Increase in cholinergic modulation with pyridostigmine induces anti-inflammatory cell recruitment soon after acute myocardial infarction in rats

Abstract

We tested the hypothesis that an increase in the anti-inflammatory cholinergic pathway, when induced by pyridostigmine (PY), may modulate subtypes of lymphocytes (CD4+, CD8+, FOXP3+) and macrophages (M1/M2) soon after myocardial infarction (MI) in rats. Wistar rats, randomly allocated to receive PY (40 mg·kg(-1)·day(-1)) in drinking water or to stay without treatment, were followed for 4 days and then were subjected to ligation of the left coronary artery. The groups-denominated as the pyridostigmine-treated infarcted (IP) and infarcted control (I) groups-were submitted to euthanasia 3 days after MI; the heart was removed for immunohistochemistry, and the peripheral blood and spleen were collected for flow cytometry analysis. Noninfarcted and untreated rats were used as controls (C Group). Echocardiographic measurements were registered on the second day after MI, and heart rate variability was measured on the third day after MI. The infarcted groups had similar MI areas, degrees of systolic dysfunction, blood pressures, and heart rates. Compared with the I Group, the IP Group showed a significant higher parasympathetic modulation and a lower sympathetic modulation, which were associated with a small, but significant, increase in diastolic function. The IP Group showed a significant increase in M2 macrophages and FOXP3(+)cells in the infarcted and peri-infarcted areas, a significantly higher frequency of circulating Treg cells (CD4(+)CD25(+)FOXP3(+)), and a less extreme decrease in conventional T cells (CD25(+)FOXP3(-)) compared with the I Group. Therefore, increasing cholinergic modulation with PY induces greater anti-inflammatory cell recruitment soon after MY in rats.

Keywords: T lymphocytes; anticholinesterase drugs; cholinergic anti-inflammatory reflex; macrophage activation; myocardial infarction.

Fig. 1.

Gating strategy. Peripheral blood mononuclear cells or splenocytes were obtained and stained for activation or FOXP3-marker evaluation. Three million cells were surface-stained with CD3/CD4 or CD8/CD25/RT1B and intracellularly stained with FOXP3. Cells were acquired in a LSRII-Fortessa, and 500,000 events were recorded in the lymphocyte gate. A: cells were gated on FSC-H and FSC-A to allow the selection of single cells. B: cells were gated on the basis of size (FSC-A) and granularity (SSC-A) to select the lymphocyte population. C: subsequently, cells were gated on CD3 and either CD4 or CD8, defining CD4 or CD8 T lymphocytes, respectively. D: further gating on CD25 × RT1B allowed us to discriminate the activated T cells. E: regulatory T cells were defined on the basis of the double expression of CD25⁺ FOXP3⁺.

Fig. 2.

Echocardiography images of the left ventricle cavity delimited by the septum (s) and posterior wall (pw). A: normal contraction of the septum and the posterior wall in a control (C) rat. B: akinetic septum and normal contraction of the posterior wall in an untreated, infarcted (I) rat. C: hypokinesia of the septum and normal contraction of the posterior wall in a pyridostigmine (PY)-treated rat.

Fig. 3.

Photomicrographs of the infarcted control (I) and pyridostigmine-treated infarcted group (IP) showing the M1 macrophages within the peri-infarcted zone. The cytoplasmic stain showed no difference in the M1 cell count between the groups (P = 0.09). There were scarce M2 macrophages in the I Group and a dense diffuse infiltration of M2 cells in the infarcted zone in the IP Group (*P < 0.05). Ten microscopic fields of seven animals per group were analyzed and displayed using means ± SE. (Immunohistochemistry, diaminobenzidine: scale bar equals 50 μm).

Fig. 4.

Photomicrographs of the I and IP Groups showing few FOXP3 cells within the peri-infarcted zone of the I Group and a moderate number of positive cells in the IP Group. Right: number of the CD4⁺CD25⁺FOXP3 cells in both groups, which is significantly different (*P = 0.0045). Ten microscopic fields of seven animals per group were analyzed and displayed using means ± SE. (Immunohistochemistry, diaminobenzidine: scale bar equals 50 μm)

Fig. 5.

PY treatment reduces CD4⁺ and CD8⁺ T-cell activation. Blood and spleen cells were collected from the C Group and from the I and IP Groups and then were stained to evaluate the activation. CD25 and RT1B double expression of the CD3⁺CD4⁺ and CD3⁺CD8⁺ cells were evaluated. A and B: CD4⁺ T-cell activation in the blood and spleen, respectively. C and D: CD8⁺ T-cell activation in the blood and spleen, respectively. *P < 0.05; **P < 0.01; ***P < 0.001; NS, nonsignificant.

Fig. 6.

PY treatment increases Treg in blood cells. FOXP3 expression was evaluated in CD4⁺CD25⁺ T cells to define Treg cells in blood (A) and spleen (B). Correlations were measured between Treg cells from blood and CD4⁺ (C) and between Treg cells from blood and CD8⁺ after T-cell activation in blood cells (D) (r and P values are depicted). In the correlation plots, blue dots correspond to the IP Group, and red dots correspond to the I Group. *P < 0.05; ***P < 0.001; NS, nonsignificant.