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. 2014 May 9;114(10):1611-22.
doi: 10.1161/CIRCRESAHA.114.303204. Epub 2014 Mar 13.

Ly-6Chigh monocytes depend on Nr4a1 to balance both inflammatory and reparative phases in the infarcted myocardium

Ingo Hilgendorf  1 , Louisa M S Gerhardt, Timothy C Tan, Carla Winter, Tobias A W Holderried, Benjamin G Chousterman, Yoshiko Iwamoto, Ronglih Liao, Andreas Zirlik, Marielle Scherer-Crosbie, Catherine C Hedrick, Peter Libby, Matthias Nahrendorf, Ralph Weissleder, Filip K Swirski
Affiliations

Ly-6Chigh monocytes depend on Nr4a1 to balance both inflammatory and reparative phases in the infarcted myocardium

Ingo Hilgendorf et al. Circ Res. .

Abstract

Rationale: Healing after myocardial infarction involves the biphasic accumulation of inflammatory lymphocyte antigen 6C (Ly-6C)(high) and reparative Ly-6C(low) monocytes/macrophages (Mo/MΦ). According to 1 model, Mo/MΦ heterogeneity in the heart originates in the blood and involves the sequential recruitment of distinct monocyte subsets that differentiate to distinct macrophages. Alternatively, heterogeneity may arise in tissue from 1 circulating subset via local macrophage differentiation and polarization. The orphan nuclear hormone receptor, nuclear receptor subfamily 4, group a, member 1 (Nr4a1), is essential to Ly-6C(low) monocyte production but dispensable to 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/MΦ 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.

Keywords: hormone receptors; macrophages; monocytes; myocardial infarction; nuclear.

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Figures

Figure 1
Figure 1. Nr4a1 expressing myeloid cells accumulate in myocardial infarction tissue
A. Cross sections of heart tissue 3 and 7 days after permanent ligation of the left anterior descending coronary artery (LAD) or sham surgery. Overview and high magnification images of immunohistochemical staining for Nr4a1. B. Immunofluorescence co-staining for Nr4a1 and CD11b of MI tissue 7 days after coronary artery ligation. Representative images from one out of three samples are shown.
Figure 2
Figure 2. Adverse cardiac remodeling in Nr4a1−/− chimeric mice 7 days after myocardial infarction
Immunohistochemical staining of MI tissue for CD11b, Collagen 1 (Col1), CD31, and non-vascular smooth muscle alpha actin (SMA) in wild-type (WT) and Nr4a1−/− chimeric mice 7 days post MI Quantification of 10 randomly selected fields of view per sample. Results are presented as mean ± SEM, * p ≤ 0.05, n =5 per group.
Figure 3
Figure 3. Enhanced accumulation of Nr4a1−/− monocytes and macrophages in myocardial infarct tissue
A. Representative images for flow cytometric analysis of MI tissue cell suspensions before, 3 and 7 days after permanent LAD ligation in WT and Nr4a1−/− mice. B. Flow cytometry and analogous gating strategy identifying blood monocyte subsets in WT and Nr4a1−/− chimeras at steady state. C. Flow cytometry based quantification of monocyte and macrophage numbers in MI tissue of WT versus Nr4a1−/− mice before and 1, 3, 7, and 21 days post MI. Results are presented as mean ± SEM, * p ≤ 0.05, n ≥ 4 per group and time point. D. Graphs illustrating flux of monocyte and macrophage subsets during phase 1 (≤ 4 days) and phase 2 (days 4–21) of infarct healing.
Figure 4
Figure 4. Nr4a1low Ly-6Chigh monocytes give rise to Nr4a1high Ly-6Clow macrophages in myocardial infarct tissue
A. Identification of Ly-6Chigh and Ly-6Clow monocyte subsets in pooled CD45.2+ GFP+ blood and spleen samples for flow assisted cell sorting. B. Illustration of experimental approach with separate adoptive transfer of sorted GFP+ Ly-6Chigh monocytes (1×106) and GFP+ Ly-6Clow monocytes (0.5×106) 3 days after permanent ligation of the LAD in CD45.1+ mice. Sacrifice 1 and 3 days after adoptive transfer. C. Flow cytometric analysis of blood from CD45.1+ recipients of GFP+ monocyte subsets on days 1 and 3 after transfer. Quantification of the percentage of GFP+ cells within the respective gates. Pooled data of two independent experiments. D. Flow cytometric analysis of MI tissue from CD45.1+ recipients of GFP+ Ly-6Chigh monocytes on days 1 and 3 after transfer. Quantification of the percentage of GFP+ cells within the respective gates. Pooled data of two independent experiments. E. Flow cytometric analysis of MI tissue from CD45.1+ recipients of GFP+ Ly-6Clow monocytes on days 1 and 3 after transfer. Quantification of the percentage of GFP+ cells within the respective gates. Pooled data of two independent experiments. F. Quantification (qRT-PCR) of Nr4a1 expression in cells sorted from the blood, MI tissue and unstimulated peritoneal lavage on days 3 and 7 after permanent LAD ligation compared to sham operated C57Bl/6 mice (0 days post MI). Results are presented as mean ± SEM, * p ≤ 0.05, n ≥ 4 per time point. G. Relative expression of VEGFα, TGFβ and IL-10 in sorted Ly-6Chigh monocytes on day 3 (phase 1) and macrophages on day 7 (phase 2) post MI compared to baseline cardiac macrophages. Results are presented as mean ± SEM, * p ≤ 0.05, n ≥ 4 per time point.
Figure 5
Figure 5. Nr4a1-deficiency promotes CCR2-mediated monocyte recruitment to the myocardial infarct tissue
A. Quantification of monocyte subsets and neutrophils in the blood of WT and Nr4a1−/− mice before and on days 1, 3, 7 and 21 post MI. Results are presented as mean ± SEM, * p ≤ 0.05, n ≥ 5 per group and time point. B. Representative dot plot of flow cytometric staining for CCR2 expression on circulating monocytes in WT and Nr4a1−/− mice 3 days post MI. C. Quantification of CCR2 expression on Ly-6Chigh monocytes by mean fluorescence intensity at indicated time points after MI. Results are shown as mean ± SEM percent change of CCR2 expression in Nr4a1−/− compared to WT control mice, * p ≤ 0.05, n ≥ 5 per group and time point. D. Quantification of serum MCP-1/CCL2 levels in WT and Nr4a1−/− mice before and on days 1, 3, 7 and 21 post MI. Results are presented as mean ± SEM, n ≥ 5 per group and time point. E. Quantification of expression of L-selectin (CD62L), P-selectin glycoprotein ligand-1 (PSGL-1) and integrin Mac1 on Ly-6Chigh monocytes by mean fluorescence intensity at indicated time points after MI. Results are shown as mean ± SEM percent change of marker expression in Nr4a1−/− compared to WT control mice, * p ≤ 0.05, n ≥ 5 per group and time point. F. Quantification of Ly-6Chigh monocyte numbers in peripheral blood and MI tissue on day 3 after permanent LAD ligation in WT and Nr4a1−/− mice treated with CCR2 antagonist (RS504393, 2mg/kg ip. b.i.d.) or vehicle (30% DMSO ip) alone. Results are presented as mean ± SEM, * p ≤ 0.05, n =5 per group.
Figure 6
Figure 6. Nr4a1-deficient macrophages are pro-inflammatory, proliferate and die in infarct tissue
A. Quantification of Ki67+ CD11b+ cells in MI tissue 7 days after permanent LAD ligation in WT and Nr4a1−/− mice on the right and representative merged images for immunofluorescent co-staining on the left. Results are presented as mean ± SEM, n =5 per group. B. Flow cytometric analysis of BrdU incorporation into WT blood and bone marrow (BM) monocytes 2 hours after iv injection of 200μg BrdU. C. Flow cytometric analysis of BrdU incorporation into WT and Nr4a1−/− Ly-6Chigh monocytes and macrophages in MI tissue 7 days after permanent LAD ligation. Results are presented as mean ± SEM, n =5 per group. D. Quantification of TUNEL+ CD11b+ cells in MI tissue 7 days after permanent LAD ligation in WT and Nr4a1−/− mice on the right and representative merged images for immunofluorescent co-staining on the left. Results are presented as mean ± SEM, n =5 per group. E. Flow cytometric analysis of Caspase 3 expression in WT and Nr4a1−/− Ly-6Chigh monocytes and macrophages in MI tissue 7 days after permanent LAD ligation. Results are presented as mean ± SEM, n =5 per group. Isotype staining is shown on the right. F. Gene expression profiling of WT and Nr4a1−/− macrophages sorted from MI tissue 7 days after permanent LAD ligation. Results are presented as mean ± SEM percent change of marker expression in Nr4a1−/− compared to WT control mice, * p ≤ 0.05, n ≥ 5 per group.
Figure 7
Figure 7. Nr4a1 limits left ventricular dysfunction after myocardial infarction
A. Representative echocardiography images with long axis B-mode (upper panel) and short axis M-mode views (lower panels) of infarcted hearts from WT and Nr4a1−/− mice on day 21 after permanent LAD ligation. Arrows and lines mark left ventricular inner diameters (LVID) in systole (dashed) and diastole (firm). B. Quantification of individual changes (Δ) in heart parameters compared to baseline on days 2 and 21 post MI. Results are presented as mean ± SEM percent change of marker expression in Nr4a1−/− compared to WT control mice, * p ≤ 0.05, n ≥ 8 per group. C. Representative images of heart cross sections 21 days post MI at increasing distance from the site of coronary artery ligation in Nr4a1−/− and WT control mice, n =5 per group. D. Quantification of scar size in heart cross sections at different levels depending on the distance from the site of coronary artery ligation. Data are presented as a fraction of the left ventricle including the septum, mean ± SEM, * p ≤ 0.05, n =5 per group. Collagen density is quantified in 5 randomly selected fields of view per sample within the scar at 2 mm distance from the site of coronary artery ligation. Results are presented as mean ± SEM, * p ≤ 0.05, n =5 per group.
Figure 8
Figure 8. Biphasic model of monocyte/macrophage accumulation in MI tissue
During the early phase of myocardial infarct healing inflammatory Nr4a1low Ly-6Chigh monocytes from the bone marrow and spleen infiltrate the MI tissue via CCR2. Consequently they differentiate into proliferating Nr4a1high Ly-6Clow macrophages that participate in the resolution of inflammation and cardiac remodeling in the second phase of infarct healing.
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References

    1. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, Abraham J, Adair T, Aggarwal R, Ahn SY, Alvarado M, Anderson HR, Anderson LM, Andrews KG, Atkinson C, Baddour LM, Barker-Collo S, Bartels DH, Bell ML, Benjamin EJ, Bennett D, Bhalla K, Bikbov B, Bin Abdulhak A, Birbeck G, Blyth F, Bolliger I, Boufous S, Bucello C, Burch M, Burney P, Carapetis J, Chen H, Chou D, Chugh SS, Coffeng LE, Colan SD, Colquhoun S, Colson KE, Condon J, Connor MD, Cooper LT, Corriere M, Cortinovis M, de Vaccaro KC, Couser W, Cowie BC, Criqui MH, Cross M, Dabhadkar KC, Dahodwala N, De Leo D, Degenhardt L, Delossantos A, Denenberg J, Des Jarlais DC, Dharmaratne SD, Dorsey ER, Driscoll T, Duber H, Ebel B, Erwin PJ, Espindola P, Ezzati M, Feigin V, Flaxman AD, Forouzanfar MH, Fowkes FG, Franklin R, Fransen M, Freeman MK, Gabriel SE, Gakidou E, Gaspari F, Gillum RF, Gonzalez-Medina D, Halasa YA, Haring D, Harrison JE, Havmoeller R, Hay RJ, Hoen B, Hotez PJ, Hoy D, Jacobsen KH, James SL, Jasrasaria R, Jayaraman S, Johns N, Karthikeyan G, Kassebaum N, Keren A, Khoo JP, Knowlton LM, Kobusingye O, Koranteng A, Krishnamurthi R, Lipnick M, Lipshultz SE, Ohno SL, Mabweijano J, MacIntyre MF, Mallinger L, March L, Marks GB, Marks R, Matsumori A, Matzopoulos R, Mayosi BM, McAnulty JH, McDermott MM, McGrath J, Mensah GA, Merriman TR, Michaud C, Miller M, Miller TR, Mock C, Mocumbi AO, Mokdad AA, Moran A, Mulholland K, Nair MN, Naldi L, Narayan KM, Nasseri K, Norman P, O’Donnell M, Omer SB, Ortblad K, Osborne R, Ozgediz D, Pahari B, Pandian JD, Rivero AP, Padilla RP, Perez-Ruiz F, Perico N, Phillips D, Pierce K, Pope CA, Porrini E, Pourmalek F, Raju M, Ranganathan D, Rehm JT, Rein DB, Remuzzi G, Rivara FP, Roberts T, De Leon FR, Rosenfeld LC, Rushton L, Sacco RL, Salomon JA, Sampson U, Sanman E, Schwebel DC, Segui-Gomez M, Shepard DS, Singh D, Singleton J, Sliwa K, Smith E, Steer A, Taylor JA, Thomas B, Tleyjeh IM, Towbin JA, Truelsen T, Undurraga EA, Venketasubramanian N, Vijayakumar L, Vos T, Wagner GR, Wang M, Wang W, Watt K, Weinstock MA, Weintraub R, Wilkinson JD, Woolf AD, Wulf S, Yeh PH, Yip P, Zabetian A, Zheng ZJ, Lopez AD, Murray CJ. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012;380:2095–2128. - PMC - PubMed
    1. Bloom DE, Cafiero ET, Jane-Llopis E, Abrahams-Gessel S, Bloom LR, Fathima S, Feigl AB, Gaziano T, Mowafi M, Pandya A, Prettner K, Rosenberg L, Seligman B, Stein AZ, Weinstein C. The Global Economic Burden of Noncommunicable Diseases. Geneva: World Economic Forum; 2011.
    1. van Amerongen MJ, Harmsen MC, van Rooijen N, Petersen AH, van Luyn MJ. Macrophage depletion impairs wound healing and increases left ventricular remodeling after myocardial injury in mice. Am J Pathol. 2007;170:818–829. - PMC - PubMed
    1. Nahrendorf M, Pittet MJ, Swirski FK. Monocytes: protagonists of infarct inflammation and repair after myocardial infarction. Circulation. 2010;121:2437–2445. - PMC - PubMed
    1. Frangogiannis NG. Regulation of the inflammatory response in cardiac repair. Circ Res. 2012;110:159–173. - PMC - PubMed

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