Fig 2: Orthogonal validation of the expression of Trem2 in the heart after MI.a Immunohistochemistry showed a gradual increase in the expression of Trem2 over time in the infarcted area after the induction of MI, almost absent in the steady-state. Results are representative of five different samples. b Western blot targeting Trem2 showed two bands: a 32 kDa sized one corresponding to the full-length Trem2 protein and an 18 kDa sized one corresponding to soluble Trem2 (sTrem2); 50 µg of infarcted heart tissue proteins were loaded onto each lane. Quantitative comparison of the full-length, soluble form, and total Trem2 expression, respectively (n = 4 for each group). Unpaired two-tailed t-test was used to determine the statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001. c Co-localization assay for Trem2 and the macrophage marker CD68 in the infarcted area according to the time-point after MI. Cells were stained with the specific antibodies anti-Trem2 (red) and anti-CD68 (green). Nuclei were counterstained with DAPI (blue). Quantification of Trem2+/CD68+ cells in the infarcted area (n = 3 for day 1 post-MI; n = 4 for days 2, 5, and 7 post-MI). One-way ANOVA with two-tailed Dunnett test was used to determine the statistical significance. d Comparison of the expression of Ccr2, 1, 3, 5, and 7 days after MI by FACS. e Comparison of the expression of Trem2 1, 3, 5, and 7 days after MI by FACS. f Bars are divided to display the percentage of F4/80+ Ccr2 or Trem2 positive cells in the different experimental days (n = 4 for steady-state, days 1 and 7 post-MI; n = 8 for day 3 post-MI; n = 6 for day 5 post-MI). g Quantitative analysis of Trem2+ F4/80+ cells. The sample size is the same as (f). One-way ANOVA with two-tailed Dunnett test was used to determine the statistical significance. **P < 0.01, ***P < 0.001. h Comparison of Arg1+ cells within F4/80+Ccr2+ and F4/80+Trem2+ cells, 5 days after MI (n = 4 for F4/80+Ccr2+; n = 8 for F4/80+Trem2+). Unpaired two-tailed t-test was used to determine the statistical significance. i Comparison of mRNA expression levels of pro-inflammatory genes in F4/80+Ccr2+ macrophages at day 1 post-MI (n = 5) and F4/80+Trem2+ macrophages at day 5 post-MI (n = 6). Unpaired two-tailed t-test was used to determine the statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001. j Comparison of mRNA expression levels of anti-inflammatory genes in F4/80+Ccr2+ macrophages at day 1 post-MI (n = 5) and F4/80+Trem2+ macrophages at day 5 post-MI (n = 6). Unpaired two-tailed t-test was used to determine the statistical significance. *P < 0.05, **P < 0.01. All of the bar charts are presented as mean ± SEM. Source data with exact P values for b, c and f–j are provided in the Source Data file.

Fig 3: CCR2 is required for LPS-driven suppression of allergen-specific Th2 cell responses(A–F) Mice were transferred with OT-II.4get cells and sensitized. Frequencies (A, C, E, and F) and numbers (B and D) of total, IL-4-GFP+, and T-bet+ OT-II cells.(G–L) Mice were treated with Alexa Fluor 647-labeled HDM ± LPS. Frequencies of HDM+ cells (G) and expression of the indicated markers on HDM+ cells and monocytic cells (H). Frequencies (I) and numbers (J–L) of HDM+ mDCs (J), mDC2s (K), and mDC1s (L).(M–S) Frequencies (M and N) and numbers (O) of IL-12p70+ mDC2s, and frequencies (P and Q) of T-bet+ mDC2s in the mLN of HDM ± LPS-treated mice. (R and S) Day-3 mLN CD11c+ mDCs from sensitized mice were co-cultured for 4 days with CTV-labeled OT-II.4get cells. Frequencies of CTVloOTII cells (R), frequencies of IL-4-GFP expression in CTVloOTII cells (S). Values in triplicate.Data are representative of three independent experiments (mean ± SD, n = 4–5, two-way ANOVA). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See Figures S5-S7.

Fig 4: Infiltrating Monocytes Contribute to Three Subsets of Macrophages during Myocarditis(A) Schematics of parabiosis mice.(B) Representative images of H&E-stained heart sections of the median CD45.1 (day 21 EAM) and CD45.2 (non-EAM) mice parabionts. Scale bars: 100 µm.(C) Comparison of total grafted CD11b+Ly6G-myeloid cell counts between parabionts.(D) Comparison of total cardiac macrophage counts between parabionts.(E) Flow cytometry plots showing (top left) percentage of CD45.2+CD11b+Ly6G-Lineage (CD3e, B220, NKp46, CD90.2, and Ter119)- grafted cells infiltrating the CD45.1 EAM hearts; (top middle) percentage of grafted cells in the hearts that differentiated into macrophages or remained as monocytes; (top right) percentage of grafted MDM subsets; (bottom left) percentage of CD45.1+CD11b+Ly6G-Lineage grafted cells infiltrating the CD45.2 non-EAM hearts; (bottom middle) percentage of grafted cells in the hearts that differentiated into macrophages or remained as monocytes; and (bottom right) percentage of grafted MDM subsets.(F) Percentages of grafted MDMs out of total number of grafted CD11b+Ly6G-Lineage- myeloid cells.(G) Percentages of grafted Ly6Chi and Ly6Clo cells out of total number of grafted F4/80-CD64+ monocytes.(H) Comparison of grafted MDM subsets defined by CCR2 and MHCII expressions between parabionts. Data are representative of two independent experiments with biological triplicates. n = 3.(C, D, F, G) Groups were compared using Student’s t test. *p < 0.05. All data were presented as mean ± SD.See also Figure S1.

Fig 5: Neutrophil and dendritic cell subsets.a The UMAP visualization of 5135 neutrophils identified five distinct subsets (left panel). Each point represents a single cell, colored according to the sub-cluster assigned. In the right panel, the heatmap shows the top 10 most differentially expressed genes in each sub-cluster. Blue and red indicate lower and higher expression, respectively. Typical markers strongly and specifically associated with each sub-cluster are shown on the left. b Bar plot showing the proportions of cells in each of the 5 sub-clusters according to the time-point after MI. c Violin plots of the expression of Fpr1, Tnf1, Ifitm2, Isg15, and Manf in neutrophil sub-clusters (Cluster 1: 1864 cells; Cluster 2: 1243 cells; Cluster 3: 844 cells; Cluster 4: 698 cells; Cluster 5: 486 cells). d The UMAP visualization of 1921 dendritic cells identified 6 distinct subsets (left panel). Each point represents a single cell, colored according to the sub-cluster assigned. In the right panel, the heatmap shows the top 10 most differentially expressed genes in each sub-cluster. Blue and red indicate lower and higher expression, respectively. Typical markers strongly and specifically associated with each sub-cluster are shown on the left. e Bar plot showing the proportions of cells in each of the 6 sub-clusters according to the time-point after MI. f Violin plots of the expression of Ms4a7, Ccr2, Cd209a, Xcr1, Fscn1, and Siglech in DC sub-clusters. (Cluster 1: 541 cells; Cluster 2: 507 cells; Cluster 3: 377 cells; Cluster 4: 264 cells; Cluster 5: 184 cells; Cluster 6: 48 cells). Regarding the box polts of c and f, the lower whisker, lower hinge, box center, upper hinge, and upper whisker represent the minimum, lower quartile, median, upper quartile, and maximum calculated without outlier values which are more than 1.5× interquartile range of the lower and upper quartiles. Source data for b, c and e, f are provided in the Source Data file.