Katharina Leucht

Crohn's disease-associated polymorphism within the PTPN2 gene affects muramyl-dipeptide-induced cytokine secretion and autophagy.

Background: The single nucleotide polymorphism (SNP) rs2542151 within the gene locus region encoding protein tyrosine phosphatase non‐receptor type 2 (PTPN2) has been associated with Crohns disease (CD), ulcerative colitis (UC), type‐I diabetes, and rheumatoid arthritis. We have previously shown that PTPN2 regulates mitogen‐activated protein kinase (MAPK) signaling and cytokine secretion in human THP‐1 monocytes and intestinal epithelial cells (IEC). Here, we studied whether intronic PTPN2 SNP rs1893217 regulates immune responses to the nucleotide‐oligomerization domain 2 (NOD2) ligand, muramyl‐dipeptide (MDP). Materials and Methods: Genomic DNA samples from 343 CD and 663 non‐IBD control patients (male and female) from a combined German, Swiss, and Polish cohort were genotyped for the presence of the PTPN2 SNPs, rs2542151, and rs1893217. PTPN2‐variant rs1893217 was introduced into T84 IEC or THP‐1 cells using a lentiviral vector. Results: We identified a novel association between the genetic variant, rs1893217, located in intron 7 of the PTPN2 gene and CD. Human THP‐1 monocytes carrying this variant revealed increased MAPK activation as well as elevated mRNA expression of T‐bet transcription factor and secretion of interferon‐&ggr; in response to the bacterial wall component, MDP. In contrast, secretion of interleukin‐8 and tumor necrosis factor were reduced. In both, T84 IEC and THP‐1 monocytes, autophagosome formation was impaired. Conclusions: We identified a novel CD‐associated PTPN2 variant that modulates innate immune responses to bacterial antigens. These findings not only provide key insights into the effects of a functional mutation on a clinically relevant gene, but also reveal how such a mutation could contribute to the onset of disease. (Inflamm Bowel Dis 2011;)

BCL-2 Modifying Factor (BMF) Is a Central Regulator of Anoikis in Human Intestinal Epithelial Cells

BCL-2 modifying factor (BMF) is a sentinel considered to register damage at the cytoskeleton and to convey a death signal to B-cell lymphoma 2. B-cell lymphoma 2 is neutralized by BMF and thereby facilitates cytochrome C release from mitochondria. We investigated the role of BMF for intestinal epithelial cell (IEC) homeostasis. Acute colitis was induced in Bmf-deficient mice (Bmf−/−) with dextran sulfate sodium. Colonic crypt length in Bmf−/− mice was significantly increased as compared with WT mice. Dextran sulfate sodium induced less signs of colitis in Bmf−/− mice, as weight loss was reduced compared with the WT. Primary human IEC exhibited increased BMF in the extrusion zone. Quantitative PCR showed a significant up-regulation of BMF expression after initiation of anoikis in primary human IEC. BMF was found on mitochondria during anoikis, as demonstrated by Western blot analysis. RNAi mediated knockdown of BMF reduced the number of apoptotic cells and led to reduced caspase 3 activity. A significant increase in phospho-AKT was determined after RNAi treatment. BMF knockdown supports survival of IEC. BMF is induced in human IEC by the loss of cell attachment and is likely to play an important role in the regulation of IEC survival.

Lack of transketolase-like (TKTL) 1 aggravates murine experimental colitis.

Transketolase-like (TKTL) 1 indirectly replenishes NADPH preventing damage induced by reactive oxygen species (ROS) formed upon intestinal inflammation. We investigated the function of TKTL1 during murine colitis and ROS detoxification for prevention of tissue damage. Mucosal damage in TKTL1(-/-) and wild-type (WT) mice was assessed by miniendoscopy and histology during dextran sodium sulfate (DSS) colitis. mRNA levels of interferon (IFN)-γ, inducible nitric oxide synthase (iNOS), interleukin (IL)-6, tumor necrosis factor (TNF), transketolase (TKT), and TKTL2 were determined by PCR and/or Western blotting. To assess oxidative and nitrosative stress nitrosylation, carbonylation and antioxidative enzymes catalase (Cat), superoxide dismutase 1 and 2, as well as glutathione (GSH) were determined. Myeloperoxidase (MPO) was determined for assessment of tissue neutrophils. TKTL1 knockout or DSS treatment did not influence TKT and TKTL2 mRNA or protein expression. Mucosal damage was significantly increased in TKTL1(-/-) mice indicated by miniendoscopy as well as a significantly shorter colon and more severe histological scores compared with WT mice during DSS colitis. This was associated with higher mRNA levels of IFN-γ, iNOS, IL-6, and TNF. In addition, iNOS protein expression was significantly enhanced in TKTL1(-/-) mice as well as MPO activity. Protein modification by nitric oxide (nitrotyrosine) was induced in TKTL1(-/-) mice. However, introduction of carbonyl groups by ROS was not induced in these mice. The expression of SOD1, SOD2, Cat, as well as GSH content was not significantly changed in TKTL1(-/-) mice. We conclude that induced colitis in TKTL1(-/-) mice was more severe compared with WT. This indicates a role of TKTL1 during mucosal repair and restoration.

Sphingomyelin and phosphatidylcholine contrarily affect the induction of apoptosis in intestinal epithelial cells.

SCOPE The major alimentary sources for the plasma membrane lipid sphingomyelin (SM) are dairy products, eggs, and meat. We recently reported that the SM metabolite ceramide induces cathepsin D mediated apoptosis in murine intestinal epithelial cells (IECs) and increases inflammation in acute colitis. We investigated the impact of SM and phosphatidylcholine on apoptosis in human IECs and point out BH3-interacting death agonist (BID) as link between cathepsin D and apoptosis. METHODS AND RESULTS HT-29 and isolated human IECs were stimulated with SM or phosphatidylcholine. SM treatment resulted in increased apoptosis. Phosphatidylcholine showed contrary effects. Western revealed higher amounts of cathepsin D and BID activation upon lipid stimulation. Western blotting revealed BID activation through SM in both an induced and a spontaneous mouse model of colitis. CONCLUSION Dietary phospholipids may induce or abolish apoptosis in IECs and seem to play a role in the pathogenesis of inflammatory bowel diseases. This nutritional factor might be considered when evaluating the pathogenesis of inflammatory bowel diseases. Effects of SMase- and SM treatment on inflammation in dextran sulfate sodium induced animal models of colitis and in vitro experiments are discussed as controversial. Variable sources of SM, feeding techniques, and mouse strains might play a role.

Knock-Out of β-Glucosidase 2 Has No Influence on Dextran Sulfate Sodium-Induced Colitis

Background/Aims: The non-lysosomal glucosylceramidase, β-glucosidase (Gba2), hydrolyzes glucosylceramide to glucose and ceramide (Cer). Cer is a potent second-messenger lipid that plays an important role in signaling cascades involved in apoptosis. The aim of this study was to investigate whether Gba2 knock-out (Gba2–/–) affects the extent of dextran sulfate sodium (DSS)-induced colitis in mice. Methods: Acute colitis was induced in wild-type (WT) and Gba2–/– mice by administration of 2% DSS in drinking water. After 7 days, mice underwent colonoscopy and were sacrificed. Results: Both DSS-treated WT (n = 10) and Gba2–/– (n = 12) mice showed elevated histological and endoscopic scores compared to respective H2O controls (n = 9 each). However, no significant differences between the DSS groups were detected. Flow cytometric analysis of propidium iodide staining, cleavage of caspases-3 and -8, indicative for apoptosis, as well as Cer levels were not altered in DSS-treated WT or Gba2–/– mice. Gba2–/– resulted in slightly decreased expression of glucocerebrosidase (Gba1) as well as in upregulation of proteins being involved in cellular regeneration, such as STAT3 (signal transducer and activator of transcription), JNK and iNOS, upon DSS treatment. Conclusion: We demonstrate that Gba2–/– does not affect the extent of DSS-induced inflammation in mice, however, it might be involved in tissue regeneration in response to toxic agents.

The Proton-activated Receptor GPR4 Modulates Intestinal Inflammation

Background and Aims During active inflammation, intraluminal intestinal pH is decreased in patients with inflammatory bowel disease [IBD]. Acidic pH may play a role in IBD pathophysiology. Recently, proton-sensing G-protein coupled receptors were identified, including GPR4, OGR1 [GPR68], and TDAG8 [GPR65]. We investigated whether GPR4 is involved in intestinal inflammation. Methods The role of GPR4 was assessed in murine colitis models by chronic dextran sulphate sodium [DSS] administration and by cross-breeding into an IL-10 deficient background for development of spontaneous colitis. Colitis severity was assessed by body weight, colonoscopy, colon length, histological score, cytokine mRNA expression, and myeloperoxidase [MPO] activity. In the spontaneous Il-10-/- colitis model, the incidence of rectal prolapse and characteristics of lamina propria leukocytes [LPLs] were analysed. Results Gpr4-/- mice showed reduced body weight loss and histology score after induction of chronic DSS colitis. In Gpr4-/-/Il-10-/- double knock-outs, the onset and progression of rectal prolapse were significantly delayed and mitigated compared with Gpr4+/+/Il-10-/- mice. Double knock-out mice showed lower histology scores, MPO activity, CD4+ T helper cell infiltration, IFN-γ, iNOS, MCP-1 [CCL2], CXCL1, and CXCL2 expression compared with controls. In colon, GPR4 mRNA was detected in endothelial cells, some smooth muscle cells, and some macrophages. Conclusions Absence of GPR4 ameliorates colitis in IBD animal models, indicating an important regulatory role in mucosal inflammation, thus providing a new link between tissue pH and the immune system. Therapeutic inhibition of GPR4 may be beneficial for the treatment of IBD.

Exogenous Heat Shock Protein gp96 Ameliorates CD4+CD62L+ T-Cell–mediated Transfer Colitis

Background:The heat shock protein gp96 is an endoplasmic reticulum chaperone involved in endoplasmic reticulum stress reactions. gp96 binds antigens and is secreted into extracellular space on cell stress. After reinternalization by antigen presenting cells, antigens can be transferred to major histocompatibility complex molecules. In recent studies, we found induction of gp96 during differentiation of intestinal macrophages, whereas it was absent in intestinal macrophages of patients with Crohns disease. Methods:To study immuno-modulating effects of gp96 in T-cell transfer colitis BALB/c donor mice were injected with 2 × 100 &mgr;g gp96. After 1 week, 2.5 × 105 CD4+CD62L+ cells were isolated from spleens and injected into severe combined immunodeficiency recipients. Another group received cells from untreated donors and was treated with 100 &mgr;g gp96 after transfer. Control groups received cells from untreated donors, or buffer alone. Results:After transfer of CD4+CD62L+ T cells from gp96-pretreated donors, mice (TBT gp96) showed an initial weight loss, but after 3 weeks, they recovered and reached the starting weight after 5 weeks. Mice treated with gp96 after transfer (TAT gp96) showed a delayed weight loss in comparison with the CD4+CD62L+ group. The histological scores in CD4+CD62L+ mice were 2.6 ± 0.1, in TBT gp96 mice 1.3 ± 0.3 (CD4+CD62L+ versus TBT gp96: P < 0.05) and in mice treated after transfer 1.9 ± 0.1 (CD4+CD62L+ versus TAT gp96: P < 0.05). Conclusions:These findings indicate an essential role of gp96 in the maintenance of tolerance against luminal antigens in the intestinal mucosa. The absence of gp96 in intestinal macrophages of patients with Crohns disease might provoke loss of this tolerance mediating mechanism.

Mo1976 Knock-out of BCL-2 Interacting Mediator of Cell Death (Bim) Aggravates Chronic DSS-Induced Colitis

G A A b st ra ct s was 15.2% and 11.9% had 1 2 tubular adenomas. In comparison the control AS-Group had ADR of 19.2%%, and 17.7% had 1 2 tubular adenomas. In the FOBT+ group, CRC was detected in 5.1% which was significantly higher than the AS-Group in which CRC was detected in 1.7% (p 0.03). There were no significant differences in other parameters among groups. Conclusions: Selecting elderly subjects for colonoscopy using FOBT as a screening tool may improve the yield of colonoscopy. The benefit in the risk benefit analysis of screening the elderly appears improved by prescreening with an inexpensive tool. This approach can also be valuable is preventing morbidity and mortality associated with colonoscopy in elderly at risk subjects.

S1734 Knock-out of β-Glucosidase 2 Prevents the Intestinal Epithelium From Dextran Sodium Sulfate-Induced Cell Damage

DSS-induced histopathology of individual (or aggregate) scores for inflammation, crypt damage, ulcerations indicates less mucosal damage in A3AR mice than WT (p<0.01). A3 AR mice are resistant to developing colitis. At 3% DSS A3AR mice lose 5% body-weight compared to 20% in WT and recover faster (p<0.01); at 1.5%, A3AR prevents weight-loss (p<0.05). There is a 2-fold reduction in MPO in A3AR mice (p<0.001); it prevented increase in CD4+ T-cells in lamina propria (p=0.009) and attenuated DSS-induced changes in colon length/weight ratio (p<0.01). There is lower incidence of occult-blood and diarrhea in A3 AR-treated mice (p<0.03; p=0.001). Conclusions: The A3AR modulates colonic motility and evacuation. Disruption of A3AR protects against colitis suggesting activation of A3AR by endogenous adenosine may be injurious in DSS-colitis. The A3AR mechanism is unknown (NIH DK 44179,ARRA-3R01 DK044179-14S).