Targeting MK attenuates leukocyte infiltration into the heart during EAM. (a and b) Semiquantitative histological analysis of leukocyte infiltration (a) and evaluation of the heart/body weight ratio (b) at day 21 after sham immunization (sham: a, mean EAM score, 0.2 ± 0.1; b, mean heart/body weight ratio, 0.56 ± 0.01) or induction of EAM. EAM mice were treated with an anti-N-MK blocking Ab (EAM + anti-N-MK: a, mean EAM score, 1.4 ± 0.27; b, mean heart/body weight ratio, 0.64 ± 0.03) or vehicle alone (EAM + vehicle: a, mean EAM score, 2.5 ± 0.26; b, mean heart/body weight ratio, 0.74 ± 0.02) as depicted. n = 10 for sham group; n = 25 for EAM groups. (c and d) Flow cytometric analysis of leukocyte subpopulations in the cardiac tissue on day 21 after induction of EAM. Diagrams show the percentage of leukocyte subpopulations of all cells after blocking the N-terminal domain (EAM + anti-N-MK, c; CD45⁺: isotype ctrl, 5.28 ± 0.76 cells [%], anti-N-MK, 2.12 ± 0.42 cells [%]; CD45⁺/CD11b⁺/Gr-1⁺: isotype ctrl, 1.59 ± 0.58 cells [%], anti-N-MK, 0.45 ± 0.08 cells [%]; Ly6G⁺: isotype ctrl, 1.74 ± 0.39 cells [%], anti-N-MK, 0.99 ± 0.28 cells [%]; CD45⁺/CD11b⁺/Gr-1⁻: isotype ctrl, 1.84 ± 0.41 cells [%], anti-N-MK, 1.37 ± 0.29 cells [%]; CD4⁺: isotype ctrl, 1.86 ± 0.23 cells [%], anti-N-MK, 1.02 ± 0.07 cells [%]) or the C-terminal domain of MK (EAM + anti-C-MK, d; CD45⁺: isotype ctrl, 4.86 ± 0.72 cells [%], anti-C-MK, 5.29 ± 0.86 cells [%]; CD45⁺/CD11b⁺/Gr-1⁺: isotype ctrl, 1.47 ± 0.51 cells [%], anti-C-MK, 1.28 ± 0.31 cells [%]; CD45⁺/CD11b⁺/Gr-1⁻: isotype ctrl, 1.61 ± 0.25 cells [%], anti-C-MK, 1.86 ± 0.30 cells [%]; CD4⁺: isotype ctrl, 1.50 ± 0.12 cells [%], anti-C-MK, 1.41 ± 0.15 cells [%]) compared with the matching isotype controls (EAM + isotype ctrl). n = 18. (e) Representative immunofluorescence images of cardiac sections of sham-treated mice for control (top) or EAM mice at day 21 after induction of EAM (bottom). Images show staining of CD31, MK, and DAPI as well as the merged image. Arrows indicate a blood vessel. Arrowheads show MK expression in the perivascular compartment. Bars, 100 µm. To determine P values, ANOVA on ranks with Dunn’s multiple comparisons test was performed for a and b, and a Mann-Whitney rank sum test was used for c and d. For all panels, *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant. Data are presented as mean ± SEM or mean ± individual data points.