Class II major histocompatibility complex plays an essential role in obesity-induced adipose inflammation

Abstract

Adipose-resident T cells (ARTs) regulate metabolic and inflammatory responses in obesity, but ART activation signals are poorly understood. Here, we describe class II major histocompatibility complex (MHCII) as an important component of high-fat-diet (HFD)-induced obesity. Microarray analysis of primary adipocytes revealed that multiple genes involved in MHCII antigen processing and presentation increased in obese women. In mice, adipocyte MHCII increased within 2 weeks on HFD, paralleling increases in proinflammatory ART markers and decreases in anti-inflammatory ART markers, and preceding adipose tissue macrophage (ATM) accumulation and proinflammatory M1 polarization. Mouse 3T3-L1 and primary adipocytes activated T cells in an antigen-specific, contact-dependent manner, indicating that adipocyte MHCII is functional. HFD-fed MHCII(-/-) mice developed less adipose inflammation and insulin resistance than did wild-type mice, despite developing similar adiposity. These investigations uncover a mechanism whereby a HFD-induced adipocyte/ART dialog involving MHCII instigates adipose inflammation and, together with ATM MHCII, escalates its progression.

Fig. 1. Adipocyte MHCII mRNA and protein expression is markedly increased with obesity

RT-PCR analysis of MHCII family genes in A) subcutaneous (SQA) and visceral (VA) adipocytes and B) SQ ATMs of lean and obese women (N=5-19/group). C) Representative flow cytometry images of human SVF and adipocyte samples hybridized with fluorescent-antibodies specific for human MHCII (pan HLA-DR), CD36 and CD45. Arrows indicate non-specific signal from residual magnetic beads not removed during the CD45-depletion procedure. RT-PCR of MHCII family genes in D) SQ and V adipocytes and E) V adipocytes and V ATMs (N=4/group) of male C57BL/6 mice fed 3 months chow (lean) or HFD (obese). Western blot analysis of MHC family proteins in F) V adipocytes from lean and obese male C57BL/6 mice and G) V adipocytes and V ATMs from obese male C57BL/6 mice (N=3 samples (2-3 mice/sample)/group; p<0.05 vs. matching lean (*) or SQA (†) sample by Mann-Whitney (A-E) or Welch’s T-test (F,G). Each mouse sample represents pooled material obtained from 4 (adipocytes) or 8 (ATMs) mice. Data represent Means±SEM.

Fig. 2. Leptin-induced IFNγ from T-cells stimulates adipocyte MHCII expression

MHCII family gene induction in A,B) differentiated primary human adipocytes and C,D) mouse 3T3-L1 preadipocytes and adipocytes cultured for 24hrs with PBS (NC, vehicle), IFNγ, TNFα, IL-1β or IL-6. E) IFNγ dose-response of CIITA expression in 3T3-L1 adipocytes. F) IFNγ secretion by C57BL/6 splenic T-cells incubated 24hrs with 1μg/ml leptin. G) 3T3-L1 adipocyte MHCII induction by supernatant of splenic T-cells incubated with PBS (NC), leptin + control IgG (IgG), or leptin + IFNγ-neutralizing antibody (αIFN). H) Correlation of z-skew normalized human SQ adipocyte CIITA mRNA (zSA.CIITA) with plasma leptin (zLeptin) and SQ adipocyte leptin mRNA (zSA.Lep). (N=35-40; dashed line indicates the 95% confidence interval of the regression line). I) MHCII expression in peritoneal macrophages (PM) and 3T3-L1 adipocytes treated 24hrs with PBS (NC) or IFNγ ± 4hrs pre-treatment with IL-10. J) IL-10Rα/β expression in PMs, 3T3-L1 adipocytes and mouse primary ATM and adipocytes. Data represent Means±SEM. (A-G, I-J: N= 2-4/group; p<0.05 vs. matching NC (*), or preadipocyte(†) or PM (‡) treatment group by T-test or 1-way ANOVA.)

Fig. 3. Adipocyte MHCII and ARTs changes occur early after HFD, while ATM changes occur later

RT-PCR analysis of A) adipose tissue, B) CD45-depleted adipocytes, C) ARTs and D) ATMs of male C57BL/6 mice fed HFD for 1, 2, 3, 4 or 12 weeks; and E) adipose tissue, F) CD45-depleted adipocytes, G) ARTs and H) ATMs of similar weight male ob/ob mice and 12wk HFD-fed C57BL/6 mice. (N=4-8/group, p<0.05 and >1.5-fold absolute change vs. matching chow control (*) or ob/ob mouse (†) expression by Mann-Whitney tests.) ART and ATM samples represent pooled material obtained from 4 (adipocytes) or 8 (ARTs and ATMs) mice. All data are normalized against the expression of matching chow-fed lean control mice (red line). RT-PCR analysis of human SVF expression of I) IFN and J) CD3-normalized expression of the ART lineage markers TBX21, FOXP3 and GATA3 (N=6-15, *p<0.05 by Welch’s T-test.) Data represent Means±SEM.

Fig. 4. Adipocytes can present MHCII antigens to activate CD4+ T-cells

Chow- and HFD-fed C57BL/6J mouse adipocytes (Adip) and ovalbumin (OVA)-specific CD4+ T-cells (T), were cultured alone or co-cultured in direct contact (Adip+T) or in separate chambers of a transwell plate (Adip//T). Cultures were incubated 48hrs ±OVA, after which supernatants were analyzed for A) IL-2 and B) IFNγ or C) incubated with ³H-thymidine for 16hrs to assess antigen-stimulated T-cell replication. (N=2-3/group, 3 mice/adipocyte sample, p<0.05 vs. T-cells±OVA and adipocytes without OVA (*) or matching chow (†) control by T-test.) 3T3-L1 adipocyte cultures expressing negative control (shNC) or Ciita (shCiita) shRNAs were treated 24hrs ± INFγ, co-cultured for 48hrs with CD4+ T-cells of OVA-immunized mice ±OVA, and then analyzed for D) IL-2 and E) IFNγ secretion, and analyzed for F) CIITA protein expression. (N=2-3/group, p<0.05 vs. matching NC (*) or matching shNC (†) expression by T-test.) Red line indicates the concentration of the lowest ELISA standard. C5BL/6J mouse preadipocytes (preAd), differentiated adipocytes (Ad) and BMDMs treated with or without IFNγ, incubated ±OVA for and OVA-specific T-cells for 48hrs and assessed for F) IL-2 and G) IFNγ production. (N=4/group, p<0.05 vs. matching T+OVA(†) by t-test or vs. preAd (*) or preAd and Ad (‡) by ANOVA). Data represent Means±SEM.

Fig. 5. HFD-fed MHCII-deficient mice have less adipose inflammation and insulin resistance

A) fasting plasma glucose and insulin levels, B) intraperitoneal insulin tolerance test data of H2A^−/− and WT mice fed 3 months of chow or HFD (N=6-7/group) and liver steatosis analyzed by C) NMR- and hematoxylin and eosin staining. Adipose inflammation was assessed by D) F4/80 immunhistochemistry and E) RT-PCR, while relative F) adipocyte changes were analyzed by RT-PCR. SVF G) ART and H) ATM abundance was assessed by flow cytometry, and I) ART and J) ATM fractions were analyzed by RT-PCR. (N=4-6/group). ART and ATM samples represent pooled material obtained from 4 (adipocytes) or 8 (ARTs and ATMs) mice. (p<0.05 vs. genotype-matched (*) or diet-matched (†) expression by T-test).

Fig. 6. Model of adipose tissue cell interactions during HFD-induced inflammation

Nutrient excess increases 1) adipocyte leptin (1 week), inducing 2) CD4+ ART IFNγ expression (2 weeks) to stimulate 3) adipocyte CIITA and MHCII expression (2-3 weeks). 4) MHCII antigen presentation by adipocytes, or other APCs, stimulates CD4+ ART proliferation and differentiation. 5) Increased adipocyte IL-10, starting at 1 week HFD, may attenuate pro-inflammatory ATM polarization and APC function. By 12 weeks HFD challenge, ART activation/proliferation stimulates 6) ATM accumulation and polarization to escalate adipose inflammation.