Quantitative and qualitative composition of the innate immune response are equally important in wound healing after myocardial infarction
Authors: Wrobel J , Rettkowski J , Seung H , Wadle C , Stachon P , Von Zur Muehlen C , Bode C , Cabezas-Wallscheid N , Heidt T 
Published: October 2021

Abstract

Background
Emergency hematopoiesis (EH) serves as the foundation of monocyte-derived and macrophage (Mφ) driven efferocytosis and ventricular remodeling after myocardial infarction (MI). Excessive myelopoiesis, however, can stipulate maladaptive wound healing and its therapeutic reduction may be a novel approach to preserve cardiac function. All-trans retinoic acid (ATRA) is a pleiotropic modulator of EH and innate immunity shielding hematopoietic stem cells from activation and driving survival and differentiation of myeloid cells.

Purpose
This study aimed to investigate this intriguing interplay of ATRA in wound healing after MI.

Methods
MI was induced by permanent coronary ligation in C57BL/6 mice and treated with daily injections of either ATRA (30mg/kg) or DMSO (vehicle) up to five days, starting 24h after ligation. Flow cytometry (FACS) was used for cell cycle analysis and immunophenotyping of leukocytes in bone marrow (BM), blood and heart. Immunohistochemistry (IH), masson trichrome (MT) staining and echocardiography evaluated inflammatory-fibrotic and functional development. Cytokine expression was analyzed by qPCR in bulk infarct and isolated, polarized Mφ-populations of BM-derived and cardiac resident origin.

Results
On day 2 after MI, EH was significantly reduced in ATRA-treated mice as compared to vehicle controls by means of cell cycle activity (n=6–13 per group; p&lt;0,01) and myeloid cells in BM, blood and infarct tissue (n=5–13; p&lt;0,05). Consequently, mRNA-expression of key inflammatory cytokines, IL-1β and TNFα, was diminished in the infarct tissue in this early phase (n=5–12; p&lt;0,05). These changes, however, failed to preserve cardiac function and ventricular remodeling, 21 days after MI (n=10–11; not significant). By qPCR, non-canonical activation of recruited ATRA-primed monocyte-derived Mφ, was found to propagate a pro-inflammatory phenotype with higher expression of MMP2 and MMP9 in sorted cardiac Mφ (n=4–5; p&lt;0,001). Furthermore, prominent IL-1β-expression in M2-polarized BM-derived Mφ indicated an impaired anti-inflammatory phenotype after ATRA treatment (n=4–6; p&lt;0,05). Strikingly, these changes also occurred in remote myocardium where IH revealed a 2-fold increase of CD11b - positive myeloid cells accompanied by increased expression of TNFα and TGFβ (n=9; p&lt;0,001). MT-staining, performed 21 days after MI, demonstrated an almost 3-fold increase in collagen deposition in remote myocardium of ATRA treated mice in contrast to vehicle controls (n=4–6; p&lt;0,0001).

Conclusion
Despite a beneficial reduction of EH after MI, short-term treatment with ATRA induced profound and persisting changes in the cytokine expression of monocyte-derived Mφ, which significantly altered their function and thus prevented improvements in cardiac function. Our data provide evidence that quantitative and qualitative changes in innate immunity are equally important for cardiac remodeling after MI.

Funding Acknowledgement
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft