Induction of regulatory T cells decreases adipose inflammation and alleviates insulin resistance in ob/ob mice

Abstract

Leptin-deficient ob/ob mice are overweight, develop insulin resistance, and serve as a model for type 2 diabetes (T2D). Studies suggest that inflammatory pathways are linked to the development of insulin resistance and T2D both in animals and humans. We asked whether the induction of regulatory T cells (Tregs) could alleviate the pathological and metabolic abnormalities in ob/ob mice. We induced TGF-beta-dependent CD4(+) latency-associated peptide (LAP)-positive Tregs by oral administration of anti-CD3 antibody plus beta-glucosylceramide. We found a decrease in pancreatic islet cell hyperplasia, fat accumulation in the liver, and inflammation in adipose tissue, accompanied by lower blood glucose and liver enzymes. In addition, treated animals had decreased CD11b(+)F4/80(+) macrophages and TNF-alpha in adipose tissue. Adoptive transfer of orally induced CD4(+)LAP(+) Tregs ameliorated metabolic and cytokine abnormalities. Our results demonstrate the importance of inflammation in T2D and identify a unique immunological approach for treatment of T2D by the induction of Tregs.

Fig. 1.

Oral anti (a)-CD3 + GC reduces hepatic fat accumulation and pancreatic hyperplasia. (A) Thirty days after oral treatment, H&E staining of the pancreas and muscle and oil-red-O staining of the liver from ob/ob mice (10 per group) were carried out. Five fields per mouse per group were analyzed. Representative pictures are shown. All pictures were taken at a magnification of ×10. (B) Quantification of the pancreatic islet cell area was carried out by examining five fields of 10 islets per field per mouse in each treatment group. Slides were examined at a magnification of ×10 in a blindfolded fashion. Data bars represent average percentages of islet areas in each treatment group. (C) Quantification of the fat area in liver (pixels × 1,000 per field). All slides were read in a random fashion, blinded to treatment group. These experiments were repeated twice with the same results. Error bars represent SD. The ob/ob mice (eight per group) were fed PBS or a-CD3 + GC for 5 consecutive days. Glucose (D), AST (E), and cholesterol (F) levels in ob/ob mice were examined on day 30 following feeding. Data points represent individual mice. The means of each group are indicated by crossbars.

Fig. 2.

Production of TGF-β and IL-10 in the MLN, pancreas, and gut following oral administration of anti (a)-CD3 + GC. (A) TGF-β, IL-10, IL-2, and IFN-γ levels were measured in MLN cells (10 mice per group) following in vitro anti-CD3 stimulation (1 μg/mL) 5 days after the last treatment. TGF-β (B) and IL-10 (C) content in supernatants from homogenized pancreas and gut was measured 10 days after the last treatment. These experiments were repeated twice with same results. Error bars represent SD.

Fig. 3.

Oral anti (a)-CD3 + GC increases CD4⁺LAP⁺ cells and decreases NKT cells in MLN, spleen, and blood. The percentages of CD4⁺LAP⁺ T cells and NKT cells were measured by FACS analysis in MLN, spleen, and blood of ob/ob mice (10 per group) fed with a-CD3 + GC, a-CD3, GC, or PBS 5 days after the last treatment. The percentages of CD4⁺LAP⁺ T cells and NKT cells in a total of 1 × 10⁴ events are presented. The numbers shown on top of each data bar represent the total number of cells. These experiments were repeated twice with the same results. Error bars represent SD.

Fig. 4.

Adoptive transfer of CD4⁺LAP⁺ T cells ameliorates metabolic abnormalities and decreases IL-17, IFN-γ, and IL-6 in ob/ob mice. CD4⁺LAP⁺ cells (4 × 10⁴) harvested from spleens of ob/ob mice (10 per group) fed anti (a)-CD3 + GC were adoptively transferred into naive ob/ob recipients (5 per group) to measure the effect of CD4⁺LAP⁺ cells on the metabolic syndrome (A) and inflammatory cytokine patterns (B) in recipients. Cytokines were measured in splenocytes stimulated with a-CD3 antibodies. These experiments were repeated twice with the same results. Error bars represent SD.

Fig. 5.

Oral anti-CD3 + GC decreases CD11b⁺F4/80⁺ macrophages, TNF-α, and IL-1 and increases CD4⁺Foxp3⁺ cells in adipose tissue of ob/ob mice. (A) Mice (four per group) were fed PBS or anti-CD3 (5 μg) plus GC (100 μg) for 5 consecutive days. At 72 h after the last feeding, perigonadal white fat was collected and fat paraffin sections were stained with H&E. Pictures were taken at a magnification of ×100. (B) At 72 h after the last feeding (six mice per group), white fat near or surrounding MLNs was used to isolate adipocytes. Adipocytes were stained with fluorescent antibodies to CD11b and F4/80 or CD4 and were then fixed, permeabilized, and stained with antibody to Foxp3. aCD3, anti-CD3. (C) At 72 h after the last feeding, RNA of adipocytes isolated from perigonadal fat was used in quantitative RT-PCR for cytokine expression of IL-10, TNF-α, and TGF-β. (D) CD4⁺ T cells were negatively selected from spleens of PBS- or anti-CD3 + GC-fed mice and cocultured with adipocytes from control mice at a 1:1 ratio for 5 days. CD4⁺ T cells were eliminated from coculture by positive selection, leaving adipocytes for extraction of RNA used in quantitative RT-PCR for cytokine expression. These experiments were repeated three times with the same results. Error bars represent SD. (E) Culture supernatants were harvested from cocultures of CD4⁺ T cells from control or anti-CD3 + GC-fed mice and adipocytes of control mice. The amount of IL-1β was measured by ELISA.

Fig. 6.

Schematic diagram of the mechanisms of oral anti-CD3 + GC in controlling inflammation.