Role of the xenobiotic receptor in inflammatory bowel disease.

BACKGROUND: Gene-environment interplay modulates inflammatory bowel diseases (IBD). Dioxin-like compounds can activate the aryl hydrocarbon receptor (AhR) and alter macrophage function as well as T-cell polarization. We hypothesized that attenuation of the AhR signaling pathway will ameliorate colitis in a murine model of IBD. METHODS: Dextran sulfate sodium (DSS) colitis was induced in C57BL/6 AhR null mice (AhR(-/-) ), heterozygous mice (AhR(-/+) ), and their wildtype (WT) littermates. Clinical and morphopathological parameters were used to compare the groups. PATIENTS: AhR pathway activation was analyzed in biopsy specimens from 25 IBD patients and 15 healthy controls. RESULTS: AhR(-/-) mice died before the end of the treatment. However, AhR(-/+) mice exhibited decreased disease activity compared to WT mice. The AhR(-/+) mice expressed less proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) (6.1- versus 15.7-fold increase) and IL17 (23.7- versus 67.9-fold increase) and increased antiinflammatory IL-10 (2.3-fold increase) compared with the AhR(+/+) mice in the colon. Colonic macrophage infiltration was attenuated in the AhR(-/+) group. AhR and its downstream targets were significantly upregulated in IBD patients versus control (CYP1A1 -19.9, and IL8- 10-fold increase). CONCLUSIONS: Attenuation of the AhR receptor expression resulted in a protective effect during DSS-induced colitis, while the absence of AhR exacerbated the disease. Abnormal AhR pathway activation in the intestinal mucosa of IBD patients may promote chronic inflammation. Modulation of AhR signaling pathway via the diet, cessation of smoking, or administration of AhR antagonists could be viable strategies for the treatment of IBD.

4-Hydroxynonenal-induced apoptosis in rat hepatic stellate cells: mechanistic approach.

BACKGROUND AND AIM: Although lipid peroxidation products act as apoptotic signals for several cell types, including hepatic stellate cells, the underlying mechanisms are not well understood. In this study we determined if: (i) 4-hydroxy-2,3-nonenal (HNE) induces apoptosis in two rat stellate cell lines, HSC-T6 and CFSC-2G; and (ii) if apoptosis is regulated at the transcriptional and/or translational level. METHODS: HSC-T6 and CFSC-2G cells were treated with HNE and total RNA and protein extracted. mRNA and protein expression levels of pro- and antiapoptotic factors were determined. The effects of HNE on activation, morphology and cell death by apoptosis were also studied. RESULTS: HNE caused dose-dependent apoptosis in both HSC-T6 and CFSC-2G cell lines. Apoptosis in HSC-T6 cells was associated with increased mRNA expression of the pro-apoptotic adaptors/regulators FasR, FasL, Bax, and caspases-2 and -3. In contrast, CFSC-2G cells showed no changes in FasR, Bax and caspase-3 mRNA levels. Caspase-3 activity was elevated in T6 but not in 2G cells. Changes in protein expression generally paralleled the mRNA findings. CONCLUSIONS: HNE-induced apoptosis of both CFSC-2G and HSC-T6 rat hepatic stellate cells is associated with changes in mRNA and protein expression of several apoptotic adaptors/regulators. The underlying mechanism for HNE-induced apoptosis may involve both transcriptional and translational regulatory steps.

Gastroduodenal Crohn's disease is associated with NOD2/CARD15 gene polymorphisms, particularly L1007P homozygosity.

Limited data exist on the specific association between gastroduodenal Crohn's disease (GDCD) and NOD2/CARD15 gene polymorphisms. The aim of this study was to assess the association between NOD2 polymorphisms and GDCD, and to assess the specific association between each of the 3 major allelic variants G908R, L1007P, and R702W and the clinical features of Crohn's disease. We retrospectively reviewed the records of 202 patients with confirmed Crohn's disease and complete data was performed. Seventy-one patients (35%) had at least 1 allelic variant: 55 had 1 variant, 4 were homozygous for L1007fs, 2 homozygous for R702W, and 10 were compound heterozygous. Eighteen patients with confirmed GDCD were identified; 10 (56%) had wild type, 4 (22%) had 1 variant, and 4 (22%) had 2 allelic variants (2 were L1007P homozygous and 2 compound heterozygous). Compared to patients without gastroduodenal involvement, those with GDCD were more likely to have 2 allelic variants (22% vs. 6%; odds ratio [OR] 2.7; 95% confidence interval [CI] 1.6-7.3) and to be homozygous for L1007P (11% vs. 1%; OR 5.2; 95% CI 2.5-9.4). G908R heterozygosity was associated with ileal involvement (OR 1.4; 95% CI 1.1-2.9) and smoking habits (OR 2.4; 95% CI 1.2-3.8), whereas L1007P homozygosity was associated with GDCD (OR 5.8; 95% CI 2.6-10.8). L1007P variation was associated with younger age at diagnosis as well. There was no specific association between R702W homo- or heterozygosity and any of the characteristics examined. In conclusion, GDCD is associated with double dose of the NOD2/CARD15 gene variants, particularly L1007P homozygosity. There is evidence of specific variant-phenotype associations. G908R heterozygosity is associated with ileal involvement and smoking, whereas L1007P homozygosity is strongly associated with GDCD and younger age at diagnosis.

Group V sPLA2 hydrolysis of low-density lipoprotein results in spontaneous particle aggregation and promotes macrophage foam cell formation.

OBJECTIVE: Secretory phospholipase A2 (sPLA2) enzymes hydrolyze the sn-2 fatty acyl ester bond of phospholipids to produce a free fatty acid and a lysophospholid. Group V sPLA2 is expressed in cultured macrophage cells and has high affinity for phosphatidyl choline-containing substrates. The present study assesses the presence of group V sPLA2 in human and mouse atherosclerotic lesions and its activity toward low-density lipoprotein (LDL) particles. METHODS AND RESULTS: Group V sPLA2 was detected in human and mouse atherosclerotic lesions by immunohistochemical staining. Electron microscopic analysis showed that mouse group V sPLA2-modified LDL is significantly smaller (mean diameter+/-SEM=25.3+/-0.25 nm) than native LDL (mean diameter+/-SEM=27.7+/-0.29 nm). Hydrolysis by group V sPLA2 induced spontaneous particle aggregation; the extent of aggregation was directly proportional to the degree of LDL hydrolysis. Group V sPLA2 modification of LDL led to enhanced lipid accumulation in cultured mouse peritoneal macrophage cells. CONCLUSIONS: Group V sPLA2 may play an important role in promoting atherosclerotic lesion development by modifying LDL particles in the arterial wall, thereby enhancing particle aggregation, retention, and macrophage uptake.

Alcohol, but not lipopolysaccharide-induced liver apoptosis involves changes in intracellular compartmentalization of apoptotic regulators.

BACKGROUND: While alcohol-induced augmentation of liver apoptosis has been demonstrated in humans and laboratory animals, the underlying mechanisms are not fully elucidated. This study addresses the question whether alcohol and bacterial lipopolysaccharide (LPS), a putative mediator of alcohol effects on the liver, induce augmentation of liver apoptosis by intrinsic or extrinsic signaling pathways. This information may prove important for future design of therapies for alcoholic liver disease. METHODS: Male rats were fed either an alcohol-containing liquid diet or an isocaloric, control diet for 15-16 weeks. At the end of feeding period, the rats were treated with LPS (0.8 mg.kg-1 body weight) or sterile saline and killed 3 and 24 hr later. The liver and blood were sampled for histology and biochemical assays. Hepatocytes were isolated by collagenase perfusion and fractionated to yield mitochondria and cytoplasm. The propensity of mitochondria to undergo permeability transition in the presence of a Ca2+ overload was determined along with distribution of various apoptotic regulators (AIF, Smac2, Bax, cytochrome c, Bcl-XL, Bfl-1, and caspase-2) between mitochondria and cytoplasmic fractions. RESULTS: Increased liver apoptosis in alcohol-treated rats was associated with translocation of several apoptotic regulators between mitochondria and cytoplasm in a manner suggesting that alcohol induces augmentation of apoptosis by recruiting intrinsic apoptotic signals. LPS treatment of rats counteracted alcohol-induced changes in intracellular compartmentalization of apoptotic regulators despite an increased rate of apoptosis. LPS may, therefore, recruit extrinsic apoptotic signals, such as proinflammatory cytokines. CONCLUSIONS: Hepatocytes are to be able to mount an apoptotic response to both intrinsic and extrinsic signals. Alcohol increases liver apoptosis predominantly through an intrinsic signaling pathway while LPS recruits extrinsic signaling pathways.

Scavenger receptor class B type I reduces cholesterol absorption in cultured enterocyte CaCo-2 cells.

Scavenger receptor class B type I (SR-BI) mediates selective uptake of cholesteryl esters from HDL as well as efflux of cellular free cholesterol to HDL. It is unclear whether the receptor is involved in intestinal cholesterol absorption. We addressed this issue by studying [3H]cholesterol flux in differentiated CaCo-2 cells incubated at their apical side with mixed taurocholate/phosphatidylcholine/cholesterol micelles. Biotinylation and HDL binding experiments showed predominant apical expression of endogenous and overexpressed SR-BI. Mixed micellar cholesterol saturation affected the magnitude and direction of cholesterol flux with significant net uptake only from supersaturated micelles and net efflux from unsaturated micelles. Incubation with micelles that depleted cellular cholesterol resulted in a decrease of SR-BI protein, whereas incubation with cholesterol-loading micelles resulted in a significant increase of SR-BI protein. Apical cholesterol uptake by CaCo-2 cells was increased in the presence of a SR-BI-blocking antibody and by partial inhibition of SR-BI expression with small inhibitory RNA. Adenovirus-mediated overexpression of apical SR-BI did not affect cholesterol uptake but stimulated apical cholesterol efflux, even to supersaturated mixed micelles. Partial inhibition of SR-BI with small inhibitory RNA reduced apical cholesterol efflux. Our data argue against a direct role for SR-BI in micellar cholesterol uptake. However, SR-BI might be involved in cholesterol absorption by facilitating cholesterol efflux to micelles.