Claudia Hofmann

Glycogen synthase kinase 3‐β: A master regulator of toll‐like receptor‐mediated chronic intestinal inflammation

Background: 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 established chronic colitis. Glycogen synthase kinase 3‐&bgr; (GSK3‐&bgr;) 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‐&bgr; activity in chronic intestinal inflammation. Methods: Chronic colitis was induced by dextran sodium sulfate (DSS) treatment. Mice were treated intraperitoneally with phosphate‐buffered saline (PBS), CpG‐ODN, or GSK3‐&bgr; 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‐&kgr;B) 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. Results: GSK3‐&bgr; blockade significantly reduced chronic intestinal inflammation and even abolished the colitis‐intensifying effects of CpG‐ODN treatment. In vitro inhibition of GSK3‐&bgr; reduced the proinflammatory phenotype of both murine and human intestinal immune cells from chronic inflamed tissue. In vivo blockade of GSK3‐&bgr; resulted in a shift from NF‐&kgr;B activity toward CREB activity in murine MLC and LPMC. Conclusions: Blockade of GSK3‐&bgr; attenuates excessive proinflammatory TLR‐mediated immune responses. GSK3‐&bgr; inhibition therefore constitutes a promising therapeutic option for selectively reducing exaggerated intestinal immune reactions toward the luminal flora in inflammatory bowel disease. (Inflamm Bowel Dis 2010)

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

Background: 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 the antiinflammatory and antifibrotic role of CTRP‐3 in Crohns disease (CD). Methods: 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. Results: 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‐&bgr; production and the expression of CTGF and collagen I in CLPF, whereas collagen III expression remained unchanged. Conclusions: CTRP‐3 exerts potent antiinflammatory and antifibrotic effects in CLPF by antagonizing the LPS pathway and by targeting the TGF‐&bgr;–CTGF–collagen I pathway.

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

BACKGROUND & AIMSnProphylactic 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.nnnMETHODSnGerm-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.nnnRESULTSnCompared 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.nnnCONCLUSIONSnEven 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.

Bilberries and their anthocyanins ameliorate experimental colitis

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% 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.

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

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/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 Crohns 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

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

Background 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. Aims To identify the interplay between innate and adaptive immune cells finally leading to protective CpG-ODN effects in intestinal inflammation. Methods 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 5u2005days 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. Results 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. Conclusions 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.

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

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 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.

Impact of Toll-like-receptor-9 (TLR9) Deficiency on Visceral Adipose Tissue Adipokine Expression during Chronic DSS-induced Colitis in Mice

BACKGROUNDnStudies postulate an involvement of adipokines in inflammatory gastrointestinal diseases. Leptin-deficient ob/ob mice as well as TLR9-deficient mice have a more moderate course of chronic DSS-induced colitis (DSS-CC) and adipocytes do express functional TLR9 molecules.nnnMATERIAL AND METHODSnAdipokine mRNA expression in visceral adipose tissue of mice before and after the induction of DSS-CC was investigated. Experiments were performed in both TLR9(wt/wt) and TLR9(-/-) mice. In vitro, the effect of TLR9 blocking peptide on leptin and visfatin protein secretion was studied in 3T3-L1 adipocytes.nnnRESULTSnInduction of DSS-CC led to an upregulation of leptin mRNA expression in TLR9(wt/wt) mice, while TLR9(-/-) animals showed a significant reduction of leptin expression even below baseline. While visfatin expression remained unchanged in TLR9(wt/wt) animals, TLR9(-/-) mice exhibited a significant induction during DSS-CC. CTRP-3 expression was reduced after colitis induction only in TLR9(-/-) animals. Of note, IL-6 expression levels remained unchanged, while CXCL1/KC and cyclophilin A expression was reduced in DSS-CC. Inhibition of TLR9 signaling by using TLR9 blocking peptide led to reduced leptin protein secretion into cell culture supernatants in 3T3-L1 adipocytes, while visfatin protein secretion was enhanced.nnnCONCLUSIONSnDSS-CC leads to differential adipokine expression profiles in the visceral fat pad in TLR9(wt/wt) vs. TLR9(-/-) mice. In vitro, inhibition of TLR9 signaling induces visfatin secretion while inhibiting leptin secretion in adipocytes. Thus, visceral adipokines are regulated by intact TLR9 signaling pathway and a specific interplay between the leptin- and the TLR9-pathways might be of pathophysiological importance in chronic intestinal inflammation.

Leitkultur Meets German Angst: On the Role of Values and Needs in the German Debate on the Integration of Refugees and Migrants

The term ‘culture’ is an open one. Various meanings can be attached to it. This chapter focuses on the connection of certain aspects of ‘culture’ to what is perceived as individual and collective identity. The example of a German ‘guiding culture’ serves as a point of departure to address the following questions: First, how can ‘culture’ in general be defined, and how is it linked to what is perceived as individual or collective identity? Second, do we need policies defending a particular culture, and (how) can a ‘guiding culture’ be justified? Third, what has—international and national—law got to do with it? Fourth, what lies beneath cultural defence politics, and which lessons can be drawn from a more thorough understanding of these underlying motives? It is suggested to add a needs-based view to the debate about the integration of migrants and refugees in a society.