Fig 2: Endothelial Lactate Controls Macrophage Polarization and Function upon Muscle Ischemia(A) Scheme illustrating experimental set-up.(B) Gene profiling of unstimulated BMDMs (vehicle) or BMDMs stimulated with mECswt-CM, mECs?pfkfb3-CM.(C and D) Gene expression analysis of arg1 (C) and mrc1 (D) in BMDMs stimulated with fractionated mECswt-CM and mECs?pfkfb3-CM, supplemented with lactate (5 mM) where indicated.(E) Lactate concentration in mECswt-CM and mECs?pfkfb3-CM.(F) Representative images of immunostainings of F4/80 (green), CD206 (red), and hoechst (blue) in BMDMs stimulated with mECswt-CM, mECs?pfkfb3-CM with or without lactate supplementation and quantification of CD206 MFI (n = 3).(G) OCR upon injection of oligomycin (oligo), FCCP, and rotenone plus antimycin A after mECswt-CM, mECs?pfkfb3-CM, mECswt+lac-CM, and mECs?pfkfb3+lac-CM stimulation (n = 4–5).(H) RNaseq data showing OXPHOS gene expression in muscle macrophages 3 days after HLI (n = 3).(I and J) Representative images (I) and quantification (J) of proliferating MPCs upon incubation with macrophage-derived CM, measured as percentage of EdU+ nuclei (red, EdU+; blue, hoechst).(K and L) MPC fusion analysis: representative images of immunofluorescent DESMIN staining (K) (red, DESMIN; blue, hoechst) and quantification of the number of nuclei per DESMIN+ myotube (L).(M) Vegf gene expression in CD45+ cells sorted from pfkfb3WT and pfkfb3?EC muscle at 3 d. (N) VEGF secretion by BMDMs after stimulation with mECswt-CM and mECs?pfkfb3-CM with or without lactate supplementation.Scale bar, 50 µm. Student’s t test (two-tailed, unpaired) in (E) and (M). One-way ANOVA with Tukey’s multiple comparisons test in (B). Two-way ANOVA with Tukey’s multiple comparisons test in (C), (D), (G), (J), (L), and (N) (*p < 0.05) as well as in (F) (*p < 0.05 versus mECswt-CM; #p < 0.05 versus ctrl). Each dot represents a single mouse (M) or the average of an independent experiment ([B], [C], [D], [E], [J], [L], and [N]). Bar graphs represent mean ± SEM. See also Figure S5 and Table S1.

Fig 3: Endothelial PFKFB3 Is Crucial for M2-like Polarization of Macrophages in the Muscle(A–C) Total number of neutrophils (A), monocytes (B), and macrophages (C) in muscle at the indicated times after HLI determined by flow cytometry.(D) Quantification of EdU+ monocytes (% of total monocytes) at the indicated times after HLI.(E and F) Representative images of F4/80 immunostainings (red) and hoechst (blue) (E) and quantification of F4/80+ area (F) on muscle at 3 days.(G) Mean fluorescent intensity (MFI) of Ly-6C, F4/80, and MERTK in muscle macrophages at the indicated times after HLI determined by flow cytometry.(H) Representative flow cytometric analysis of CD206+ and Relma+ cells in muscle 3 days after HLI.(I and J) Quantification of Relma+CD206+ (I) and Relma-CD206+ (J) F4/80+CD11b+ macrophages.(K and L) Representative histograms (K) and quantification of CD206 MFI (L) in the total muscle macrophage population 3 days after HLI.(M and N) Representative immunostainings for F4/80 (green) and CD206 (red) on CD45+ cells (M) sorted from muscle 3 d after HLI and quantification of CD206 MFI (N).(O and P) Representative images of CD206 immunostainings (red) and hoechst (blue) (O) and quantification of CD206+ macrophages number (P) 3 days after HLI.(Q) Quantification of EdU+CD206+ macrophages (% of CD206+ macrophages) at 3 days after HLI determined by flow cytometry(R and S). Principal-component analysis (R) and Volcano plot from RNA-seq analysis showing differential gene expression (S) of macrophages isolated from pfkfb3WT and pfkfb3?EC muscles 3 days after HLI (n = 3).(T) Expression pattern of pro-inflammatory and anti-inflammatory markers genes in muscle macrophages 3 days after HLI (n = 3).Arrowheads point at CD206+ cells (O). Scale bar, 50 µm. Student’s t test (two-tailed, unpaired) in (F), (I), (J), (L), (N), (P), and (Q) (*p < 0.05). Two-way ANOVA with Tukey’s multiple comparisons test in (A), (B), (C), (D), and (G) (*p < 0.05). Each dot represents a single mouse ([A], [B], [C], [D], [F], [G], [I], [J], [L], [N], [P], and [Q]). Bar graphs represent mean ± SEM. See also Figures S2 and S3.

Fig 4: Microglial Changes during Development, Related to Figure 1(A) Gating strategy for flow cytometry purification of CD45low, CD11b+, F4/80+, CD64+, Ly6C- yolk-sac progenitors and microglia.(B) Gating strategy of flow cytometric analysis of cells from FUCCI mice showing CD45low, CD11b+, F4/80+, and Azami green+ cells.(C) The seven clusters characterizing the different developmental phases of microglia.The plots show the expression of the corresponding genes that are associated with these functions during development. Clusters 1, 2 and 3 are related to the progenitor phase, cluster 4 to embryonic phase 1, cluster 5 to embryonic phase 2 and clusters 6 and 7 to the adult stage.See also Table S1.

Fig 5: Microglia Undergo Distinct Developmental Phases(A and B) Dendrogram (A) and PCA (B) on transcriptomes of murine YS progenitors and microglia at different developmental stages. n = 3–4 replicates per stage, with each replicate obtained by pooling microglia sorted from several female and male brains. PC, principal component.(C) Heatmap of the DEGs with clusters (left), associated signaling pathways (right), and corresponding expression plots. Each row is a biological replicate.(D) Percentages of Azami green+ cells (S/G2/M cell-cycle phases) among F4/80/CD11b-positive cells from brains of FUCCI mice. Data are represented as means ± SEM; n = 3–5 per stage; one-way ANOVA with Tukey post hoc test was used to assess differences; ***p < 0.001.(E) Heatmap of the expression level of microglia sensome genes.(F) Microglial sensome gene expression in the different developmental clusters. Embryo P, embryonic phase.See also Figures 2, S1, and Table S1.