Journal of Molecular and Cellular Cardiology Treatment with anti-RANKL antibody reduces infarct size and attenuates dysfunction impacting on neutrophil-mediated injury  Federico Carbone, Lindsey A. Crowe, Aline Roth, Fabienne Burger, Sébastien Lenglet, Vincent Braunersreuther, Karim J. Brandt, Alessandra Quercioli, François Mach, Jean-Paul Vallée, Fabrizio Montecucco  Journal of Molecular and Cellular Cardiology  Volume 94, Pages 82-94 (May 2016) DOI: 10.1016/j.yjmcc.2016.03.013 Copyright © 2016 Elsevier Ltd Terms and Conditions

Fig. 1 RANKL is upregulated within infarcted hearts during reperfusion. Left ventricle ischemia was performed for 60min in male untreated C57Bl/6 mice. Intracardiac levels of RANKL and OPG were assessed at different time points of reperfusion in untreated and sham-operated mice (SHAM), sacrificed at 24h. Data are expressed as median (interquartile range). A. Representative images of RANKL- and OPG-stained consecutive middle heart sections of infarcted and sham-operated hearts. B. Quantification of RANKL stained area within the infarcted myocardium (n=5 per time point). C. Quantification of OPG stained area within the infarcted myocardium (n=5 per time point). Journal of Molecular and Cellular Cardiology 2016 94, 82-94DOI: (10.1016/j.yjmcc.2016.03.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Fig. 2 RANKL serum levels are increased in infarcted mice during reperfusion. Serum RANKL (A), OPG (B), and RANKL/OPG ratio (C) in sham-operated animals (SHAM) and in mice submitted to 60min ischemia a different times of reperfusion. Results are expressed as median (interquartile range) (Sham: n=9; 5min: n=5; 1h: n=5; 8h: n=13; 12h: n=23; 24h: n=10). Journal of Molecular and Cellular Cardiology 2016 94, 82-94DOI: (10.1016/j.yjmcc.2016.03.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Fig. 3 Treatment with anti-RANKL IgG reduces infarct size in vivo after 24h of reperfusion. Vehicle, CTL IgG (200μg/mouse) or anti-RANKL IgG (200μg/mouse) were administered in one shot at 5min after ischemia onset. Data are expressed as median (interquartile range). A. Quantification of area at risk (AAR) per ventricle surface (V) (n=10 per group). B. Quantification of infarct size (I) per AAR (n=10 per group). C. Quantification of infarct size (I) per ventricle surface (V) (n=10 per group). D. Representative images of TTC stained middle heart sections of Vehicle — CTL IgG- or anti-RANKL IgG-treated mice. The AAR was demarcated with the red line. E. Serum cardiac troponin I (cTnI) levels of Vehicle — CTL IgG- or anti-RANKL IgG-treated mice after 16h and 24h of reperfusion (n=7 per group at 16h, n=10 per group at 24h). Journal of Molecular and Cellular Cardiology 2016 94, 82-94DOI: (10.1016/j.yjmcc.2016.03.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Fig. 4 Treatment with anti-RANKL IgG improves left ventricular function at 24h of reperfusion. A, C, E. Representative magnetic resonance images (MRI) (among n=8 in Vehicle-treated, n=6 in CTL IgG-treated and n=7 in anti-RANKL IgG-treated mice) of 2-chamber, 4-chamber long axis and short axis at mid papillary muscle level of end-systole and end-diastole of Vehicle — (A), CTL IgG- (C) and anti-RANKL IgG-treated (E) mice at 24h of reperfusion. Arrows indicate the infarcted zones. B, D, F. Representative microphotographs (among n=8 in Vehicle-treated, n=6 in CTL IgG-treated and n=7 in anti-RANKL IgG-treated mice) at mid papillary muscle level of the same infarcted hearts tested with MRI showing Nitrobluetetrazolium chloride staining (arrows indicate white colored regions that are metabolically inactive). Journal of Molecular and Cellular Cardiology 2016 94, 82-94DOI: (10.1016/j.yjmcc.2016.03.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Fig. 5 Treatment with anti-RANKL IgG reduces neutrophil and MMP-9 content in infarcted hearts in vivo. A. Quantification of infiltrated neutrophils (Ly6G+ cells) per area in frozen sections of infarcted hearts at different reperfusion time points (1h, 16h, and 24h). Data are expressed as median (interquartile range). (n=4 per group at 1h, n=7 per group at 16h, n=9 per group at 24h). B. Representative images of neutrophil infiltration at 16 and 24h reperfusion are shown. C. Quantification of MMP-9 stained areas in frozen sections of infarcted hearts at different reperfusion time points (1h, 16h, and 24h). Data are expressed as median (interquartile range). (n=4 per group at 1h, n=7 per group at 16h, n=9 per group at 24h). D. Representative images of MMP-9 stained heart sections at 16 and 24h of reperfusion are shown. Journal of Molecular and Cellular Cardiology 2016 94, 82-94DOI: (10.1016/j.yjmcc.2016.03.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Fig. 6 RANK is expressed in mouse primary neutrophils. Representative agarose gel of PCR products from mouse neutrophils (isolated from four different mouse donors M_1-M_4) showing mRNA expression of RANKL receptor RANK. On the left the molecular weights of the markers are shown. Journal of Molecular and Cellular Cardiology 2016 94, 82-94DOI: (10.1016/j.yjmcc.2016.03.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Fig. 7 In vitro incubation of anti-RANKL IgG with recombinant RANKL inhibits mouse neutrophil migration towards this cytokine. A. Mouse neutrophil migration towards 200ng/ml CXCL2 or control medium in the presence or absence of control medium (NIL) or of different dilutions of serum from infarcted mice treated with CTL IgG or anti-RANKL IgG at 24h of reperfusion. Data are expressed mean±SD, n=4. B. Mouse neutrophil migration towards different doses (0.01–10ng/ml) of RANKL or control medium pre-incubated with CTL IgG (6μg/ml) or different concentrations (0.06–6μg/ml) of anti-RANKL IgG. Data are expressed mean±SD, n=4 per group. C. Mouse neutrophil migration towards 200ng/ml CXCL2 or control medium pre-incubated in the presence or absence of 6μg/ml CTL IgG or 6μg/ml of anti-RANKL IgG. Data are expressed mean±SD, n=4 per group. Journal of Molecular and Cellular Cardiology 2016 94, 82-94DOI: (10.1016/j.yjmcc.2016.03.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Fig. 8 In vitro incubation of anti-RANKL IgG with recombinant RANKL inhibits mouse neutrophil degranulation in response to this cytokine. A. Pro-MMP-9 levels in mouse neutrophil supernatants in the presence or absence of control medium (CTL), 10ng/ml phorbol-12-myristate-13-acetate (PMA, positive control) or different concentrations of mouse recombinant RANKL. Data are expressed as median (interquartile range), n=13 per group. B. Pro-MMP-9 levels in mouse neutrophil supernatants of in the presence or absence of control medium (CTL), 10ng/ml PMA or 10ng/ml RANKL pre-incubated with different concentration of anti-RANKL IgG. Data are expressed as median (interquartile range), n=13 per group. C. Pro-MMP-9 levels in mouse neutrophil supernatants of in the presence or absence of control medium (CTL), 10ng/ml PMA or 10ng/ml RANKL and pre-incubated with 6μg/ml CTL IgG. Data are expressed as median (interquartile range), n=13 per group. Journal of Molecular and Cellular Cardiology 2016 94, 82-94DOI: (10.1016/j.yjmcc.2016.03.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Journal of Molecular and Cellular Cardiology 2016 94, 82-94DOI: (10 Journal of Molecular and Cellular Cardiology 2016 94, 82-94DOI: (10.1016/j.yjmcc.2016.03.013) Copyright © 2016 Elsevier Ltd Terms and Conditions