Fig 2: The effects of DHA feeding on the cellularity and activation of T and B cells in the spleen at day 7 postinfection. A Flow cytometric analysis and enumeration of total T cells (CD45+CD3-CD19-), CD4+ T cells (CD45+CD3+CD19-CD4+CD8-), CD8+ T cells (CD45+CD3+CD19-CD4-CD8+) and B cells (CD45+CD3-CD19+) in the spleens of control versus DHA-fed mice. B Flow cytometric analysis and cumulative data indicating the expression of CD62L and CD44 in CD4+ T and CD8+ T cells between control and DHA-fed mice. For each experiment, n = 5 to 9 pooled from 2 independent experiments (A, B). Each symbol represents an individual mouse, and the blue dots and red square represent control and DHA-enriched diet-fed mice, respectively. Error bars represent interquartile ranges; *p < 0.05; **p < 0.01; ***p < 0.001

Fig 3: 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 4: Demonstration of continuous transfer of exogenous mitochondria in blood of Polg mouse model.Lineage negative cells ubiquitously expressing red-labeled nucleus (dTomato) and green-labeled mitochondria (Dendra) derived from ROSAnT-nG/PhAMexcised mice were augmented with Dendra2+ mitochondria for 21 h and injected to Polg mice dosed at age 24–27 weeks (males) and 30-34 weeks (females). At 12 h, 1 w, 1 m, and 4.5 m post-injection blood was drawn and BM was extracted and both were analyzed by flow cytometry and confocal microscopy. a Graphical presentation of experimental design and a representative image of confocal microscopy of dTomato+Dendra2+ (left) and dTomato-Dendra2+ (right) found in bone marrow 1 m post-injection. Scale bar 5 µm. b Representation of flow cytometry analysis, which is expected to detect three types of cells: Polg cells, transplanted/daughter cells (red box, dTomato+Dendra2+) and Polg cells which received mitochondrial transfer (green box, dTomato-Dendra2+). c Representative figure of flow cytometry analysis of dTomato-Dendra2+ cells within CD45+ cell population in peripheral blood 12 h, 1 w, 1 m and 4.5 m post-injection (left). Percentages of dTomato+Dendra2+ cells and dTomato-Dendra2+ cells in peripheral blood (right). d Percentages of CD3+ T cells, CD19+ B cells and CD11b+ myeloid cells within the CD45+ and dTomato-Dendra2+ populations in peripheral blood 1 m post-treatment. e Percentages of dTomato+Dendra2+ cells (left) and dTomato-Dendra2+ cells (right) in the bone marrow. f Representative image of confocal microscopy analysis of BM extracted cells at 4.5 months after treatment, demonstrating Dendra2+ mitochondria transfer between adjacent cells. Left, differential interference contrast (DIC); center, merged florescent image from DAPI, Dendra2 and dTomato channels. Scale bar, 5 µm; right, 3D reconstruction of the Z-stack images. BM bone marrow, HSPCs hematopoietic stem and progenitor cells.

Fig 5: Immunophenotyping of eWAT in mice fed chow or custom high-energy diets for 6 weeks. Stromal vascular cells from adipose tissue were analyzed by flow cytometry after initial gating on CD45+ lymphocytes (A) or CD45+ live cells (B). (A) Top panel: gating strategy for this population. Bottom panel: contour plots distinguishing NK cells (NKp46+,TCRb-), NK+T+ cells (NKp46+, TCRb+), and T cells (NKp46-, TCRb+) in eWAT. (B) Top panel: gating strategy for this population. Bottom panel: flow cytometry plots distinguishing resident macrophages (CD11b+, Ly6Cint) from infiltrating macrophages (CD11b+, Ly6Chi) in eWAT. (C) Absolute cell numbers of each cell population based on flow cytometry data. Values represent mean ± SE for n = 5 mice. *P < .05. CD45, cluster of differentiation 45; FSC-A, forward scatter-area; iMACs, infiltrating macrophages; rMACs, resident macrophages; SSC-A, side scatter-area; St Ol, starch-oleate; St Pal, starch-palmitate; Suc Ol, sucrose-oleate; Suc Pal, sucrose-palmitate; TCRß, T-cell receptor beta.