Short term high fat diet challenge promotes alternative macrophage polarization in adipose tissue via natural killer T cells and interleukin-4

Abstract

Inflammation in adipose tissue plays an important role in the pathogenesis of obesity-associated complications. However, the detailed cellular events underlying the inflammatory changes at the onset of obesity have not been characterized. Here we show that an acute HFD challenge is unexpectedly associated with elevated alternative (M2) macrophage polarization in adipose tissue mediated by Natural Killer T (NKT) cells. Upon 4d HFD feeding, NKT cells are activated, promote M2 macrophage polarization and induce arginase 1 expression via interleukin (IL)-4 in adipose tissue, not in the liver. In NKT-deficient CD1d(-/-) mice, M2 macrophage polarization in adipose tissue is reduced while systemic glucose homeostasis and insulin tolerance are impaired upon 4d HFD challenge. Thus, our study demonstrate, for the first time to our knowledge, that acute HFD feeding is associated with remarkably pronounced and dynamic immune responses in adipose tissue, and adipose-resident NKT cells may link acute HFD feeding with inflammation.

FIGURE 1.

Metabolic effects of acute 4d HFD feeding. A and B, body (A) and epididymal fat weights (B) of wild type 6-week-old C57BL/6 male mice under HFD (60%) for 4 days, compared with age-matched male mice on LFD (13% fat). n = 12–15 mice with three repeats. C, histology section of adipose tissue. D, measurement of diameters of 450 adipocytes from 3–4 mice on 4d HFD (gray) and age-matched LFD (white). E, Whisker plot of adipocyte diameters. F, serum insulin and glucose levels in WT mice (following a 6 h fast for insulin and 16 h fast for glucose) on 60% HFD for 4 days or 8 weeks, compared with age-matched mice on LFD (13% fat). n = 8–10 mice each, 2 repeats. G and H, GTT and ITT of WT mice on LFD or 4d HFD. n = 15 each cohort, three repeats. Note that glucose intolerance develops within days of HFD feeding. Mice were placed on HFD at the age of 6 weeks. Values represent mean ± S.E. *, p < 0.05; **, p < 0.01; and ***, p < 0.005.

FIGURE 2.

The effect of acute 4d HFD feeding on macrophage polarization and Arg1 expression in WAT. A, Q-PCR analysis of M1 and M2 genes (purple) in A WAT of 4d HFD or LFD mice (n = 12–15 mice each group, 3 repeats), B in the liver of mice following LFD or 4d HFD (n = 4–5 each cohort), and C in the WAT of mice following LFD or 1d HFD (n = 4–5 each cohort, 2 repeats). D, Western blot analysis of Arg1 protein in WAT of wild type mice and quantitated below. D, n = 4 mice each with two repeats (representative data from one experiment shown). E, flow cytometric analysis of intracellular Arg1 in CD45⁺ F4/80⁺ cells from adipose tissue of mice under LFD versus 4d HFD. Percent of positive cells is indicated. Values represent averages of one experiment (n = 6 mice each), two repeats. F, IL-4 ELISA of culture medium of various cell types purified from adipose tissue with purity shown in supplemental Fig. S3. Medium was collected after 12 h in culture. n = 25 mice, two repeats. Values represent mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.005.

FIGURE 3.

Adipose-resident type I NKT cells increase with 4d HFD and are predominantly CD4⁻CD8⁻. Analysis of NKT cells in epididymal adipose tissue of 6-week-old male lean mice. A and B, total number of NKT cells per gram of adipose tissue (A) and percent (B) of NKT cells in total adipose lymphocytes are shown. n = 12 mice with three repeats. C, characterization of cell-surface markers CD4-CD8 of αGalCer-CD1d-tetramer-positive NKT cells from indicated tissues. Numbers in the flow histogram indicate the percent of CD1d-tetramer-positive NKT cells. n = 10–12 mice with at least 4 repeats. Values represent mean ± S.E. *, p < 0.05.

FIGURE 4.

Metabolic phenotypes of CD1d^−/− mice with 4d HFD challenge. A, body weight and B, serum insulin level of three cohorts with LFD or 4d HFD feeding followed by a 6 h fasting. n = 8–10 mice per group, two repeats. C and D, GTT and ITT of 4d HFD cohorts. * refers to the p values comparing CD1d^−/− HFD to other groups (color-coded). n = 8–10 mice each group, 2 repeats. E, GTT of 7-week-old WT and CD1d^−/− mice on LFD. n = 10 mice each, two repeats. Values represent mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.005.

FIGURE 5.

Loss of NKT cells alters inflammatory responses in adipose tissue with 4d HFD challenge. 6-week-old WT or CD1d^−/− mice were placed on LFD or HFD for 4 days. A, Q-PCR analysis of M1 and M2 (purple) genes in the WAT (n = 12–15 mice each group, three repeats). B and C, Western blot analysis of Arg1 expression in WAT (upper) and liver (lower) of WT and CD1d^−/− cohorts on either LFD or 4d HFD. n = 3–4 mice each with two repeats. Quantitation shown in C. D, flow cytometric analysis of intracellular Arg1 in CD45⁺ F4/80⁺ cells of adipose tissue of CD1d^−/− mice under LFD versus 4d HFD. Percent of positive cells is indicated. Values represent averages of one experiment (n = 6 mice each) with two repeats. Values represent mean ± S.E. n.s., not significant; *, p < 0.05 and **, p < 0.01 comparing the two groups or groups included in brackets.

FIGURE 6.

4d HFD feeding increases IL-4 levels in WAT via NKT cells. A, Q-PCR analysis of IL-4 and IFN-γ mRNA levels following 4d HFD feeding in adipose tissue of WT and CD1d^−/− mice (n = 12–15 mice each group, three repeats). B, Western blot analyses of p-Y STAT proteins in IL-4-treated purified primary adipocytes and SVC of adipose tissue from WT mice (two repeats). Values represent mean ± S.E. **, p < 0.01.

FIGURE 7.

Loss of IL-4 alters inflammatory responses in adipose tissue with 4d HFD challenge. A, Q-PCR analysis of M1 and M2 (purple) genes in the WAT of 4 cohorts (n = 4–5 mice each cohort). B, Western blot analysis of Arg1 expression in WAT of WT and IL-4^−/− cohorts on either LFD or 4d HFD with quantitation shown below. n = 3–4 mice each, two repeats. C, flow cytometric analysis of intracellular Arg1 in CD45⁺ F4/80⁺ cells of adipose tissue of IL-4^−/− mice under LFD versus 4d HFD. Percent of positive cells is indicated. Values represent averages of one experiment (n = 6 mice each), two repeats. Values represent mean ± S.E. *, p < 0.05. D, proposed function of adipose-resident NKT cells in response to acute HFD feeding. Upon acute 4d HFD challenge, NKT cells promote M2 polarization and Arg1 expression via IL-4 in adipose tissue, thereby promoting systemic insulin sensitivity and glucose tolerance.