Normalization of obesity-associated insulin resistance through immunotherapy - PubMed (original) (raw)
doi: 10.1038/nm.2001. Epub 2009 Jul 26.
Yin Chan, Geoffrey Paltser, Dorothy Truong, Hubert Tsui, Jasmine Bahrami, Ruslan Dorfman, Yongqian Wang, Julian Zielenski, Fabrizio Mastronardi, Yuko Maezawa, Daniel J Drucker, Edgar Engleman, Daniel Winer, H-Michael Dosch
Affiliations
- PMID: 19633657
- PMCID: PMC3063199
- DOI: 10.1038/nm.2001
Normalization of obesity-associated insulin resistance through immunotherapy
Shawn Winer et al. Nat Med. 2009 Aug.
Abstract
Obesity and its associated metabolic syndromes represent a growing global challenge, yet mechanistic understanding of this pathology and current therapeutics are unsatisfactory. We discovered that CD4(+) T lymphocytes, resident in visceral adipose tissue (VAT), control insulin resistance in mice with diet-induced obesity (DIO). Analyses of human tissue suggest that a similar process may also occur in humans. DIO VAT-associated T cells show severely biased T cell receptor V(alpha) repertoires, suggesting antigen-specific expansion. CD4(+) T lymphocyte control of glucose homeostasis is compromised in DIO progression, when VAT accumulates pathogenic interferon-gamma (IFN-gamma)-secreting T helper type 1 (T(H)1) cells, overwhelming static numbers of T(H)2 (CD4(+)GATA-binding protein-3 (GATA-3)(+)) and regulatory forkhead box P3 (Foxp3)(+) T cells. CD4(+) (but not CD8(+)) T cell transfer into lymphocyte-free Rag1-null DIO mice reversed weight gain and insulin resistance, predominantly through T(H)2 cells. In obese WT and ob/ob (leptin-deficient) mice, brief treatment with CD3-specific antibody or its F(ab')(2) fragment, reduces the predominance of T(H)1 cells over Foxp3(+) cells, reversing insulin resistance for months, despite continuation of a high-fat diet. Our data suggest that the progression of obesity-associated metabolic abnormalities is under the pathophysiological control of CD4(+) T cells. The eventual failure of this control, with expanding adiposity and pathogenic VAT T cells, can successfully be reversed by immunotherapy.
Figures
Figure 1. Phenotype of Tfat (all mice 14–16 wk old, each contour plot represents 3–4 independent experiments)
a) CD4 and CD8 expression on CD3+ cells in spleen and SAT and VAT of B6 high fat diet (HFD) and normal chow diet (NCD) mice. b) Proportion of CD4+IFNγ+ (Th1) and CD4+IL-17+ (Th17) T-cells in spleen, SAT and VAT. c) Proportion of CD4+Foxp3+ (Treg) cells in spleen, SAT and VAT (upper panel: FACS plots; lower panel: pooled data; *p<0.03, t-test). **d)** Total number of CD3+CD4+, CD4+IFNγ+ (Th1), CD4+IL-17+ (Th17), CD4+Foxp3+ (Treg) T cells/g of SAT and VAT in normal chow diet and HFD mice (n=4–5/group, *p<0.05, t-test). **e)** T-bet (Th1):Foxp3 ratio in human VAT (upper left panel, r2=0.62, p<0.05). Lower left panel: VAT T-bet:Foxp3 ratio in patients with BMI>30 _vs. <25_ (*p<0.02, t-test). Upper right panel: Foxp3 (brown) and T-bet (blue, Th1 cells) in human VAT (BMI>30); lower right panel: BMI<25, bar 50 μm; T-bet:Foxp3 ratios from >200 stained cells/2 tissue levels. f) CD3+CD4+ T cells (%) with secondary re-rearranged. non-OVA TCRα detected by reduced OT2 TCRα:CD3 ratio in spleens, SAT and VAT of 6 or 16 wk old, regular diet or HFD B6 OT2 mice. g) TCRVα gene usage of CD4+ T cells isolated from spleens of 16 wk old obese wild type (WT) or OT2 mice, and from VAT of HFD and NCD WT and OT2 mice. CD4+ OT2 T cells were identified and sorted as in (f).
Figure 2. Impact of lymphocyte deficiency on weight gain, fat distribution, glucose tolerance and insulin resistance
a) Body weights of >6 wk old WT and RAGnull B6 mice on NCD (n=20/group) or HFD (n=20/group, *p<0.03, Mann-Whitney, WT vs. RAGnull). b) Weights of single epididymal VAT and inguinal SAT pads from 14–15 wk WT and RAGnull mice on NCD or HFD (left panel: n=6 mice/group, *p<0.03, t-test), right panel: VAT:SAT ratio, *p<0.001, t-test. c) Relative fat cell diameter (see methods, n=3 mice/group, *p<0.0001, Mann-Whitney). d) Food intake (top panel) and respiratory exchange ratio (RER, bottom panel) in HFD WT or RAGnull mice (n=4/group). e) Glucose tolerance of RAGnull or WT mice on NCD or HFD (n=10/group, *p<0.02, 2-way ANOVA). f) Fasting glucose (left panel) or insulin blood levels (right panel) in 14 wk old HFD and NCD WT or RAGnull mice (n=10 mice/group, *p<0.05, t-test). g) Insulin tolerance test (ITT) in 14 wk old WT RAGnull mice on NCD or HFD (n=6–10/group, *p<0.05, two-way ANOVA).
Figure 3. CD4+ T cell grafts reverse obesity-associated metabolic abnormalities in RAGnull mice
a) FACS plots analyzing purity of CD3+CD8+ (top) and CD3+CD4+ (bottom) T cells in spleen, SAT, and VAT, 2 wk following transfer into 12 wk old HFD RAGnull recipients. All subsequent experiments were performed 2–4 wk _post_-transfer and compared to non-transferred, age-matched, HFD RAGnull mice. b) Left panel: body weights (n=5/group) of recipient mice were monitored post-transfer (*p<0.05, t-test, data from one of four similar experiments). Right panel: weights of individual VAT and SAT pads (n=5/group). CD4+ T cell transfer reduces VAT mass (*p<0.05, t-test). c) Adipocyte diameter following transfer of CD4+ or CD8+ T cells into HFD RAGnull mice (n=3 mice/group, *p<0.01, Mann-Whitney). d) CD4+ T cell transfer improves glucose tolerance in HFD RAGnull recipients (n=10/group, *p<0.0001, 2-way ANOVA). e) Fasting glucose (left panel) and insulin (right panel) in CD4+ or CD8+ transferred HFD RAGnull mice (n=5–10 mice/group, *p<0.03, **P<0.01, t-test). f) HFD RAGnull mice reconstituted with CD4+ T-cells show improved insulin tolerance (n=10, *p<0.05, 2-way ANOVA). g) FACS plots of OT2 TCRVα re-rearrangements in spleen and VAT, 2 wk following transfer. The purity of CD4+OT2hi cells transferred was 99.5%. h) CD4+OT2:TCRαhi (CD4+OT2hi) T cells fail to improve glucose and (i) insulin tolerance following transfer (n=5/group, *p<0.0001, 2-way ANOVA). j) Fasting glucose (left panel) and fasting insulin (middle panel) in CD4+ or CD4+OT2:TCRαhi (OT2hi) transferred HFD RAGnull mice (n=5 mice/group, *p<0.05, t-test). Right panel, CD4+OT2hi:TCRαhi T cells fail to improve weight post transfer (n=5 mice/group, *p<0.004, t-test).
Figure 4. CD4+Foxp3− T cells reverse metabolic abnormalities following transfer
a) FACS plots of CD4+Foxp3+ T cells (%) in spleen and VAT, 3 wk after transfer of purified CD4+ or purified CD4+Foxp3− (EGFPneg, 99.1% pure) cells, one of two similar plots shown. b) Change of HFD-RAGnull body weight 3 wk post transfer of CD4+, CD4+Foxp3−, and CD4+IL-10null T cells (n=5/group, *p<0.01, t-test). **c)** GTT (n=5/group, *p<0.0001, 2-way ANOVA). **d**) fasting glucose (left panel, n=5 mice/group, *p<0.01, t-test) and fasting insulin (right panel, n=5 mice/group, *p<0.01, t-test), 3 wk _post_ transfer of CD4+, CD4+Foxp3−, or CD4+IL-10null T cells into HFD-RAGnull recipients. **e**) IL-4 (left panel) and IL-13 (right panel) produced by anti-CD3 _plus_ anti-CD28 stimulated VAT T cells of 16 wk old HFD WT, and HFD RAGnull recipients 3 wk _post_ transfer of CD4+ T cells (n=3/group). **f**) FACS plots for CD4+ gated T cells from VAT of HFD RAGnull mice that received either CD4+STAT6null (left panel) or CD4+WT (right panel) T cells 3 wk previously. **g**) Glucose tolerance 3 wk following transfer of CD4+ or CD4+STAT6null T cells (n=5/group, *p<0.05, 2-way ANOVA). **h**) Fasting glucose (left panel), fasting insulin (middle panel), and weight change (right panel) in HFD RAGnull recipients 3 wk _post_ transfer of purified CD4+ or CD4+STAT6null T cells (n=5 mice/group, *p<0.01, t-test). **i,** Left panel: representative FACS plot CD4+GATA-3+ (%Th2) cells in spleen and VAT of 14–16 wk old regular diet and HFD B6 mice. **j,** Left panel: pooled data from (i, n=4/group, *p<0.03, t-test), right panel: total number of CD4+GATA-3+ (Th2) T cells/g of VAT in regular diet and HFD B6 mice (n=4/group, p>0.4, t-test).
Figure 5. Anti-CD3 and its F(ab′)2 fragment improve obesity-induced insulin resistance
16 wk old obese (HFD) B6 mice received anti-CD3 antibody (αCD3**, a–e**), Isotype-matched control IgG or anti-CD3-(F(ab′)2 (f–j), maintaining HFD for 6 or 9 wk. a. FACS plots (%) of CD4+Foxp3+ Treg cells in spleen and VAT 9 weeks post αCD3 (one of 3 similar experiments shown). b) Fasting glucose (top panel) and insulin (bottom panel) after αCD3 (n=8, *p<0.05, t-test). c) Glucose tolerance (n=8, *p<0.0004, 2-way ANOVA) and d) ITT (n=8, *p<0.0005, 2-way ANOVA) post αCD3. e) Left panel: body weights of HFD mice following αCD3 (n=8, *p<0.05, t-test). Right panel: adipocyte diameter post αCD3 (n=3 mice/group). f) Glucose tolerance (left panel) 6 wk post F(ab′)2 (n=5, *p<0.01, 2-way ANOVA, one of 2 similar experiments). Fasting insulin (middle panel) and glucose (right panel) in HFD mice 6 wk following F(ab′)2 (n=5, *p<0.04, t-test). g) Body weights of HFD mice following F(ab′)2. h) CD4+Foxp3+ Treg cells in spleen and VAT of HFD B6 mice 6 wk post F(ab′)2. (one of 3 similar experiments). i) IFNγ, IL-17, and IL-13, levels following stimulation of B6 VAT T cells with αCD3 plus anti-CD28, 8 wk post F(ab′)2 (n=3/group). j) FACS plot of CD4+ gated, GATA-3 stained VAT T cells, 8 wk post F(ab′)2).
Figure 6. F(ab′)2 therapy alters VAT resident macrophage phenotype
a) MMRhi (%, upper gate), MMRlo (middle gate), and MMR− (lower gate) macrophages from VAT of 16 wk old HFD or lean NCD B6 mice (representative data from 4 similar experiments). b) IL-10 (left panel), MCP-1 (middle panel) and TNFα (right panel) produced by LPS stimulated, F4/80+ macrophages sorted from VAT of HFD (top panel) or NCD (lower panel) mice into MMR−, MMRlo, and MMRhi cell populations (one of 4 similar experiments). We consistently failed to obtain sufficient cell numbers for analysis of MMR− macrophages from VAT from lean mice. c) FACS plots from 2 independent experiments, measuring MMR expression in F4/80+ macrophages from HFD B6 VAT, 6 wk post F(ab′)2. d) IL-10 (left panel), MCP-1 (middle panel) and TNFα (right panel) produced by LPS stimulated F4/80+ macrophages sorted from VAT 6 wk post F(ab′)2 (n=3/group, *p<0.05, t-test).
Comment in
- T-ing up inflammation in fat.
Lumeng CN, Maillard I, Saltiel AR. Lumeng CN, et al. Nat Med. 2009 Aug;15(8):846-7. doi: 10.1038/nm0809-846. Nat Med. 2009. PMID: 19661987 No abstract available.
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