Splenic follicular B GFP−Tomato+ cells were FACS-sorted from ChAT-GFP− ChAT.Cre+TomatoLSL+ and ChAT-GFP+ ChAT.Cre+Tomato+ and stimulated (16 h) with media, LPS (10 μg/mL), or R848 (1 μg/mL). As a control, B220+CD23+ GFP− cells from ChAT-GFP+ ChAT.Cre+Tomato− mice were sorted and stimulated. (E) FACS-sorted B2 GFP− or GFP+ cells were treated with LPS ± NVP (1 μM) for either 0.5 h or 16 h and were assessed by MS [n = 3 mice per group, representative of three experiments (A–C) and two experiments (D)]. *P < 0.05 by ANOVA; **P < 0.01; #P < 0.001.
Fig. 5.
Fig. 5.
Specific stimuli induce release of ACh. Release of ACh was determined from FACS-sorted B220+CD23+ ChAT-GFP− or ChAT-GFP+ splenic B cells, following stimulation with CCK (A) or NE (B) with 10 μM physostigmine bromide (20 min). Supernatants were mixed 1:1 with methanol (MeOH) and assessed by MS. As a control, sorted ChAT-GFP+ and ChAT-GFP− cells were lysed with MeOH and mixed 1:1 with a PBS “pellet” (n = 3, measured in duplicate). *P < 0.05 by ANOVA.
Fig. 6.
Fig. 6.
B cell-derived ACh is required for neutrophil recruitment. Peritoneal B1 or splenic B2 cells were enriched and FACS-sorted based on ChAT-GFP expression. Cells were transferred (2 × 105, i.p.) to Rag1−/− recipients, and LPS (0.2 mg/kg) was injected 16 h after transfer. (A) Neutrophils were assessed 3 h after challenge with LPS. Abs. No., absolute number. (B) Serum was assessed for cytokine production by cytometric bead assay. (C) Rag1−/− sham or vagotomized (VGX) mice received FACS-sorted GFP− or GFP+ peritoneal B1 cells. Mice were challenged with LPS (0.2 mg/kg) and assessed for peritoneal neutrophil recruitment (C) and serum chemokines (D) (n = 3–7 mice per group in two separate experiments). *P < 0.05, two-tailed t test (A) and two-way ANOVA (C).
Fig. 7.
Fig. 7.
Endothelial adhesion molecules are down-regulated by ACh. Bend3 endothelial cells were pretreated (16 h) with physostigmine bromide (10 μM) and ACh (0–1,000 nM) ± atropine (10 μM) before LPS stimulation (1 ng/mL, 4 h). Cells were trypsinized and assessed for surface ICAM-1 (A) and VCAM (B) by flow cytometry (n = 3 separate experiments). *P < 0.05 (vs. “0 ACh”).
See this image and copyright information in PMC

Comment in

  • Neuroimmunology: ChATty B cells.
    Papatriantafyllou M. Papatriantafyllou M. Nat Rev Immunol. 2013 Feb;13(2):70. doi: 10.1038/nri3396. Nat Rev Immunol. 2013. PMID: 23348411 No abstract available.

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