Serge Dionne

Direct and Indirect Induction by 1,25-Dihydroxyvitamin D3 of the NOD2/CARD15-Defensin β2 Innate Immune Pathway Defective in Crohn Disease

Vitamin D signaling through its nuclear vitamin D receptor has emerged as a key regulator of innate immunity in humans. Here we show that hormonal vitamin D, 1,25-dihydroxyvitamin D3, robustly stimulates expression of pattern recognition receptor NOD2/CARD15/IBD1 gene and protein in primary human monocytic and epithelial cells. The vitamin D receptor signals through distal enhancers in the NOD2 gene, whose function was validated by chromatin immunoprecipitation and chromatin conformation capture assays. A key downstream signaling consequence of NOD2 activation by agonist muramyl dipeptide is stimulation of NF-κB transcription factor function, which induces expression of the gene encoding antimicrobial peptide defensin β2 (DEFB2/HBD2). Pretreatment with 1,25-dihydroxyvitamin D3 synergistically induced NF-κB function and expression of genes encoding DEFB2/HBD2 and antimicrobial peptide cathelicidin in the presence of muramyl dipeptide. Importantly, this synergistic response was also seen in macrophages from a donor wild type for NOD2 but was absent in macrophages from patients with Crohn disease homozygous for non-functional NOD2 variants. These studies provide strong molecular links between vitamin D deficiency and the genetics of Crohn disease, a chronic incurable inflammatory bowel condition, as Crohns pathogenesis is associated with attenuated NOD2 or DEFB2/HBD2 function.

Butyrate induced Caco-2 cell apoptosis is mediated via the mitochondrial pathway

Background:During the process of tumorigenesis most colon cancer cells acquire resistance to apoptosis. The short chain fatty acid butyrate is well established as an antitumour agent which selectively induces apoptosis in colon cancer cells but not in normal intestinal epithelial cells. Aims: To analyse the signalling pathway of butyrate induced apoptosis. Methods: Using Caco-2 cells we focused on the bcl family of proteins, mitochondrial pathway, and caspase signalling cascade involved in butyrate induced apoptosis. Techniques employed included western blots, immunofluorescence, as well as experiments with peptide inhibitors of specific caspases. Results: Butyrate induced a clear shift of the mitochondrial bcl rheostat towards a proapoptotic constellation, as demonstrated by upregulation of proapoptotic bak accompanied by reduced antiapoptotic bcl-xL levels. This was associated with translocation of cytochrome-c from the mitochondria to the cytosol, resulting in activation of the caspase cascade via caspase-9. Key executioner enzymes were caspases-3 and -1. No effect of butyrate on regulatory proteins of the inhibitor of apoptosis family was observed. Conclusions: Butyrate induced Caco-2 cell apoptosis via the mitochondrial pathway. Upregulation of bak and translocation of cytochrome-c were upstream of the caspase cascade. Subsequently, this cascade was activated via the formation of an apoptosome.

Butyrate mediates Caco-2 cell apoptosis via up-regulation of pro-apoptotic BAK and inducing caspase-3 mediated cleavage of poly-(ADP-ribose) polymerase (PARP).

Butyrate exerts potent anti-tumor effects by inhibiting cancer cell growth and inducing apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. Using the Caco-2 cell line, a well established model of colon cancer cells, our data show that butyrate induced apoptosis (maximum 79%) is mediated via activation of the caspase-cascade. A key event was the proteolytic activation of caspase-3, triggering degradation of poly-(ADP-ribose) polymerase (PARP). Inactivation of caspase-3 with the tetrapeptide zDEVD-FMK completely inhibited the apoptotic response to butyrate. In parallel, butyrate potently up-regulated the expression of the pro-apoptotic protein bak, without changing Caco-2 cell bcl-2 expression. Butyrate-induced Caco-2 cell apoptosis was completely blocked by the addition of cycloheximide, indicating the necessity of protein synthesis. However, when this inhibitor was added at a time point where bak expression was already enhanced (12–16 h after butyrate stimulation), it failed to protect Caco-2 cells against apoptosis. Taken together, these data provide evidence that the molecular events involved in butyrate induced colon cancer cell apoptosis include the caspase-cascade and the mitochondrial bcl-pathway.

Lipopolysaccharide modulation of normal enterocyte turnover by toll-like receptors is mediated by endogenously produced tumour necrosis factor alpha.

Background: Circulating levels of endotoxin (or lipopolysaccharide (LPS)) and anti-endotoxin antibodies are increased in patients with inflammatory bowel disease, supporting the hypothesis of a role for endogenous bacterial products in the pathogenesis of these disorders. Aim: The aim of this study was to analyse the direct effects of LPS on intestinal epithelial cell turnover. Methods and Results: LPS significantly inhibited growth of the human non-transformed immature crypt cell line (HIEC), whereas IEC-6 cell proliferation was stimulated by LPS. As LPS is a physiological inducer of tumour necrosis factor α (TNFα) in various cell systems and this cytokine exerted similar anti-proliferative (HIEC) or growth stimulatory (IEC-6 cells) effects, the study thus tested the hypothesis that endogenously produced TNFα in response to LPS mediates this growth modulatory effect in an autoparacrine/paracrine way. Therefore, during LPS stimulation, the biological activity of TNFα was blocked using neutralising anti-TNFα antibodies, as well as inhibitory, antagonistic antibodies directed against the p55 TNF receptor, signalling the antimitotic TNFα effect in HIEC. Both experimental approaches completely abolished the growth modulatory effects of LPS in HIEC/IEC-6 cells. Production and secretion of TNFα by HIEC/IEC-6 cells in response to LPS was confirmed on mRNA and protein level by reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay. LPS signalling was independent of CD14 in HIEC, as these cells lack this receptor. However, HIEC expressed TLR4 and MD2 resulting in a fully functional signalling complex as demonstrated by RT-PCR, western blot, and immunofluorescence analyses. Conclusion: These results support the hypothesis that LPS induced changes of intestinal epithelial cell turnover may directly contribute to the pathogenesis of inflammatory epithelial cell lesions by endogenous TNFα production by enterocytes.

Susceptibility to FAS-induced apoptosis in human nontumoral enterocytes: Role of costimulatory factors

FAS–FAS ligand interaction has been implicated in increased enterocyte apoptosis seen in immune‐mediated bowel injury. However, scant information exists on the role of FAS in physiological enterocyte turnover. In the present study, the regulation of enterocyte FAS and FAS ligand expression by cytokines and its functional role in human intestinal epithelial cell apoptosis and proliferation were analyzed with two different models: a nontransformed human intestinal epithelial cell line (HIEC) and normal colonic explant cultures. HIEC constitutively expressed FAS, as analyzed by flow cytometry. However, stimulation with agonistic anti‐FAS antibody (1–500 ng/ml) did not induce HIEC apoptosis. In contrast, in the presence of tumor necrosis factor α (TNFα) and/or interferon γ (IFNγ), HIEC became highly susceptible to FAS‐induced apoptosis. The sensitizing effect to FAS‐induced apoptosis was mediated via TNFα‐ and IFNγ‐induced upregulation of FAS expression (maximally 348%). Receptor studies showed that the effect of TNFα on FAS was mediated via the p55 TNF receptor. In colonic organ cultures, IFNγ and TNFα also enhanced colonocyte FAS expression, resulting in a markedly increased apoptotic response to stimulation of this receptor, as shown by in situ terminal deosyuridine triphosphate nick‐end staining. Neither FAS ligand expression nor its induction by cytokines was observed in HIEC or colonic explants. Proliferation studies showed that FAS is not implicated in regulating HIEC growth. These findings suggest that, despite the fact that normal human enterocytes express FAS, costimulatory factors, such as TNFα or IFNγ, abundantly secreted under inflammatory conditions, are necessary to sensitize intestinal epithelial cells to FAS‐induced apoptosis by upregulating this receptor. J. Cell. Physiol. 181:45–54, 1999.

Modulation of cytokine release from colonic explants by bacterial antigens in inflammatory bowel disease

The intestinal flora play an important role in experimental colitis and inflammatory bowel disease (IBD). Using colonic explant cultures from 132 IBD and control subjects, we examined tumour necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1 and interleukin‐1 receptor antagonist (IL‐1RA) production in vitro in response to bacterial activators. Unstimulated TNF‐α release was increased significantly in rectal biopsies from involved IBD tissue, correlating with inflammation severity. Whereas lipopolysaccharide (LPS) only moderately stimulated TNF‐α production from inflamed tissue, pokeweed mitogen (PWM) induced its release in all groups, with a stronger response in involved IBD tissue. Superantigen staphylococcal enterotoxin A (SEA) had a similar, but weaker effect. SEB was observed to be the strongest inducer of TNF‐α for all groups, again with a more marked response in inflamed tissue. Stimulated release of IL‐1 was considerably less than for TNF‐α. The superantigens’ superior potency over LPS was not as marked for IL‐1 as it was for TNF‐α. In addition to IL‐1, IL‐1RA release was also triggered by the bacterial products. The net effect of activation on the IL‐1RA/IL‐1 ratio was relatively modest. Release of the proinflammatory cytokines TNF‐α and IL‐1, as well as that of the anti‐inflammatory cytokine IL‐1RA was increased by incubation of colonic tissue with bacterial factors. TNF‐α production and release was increased significantly in involved colonic explants from IBD. SEB was even capable of inducing TNF‐α release from uninvolved colonic tissue.

Phase I and phase II metabolism of lithocholic acid in hepatic acinar zone 3 necrosis: Evaluation in rats by combined radiochromatography and gas-liquid chromatograpry-mass spectrometry

In the present study, lithocholic acid (LCA) metabolism was assessed by radiochromatography and gas-liquid chromatography-mass spectrometry, and its relationship to cholestasis was investigated. In addition, the role of the perivenous zone in LCA-induced cholestasis and LCA biotransformation was examined by using bromobenzene (BZ), a chemical that causes selective necrosis of hepatocytes in this zone. LCA injection induced cholestasis of comparable amplitude in both control and BZ-treated rats. The biliary recovery of bile salts (BS) was 65-70% 2 hr after LCA injection. Excretion of LCA and its cholestatic metabolite, LCA glucuronide, was similar in both groups, although LCA excretion was delayed in BZ-treated animals. The appearance of LCA and LCA glucuronide in bile occurred early, and their proportion decreased with time. Concentrations of choleretic hydroxylated metabolites were low immediately after LCA injection but increased with time. 3 alpha,6 beta-Dihydroxy-5 beta-cholanoic and 3 alpha,6 beta,7 beta-trihydroxy-5 beta-cholanoic acids were the major species arising from LCA, indicating the importance of 6 beta hydroxylation in LCA detoxification in rats. Other metabolites were found, but their contribution was either minor or negligible. Overall amounts of hydroxylated metabolites were comparable in both groups, but trihydroxylated metabolites predominated over their dihydroxylated counterparts in control rats, whereas the production of dihydroxylated forms was more pronounced in BZ-treated animals. These results suggest that the destruction of perivenous hepatocytes does not exacerbate LCA-induced cholestasis, and that there may be an acinar zonation of LCA biotransformation to trihydroxylated metabolites in the rat liver.

No evidence of persisting measles virus in the intestinal tissues of patients with inflammatory bowel disease

The hypothesis that measles virus (MV) is implicated in the pathogenesis of Crohn’s disease (CD) was initiated by Wakefield et al 1 in the early 1990s. Several other reports soon followed, which suggested the presence of MV in CD samples using electron microscopy, immunohistochemical analysis, in situ hybridisation and nested PCR.2–4 Controversy in response to this claim resulted in falling measles vaccination rates, mainly in the UK. Immediate concerns were raised regarding several aspects of these studies, including reagent specificity.5 In addition, several groups were unable to replicate their findings using a range of PCR techniques.6–9 In 1998 and 2002, the initial investigators again raised concerns over MV persistence in bowel tissue, this time in children with autism.10 These allegations further damaged confidence in MV vaccination programmes. As the primer pairs that had been used to implicate MV in inflammatory bowel disease (IBD; Kawashima)4 and autism (Uhlmann)10 were not included in the negative studies,6–9 it could still be argued that the negative results arose due to the use of incorrect or suboptimal primers. We therefore used the Kawashima and Uhlmann primers in an attempt …

The effect of inflammation severity and of treatment on the production and release of TNFα by colonic explants in inflammatory bowel disease

Despite its pivotal role in mucosal inflammation, data on TNFα levels in inflammatory bowel diseases have been contradictory.

The influence of vitamin D on M1 and M2 macrophages in patients with Crohn's disease

Defective bacterial clearance by macrophages plays an important role in Crohn’s disease (CD). Phenotypes and functions of inflammatory M1 and anti-inflammatory M2 have not been studied in CD. Vitamin D supplementation reduces the severity of CD by unclear mechanisms. We studied macrophage characteristics in CD and controls and the effects of 1,25 vitamin D (1,25D). PBMC were isolated from CD patients and controls. M1 and M2 were generated by culturing of monocytes with GM-CSF and M-CSF, respectively. CD M1 and M2 showed normal phagocytosis and chemotaxis to CCL2 and fMLP. LPS-induced production of TNF-α, IL-12p40 and IL-10 was comparable between groups. Phagocytosis was unaltered with 1,25D; migration only increased marginally. M1 produced more IL-12p40 and TNF-α; IL-10 was greater in M2. 1,25D markedly decreased IL-12p40 by M1 and M2. 1,25D decreased TNF-α in CD M1; IL-10 levels were unaffected. M2 express F13A1, PTGS2, CD163, CXCL10, CD14 and MMP2, whereas TGF-β, CCL1 and CYP27B1 expression was higher in M1. Marker expression was similar between CD and controls. M1 and M2 markers were not differentially modulated by 1,25D. CD macrophages are not functionally or phenotypically different vs. controls. 1,25D markedly decreased pro-inflammatory M1 cytokines but did not modulate polarization to anti-inflammatory M2 phenotype.