High-fat diet-induced adipose tissue-resident macrophages, T cells, and dendritic cells modulate chronic inflammation and adipogenesis during obesity

Figures

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  Figure 1. Feeding mice on a high-fat diet (HFD) increased body weight, blood glucose levels, epididymal adipose tissue weight, and adipocyte size, and developed more crown-like structures (CLS). Wildtype (WT) mice were fed a normal diet (ND) or high-fat diet for twelve weeks. (A) Average food consumption of a mouse per week. (B) Body weight from weeks 0 to 12. (C) Percent (%) body weight change from weeks 0 to 12. (D) Blood glucose levels before (week 0) and after 8 weeks of feeding ND and HFD. (E) Epididymal adipose tissue (eAT) weight. (F) Percentage of eAT weight relative to body weight. (G) Alteration of adipocyte size and numbers of CLS (marked by black arrow) in epididymal adipocytes visible on histological sections stained with hematoxylin and eosin (H&E); scale bar, 100 μm. (A-C), n = 15; (D), n = 5; (E, F), n = 22. Data are presented as mean values ± SEM; statistical analyses were performed by unpaired t-test: **p < 0.01, ***p < 0.001, and ****p < 0.0001.

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  Figure 2. T cells from HFD-fed mice regulated gene and protein expression in macrophages from ND-fed mice. Mice were fed ND or HFD for twelve weeks. T cells and macrophages were isolated from the epididymal AT SVF of HFD and ND mice, respectively. HFD T cells and ND macrophages were co-cultured at a ratio of 0:1, 0.5:1, 1:1, and 2:1 for 72 h, with cells cultured at a 0:1 ratio serving as a macrophage control. Cell culture conditioned medium (CM) was collected and stored for later measurement of cytokine and chemokine levels, cells were harvested, and total RNA was isolated for (A) analysis of macrophage-specific genes and inflammatory markers by RT-qPCR. (B, C) For cells co-cultured at a 0:1 and 1:1 ratio, levels of (B) cytokines and (C) chemokines in the culture medium were measured. Data are presented as mean values ± SEM; statistical analysis was performed using (A) one-way ANOVA then Dunnett’s post hoc test or (B, C) unpaired t-test (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. HT, AT-derived T-cells from HFD-fed mice; NM, AT-derived macrophages from ND-fed mice.

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  Figure 3. Macrophages from HFD-fed mice stimulated the expression of inflammatory genes and proteins in T cells from ND-fed mice. Mice were fed ND or HFD for twelve weeks. Macrophages and T cells were isolated from the epididymal AT SVF of HFD and ND mice, respectively. Macrophages from HFD-fed mice and T cells from ND-fed mice were co-cultured for 72 h at a ratio of 0:1, 0.5:1, 1:1, and 2:1 for 72 h, with cells cultured at a 0:1 ratio serving as a T cell control. CM was collected and stored for later measurement of cytokine and chemokine levels, cells were harvested, and total RNA was isolated for (A) analysis of T cell-specific genes and inflammatory markers by RT-qPCR. (B, C) For cells co-cultured at a 0:1 and 1:1 ratio, levels of (B) cytokines and (C) chemokines in CM were measured. Data are presented as mean values ± SEM; statistical analysis was performed using (A) one-way ANOVA then Dunnett’s post hoc test or (B, C) unpaired t-test (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. HM, AT-derived macrophages from HFD-fed mice; NT, AT-derived T cells from ND-fed mice.

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  Figure 4. Macrophages from HFD-fed mice induced gene and protein expression in cells from the stromal vascular fraction of ND-fed mice. Mice were fed ND or HFD for twelve weeks. Macrophages were isolated from the epididymal AT SVF of HFD-fed mice and co-cultured for 72 h with cells of the SVF of ND-fed mice at a ratio of 0:1, 0.5:1, and 1:1, with cells cultured at a 0:1 ratio serving as a control. CM was collected and stored for later measurement of cytokine and chemokine levels, cells were harvested and total RNA was isolated for (A) analysis of inflammatory and adipogenic markers by RT-qPCR. (B, C) For cells co-cultured at a 0:1 and 1:1 ratio, levels of (B) cytokines and (C) chemokines in CM were measured. Data are presented as mean values ± SEM; statistical analysis was performed using (A) one-way ANOVA then Dunnett’s post hoc test or (B, C) unpaired t-test (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. HM, AT-derived macrophages from HFD-fed mice; NSVF, AT-derived SVF from ND-fed mice.

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  Figure 5. Macrophages from HFD-fed mice induced lipid accumulation, adipogenesis, and inflammation in 3T3-L1 adipocytes. Mice were fed HFD for twelve weeks. Macrophages were isolated from SVF of epididymal AT of HFD. 3T3-L1 pre-adipocytes were cultured and differentiated into adipocytes. HFD macrophage and 3T3-L1 adipocytes were co-cultured in the ratio of (0:1, 0.5:1, and 1:1) for 72 h, and 0:1 ratio was considered as control. CM was collected and stored for later measurement of cytokine and chemokine levels and (A) the cells co-cultured at a ratio of 0:1, and 1:1 were stained with Oil Red O and visualized by microscopy, representative images from Oil Red O-stained cells are shown; scale bar, 100 μm. (B) The cells were harvested and total RNA was isolated for analysis of adipogenic, browning, and inflammatory markers by RT-qPCR. (C-D) For cells co-cultured at a 0:1 and 1:1 ratio, levels of (C) cytokines and (D) chemokines in CM were measured. Data are presented as mean values ± SEM; statistical analysis was performed using (B) one-way ANOVA then Dunnett’s post hoc test or (C, D) unpaired t-test (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. HM, AT-derived macrophages…

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  Figure 6. T cells from HFD-fed mice triggered lipid accumulation, adipogenesis, and inflammation in 3T3-L1 adipocytes. Mice were fed HFD for twelve weeks. T cells were isolated from the SVF epididymal AT of HFD-fed mice and cultured 3T3-L1 pre-adipocytes were differentiated into adipocytes and the two cell types were co-cultured for 72 h at a ratio of 0:1, 0.5:1, and 1:1, with the 0:1 ratio serving as a control. CM was collected and stored for later determination of cytokine and chemokine levels and the cells were stained with Oil Red O and visualized by microscopy. Total RNA was isolated for analysis by RT-qPCR. (A) Representative images from Oil red O-stained 3T3-L1 adipocyte (0:1, and 1:1) (scale bar 100 μm). (B) RT-PCR analysis of adipogenic, browning, and inflammatory markers. (C-D) For cells co-cultured at a 0:1 and 1:1 ratio, levels of (C) cytokines and (D) chemokines in CM were measured. Data are presented as mean values ± SEM; statistical analysis was performed using (B) one-way ANOVA then Dunnett’s post hoc test or (C-D) unpaired t-test (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. HT, AT-derived T-cells from HFD-fed mice; D3T3-L1, differentiated 3T3-L1…

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  Figure 7. Dendritic cells (DCs) from HFD-fed mice accelerated lipid synthesis, adipogenesis, and inflammation in 3T3-L1 adipocytes. Mice were fed HFD for twelve weeks. DCs were isolated from the eAT SVF of HFD-fed mice, 3T3-L1 pre-adipocytes were cultured and differentiated into adipocytes, and the two cell types were co-cultured for 72 h at a ratio of 0:1, 0.5:1, and 1:1, with the 0:1 ratio serving as a control. CM was collected and stored for later determination of cytokine and chemokine levels; the cells were harvested and total cellular RNA was isolated for (A) RT-qPCR analysis of adipogenic, and inflammatory markers. (B, C) For cells co-cultured at a 0:1 and 1:1 ratio, levels of (B) cytokines and (C) chemokines in CM were measured. Data are presented as mean values ± SEM; statistical analysis was performed using (A) one-way ANOVA then Dunnett’s post hoc test or (B, C) unpaired t-test (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. HDC, AT-derived dendritic cells from HFD-fed mice; D3T3-L1, differentiated 3T3-L1 adipocytes.

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  Figure 8. Macrophages, T cells, and dendritic cells from HFD-fed mice directly promoted differentiation of pre-adipocytes to adipocytes. Mice were fed HFD for twelve weeks. Macrophages, T cells, and dendritic cells (DCs) were isolated from the eAT SVF of HFD-fed mice and were co-cultured for 6 days with confluent 3T3-L1 pre-adipocytes either with differentiation induction cocktail or without external differentiating agents; a 0:1 ratio was used as a control. The cells were visualized under a phase contrast microscope in unstained live condition and stained with Oil Red O and imaged under a bright field microscope. (A-C) 3T3-L1 cells were co-cultured in the presence of a differentiation cocktail with a combination of (A) HFD macrophages:3T3-L1 (0:1, 0.5, and 1:1 ratios), (B) HFD T cells:3T3-L1 (0:1, 0.5, and 1:1 ratios), and (C) HFD DCs:3T3-L1 (0:1, and 0.125:1 ratios). (D-F) 3T3-L1 cells were co-cultured in the absence of a differentiation cocktail with (D) HFD macrophages:3T3-L1 (0:1, 0.5, 1:1, and 2:1), (E) HFD T cells:3T3-L1 (0:1, 0.25:1 and 0.5:1), and (F) HFD DCs:3T3-L1 (0:1, 0.5:1, and 1:1). Representative images are shown; scale bars, 100 μm. Lipid-containing 3T3-L1 adipocytes are…