Expression of peroxisome proliferator-activated receptor-gamma in macrophage suppresses experimentally induced colitis

Abstract

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has been shown to be a protective transcription factor in mouse models of inflammatory bowel disease (IBD). PPAR-gamma is expressed in several different cell types, and mice with a targeted disruption of the PPAR-gamma gene in intestinal epithelial cells demonstrated increased susceptibility to dextran sulfate sodium (DSS)-induced IBD. However, the highly selective PPAR-gamma ligand rosiglitazone decreased the severity of DSS-induced colitis and suppressed cytokine production in both PPAR-gamma intestinal specific null mice and wild-type littermates. Therefore the role of PPAR-gamma in different tissues and their contribution to the pathogenesis of IBD still remain unclear. Mice with a targeted disruption of PPAR-gamma in macrophages (PPAR-gamma(DeltaMphi)) and wild-type littermates (PPAR-gamma(F/F)) were administered 2.5% DSS in drinking water to induce IBD. Typical clinical symptoms were evaluated on a daily basis, and proinflammatory cytokine analysis was performed. PPAR-gamma(DeltaMphi) mice displayed an increased susceptibility to DSS-induced colitis compared with wild-type littermates, as defined by body weight loss, diarrhea, rectal bleeding score, colon length, and histology. IL-1beta, CCR2, MCP-1, and inducible nitric oxide synthase mRNA levels in colons of PPAR-gamma(DeltaMphi) mice treated with DSS were higher than in similarly treated PPAR-gamma(F/F) mice. The present study has identified a novel protective role for macrophage PPAR-gamma in the DSS-induced IBD model. The data suggest that PPAR-gamma regulates recruitment of macrophages to inflammatory foci in the colon.

Figure 1. Macrophage-specific disruption of PPARγ

(A) PCR analysis of the recombination of PPARγ allele in macrophage (Mφ), dendritic cells (DC) or neutrophil (neut) genomic DNA isolated from PPARγ^ΔMφ or PPARγ^F/F mice or from heart, kidney, liver or brown adipose tissue (BAT) from PPARγ^ΔMφ mice. (B) Northern blot analysis measuring PPARγ expression in total RNA from macrophage cells isolated from PPARγ^ΔMφ or PPARγ^F/F mice. Expression was normalized to 36B4 gene expression. (C) Western blot analysis measuring PPARγ expression in 10μg nuclear lysate from macrophage cells isolated from PPARγ^ΔMφ or PPARγ^F/F mice. Expression was normalized to GAPDH protein expression, and CV-1 cells transfected with PPARγ served as positive control. (D) qPCR analysis of cd36 and aP2 mRNA in macrophages isolated from PPARγ^ΔMφ or PPARγ^F/F mice. Expression was normalized to 36B4 and each bar represents the mean value ± S.D. (*)= P<.05 compared to macrophage isolated from PPARγ^F/F mice.

Figure 2. Cytokine expression in macrophages isolated from PPARγ^ΔMφ or PPARγ^F/F mice

iNOS, CCR2, IL-1β, Il-10, IL-6, TNFα, MCP-1 and IFNγ mRNA expression was assessed by qPCR in macrophages isolated from PPARγ^ΔMφ or PPARγ^F/F incubated with vehicle (Veh) (black bars) or 1 μM rosiglitazone (Rosi) for 24 hours (open bars). Expression was normalized to 36B4 and each bar represents the mean value ± S.D. (*)= p < 0.05 compared to vehicle incubated macrophages isolated from PPARγ^F/F mice. (†)= p < 0.05 compared to vehicle incubated macrophages isolated from PPARγ^ΔMφ mice.

Figure 3. Clinical assessment of DSS-induced IBD in PPARγ^ΔMφ or PPARγ^F/F mice

(A) Body weight changes following DSS-induction of colitis, (B) colon length, (C) diarrhea score, (D) bleeding score, (E) representative H & E stained colon sections (F) and histology score. Data represent the mean value ± S.D of n=21 PPARγ^ΔMφ and n=21 PPARγ^F/F mice, (*)= p < 0.05 compared to PPARγ^F/F DSS treated mice.

Figure 4. Cytokine expression from colonic tissue following seven-day DSS or control treatment from PPARγ^ΔMφ or PPARγ^F/F mice

iNOS, CCR2, IL-1β, Il-10, IL-6, TNFα, MCP-1 and IFNγ mRNA expression was assessed by qPCR in from colonic tissue in PPARγ^ΔMφ or PPARγ^F/F mice given normal drinking water (Con) or water containing 2.5% DSS for seven days (DSS). Expression was normalized to 36B4 and each bar represents the mean value ± S.D. (*)= p < 0.05 compared to colons from PPARγ^F/F mice following 7-day DSS treatment.

Figure 5. Chemotatic response of macrophages isolated from PPARγ^ΔMφ or PPARγ^F/F mice

(A) In-vitro migration activity of macrophages incubated with vehicle (Veh), 100ng MCP-1 (Tam) or 1μM rosiglitazone (Rosi) or co-incubated with MCP-1 and Rosi for 4 hours each bar represents the mean value ± S.D. (*)= p < 0.05 compared to macrophages incubated with MCP-1 from PPARγ^F/F mice. (B) Immunostaining of MARCO in colonic tissue following 7 day DSS treatment from PPARγ^ΔMφ or PPARγ^F/F mice. Inset is an increased magnification. (C) Macrophage specific CD68 marker assessed by qPCR. Expression normalized to 36B4 and each bar represents the mean value ± S.D. (†)= p < 0.05 compared to PPARγ^F/F mice given normal drinking water (Con). (*)= p < 0.05 compared to PPARγ^F/F given water containing 2.5% DSS.