Macrophages secrete murinoglobulin-1 and galectin-3 to regulate neutrophil degranulation after myocardial infarction

Abstract

Inflammation presides early after myocardial infarction (MI) as a key event in cardiac wound healing. Ischemic cardiomyocytes secrete inflammatory cues to stimulate infiltration of leukocytes, predominantly macrophages and neutrophils. Infiltrating neutrophils degranulate to release a series of proteases including matrix metalloproteinase (MMP)-9 to break down extracellular matrix and remove necrotic myocytes to create space for the infarct scar to form. While neutrophil to macrophage communication has been explored, the reverse has been understudied. We used a proteomics approach to catalogue the macrophage secretome at MI day 1. Murinoglobulin-1 (MUG1) was the highest-ranked secreted protein (4.1-fold upregulated at MI day 1 vs. day 0 pre-MI cardiac macrophages, p = 0.004). By transcriptomics evaluation, galectin-3 (Lgals3) was 2.2-fold upregulated (p = 0.008) in MI day 1 macrophages. We explored the direct roles of MUG1 and Lgals3 on neutrophil degranulation. MUG1 blunted while Lgals3 amplified neutrophil degranulation in response to phorbol 12-myristate 13-acetate or interleukin-1β, as measured by MMP-9 secretion. Lgals3 itself also stimulated MMP-9 secretion. To determine if MUG1 regulated Lgals3, we co-stimulated neutrophils with MUG1 and Lgals3. MUG1 limited degranulation stimulated by Lgals3 by 64% (p < 0.001). In vivo, MUG1 was elevated in the infarct region at MI days 1 and 3, while Lgals3 increased at MI day 7. The ratio of MUG1 to Lgals3 positively correlated with infarct wall thickness, revealing that MUG1 attenuated infarct wall thinning. In conclusion, macrophages at MI day 1 secrete MUG1 to limit and Lgals3 to accentuate neutrophil degranulation to regulate infarct wall thinning.

Fig. 1. Experimental Design. D-day; MI-myocardial infarction.

Fig. 2. MI D1 Macrophage secretome analysis revealed MUG1 as the highest upregulated protein and USP48 as the highest downregulated protein. (A) Heat map ranking of proteins by p value and fold-change at MI D1 vs. D0. (B) By Volcano plot, MUG1 was the most upregulated and USP-48 the most downregulated protein. (C) PLS-DA 2D scores plot shows a significant shift in proteome of D0 vs. D1 macrophage secretome. (D) Enrichment analysis shows that for MI D1 upregulated proteins (top), IL-17 signaling was the most enriched pathway. Of the downregulated proteins (bottom), PPAR signaling was the most enriched pathway. Sample sizes were n = 4 pooled M sets; analysis by unpaired Students t-test and Enrichr pathway analysis.

Fig. 3. D1 MI macrophage secretome reduced neutrophil degranulation through MUG1. (A) Experimental design. (B) Neutrophils treated with phorbol 12-myristate-13-acetate (PMA) induced MMP-9 release, a surrogate marker of degranulation. Treatment with MI D1 macrophage secretome (10%, by volume) significantly reduced MMP-9 release, an effect reversed by addition of a MUG1 blocking antibody. (C) Heatmap of the top 25 proteins ranked by p value for the comparison of PMA + MI D1 macrophage secretome + MUG1 Ab vs. PMA + MI D1 macrophage secretome. (D) For the proteins downregulated by the D1 MI macrophage secretome, cytokine-cytokine receptor interaction signaling was most enriched (top). For the proteins upregulated by addition of a MUG1 blocking Ab to PMA+ MI D1 macrophage secretome, cytokine-cytokine receptor interaction was most enriched (bottom). Sample sizes were n = 4M per paired group; analysis by one way ANOVA with Student Newman–Keuls post-hoc test, paired Students t-test, and Enrichr pathway analysis.

Fig. 4. MUG1 downregulated neutrophil degranulation and pro-inflammatory cytokine release. (A) Experimental design. (B) Neutrophils treated with IL-1β induced MMP-9 release, a surrogate marker of neutrophil degranulation. IL-1β stimulated release of MMP-9 was attenuated by supplementation of MUG1. (C) Heatmap ranking of the 13 IL-1β stimulated neutrophil proteins decreased by MUG1. (D) By enrichment analysis, IL-17 signaling was the most enriched pathway for the MUG1 downregulated proteins (top). By volcano plotting, MMP-9, MPO, and NGAL were the IL-1β induced neutrophil proteins most downregulated by MUG1 (bottom). Sample sizes were n = 8, 4M/4F per paired group; analysis done by one way ANOVA with Student Newman–Keuls post-hoc test, paired Students t-test, and Enrichr pathway analysis.

Fig. 5. Lgals3 was upregulated in the D1 MI macrophage and induced MMP-9 release from neutrophils. (A) Lgals3 mRNA expression increased 2.2-fold in MI D1 macrophages compared to D0 no MI controls. (B) Experimental design. (C) Neutrophils treated with IL-1β induced MMP-9 release, a surrogate marker of neutrophil degranulation. Lgals3 independently induced MMP-9 release. Co-stimulating Lgals3 with IL-1β did not have an additive effect (p = 0.885 for IL-1β vs. IL-1β + Lgals3). (D) Heatmap showing the top 9 proteins increased by IL-1β compared to unstimulated controls, ranked by p value. (E) Enrichment analysis of proteins upregulated by Lgals3 revealed leukocyte transendothelial migration as the most upregulated pathway (top). By volcano plot, MPO was the most upregulated neutrophil protein stimulated by Lgals3. Sample sizes were n = 8, 4M/4F each paired group; analysis done by one way ANOVA with Student Newman–Keuls post-hoc test, paired Students t-test, and Enrichr pathway analysis.

Fig. 6. MUG1 inhibited LGALS3 induced neutrophil degranulation. By immunoblotting for MMP-9 release into the neutrophil conditioned media, MUG1 inhibited neutrophil degranulation induced by Lgals3 by 64%. Sample sizes were n = 8, 4M/4F each paired group; analysis done by one-way ANOVA with Student Newman–Keuls post-hoc test.

Fig. 7. In the LV infarct region, MUG1 increased at MI D1-D3 and tracked with preserved LV wall thickness, while LGALS3 peaked at MI D7. (A) Cardiac physiology after MI at days 1, 3, and 7, including representative infarct size images for each day. (B) Representative MUG1 immunoblot of LV infarct samples (top). Representative Lgals3 immunoblot of LV infarct samples (bottom). (C) MUG1 increased after MI, peaking at MI D3 (6-fold increase vs. D0) and returned to D0 levels by MI D7. Lgals3 increased from MI D3 and peaked at MI D7 of MI (634-fold compared to D0). The MUG1 to Lgals3 ratio peaked at MI D1, and the MUG1 to Lglas3 ratio positively correlated with infarct wall thickness. Sample sizes were n = 7–8, 3–4M/4F per group; analysis done by one way ANOVA with Student Newman–Keuls post-hoc test.

Fig. 8. Macrophage coordination of neutrophil physiology. Macrophages inhibit neutrophil degranulation by secretion of MUG1 and induce neutrophil degranulation by secretion of Lgals3 to coordinate LV wall thinning and early MI wound healing. Release of MMP-9 provides an additional feedback to proteolyze and activate Lgals3.