Bone marrow MyD88 signaling modulates neutrophil function and ischemic myocardial injury

Abstract

Myeloid differentiation factor 88 (MyD88), an adaptor critical for innate immune function, plays a role in neutrophil recruitment and myocardial injury after transient ischemia. However, how MyD88 signaling modulates neutrophil function and myocardial injury remains unclear. In an in vivo model of neutrophil migration and a chimeric model of MyD88 deletion, we demonstrated that Gr-1-positive (Gr-1(+)) neutrophil migration was significantly decreased by 68% in MyD88-deficient (Myd88(-/-)) mice and by 33% in knockout→wild-type (KO→WT; donor→recipient) chimeric mice, which lacked MyD88 in bone marrow cells but maintained normal MyD88 expression in the heart. This marked attenuation in neutrophil migration was associated with decreased peritoneal neutrophil CXCR2 expression and lower peritoneal KC, a neutrophil chemoattractant, in MyD88(-/-) mice. Moreover, in vitro, KC induces significantly more downregulation of CXCR2 expression in MyD88(-/-) than WT neutrophils. In an in vivo model of myocardial ischemia-reperfusion (I/R) injury, KO→WT chimeric mice had significantly smaller infarct sizes compared with the WT→WT mice. While there was a marked increase in proinflammatory cytokine/chemokine expression in the myocardium following I/R, there was no significant difference between WT→WT and KO→WT mice. In contrast, Gr-1(+) neutrophil recruitment in the myocardium was markedly attenuated in MyD88(-/-) mice. Deletion of Toll-interleukin-1 receptor (TIR)-domain-containing adaptor protein-inducing interferon-β-mediated transcription factor (Trif), another innate immune adaptor, had no effect on the KC-mediated CXCR2 downregulation or on myocardial neutrophil recruitment after I/R. Taken together, these findings suggest that MyD88 signaling is essential for maintaining neutrophil migratory function and chemokine receptor expression. MyD88 signaling in bone marrow-derived circulating cells may play a specific and critical role in the development of myocardial I/R-induced injury.

Fig. 1.

Knockout (KO) and KO→wild-type (KO→WT) mice have impaired neutrophil mobility and reduced peritoneal cytokine/chemokine levels. Mice were injected intraperitoneally with thioglycollate. Ten hours after the injection, 3 ml of normal saline was injected and mixed well. The peritoneal lavage fluid and leukocytes were isolated and analyzed. A: total numbers of cells recruited into the peritoneal cavity of WT and KO mice. B: total neutrophils recruited into the peritoneal cavity of WT and KO mice. Each error bar represents mean ± SD of 3 mice. C: a representative example of flow cytometry plots of peritoneal neutrophils from WT and myeloid differentiation factor 88 (MyD88)-deficient mice (MyD88^−/−). The cells were gated on CXCR2 and Gr-1. The cells within the circle represent CXCR2⁺/Gr-1⁺ neutrophils, and the percentage of neutrophils is shown. D: total numbers of cells recruited into the peritoneal cavity of the chimeric mice. E: total neutrophils recruited into the peritoneal cavity of the chimeric mice. Each error bar represents mean ± SD of 3 mice. F: a representative example of flow cytometry plots of peritoneal neutrophils from WT→WT and KO→WT chimeric mice. G: peritoneal lavage was analyzed for cytokine production using Luminex multiplex immunoassay. The bars in each dot plot represent median values of the measured cytokines. Some cytokine values were overlapping. n = 4–7. MCP-1, monocyte chemoattractant protein 1; MIP-2, macrophage inflammatory protein 2.

Fig. 2.

KO and KO→WT mice have decreased neutrophil CXCR2. A: CXCR2 expression of neutrophils recruited to the peritoneum in WT and MyD88^−/− mice. The y-axis represents CXCR2 expression as measured by mean fluorescence. Each error bar represents mean ± SD of 3–6 mice. B: a representative example of FACS plots of neutrophil CXCR2 expression in WT (red) or MyD88^−/− mice (green). C: CXCR2 expression on neutrophils recruited to the peritoneal space in WT→WT and KO→WT mice. Each error bar represents mean ± SD of 3–6 mice. D: a representative example of FACS of neutrophil CXCR2 expression in WT→WT (red) and KO→WT (green) mice.

Fig. 3.

MyD88 deficiency enhances the KC-induced downregulation of neutrophil CXCR2 expression. Bone marrow-derived neutrophils from both WT and MyD88^−/− mice were incubated with or without the indicated KC concentrations at 37°C for 30 min. Cells were stained and analyzed in flow cytometry and gated on Gr-1 and CXCR2. A: accumulated data of neutrophil CXCR2 expression presented as the percentage of the control cells without KC treatment. Each error bar represents mean ± SD of 4 separate neutrophil preparations. B: a representative plot of flow cytometry illustrating neutrophil CXCR2 expression in the control cells and the cells treated with 50 nM KC from WT and MyD88^−/− mice.

Fig. 4.

KO→WT mice have smaller myocardial infarction (MI) size compared with WT→WT mice. Mice were subjected to 30 min of ischemia and 24 h of reperfusion. At the end of reperfusion, animals were euthanized, and area-at-risk (AAR) and MI were analyzed. A: representative pictures of AAR and MI from the 4 groups of mice. B: cumulative data of AAR/left ventricle (LV). C: cumulative data of MI/AAR. Each error bar represents mean ± SD of 6–9 mice.

Fig. 5.

Bone marrow MyD88 deficiency has no impact on myocardial cytokine levels during ischemia-reperfusion (I/R). Both WT→WT and KO→WT chimeric mice were subjected to sham operation or I/R. The hearts were isolated, and myocardial cytokines were measured using Luminex multiplex immunoassay. The bars in each dot plot represent median values of the measured cytokines. Some cytokine values were overlapping. n = 5–6.

Fig. 6.

MyD88^−/− mice have marked attenuation in myocardial neutrophil recruitment after I/R. Twenty-four hours after 60 min of left anterior descending coronary artery (LAD) ligation, the hearts were isolated, perfused, and digested. After removal of the large cardiomyocytes through filtration, 50% of total cells were loaded onto flow cytometry and gated on Gr-1 and CXCR2. A: total Gr-1⁺ cells as measured by flow cytometry. Each error bar represents mean ± SD of 4 mice. A small number of neutrophils were recovered from the sham-operated hearts as indicated by the line. B: a representative example of flow cytometry plots of myocardial infiltrating cells from sham, WT-I/R, and KO-I/R mice. FSC, forward scatter; SSC, side scatter.

Fig. 7.

Toll-interleukin-1 receptor (TIR)-domain-containing adaptor protein-inducing interferon-β-mediated transcription factor (Trif) deficiency has no impact on myocardial neutrophil recruitment and KC-induced CXCR2 downregulation. Twenty-four hours after 60 min of LAD ligation, the isolated hearts were perfused and digested. After removal of the large cardiomyocytes, 50% of total cells were loaded onto flow cytometry and gated on Gr-1 and CXCR2. A: total Gr-1⁺ cells as measured by flow cytometry from the hearts subjected to I/R. Each error bar represents mean ± SD of 3 mice. B: a representative example of flow cytometry plots of myocardial infiltrating cells from WT-I/R and Trif-KO-I/R mice. C: accumulated data of neutrophil CXCR2 expression presented as the percentage of the control cells without KC treatment. Each error bar represents mean ± SD of 3 separate neutrophil preparations. D: a representative plot of flow cytometry illustrating neutrophil CXCR2 expression in the control cells and the cells treated with 50 nM KC from WT and Trif-KO mice.