Claudia Hofmann

Publications of Claudia Hofmann

• IL-33 attenuates development and perpetuation of chronic intestinal inflammation.

  Authors: Philipp Groβ, Kristina Doser, Werner Falk, Florian Obermeier, Claudia Hofmann

  Inflammatory bowel diseases. 04/2012;

  BACKGROUND: Interleukin-33 (IL-33) is a member of the IL-1 family. Recent evidence shows the importance of IL-33 in autoimmune and inflammatory diseases. To elucidate its impact on inflammatory bowelBACKGROUND: Interleukin-33 (IL-33) is a member of the IL-1 family. Recent evidence shows the importance of IL-33 in autoimmune and inflammatory diseases. To elucidate its impact on inflammatory bowel disease we studied the effects of exogenous IL-33 during the induction of acute dextran sodium sulfate (DSS)-induced colitis, the induction period of chronic DSS colitis, and after establishment of chronic inflammation. METHODS: For induction of acute colitis mice received DSS in their drinking water for 7 days and were killed at day 8 or 14 after first DSS administration. Chronic colitis was induced by four cycles of DSS. Animals were treated with IL-33 between the DSS cycles (intermediate treatment) or after onset of chronic disease (posttreatment). Colons and mesenteric lymph nodes were isolated for histology and cytokine secretion, flow cytometric analysis, determination of myeloperoxidase, and transcription factor activity. RESULTS: While IL-33 in acute colitis led to slight aggravation of inflammation, both chronic colitis approaches resulted in a significant reduction of inflammatory colon contraction, amelioration of disease scores, suppression of interferon-gamma (IFN-γ), and a shift to T helper (Th)2-associated cytokines. Examination of colon tissue revealed increased Ly6g-mRNA levels and myeloperoxidase (MPO) activity in IL-33-treated animals. Evaluation of bacterial translocation revealed decreased translocation incidence in IL-33-treated mice. CONCLUSIONS: In summary, IL-33 has extenuating effects in chronic DSS-induced colitis: Excessive Th1-directed cytokine responses are shifted toward Th2-like immune reactions and general inflammation parameters are reduced. IL-33-induced neutrophil influx during chronic inflammation reduced translocation of pathogenic bacteria across damaged epithelium. (Inflamm Bowel Dis 2012;).

• Intestinal bacterial translocation in rats with cirrhosis is related to compromised paneth cell antimicrobial host defense.

  Authors: Zora Teltschik, Reiner Wiest, Julia Beisner, Sabine Nuding, Claudia Hofmann, Juergen Schoelmerich, Charles L Bevins, Eduard F Stange, Jan Wehkamp

  Hepatology (Baltimore, Md.). 11/2011;

  Liver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrierLiver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrier function. We hypothesized that diminished mucosal antimicrobial host defense could predispose to BT. Two rodent models of portal hypertension with increased BT were used, CCl(4) -induced ascitic cirrhosis and 2-day portal vein-ligated (PVL) animals. BT was assessed by standard microbiological techniques on mesenteric lymph nodes. Total RNA was isolated systematically throughout the intestinal tract, and expression of Paneth cell α-cryptdins and β-defensins was determined by real-time quantitative polymerase chain reaction (qPCR). To determine functional consequences, mucosal antimicrobial activity was assessed with a fluorescence-activated cell sorting assay. BT was detectable in 40% of rats with cirrhosis. Compared with the group without BT, these animals exhibited diminished intestinal Paneth cell α-cryptdin 5 and 7 expression. In contrast, PVL was associated with BT in all animals but did not affect antimicrobial peptides. The decrease in Paneth cell antimicrobials was most pronounced in the ileum and the coecum. Other antimicrobials showed no changes or even an induction in the case of BT at different sites. Antimicrobial activity toward different commensal strains was reduced, especially in the distal ileum and the cecum in experimental cirrhosis with BT (excluding PVL). Conclusion: Compromised Paneth cell antimicrobial host defense seems to predispose to BT in experimental cirrhosis. Understanding this liver-gut axis including the underlying mechanisms could help us to find new treatment avenues. (HEPATOLOGY 2011).

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

  Authors: Michael Scharl, Jessica Mwinyi, Anne Fischbeck, Katharina Leucht, Jyrki J Eloranta, Joba Arikkat, Theresa Pesch, Silvia Kellermeier, Alma Mair, Gerd A Kullak-Ublick, Kaspar Truninger, Faiza Noreen, Jaroslaw Regula, Pawel Gaj, Valerie Pittet, Christoph Mueller, Claudia Hofmann, Michael Fried, Declan F McCole, Gerhard Rogler

  Inflammatory bowel diseases. 10/2011;

  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 Crohn's diseaseBACKGROUND: The single nucleotide polymorphism (SNP) rs2542151 within the gene locus region encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) has been associated with Crohn's 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 T(84) 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-γ in response to the bacterial wall component, MDP. In contrast, secretion of interleukin-8 and tumor necrosis factor were reduced. In both, T(84) 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;).

• Bilberries and their anthocyanins ameliorate experimental colitis.

  Authors: Heidi Piberger, Anett Oehme, Claudia Hofmann, Andrea Dreiseitel, Philipp G Sand, Florian Obermeier, Juergen Schoelmerich, Peter Schreier, Gerhard Krammer, Gerhard Rogler

  Molecular nutrition & food research. 09/2011; 55(11):1724-9.

  Bilberries have positive effects in acute and chronic diarrhea. Patients with inflammatory bowel disease (IBD) report on improved symptoms upon ingestion. Bilberries contain approximately 10% ofBilberries have positive effects in acute and chronic diarrhea. Patients with inflammatory bowel disease (IBD) report on improved symptoms upon ingestion. Bilberries contain approximately 10% of anthocyanins (ACs), which have anti-oxidative, anti-carcinogenic, and anti-inflammatory properties. We investigated whether experimental colitis can be ameliorated by dried bilberries or ACs. Acute and chronic dextrane sodium sulphate (DSS) colitis were induced in Balb/c mice by 2.5% DSS in the drinking water. Mice were fed with dried bilberries or ACs, respectively. Cytokines were determined in supernatants from mesenteric lymph nodes (MLNs) by ELISA and apoptosis was investigated by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assays. Oral administration of bilberries during acute DSS-induced colitis ameliorated disease severity and reduced secretion of IFN-γ and tumor necrosis factor from mesenteric lymph node cells. Dried bilberries also improved chronic DSS-colitis. Ingestion of ACs reduced intestinal inflammation in acute and chronic DSS-colitis with decreased histological scores and cytokine secretion. Both bilberries and ACs prevented inflammation-induced apoptosis in colonic epithelial cells. Taken together, ingestion of dried bilberries had positive effects on various parameters especially in acute DSS-colitis. Oral administration of ACs resulted in an amelioration of acute colitis as well as chronic colitis. These promising results justify a clinical study on their therapeutic effect in inflammatory bowel disease patients.

• BCL-2 modifying factor (BMF) is a central regulator of anoikis in human intestinal epithelial cells.

  Authors: Martin Hausmann, Katharina Leucht, Christian Ploner, Stephan Kiessling, Andreas Villunger, Helen Becker, Claudia Hofmann, Werner Falk, Michaela Krebs, Silvia Kellermeier, Michael Fried, Jürgen Schölmerich, Florian Obermeier, Gerhard Rogler

  The Journal of biological chemistry. 06/2011; 286(30):26533-40.

  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 therebyBCL-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.

• DSS induced colitis increases portal LPS levels and enhances hepatic inflammation and fibrogenesis in experimental NASH.

  Authors: Erwin Gäbele, Karin Dostert, Claudia Hofmann, Reiner Wiest, Jürgen Schölmerich, Claus Hellerbrand, Florian Obermeier

  Journal of hepatology. 04/2011; 55(6):1391-9.

  Intestinal bacterial overgrowth and increased permeability are features of non alcoholic steatohepatitis (NASH). Bacterial endotoxin has been shown to promote NASH progression. Application of dextranIntestinal bacterial overgrowth and increased permeability are features of non alcoholic steatohepatitis (NASH). Bacterial endotoxin has been shown to promote NASH progression. Application of dextran sulfate sodium (DSS) is a colitis model in mice characterized by damage of the intestinal barrier. This study was designed to investigate if application of DSS aggravates experimental NASH. Male C57bl/6 mice were allocated into four experimental groups receiving either (I) standard chow (SC), (II) a high fat (HF) diet, (III) SC+DSS (1% in the drinking water), and (IV) HF+DSS for 12 weeks. DSS treatment caused inflammation and proinflammatory gene expression (IL-1β, IL-17, TNF) in the colon. Expression of colonic antimicrobial peptide Cramp was significantly induced in SC+DSS mice, whereas expression was blocked in the HF+DSS group. Endotoxin levels were elevated in SC+DSS and HF mice but further augmented in the HF+DSS group. In line with this, increased hepatic TLR4 and TLR9 mRNA levels were detected in HF+DSS mice. The histological analysis revealed hepatic steatosis in both HF groups. Hepatic inflammation was more severe in HF+DSS mice, reflected by histology and analysis of proinflammatory gene expression (TNF and MCP-1). HF+DSS mice showed increased hepatic fibrosis by sirius red staining, hepatic collagen I expression, and α-SMA positive cells accompanied by higher p47(phox), TIMP-1, TGF-β, Pai-1, and α-SMA mRNA expression. Induction of an intestinal inflammation in experimental NASH promotes LPS translocation, hepatic inflammation, and fibrogenesis probably due to inhibition of intestinal antimicrobial peptides. These findings underscore the pathophysiological role of the gut-liver axis in the progression of NASH.

• Mucosal immunosuppression and epithelial barrier defects are key events in murine psychosocial stress-induced colitis.

  Authors: Stefan O Reber, Sebastian Peters, David A Slattery, Claudia Hofmann, Jürgen Schölmerich, Inga D Neumann, Florian Obermeier

  Brain, behavior, and immunity. 03/2011; 25(6):1153-61.

  Chronic psychosocial stress is a risk factor for many affective and somatic disorders, including inflammatory bowel diseases. In support chronic subordinate colony housing (CSC, 19 days), anChronic psychosocial stress is a risk factor for many affective and somatic disorders, including inflammatory bowel diseases. In support chronic subordinate colony housing (CSC, 19 days), an established mouse model of chronic psychosocial stress, causes the development of spontaneous colitis. However, the mechanisms underlying the development of such stress-induced colitis are poorly understood. Assessing several functional levels of the colon during the initial stress phase, we show a pronounced adrenal hormone-mediated local immune suppression, paralleled by impaired intestinal barrier functions, resulting in enhanced bacterial load in stool and colonic tissue. Moreover, prolonged treatment with broad-spectrum antibiotics revealed the causal role of these early maladaptations in the development of stress-induced colitis. Together, we demonstrate that translocation of commensal bacteria is crucial in the initiation of stress-induced colonic inflammation. However, aggravation by the immune-modulatory effects of fluctuating levels of adrenal hormones is required to develop this into a full-blown colitis.

• C1q/TNF-related protein-3 (CTRP-3) is secreted by visceral adipose tissue and exerts antiinflammatory and antifibrotic effects in primary human colonic fibroblasts.

  Authors: Claudia Hofmann, Ning Chen, Florian Obermeier, Gisela Paul, Christa Büchler, Andrea Kopp, Werner Falk, Andreas Schäffler

  Inflammatory bowel diseases. 02/2011; 17(12):2462-71.

  The adipokine CTRP-3 (C1q/TNF-related protein-3) belongs to the C1q/TNF-related protein family which antagonizes the effects of lipopolysaccharide (LPS). The aim was to investigate theThe adipokine CTRP-3 (C1q/TNF-related protein-3) belongs to the C1q/TNF-related protein family which antagonizes the effects of lipopolysaccharide (LPS). The aim was to investigate the antiinflammatory and antifibrotic role of CTRP-3 in Crohn's disease (CD). Mesenteric adipose tissue (MAT) of patients with CD or colonic cancer (CC) was resected. Human primary colonic lamina propria fibroblasts (CLPF) were isolated from controls and CD patients. Concentrations of chemokines and cytokines in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Expression of connective tissue growth factor (CTGF), collagen I, and collagen III was analyzed by real-time polymerase chain reaction (PCR). Recombinant CTRP-3 expressed in insect cells was used for stimulation experiments. CTRP-3 is synthesized and secreted by MAT resected from patients with CD, ulcerative colitis (UC), CC, and sigma diverticulitis as well as by murine and human mature adipocytes. CTRP-3 had no effect on the basal secretion of MCSF, MIF, or RANTES in MAT of CD and control patients. LPS-stimulation (10 ng/mL) significantly increased IL-8 release in CLPF of CD patients and, to a lesser extent, in cells of controls and of fibrotic CD tissue. CTRP-3 significantly and dose-dependently reduced LPS-induced IL-8 secretion in CLPF within 8 hours after LPS exposure, whereas LPS-induced IL-6 and TNF release was not affected. CTRP-3 inhibited TGF-β production and the expression of CTGF and collagen I in CLPF, whereas collagen III expression remained unchanged. CTRP-3 exerts potent antiinflammatory and antifibrotic effects in CLPF by antagonizing the LPS pathway and by targeting the TGF-β-CTGF-collagen I pathway.

• Glycogen synthase kinase 3-β: a master regulator of toll-like receptor-mediated chronic intestinal inflammation.

  Authors: Claudia Hofmann, Nadja Dunger, Jürgen Schölmerich, Werner Falk, Florian Obermeier

  Inflammatory bowel diseases. 11/2010; 16(11):1850-8.

  A disturbed regulation of Toll-like receptor (TLR) signal transduction resulting in the exclusive activation of proinflammatory signaling pathways may be critical for the perpetuation of establishedA disturbed regulation of Toll-like receptor (TLR) signal transduction resulting in the exclusive activation of proinflammatory signaling pathways may be critical for the perpetuation of established chronic colitis. Glycogen synthase kinase 3-β (GSK3-β) was recently identified as an important regulator of TLR signaling mediating excessive inflammatory responses. The aim of this study was to assess the role of GSK3-β activity in chronic intestinal inflammation. Chronic colitis was induced by dextran sodium sulfate (DSS) treatment. Mice were treated intraperitoneally with phosphate-buffered saline (PBS), CpG-ODN, or GSK3-β inhibitors (SB216763, LiCl). Intestinal inflammation was evaluated by histologic analysis and cytokine secretion of mesenteric lymph node cells (MLC). Nuclear extracts of MLC and lamina propria mononuclear cells (LPMC) were analyzed for nuclear factor kappaB (NF-κB) and CREB activity. Murine and human intestinal immune cells were stimulated in vitro with CpG-ODN, lipopolysaccharide (LPS), or anti-CD3 with or without LiCl. GSK3-β blockade significantly reduced chronic intestinal inflammation and even abolished the colitis-intensifying effects of CpG-ODN treatment. In vitro inhibition of GSK3-β reduced the proinflammatory phenotype of both murine and human intestinal immune cells from chronic inflamed tissue. In vivo blockade of GSK3-β resulted in a shift from NF-κB activity toward CREB activity in murine MLC and LPMC. Blockade of GSK3-β attenuates excessive proinflammatory TLR-mediated immune responses. GSK3-β inhibition therefore constitutes a promising therapeutic option for selectively reducing exaggerated intestinal immune reactions toward the luminal flora in inflammatory bowel disease.

• T cell-dependent protective effects of CpG motifs of bacterial DNA in experimental colitis are mediated by CD11c+ dendritic cells.

  Authors: Claudia Hofmann, Nadja Dunger, Nicole Grunwald, Günter J Hämmerling, Petra Hoffmann, Jürgen Schölmerich, Werner Falk, Florian Obermeier

  Gut. 10/2010; 59(10):1347-54.

  Oligodeoxynucleotides (ODNs) containing unmethylated cytosine-guanosine (CpG) sequence motifs constitute the immunostimulatory components of bacterial DNA which potently activate innate immunity.Oligodeoxynucleotides (ODNs) containing unmethylated cytosine-guanosine (CpG) sequence motifs constitute the immunostimulatory components of bacterial DNA which potently activate innate immunity. Administration of CpG-ODNs before the onset of experimental colitis prevents intestinal inflammation by induction of colitis-suppressing T cells. To identify the interplay between innate and adaptive immune cells finally leading to protective CpG-ODN effects in intestinal inflammation. Total splenic cells or purified selected cell types (CD4(+)CD62L(+) T cells alone or with B cells or dendritic cells (DCs)) from BALB/c mice were (co)-incubated in vitro with CpG-ODN for 5 days and CD4(+)CD62L(+) cells were injected intraperitoneally into C.B.-17 SCID (severe combined immunodeficiency) mice. Splenic CD4(+)CD62L(+) T cells were isolated from transgenic donor mice in which CD11c(+) DCs were depleted by diphtheria toxin administration during CpG-ODN treatment and injected into C57BL/6 Rag2(-/-) recipients. Intestinal inflammation was evaluated by histological scoring and cytokine secretion of mesenteric lymph node cells. CpG-ODN treatment of total splenic cells but not of purified CD4(+)CD62L(+) cells reduced the colitogenic potential of transferred T cells. While CpG-ODN stimulation of co-cultured CD4(+)CD62L(+) and B-cells did not alter the colitogenic potential of T cells, co-incubation of CpG-ODN-stimulated DCs and CD4(+)CD62L(+) cells reduced the colitogenic potential of the T cell population. Depletion of CD11c(+) DCs during CpG-ODN administration in vivo abolished the protective CpG-ODN effects. CpG-ODN-dependent protective effects in experimental colitis act indirectly on CD4(+)CD62L(+) T cells. While the involvement of B cells could be excluded, CD11c(+) DCs were identified as key mediators of CpG-ODN-induced protection in experimental colitis.

• Inflammatory bowel diseases: when natural friends turn into enemies-the importance of CpG motifs of bacterial DNA in intestinal homeostasis and chronic intestinal inflammation.

  Authors: Florian Obermeier, Claudia Hofmann, Werner Falk

  International journal of inflammation. 01/2010; 2010:641910.

  From numerous studies during the last years it became evident that bacteria and bacterial constituents play a decisive role both in the maintenance of intestinal immune homeostasis as well as in theFrom numerous studies during the last years it became evident that bacteria and bacterial constituents play a decisive role both in the maintenance of intestinal immune homeostasis as well as in the development and perpetuation of chronic intestinal inflammation. In this review we focus on the role of bacterial DNA which is a potent immunomodulatory component of the bacterial flora. Bacterial DNA has been shown to be protective against experimental colitis. In contrast bacterial DNA essentially contributes to the perpetuation of an already established chronic intestinal inflammation in a Toll-like receptor (TLR)9-dependent manner. This dichotomic action may be explained by a different activation status of essential regulators of TLR signaling like Glycogen synthase kinase 3-β (GSK3-β) depending on the pre-activation status of the intestinal immune system. In this review we suggest that regulators of TLR signaling may be interesting therapeutic targets in IBD aiming at the restoration of intestinal immune homeostasis.

• Primary human colonic epithelial cells are transiently protected from anoikis by a Src-dependent mechanism.

  Authors: Claudia Hofmann, Elisabeth Lippert, Werner Falk, Jürgen Schölmerich, Gerhard Rogler, Florian Obermeier

  Biochemical and biophysical research communications. 10/2009;

  Complete loss of cell anchorage triggers apoptosis in primary human colonic epithelial cells (CEC), a phenomenon known as anoikis. Besides the induction of pro-apoptotic events, activation ofComplete loss of cell anchorage triggers apoptosis in primary human colonic epithelial cells (CEC), a phenomenon known as anoikis. Besides the induction of pro-apoptotic events, activation of survival pathways was observed in detached intestinal epithelial cell lines, providing a transient apoptosis protection. However, nothing is known about molecular mechanisms protecting primary CEC from anoikis. In this study intact CEC crypts were isolated and kept in suspension, a condition which leads to the loss of cell-cell anchorage and induces anoikis. To reconstitute cell-cell contacts, cells were centrifuged to form cell aggregates. Induction of apoptosis was assessed by caspase-3 activity assay; activation of survival pathways was analyzed by Western blot. Immediately after loss of cell anchorage a rapid activation of survival proteins was observed before active caspase-3 could be detected. Src hyperactivation significantly contributed to transient protection from anoikis in CEC because its inhibition reversed the protecting effect of re-establishment of cell contacts. Basal levels of active Src in CEC from patients with inflammatory bowel disease were markedly reduced compared to control patients. These results demonstrate that loss of cell anchorage activates survival pathways in primary human CEC providing transient anoikis protection. Src is an important mediator of this mechanism and therefore constitutes a key regulatory molecule coordinating survival signals mediated by cell adhesion in primary human CEC.

• Analysis of Cd14 as a genetic modifier of experimental inflammatory bowel disease (IBD) in mice.

  Authors: Maike F de Buhr, Hans J Hedrich, Astrid M Westendorf, Florian Obermeier, Claudia Hofmann, Nils-H Zschemisch, Jan Buer, Dirk Bumann, Sanna M Goyert, Andre Bleich

  Inflammatory bowel diseases. 08/2009;

  BACKGROUND AND AIM:: By combining QTL and gene expression analyses, we have previously identified Cd14 as a potential candidate gene contributing to the differential IBD susceptibility of C3H/HeJBirBACKGROUND AND AIM:: By combining QTL and gene expression analyses, we have previously identified Cd14 as a potential candidate gene contributing to the differential IBD susceptibility of C3H/HeJBir (C3/J)-Il10(-/-) mice [carrying IBD-resistance alleles at this QTL (Cdcs6)] and C57BL/6J (B6)-Il10(-/-) mice, corroborating studies that showed an association of a CD14-promoter polymorphism with Crohn's disease and ulcerative colitis. The aim of the present study was to analyze the molecular mechanisms leading to differential intestinal expression of Cd14 and its contribution to IBD development. METHODS:: Intestinal CD14 expression was assessed by FACS, immunohistochemistry, and ELISA on supernatants of primary epithelial cell and tissue cultures. RAW264.7 cells were stimulated with LPS and PGN in the presence or absence of CD14. Cd14 alleles were sequenced and promoters cloned for luciferase assays in transfected RAW264.7 cells. The severity of typhlocolitis between Cd14(-/-) and wild-type mice was compared in 2 distinct mouse models of IBD (acute DSS and Il10(-/-)). RESULTS:: In the gut, CD14 was detected mainly in its soluble form (sCD14), with higher expression in C3/J-Il10(-/-) mice. Polymorphisms in C3/J mice caused higher activity of the Cd14 promoter (luciferase assays). Intestinal sCD14 concentrations influenced the LPS and PGN responses of RAW264.7 cells. In vivo, genetic deletion of Cd14 aggravated colitis in both mouse models of IBD. CONCLUSIONS:: Our study shows that Cd14-promoter polymorphisms affect CD14 expression and confirms the protective effect of CD14 against experimental IBD, potentially mediated by TLR2- and TLR4-dependent effects on intestinal barrier function. These findings support the concept that human CD14-promoter polymorphisms contribute to disease development. Inflamm Bowel Dis 2009.

• Transforming growth factor-beta 1 induces intestinal myofibroblast differentiation and modulates their migration.

  Authors: Julia Brenmoehl, Sandra Nicole Miller, Claudia Hofmann, Daniela Vogl, Werner Falk, Jürgen Schölmerich, Gerhard Rogler

  World journal of gastroenterology : WJG. 04/2009; 15(12):1431-42.

  AIM: To investigate the effects of transforming growth factor beta 1 (TGF-beta 1) on the differentiation of colonic lamina propria fibroblasts (CLPF) into myofibroblasts in vitro. METHODS: PrimaryAIM: To investigate the effects of transforming growth factor beta 1 (TGF-beta 1) on the differentiation of colonic lamina propria fibroblasts (CLPF) into myofibroblasts in vitro. METHODS: Primary CLPF cultures were incubated with TGF-beta 1 and analyzed for production of alpha-smooth muscle actin (alpha-SMA), fibronectin (FN) and FN isoforms. Migration assays were performed in a modified 48-well Boyden chamber. Levels of total and phosphorylated focal adhesion kinase (FAK) in CLPF were analyzed after induction of migration. RESULTS: Incubation of CLPF with TGF-beta 1 for 2 d did not change alpha-SMA levels, while TGF-beta 1 treatment for 6 d significantly increased alpha-SMA production. Short term incubation (6 h) with TGF-beta 1 enhanced CLPF migration, while long term treatment (6 d) of CLPF with TGF-beta 1 reduced migration to 15%-37% compared to untreated cells. FN and FN isoform mRNA expression were increased after short term incubation with TGF-beta 1 (2 d) in contrast to long term incubation with TGF-beta 1 for 6 d. After induction of migration, TGF-beta 1-preincubated CLPF showed higher amounts of FN and its isoforms and lower levels of total and phosphorylated FAK than untreated cells. CONCLUSION: Long term incubation of CLPF with TGF-beta 1 induced differentiation into myofibroblasts with enhanced alpha-SMA, reduced migratory potential and FAK phosphorylation, and increased FN production. In contrast, short term contact (6 h) of fibroblasts with TGF-beta 1 induced a dose-dependent increase of cell migration and FAK phosphorylation without induction of alpha-SMA production.

• Transforming growth factor-1 induces intestinal myofibroblast differentiation and modulates their migration

  Authors: Julia Brenmoehl, Sandra Nicole Miller, Claudia Hofmann, Daniela Vogl, Werner Falk, Jürgen Schölmerich, Gerhard Rogler

  World Journal of Gastroenterology. 01/2009;

  AIM: To investigate the effects of transforming growth factor β1 (TGF-β1) on the differentiation of colonic lamina propria fibroblasts (CLPF) into myofibroblasts in vitro.METHODS: Primary CLPFAIM: To investigate the effects of transforming growth factor β1 (TGF-β1) on the differentiation of colonic lamina propria fibroblasts (CLPF) into myofibroblasts in vitro.METHODS: Primary CLPF cultures were incubated with TGF-β1 and analyzed for production of α-smooth muscle actin (α-SMA), fibronectin (FN) and FN isoforms. Migration assays were performed in a modified 48-well Boyden chamber. Levels of total and phosphorylated focal adhesion kinase (FAK) in CLPF were analyzed after induction of migration.RESULTS: Incubation of CLPF with TGF-β1 for 2 d did not change α-SMA levels, while TGF-β1 treatment for 6 d significantly increased α-SMA production. Short term incubation (6 h) with TGF-β1 enhanced CLPF migration, while long term treatment (6 d) of CLPF with TGF-β1 reduced migration to 15%-37% compared to untreated cells. FN and FN isoform mRNA expression were increased after short term incubation with TGF-β1 (2 d) in contrast to long term incubation with TGF-β1 for 6 d. After induction of migration, TGF-β1-preincubated CLPF showed higher amounts of FN and its isoforms and lower levels of total and phosphorylated FAK than untreated cells.CONCLUSION: Long term incubation of CLPF with TGF-β1 induced differentiation into myofibroblasts with enhanced α-SMA, reduced migratory potential and FAK phosphorylation, and increased FN production. In contrast, short term contact (6 h) of fibroblasts with TGF-β1 induced a dose-dependent increase of cell migration and FAK phosphorylation without induction of α-SMA production.

• Lipid alterations in experimental murine colitis: role of ceramide and imipramine for matrix metalloproteinase-1 expression.

  Authors: Jessica Bauer, Gerhard Liebisch, Claudia Hofmann, Christian Huy, Gerd Schmitz, Florian Obermeier, Jürgen Bock

  PloS one. 01/2009; 4(9):e7197.

  BACKGROUND: Dietary lipids or pharmacologic modulation of lipid metabolism are potential therapeutic strategies in inflammatory bowel disease (IBD). Therefore, we analysed alterations of bioactiveBACKGROUND: Dietary lipids or pharmacologic modulation of lipid metabolism are potential therapeutic strategies in inflammatory bowel disease (IBD). Therefore, we analysed alterations of bioactive lipids in experimental models of colitis and examined the functional consequence of the second messenger ceramide in inflammatory pathways leading to tissue destruction. METHODOLOGY/PRINCIPAL FINDINGS: Chronic colitis was induced by dextran-sulphate-sodium (DSS) or transfer of CD4(+)CD62L(+) cells into RAG1(-/-)-mice. Lipid content of isolated murine intestinal epithelial cells (IEC) was analysed by tandem mass spectrometry. Concentrations of MMP-1 in supernatants of Caco-2-IEC and human intestinal fibroblasts from patients with ulcerative colitis were determined by ELISA. Imipramine was used for pharmacologic inhibition of acid sphingomyelinase (ASM). Ceramide increased by 71% in chronic DSS-induced colitis and by 159% in the transfer model of colitis. Lysophosphatidylcholine (LPC) decreased by 22% in both models. No changes were detected for phosphatidylcholine. Generation of ceramide by exogenous SMase increased MMP-1-protein production of Caco-2-IEC up to 7-fold. Inhibition of ASM completely abolished the induction of MMP-1 by TNF or IL-1beta in Caco-2-IEC and human intestinal fibroblasts. CONCLUSIONS/SIGNIFICANCE: Mucosal inflammation leads to accumulation of ceramide and decrease of LPC in the intestinal epithelium. One aspect of ceramide generation is an increase of MMP-1. Induction of MMP-1 by TNF or IL-1beta is completely blocked by inhibition of ASM with imipramine. Therefore, inhibition of ASM may offer a treatment strategy to reduce MMP-1 expression and tissue destruction in inflammatory conditions.

• CpG Motifs of Bacterial DNA Exert Protective Effects in Mouse Models of IBD by Antigen-Independent Tolerance Induction.

  Authors: Andre Bleich, Lydia M Janus, Anna Smoczek, Astrid M Westendorf, Ulrike Strauch, Michael Mähler, Hans J Hedrich, Stefan Fichtner-Feigl, J U ̈rgen Schölmerich, Werner Falk, Claudia Hofmann, Florian Obermeier

  Gastroenterology. 09/2008;

  BACKGROUND & AIMS: Prophylactic treatment of mice with CpG motifs of bacterial DNA protects from experimental inflammatory bowel disease, at least partly via induction of inhibitory T cells. The aimBACKGROUND & AIMS: Prophylactic treatment of mice with CpG motifs of bacterial DNA protects from experimental inflammatory bowel disease, at least partly via induction of inhibitory T cells. The aim of this study was to elucidate whether these CpG-dependent protective effects require presence of bacterial flora suggesting antigen-specific regulatory activity. METHODS: Germ-free BALB/c and IL-10(-/-) mice were treated with CpG-oligodeoxynucleotides (ODN), control-ODN, or PBS. CD4(+)CD62L(+) cells of these mice were transferred into SCID recipients. CpG-ODN-treated germ-free IL-10(-/-) mice were transferred into colitogenic environment. Monoclonal antibodies were used to neutralize TGF-beta and IFN-alpha/beta during CpG-ODN treatment. CD4(+)CD62L(+) cells of donors were evaluated for cytokine secretion and FOXP3, PD-1, and CD25 expression. RESULTS: Compared to PBS or control-ODN treatment, CpG-ODN application to germ-free donors led to decreased intestinal inflammation as indicated by histology, decreased proinflammatory cytokines, and increased IL-10 secretion. Protection was also observed after cotransfer of cells from PBS and CpG-ODN treated donors. Anti-TGF-beta and anti-INF-alpha/beta partly reversed the protective CpG-ODN effect. CpG-ODN-treated germ-free IL-10(-/-) mice transferred into colitogenic environment developed significantly less colitis than controls but not recipients of IL-10(-/-)CD4(+)CD62L(+)cells. CD4(+)CD62L(+)cells of CpG-treated germ-free animals displayed increased expression of regulatory markers. CONCLUSIONS: Even without pre-existence of bacterial flora CpG-ODN exposition induces tolerance, indicating that CpG-ODN-induced regulatory T cells are not bacterial antigen specific. TGF-beta and IFN-alpha/beta play major roles in induction of regulatory cells, and although IL10-independent mechanisms play a role in CpG-ODN protection, this cytokine likely is important for the effector mechanism of CpG-ODN-induced regulatory T cells.

• Cell-cell contacts prevent anoikis in primary human colonic epithelial cells.

  Authors: Claudia Hofmann, Florian Obermeier, Monika Artinger, Martin Hausmann, Werner Falk, Juergen Schoelmerich, Gerhard Rogler, Johannes Grossmann

  Gastroenterology. 02/2007; 132(2):587-600.

  BACKGROUND & AIMS: Colonic epithelial cells (CECs) receive important survival signals from the extracellular matrix and undergo detachment-induced apoptosis (anoikis) as soon as they lose theirBACKGROUND & AIMS: Colonic epithelial cells (CECs) receive important survival signals from the extracellular matrix and undergo detachment-induced apoptosis (anoikis) as soon as they lose their cell-matrix anchorage. In contrast to the established role of cell-matrix contact, the role of cell-cell contacts as a physiologic survival factor for CECs is less clear. METHODS: Intact CEC crypts gently centrifuged to form a cell aggregate in which cell-cell contacts were maintained. Induction of apoptosis was assessed by Western Blot analysis, colorimetric assays, DNA electrophoresis, 4',6-diamidino-2-phenylindole staining, and flow cytometry. Activation of survival pathways was analyzed by Western blot. The role of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (Erk)1/2, epidermal growth factor receptor, phosphatidylinositol 3-kinase (PI3-K), and Src signaling was investigated using specific inhibitors. RESULTS: Despite a complete loss of cell-matrix adhesion after CEC isolation, activation of caspases was blocked and anoikis was prevented when cell-cell contacts were preserved. CECs with preserved cell-cell contacts exhibited a rapid dephosphorylation of focal adhesion kinase. Aggregated CECs had stable levels of active beta-catenin and phosphorylated Akt, Erk1/2, and epidermal growth factor receptor, but CECs undergoing anoikis rapidly degraded beta-catenin and dephosphorylated Akt. Inhibition of Src- and PI3-K-dependent signaling reversed the antiapoptotic effect of cell-cell contact preservation, while inhibition of the MEK pathway had no effect. CONCLUSIONS: Integrity of cell-cell contacts compensates for the loss of cell-matrix contact-mediated survival signals in CECs and prevents apoptosis. Cell-cell contact-triggered CEC survival involves antiapoptotic signaling through beta-catenin-, Src-, and PI3-K/Akt- but not through MEK- and focal adhesion kinase-dependent pathways.

• CpG motifs of bacterial DNA essentially contribute to the perpetuation of chronic intestinal inflammation.

  Authors: Florian Obermeier, Nadja Dunger, Ulrike G Strauch, Claudia Hofmann, Andre Bleich, Nicole Grunwald, Hans J Hedrich, Elisabeth Aschenbrenner, Brigitte Schlegelberger, Gerhard Rogler, Jürgen Schölmerich, Werner Falk

  Gastroenterology. 10/2005; 129(3):913-27.

  BACKGROUND & AIMS: Recently, we demonstrated a proinflammatory effect of cytosin-guanosin dinucleotide (CpG)-oligodeoxynucleotide (ODN) treatment in established dextran sulphate sodium (DSS)-inducedBACKGROUND & AIMS: Recently, we demonstrated a proinflammatory effect of cytosin-guanosin dinucleotide (CpG)-oligodeoxynucleotide (ODN) treatment in established dextran sulphate sodium (DSS)-induced colitis. Here, we investigated whether DNA derived from luminal bacteria plays a role in the perpetuation of chronic intestinal inflammation. METHODS: Toll-like receptor (TLR9)-deficient and wild-type (wt) control mice were used for the induction of chronic DSS colitis. Moreover, mice with established chronic colitis using different experimental models were treated with adenoviral ODN (AV-ODN) known to block CpG effects. Colonic inflammation was scored and cytokine production was quantified both in colonic tissue and draining mesenteral lymph node cells (MLC). RESULTS: Eight weeks after induction of chronic DSS colitis in TLR9-deficient mice, intestinal inflammation was significantly lower (-68%), and proinflammatory cytokine production was drastically reduced. Treatment of wt mice with chronic DSS-induced colitis with AV-ODN resulted in a significant amelioration of disease with a reduced histologic score (-43%) and reduced cytokine production of MLC (interleukin [IL]-6: -68%; interferon [IFN]-gamma: -48%) and RNA expression of the T helper (Th)1-specific transcription factor T-bet (-62%) in colonic tissue. Qualitatively, the same results were obtained in the severe combined immunodeficiency disease (SCID) transfer model of colitis and in spontaneous colitis in IL-10-deficient mice. CONCLUSIONS: Bacterial DNA derived from luminal bacteria contributes significantly to the perpetuation of chronic intestinal inflammation. Inhibition of the immune-stimulating properties of bacterial DNA using AV-ODN may offer a novel and specific tool for the treatment of inflammatory bowel disease.

• RNA expression profiling of normal and tumor cells following photodynamic therapy with 5-aminolevulinic acid-induced protoporphyrin IX in vitro.

  Authors: Peter J Wild, Rene C Krieg, Juergen Seidl, Robert Stoehr, Kerstin Reher, Claudia Hofmann, Jari Louhelainen, André Rosenthal, Arndt Hartmann, Christian Pilarsky, Anja K Bosserhoff, Ruth Knuechel

  Molecular cancer therapeutics. 05/2005; 4(4):516-28.

  Photodynamic therapy using 5-aminolevulinic acid-induced protoporphyrin IX synthesis as a photosensitizing reagent is an encouraging modality for cancer treatment. Understanding the mechanism ofPhotodynamic therapy using 5-aminolevulinic acid-induced protoporphyrin IX synthesis as a photosensitizing reagent is an encouraging modality for cancer treatment. Understanding the mechanism of tumor phototoxicity is important to provide a basis for combinatory therapy regimens. A normal cell line (UROtsa, urothelial) and two tumor cell lines (RT4, urothelial; HT29, colonic) were treated with cell line-specific LD50 doses of light after exposure to 5-aminolevulinic acid (100 microg/mL), and harvested for RNA extraction 0, 10, and 30 minutes after irradiation. The RNA was hybridized to the metg001A Affymetrix GeneChip containing 2,800 genes, focusing on cancer-related and growth regulatory targets. Comparing the gene expression profiles between the different samples, 40 genes (e.g., SOD2, LUC7A, CASP8, and DUSP1) were identified as significantly altered in comparison with the control samples, and grouped according to their gene ontology. We selected caspase-8 (CASP8) and dual specificity phosphatase 1 (DUSP1) for further validation of the array findings, and compared their expression with the expression of the immediate early gene FOS by quantitative reverse transcription-PCR. RNA expression of CASP8 stayed unchanged whereas DUSP1 RNA was up-regulated in normal and tumor cells starting 30 minutes after irradiation. In contrast, FOS RNA was found continuously up-regulated over time in all three cell lines. Induction of DUSP1 protein expression was clearly shown after 1 hour using Western blot analysis. Interestingly, no changes of caspase-8 protein expression but activation of catalytic activity was detected only in UROtsa cells starting 1 hour after photodynamic therapy, whereas no changes were seen in both tumor cell lines. According to caspase-8, the active caspase 3 fragment was found only in the normal urothelial cell line (UROtsa) 1 hour after photodynamic therapy. Combined data analysis suggests that photodynamic therapy in vitro (LD50) leads to apoptosis in UROtsa and to necrosis in the tumor cell lines, respectively. RNA expression profiling of normal and tumor cell lines following photodynamic therapy with 5-aminolevulinic acid gave insight into the major molecular mechanisms induced by photodynamic therapy.