Interferon-gamma, a Th1 cytokine, regulates fat inflammation: a role for adaptive immunity in obesity

Abstract

Adipose tissue (AT) can accumulate macrophages and secrete several inflammatory mediators. Despite its pivotal role in the progression of chronic inflammatory processes such as atherosclerosis, the adaptive role of immunity in obesity remains poorly explored. Visceral AT of diet-induced obese C57BL/6 mice had higher numbers of both CD4(+) and CD8(+) T cells than lean controls, monitored by flow cytometry. When stimulated in vitro, T cells from obese AT produced more interferon (IFN)gamma than those from controls. AT from obese animals also had more cells expressing I-A(b), a mouse class II histocompatibility marker implicated in antigen presentation, as determined by immunostaining. Differentiated 3T3-L1 cells stimulated with recombinant IFNgamma or T-helper 1-derived supernatant produced several chemokines and their mRNAs. Obese IFNgamma-deficient animals had significantly reduced AT expression of mRNA-encoding inflammatory genes such as tumor necrosis factor-alpha and monocyte chemoattractant protein-1, decreased AT inflammatory cell accumulation, and better glucose tolerance than control animals consuming the same diet. Obese mice doubly deficient for IFNgamma receptor and apolipoprotein (Apo)E on a mixed 129SvEv/C57BL/6 (129/B6) genetic background, despite exhibiting similar AT mRNA levels of tumor necrosis factor-alpha and monocyte chemoattractant protein-1 as 129/B6-ApoE(-/-) controls, had decreased expression of important T cell-related genes, such as IFNgamma-inducible protein-10 and I-A(b), and lower plasma triglycerides and glucose. These results indicate a role for T cells and IFNgamma, a prototypical T-helper 1 cytokine, in regulation of the inflammatory response that accompanies obesity.

Figure 1. Visceral AT from obese mice contains more inflammatory cells than that from lean controls

SVCs from peri-epididymal AT from lean and obese C57BL/6J animals were labeled with conjugated antibodies to F4/80, CD3 (I), CD4, CD8 (II), CD11c, and B220 (III), and quantified by flow cytometry. The graphs represent differences between mice on LF (lean) and HF (obese) diets, calculated by Student’s t-test. *p<0.05 vs LF. Immunostaining of peri-epididymal fat tissue with rat anti-mouse CD45 (A–C), Mac-3 (E–G), CD3 (I–L), and I-A^b (N-P) antibodies was performed as described. Graphs (D, H, M, Q) represent the numbers of positive cells in each group, and the differences were calculated by Student’s t-test. Original magnification: x100 for A–B, E–F, I–J, N–O; x400 for C, G, L, and P. #p<0.05 vs LF; n=5–8.

Figure 2. IFNγ expression in AT increases with diet-induced obesity in mice

RNA was extracted from visceral AT from C57BL/6J mice on LF or HF diet for 21 weeks. RT-qPCR measured mRNA levels of IFNγ over GAPDH in both groups (n=10 in each) (2A). SVCs isolated from AT from C57BL/6J mice fed a LF or HF diet for 15 weeks were labeled with CD3 and IFNγ antibodies after incubation with (2B, right) or without (2B, left) PMA and ionomycin. The graph (2C) represents the difference of IFNγ production by T cells and other cells between lean and obese groups, calculated by Student’s t-test. *p<0.05 vs LF.

Figure 3. IFNγ stimulates the expression of chemokines in 3T3-L1 cells

In 3A, 3T3-L1 cells were incubated with different dilutions of conditioned media from Th1 cells, with or without 10 μg/ml of anti-IFNγ. Levels of IP-10 were measured in the supernatants. Differences were calculated in pairs by Student’s t-test. *p<0.05 vs control (Th1 0/anti-IFNγ 0); #p<0.05 vs preceding bar. In 3B, 3T3-L1 cells were stimulated with different concentrations of mouse recombinant IFNγ. MCP-1, IP-10, and MIG protein levels were measured in the supernatants. In 3C, 3T3-L1 cells were stimulated with 100 U/ml of IFNγ or left untreated. After 4 and 24 h, cells were harvested and mRNA was used in a microarray screening. The white bars represent the T statistic values after 4 h and the black bars, after 24 h of stimulation with IFNγ, relative to expression in untreated cells; n=5–6; *p<0.05 vs untreated.

Figure 4. IFNγ induces expression of cytokines by AT in culture

Minced peri-epididymal AT was incubated with media alone or with 100U/ml of IFNγ for 6 h at 37°C and protein levels of IP-10, MIG and TNFα (A–C) were measured; n=3; *p≤0.01 vs untreated AT, calculated by Student’s t-test.

Figure 5. IFNγ deficiency does not change total body weight, but improves glucose tolerance (GT) in obese mice

5A–B: the bars represent average numbers of each group, and differences were calculated by Student’s t-test. In 5C, average GT curves from both groups under LF diet and in 5D, from groups under HF diet. Area under the GT curve was calculated for each mouse and the average of each group represented in 5E. Differences between groups were calculated by Student’s t-test. *p≤0.05 vs WT/LF; #p≤0.05.; n=5–6.

Figure 6. IFNγ regulates inflammatory gene expression in visceral fat of obese mice

mRNA levels of TNFα, CD68, MCP-1, RANTES, IL-10, and STAT-1 (A–F, respectively) in peri-epididymal AT were quantified by RT-qPCR and normalized to GAPDH. Fold change was calculated relative to WT/LF. *p<0.01 vs WT/LF; #p<0.05 vs WT/LF; §p<0.05; n=5–6.

Figure 7. IFNγ deficiency limits inflammatory cell accumulation in obese visceral AT

Fixed and paraffin-embedded AT was stained with anti-Mac-3 antibody, and positive cells were counted in 10 consecutive fields in each slide. A representative picture from each group is shown (7A–D). Numbers from each group were plotted in 7E. Differences were calculated by Student’s t-test. #p<0.05 vs WT/LF; *p<0.05; n=5–6..

Figure 8. IFNγ deficiency regulates inflammation in visceral fat of obese ApoE −/− mice

mRNA levels of IP-10, MIG, I-A^b, RANTES, CXCR3, and CD3 (A–F) in peri-epididymal AT were quantified by RT-qPCR and normalized to GAPDH. Fold change was calculated relative to ApoE^−/−. *p<0.05 vs ApoE^−/− **p<0.01 vs ApoE^−/−; n=5–8.