Fig 1: Cellular infiltration and pro-inflammatory macrophage activity are abrogated in neutrophil depleted mice during NASH development. IHC for detection of monocyte derived macrophages and other CD11b+ infiltrating myeloid cells (a), hepatic resident F4/80+ Kupffer cells (b) and pro-inflammatory Ly6Chi monocytes/macrophages was performed and quantified for comparison among the control diet receiving group and the mice receiving either IgG or anti-Ly6G antibodies during ongoing FFC diet . mRNA expression was analyzed via RT-qPCR and fold-change was compared between the mentioned groups regarding the main monocyte chemotactic pathway CCL2/CCR2 (d) and pro-inflammatory macrophage activity markers such as components of the NLRP3 inflammasome pathway, chitinase-3 like 1 protein (CHI3L1) and TNF-a (e). Data was compared by using ANOVA analysis followed by the Turkey post hoc test. Western Blots targeting components of the NLRP3 downstream cascade were performed to compare the mentioned groups in a protein expression level (f). a-tubulin (band imported from Supplementary Fig. 1b) was used as housekeeping protein in both assessments. Full-length blottings of pro-caspase 1 and pro-IL-1ß are shown in Supplementary Fig. 1b and 2, respectively. Ccl2 C–C motif chemokine ligand 2, ccr2 C–C motif chemokine receptor 2, NLRP3 NLR family pyrin domain containing 3, CHI3L1 chitinase 3-like 1, TNF-a/tnfa tumor necrosis factor alpha.
Fig 2: 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.
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: Lactate-Induced Macrophage Polarization Is MCT1 Dependent(A) Lactate uptake in macrophages isolated from muscle 3 days after HLI.(B) Lactate uptake in BMDMs after stimulation with mECs-CM. mECs-CM was supplemented with vehicle (ctrl), lactate, or MCT1 inhibitor AZD3965 (AZD) (mECswt+AZD-CM, mECs?pfkfb3+AZD-CM).(C) Lactate oxidation in BMDMs upon stimulation with mECswt-CM, mECs?pfkfb3-CM, mECswt+lac-CM, mECs?pfkfb3+lac-CM, mECswt+AZD-CM, and mECs?pfkfb3+AZD-CM.(D) Representative images of immunostainings for F4/80 (green), CD206 (red), and hoechst (blue) in BMDMs stimulated with mECswt+AZD-CM or mECs?pfkfb3+AZD-CM and flow cytometry quantification of CD206 MFI.(E and F) Representative images (red, EdU+; blue, hoechst) (E) and quantification (F) of EdU+ MPCs.(G and H) Representative DESMIN staining (red, DESMIN; blue, hoechst) (G) and fusion analysis (H) upon stimulation with mECs-CM ? BMDMs-CM.(I) VEGF levels in mECs-CM ? BMDMs-CM.Scale bar, 50 µm. Student’s t test (two-tailed, unpaired) in (A) (*p < 0.05). Two-way ANOVA with Tukey’s multiple comparisons test in (B), (C), (F), (H), and (I) (*p < 0.05) test and in (D) (*p < 0.05 versus mECswt-CM; #p < 0.05 versus ctrl). Each dot represents a single mouse (A) or the average of an independent experiment ([B], [C], [F], [H], and [I]). Bar graphs represent mean ± SEM.
Fig 5: 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.
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