Immunomodulatory role of nonneuronal cholinergic signaling in myocardial injury
Cibele Rocha-Resende, Carla Weinheimer, Geetika Bajpai, Luigi Adamo, Scot J. Matkovich, Joel Schilling, Philip M. Barger, Kory J. Lavine, Douglas L. Mann
Cibele Rocha-Resende, Carla Weinheimer, Geetika Bajpai, Luigi Adamo, Scot J. Matkovich, Joel Schilling, Philip M. Barger, Kory J. Lavine, Douglas L. Mann
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Research Article Cardiology Inflammation

Immunomodulatory role of nonneuronal cholinergic signaling in myocardial injury

Abstract

Whereas prior studies have demonstrated an important immunomodulatory role for the neuronal cholinergic system in the heart, the role of the nonneuronal cholinergic system is not well understood. To address the immunomodulatory role of the nonneuronal cholinergic system in the heart, we used a previously validated diphtheria toxin–induced (DT-induced) cardiomyocyte ablation model (Rosa26-DTMlc2v-Cre mice). DT-injected Rosa26-DTMlc2v-Cre mice were treated with diluent or pyridostigmine bromide (PYR), a reversible cholinesterase inhibitor. PYR treatment resulted in increased survival and decreased numbers of MHC-IIloCCR2+ macrophages in DT-injected Rosa26-DTMlc2v-Cre mice compared with diluent-treated Rosa26-DTMlc2v-Cre mice. Importantly, the expression of CCL2/7 mRNA and protein was reduced in the hearts of PYR-treated mice. Backcrossing Rosa26-DTMlc2v-Cre mice with a transgenic mouse line (Chat-ChR2) that constitutively overexpresses the vesicular acetylcholine transporter (VAChT) resulted in decreased expression of Ccl2/7 mRNA and decreased numbers of CD68+ cells in DT-injured Rosa26-DTMlc2v-Cre/Chat-ChR2 mouse hearts, consistent with the pharmacologic studies with PYR. In vitro studies with cultures of LPS-stimulated peritoneal macrophages revealed a concentration-dependent reduction in CCL2 secretion following stimulation with acetylcholine, nicotine, and muscarine. To our knowledge, these findings reveal a previously unappreciated immunomodulatory role for the nonneuronal cholinergic system in regulating homeostatic responses in the heart following tissue injury.

Authors

Cibele Rocha-Resende, Carla Weinheimer, Geetika Bajpai, Luigi Adamo, Scot J. Matkovich, Joel Schilling, Philip M. Barger, Kory J. Lavine, Douglas L. Mann

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Figure 5

CCL2 and TNFs secretion in cultures of lipopolysaccharide-stimulated intraperitoneal macrophages.

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CCL2 and TNFs secretion in cultures of lipopolysaccharide-stimulated int...
(A) CCL2 protein levels in the supernatant of cultures of lipopolysaccharide-stimulated (LPS-stimulated) intraperitoneal macrophages (ipMACs) treated in the presence and absence of increasing concentrations of acetylcholine (ACh), muscarine, and nicotine (n = 3–14 biological replicates per group). (B) TNF protein levels in the supernatant of cultures of LPS-stimulated ipMACs treated in the presence and absence of increasing concentrations of acetylcholine, muscarine and nicotine (n = 3–9 biological replicates per group). Data are shown as percentage of experimental control (LPS) per concentration ± SEM, analyzed by 2-way ANOVA. For A and B, *P < 0.05, **P < 0.01, ***P < 0.001, ****P ≤ 0.0001 in comparison with LPS. The graphs were produced using GPLOT procedure of SAS. For each concentration in the x axis, the graph reports on the y axis the mean analyte level expressed as percentage of control (LPS) ± SEM. (C) Representative immunofluorescence staining of M1 and M3 ACh receptors in cultures of ipMACs. Scale bar: 20 μm. Green, M1 or M3; blue, DAPI. (D) CCL2 protein levels in the supernatant of cultures of LPS-stimulated ipMACs in the presence and absence of muscarine, 4-DAMP (M3 receptor antagonist), and telenzepine (M1 receptor antagonist, n = 9–24 biological replicates per group). Diluent, PBS. For D, *P < 0.0001 in comparison with LPS-treated cells; †P < 0.0001 in comparison with diluent (nonstimulated ipMACs) and LPS-stimulated cells; ‡P < 0.01 in comparison with cells treated with 4-DAMP, muscarine, and LPS. P value was calculated using 2-way ANOVA using generalized linear models procedure with the option ADJUST = TUKEY to account for any missing values in A and B and with 1-way ANOVA followed by the Tukey post hoc test for D.