Integrative Ly-6C high Monocytes Depend on Nr4a1 to Balance Both Inflammatory and Reparative Phases in the Infarcted Myocardium

Ly-6C low macrophage differentiation; dependence on Nr4a1 can thus discriminate between systemic and local origins of macrophage heterogeneity. Objective: This study tested the role of Nr4a1 in myocardial infarction in the context of the 2 Mo/M Φ accumulation scenarios. Methods and Results: We show that Ly-6C high monocytes infiltrate the infarcted myocardium and, unlike Ly-6C low monocytes, differentiate to cardiac macrophages. In the early, inflammatory phase of acute myocardial ischemic injury, Ly-6C high monocytes accrue in response to a brief C–C chemokine ligand 2 burst. In the second, reparative phase, accumulated Ly-6C high monocytes give rise to reparative Ly-6C low F4/80 high macrophages that proliferate locally. In the absence of Nr4a1, Ly-6C high monocytes express heightened levels of C–C chemokine receptor 2 on their surface, avidly infiltrate the myocardium, and differentiate to abnormally inflammatory macrophages, which results in defective healing and compromised heart function. Conclusions: Ly-6C high monocytes orchestrate both inflammatory and reparative phases during myocardial infarction and depend on Nr4a1 to limit their influx and inflammatory cytokine expression. ( Circ Res . 2014;114:1611-1622.) and interpreted as evidence for sequential monocyte subset recruitment. We used Nr4a1, which is essential to Ly-6C low monocytes but dispensable to Ly-6C low macrophages, as a tool to discriminate between sequential recruitment and local macrophage differentiation. Combining advanced flow cytometry and fate-mapping approaches, we show that the biphasic response originates from Ly-6C high monocytes, which accumulate in the infarct early and, over time, as they differentiate to macrophages, lose Ly-6C expression. Although this revised model agrees with the initial description of biphasic macrophage accumulation, it departs from it in 1 critical interpreta-tion: the first Ly-6C high monocyte-dominant phase is followed not by a Ly-6C low monocyte but by a Ly-6C high monocyte-de-rived Ly-6C low macrophage-dominant phase. This expanded concept highlights the importance of the local environment in orchestrating inflammation and healing. monocyte/macrophage response during myocardial infarction. Histology. Histology. Hearts were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA), frozen in ice-cold 2-Methylbutane (Fisher Scientific, Fair Lawn, NJ) and sectioned into 6 µm slices yielding 30-40 sections per mouse. The following antibodies were used for immunohistology: Anti-Nur77 (clone M-210, Santa Cruz Biotechnology, Inc.), anti-CD11b (clone M1/70, BD Biosciences), anti-Collagen I (ab21286, Abcam), anti-CD31 (clone MEC13.3, BD Biosciences) , anti- α -smooth muscle actin (ab5694; Abcam) for smooth muscle cells (SMA). Biotinylated secondary antibodies followed by VECTASTAIN ABC reagent (Vector Laboratories, Inc. Burlingame, CA) were applied and the color development was performed using AEC substrate (Dako North America, Inc. Carpinteria, CA). Masson’s Trichrome staining was performed in cross sections of paraffin-embedded hearts 21 days post MI. For immunofluorescence staining anti-Ki67-FITC (clone SP6, Abcam), anti-CD11b (clone M1/70, BD Biosciences), anti-CD3 (clone 17A2, BD Biosciences), anti-CD31 (clone MEC 13.3, BD Biosciences), secondary biotinylated anti-rat IgG and streptavidin DyLight 594 (Vector Laboratories), and Vectashield mounting medium with DAPI (Vector Laboratories) were used. TUNEL staining was carried out using DeadEnd Fluorometric TUNEL System (Promega, Madison, WI) according to the manufacture's instructions. Images capture was performed using a Nanozoomer 2.0RS (Hamamatsu, Japan) and Olympus BX63 (Olympus America Inc., Center Valley, PA) equipped with a ANDOR Neo sCMOS Monochrome Camera (ANDOR technology, Northern Ireland). Images were analyzed with ImageJ. Flow Cytometry. Cell suspensions were stained in PBS supplemented with sterile 2% FBS and 0.5% BSA. The following monoclonal antibodies were used for flow cytometric analysis: anti-Ly6C (clone AL-21, BD Biosciences), anti-CD45.1 (clone A20, Biolegend), anti-CD45.2 (clone 104, BD Biosciences), anti-CD3e (clone 145-2C11, ebioscience), anti-CD90.2 (clone 53-2.1, BD Biosciences), anti-CD19 (clone 6D5, Biolegend), anti-B220 (clone RA3-6B2, BD Biosciences), anti-MHCII (clone AF6-120.1, BD Biosciences), anti-F4/80 (clone BM8, Biolegend), anti-CD49b (clone DX5, BD Biosciences), anti-NK1.1 (clone PK136, BD Biosciences), anti-Ly6G (clone 1A8, BD Biosciences), anti-CD11b (clone M1/70, BD Biosciences), anti-CD11c (clone HL3, BD Biosciences), anti-CD115 (clone AFS98, ebioscience), anti-Ter119 (clone Ter-119, BD Biosciences), anti-CD68 (clone FA-11, Biolegend), anti-CD62L (clone MEL-14, Biolegend), anti-PSGL1 (clone 2PH1, BD Biosciences), anti-CCR2 (Clone R&D systems), anti-active Caspase 3(clone C92-605, BD Biosciences), anti-BrdU (BD Biosciences), and isotype controls. cells BD Cytofix/Cytoperm cells

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