Volume 124, Issue 5, Pages 1265-1276 (May 2003) Impaired expression of peroxisome proliferator-activated receptor γ in ulcerative colitis  Laurent Dubuquoy, Emmelie Å Jansson, Samir Deeb, Sabine Rakotobe, Mehdi Karoui, Jean-Frédéric Colombel, Johan Auwerx, Sven Pettersson, Pierre Desreumaux  Gastroenterology  Volume 124, Issue 5, Pages 1265-1276 (May 2003) DOI: 10.1016/S0016-5085(03)00271-3

Figure 1 Modulation of PPARγ expression and activation by TLR4 in Caco-2 cells. (A ) Increased expression of PPARγ mRNA compared with an internal standard after stimulation of Caco-2 cells with LPS (50 ng/mL−1) or transfection with the constitutive active TLR4 (mCD4/toll). (B ) Caco-2 cells transfected with the response element for PPARγ (2XCYP) and PPARγ1 or the constitutive active TLR4 (mCD4/toll) showed a 2- or 3-fold activation, suggesting that the enhancement of PPARγ reporter gene activity might be owing to an increased expression of PPARγ. (C ) CaCo-2 cells transfected with 2XCYP and mCD4/toll displayed a 3-fold increase of the reporter gene activity. This induction was limited by the addition of 1 μg of a dnIKKβ, which blocks the NF-κB pathway. Two μg of dnIKK suppressed the effect of the TLR4 signals on PPARγ reporter gene activity. Stimulation of PPARγ with the synthetic ligand BRL 49653 (10 μmol/L) resulted in an 6- to 8-fold increased reporter gene activity. Results are expressed as fold activation (mean ± SEM) compared with cells transfected with an empty vector. Gastroenterology 2003 124, 1265-1276DOI: (10.1016/S0016-5085(03)00271-3)

Figure 2 Regulation of PPARγ expression by bacteria and TLR4 in the colon of mice. PPARγ immunostainings in colonic sections of mice (magnification ×250). (A ) PPARγ staining (green) was observed at the colonic epithelial surface of mice having an intestinal CF or HF. Almost no PPARγ staining was found in the epithelial layer of colonic sections of GF mice. (B ) PPARγ expression in epithelial cells from control mice (C3H/HeouJ, left) or TLR4 mutant mice (C3H/HeJ, right). PPARγ was expressed in epithelial cells at the top of the epithelium of C3H/HeouJ (Lpsn/Lpsn) mice but absent in the colon of C3H/HeJ (lpsd/lpsd)mice. Gastroenterology 2003 124, 1265-1276DOI: (10.1016/S0016-5085(03)00271-3)

Figure 3 Impaired mRNA expression of PPARγ in UC patients. (A ) PPARγ mRNA levels were determined by retrotranscription-competitive PCR in healthy mucosa of control patients (controls), mucosa of patients with CD (CDh, healthy) and CDi (inflamed), and of patients with UC (UCh and UCi). The number of patients as well as the statistical significance are indicated. Results are expressed as the mean value ± SEM of each subgroup. (B ) A representative RPA. PPARγ mRNA levels determined by a RPA protection assay, measuring the specific transcript of PPARγ, correlated to an internal standard, the γ-actin transcript. The autoradiograph shows 3 representative patients with CD or UC. Gastroenterology 2003 124, 1265-1276DOI: (10.1016/S0016-5085(03)00271-3)

Figure 4 Impaired PPARγ protein levels in UC patients. PPARγ protein levels were determined by Western blot analysis. Protein extracts were prepared from whole, noninflamed colonic mucosa of patients with UC, CD, and biopsy samples obtained from control patients. (A ) Immunodetection of PPARγ protein (approximately 57 kilodaltons) in the colon of 3 representative patients with UC, CD, and controls, respectively. (B ) PPARγ levels (mean OD ± SEM) in the colon of patients with UC (black), CD (grey), and controls (white). The number (n) of patients as well as the statistical significance (p) are indicated. Gastroenterology 2003 124, 1265-1276DOI: (10.1016/S0016-5085(03)00271-3)

Figure 5 UC patients display impaired expression of PPARγ in colonic epithelial cells. (A–C) PPARγ immunostainings (green) in sections obtained from (A ) healthy controls and noninflamed biopsy specimens from (B ) CD and (C ) UC patients, respectively (magnification 250×). (A ) PPARγ staining in epithelial cells from control patients. A few lamina propria mononuclear cells (LPMCs) were labeled (1% to 3%). (B ) Staining of epithelium from CD patients. Slightly more LPMCs were labeled (5% to 10%). (C ) Colon sections of patients with UC. There was no staining in the epithelium layer despite a labeling preservation in the lamina propria involving about 1% to 5% of LPMCs. (D) Western blot analysis of PPARγ protein in purified epithelial cells (as described in the Materials and Methods section) from the colonic mucosa of representative control, CD, and UC patients. Gastroenterology 2003 124, 1265-1276DOI: (10.1016/S0016-5085(03)00271-3)

Figure 6 PPARγ levels were similar in PBMCs of controls and IBD patients. (A ) PPARγ mRNA and (B ) PPARγ protein concentrations were determined respectively by retrotranscription-competitive PCR and Western blot in PBMCs of control, CD, and UC patients. The numbers of patients (n) as well as the statistical significance (p) are indicated and results are expressed as the mean ± SEM. Gastroenterology 2003 124, 1265-1276DOI: (10.1016/S0016-5085(03)00271-3)

Figure 7 Imbalance between PPARγ and TLR4 expressions in UC patients. Detection of TLR4 (visualized in red) and PPARγ (visualized in green) in the (A–F) healthy and (G–L) inflamed colonic mucosa of patients with UC, CD, and a representative control patient with diverticulitis of the sigmoid colon (control). Negative controls revealed no staining in healthy colons of patients with (A ) UC, (B ) CD, or (C ) control patient. (D–F ) A very faint TLR4 staining was observed in the healthy colon of the 3 groups of patients. The antibody directed against TLR4 revealed a strong red staining in inflamed colons of patients with (G) UC and (H ) CD compared with (I ) control. ( J ) The double TLR4 and PPARγ staining only showed a red fluorescence in patients with UC. (K ) The anti-TLR4 and anti-PPARγ antibodies revealed, respectively, a red and green staining but also a yellow fluorescence corresponding to a co-expression of these 2 receptors in inflamed colons of CD patients. (L) A predominant PPARγ staining in green was observed after double-staining analysis in control patients. Gastroenterology 2003 124, 1265-1276DOI: (10.1016/S0016-5085(03)00271-3)