A refined and translationally relevant model of chronic DSS colitis in BALB/c mice.

Inflammatory bowel diseases (IBD) are chronic relapsing disorders of the gastrointestinal tract. Several mouse models for IBD are available, but the acute dextran sulfate sodium (DSS)-induced colitis model is mostly used for preclinical studies. However, this model lacks chronicity and often leads to significant loss of mice. The aim of this study was to establish a refined and translationally relevant model of DSS chronic colitis in BALB/c mice. In the first part, we compared several standard therapeutic (ST) treatments for IBD in the acute DSS colitis model to identify the optimal treatment control for a DSS colitis model as compared to literature data. In the second part, we tested the two most effective ST treatments in a refined model of chronic DSS colitis. Cyclosporine A (CsA) and 6-thioguanine (6-TG) caused considerable reduction of clinical scores in acute DSS colitis. The clinical outcome was confirmed by the results for colon length and by histopathological evaluation. Moreover, CsA and 6-TG considerably reduced mRNA expression of several pro-inflammatory cytokines in spleen and colon. Both compounds also showed a substantial therapeutic effect in the refined model of chronic DSS colitis with regard to clinical scores and histopathology as well as the expression of inflammatory markers. The refined model of chronic DSS colitis reflects important features of IBD and is well suited to test potential IBD therapeutics.

NLRP3 tyrosine phosphorylation is controlled by protein tyrosine phosphatase PTPN22.

Inflammasomes form as the result of the intracellular presence of danger-associated molecular patterns and mediate the release of active IL-1ß, which influences a variety of inflammatory responses. Excessive inflammasome activation results in severe inflammatory conditions, but physiological IL-1ß secretion is necessary for intestinal homeostasis. Here, we have described a mechanism of NLRP3 inflammasome regulation by tyrosine phosphorylation of NLRP3 at Tyr861. We demonstrated that protein tyrosine phosphatase non-receptor 22 (PTPN22), variants in which are associated with chronic inflammatory disorders, dephosphorylates NLRP3 upon inflammasome induction, allowing efficient NLRP3 activation and subsequent IL-1ß release. In murine models, PTPN22 deficiency resulted in pronounced colitis, increased NLRP3 phosphorylation, but reduced levels of mature IL-1ß. Conversely, patients with inflammatory bowel disease (IBD) that carried an autoimmunity-associated PTPN22 variant had increased IL-1ß levels. Together, our results identify tyrosine phosphorylation as an important regulatory mechanism for NLRP3 that prevents aberrant inflammasome activation.

Identification of critical regions within the TIR domain of IL-1 receptor type I.

Interleukin-1 receptor type I (IL-1RI) belongs to a superfamily of proteins characterized by an intracellular Toll/IL-1 receptor (TIR) domain. This domain harbors three conserved regions called boxes 1-3 that play crucial roles in mediating IL-1 responses. Boxes 1 and 2 are considered to be involved in binding of adapter molecules. Amino acids possibly crucial for IL-1RI signaling were predicted via homology models of the IL-1RI TIR domain based on the crystal structure of IL-1RAPL. The role of ten of these residues was investigated by site-directed mutagenesis and a functional luciferase assay reflecting NF-κB activity in transiently transfected Jurkat cells. In particular, the mutants E437K/D438K, E472A/E473A and S465A/S470A/S471A/E472A/E473A showed decreased and the mutant E437A/D438A increased IL-1 responsiveness compared to the mouse IL-1RI wild type. In conclusion, the αC' helix (Q469-E473 in mouse IL-1RI) is probably involved in heterotypic interactions of IL-1RI with IL-1RAcP or MyD88.

Galectin-3 Modulates Experimental Colitis.

BACKGROUND: We recently identified galectin-3 (gal-3) as a new and strong fibroblast activator produced by colonic epithelial cells. Very little is known about the influence of gal-3 in inflammatory bowel disease. We, therefore, investigated the impact of gal-3 on dextran sodium sulfate (DSS)-induced colitis in a mouse model. METHODS: Colonic lamina propria fibroblasts of healthy controls were used for co-incubation studies of gal-3 with gal-1, TGF-ß1, IFNγ, IL-4 and IL-10. Acute and chronic DSS colitis was induced by 3% DSS in drinking water in female Balb/c mice weighing 20-22 g. Recombinant gal-3 was expressed by the pET vector system and used for a 5-day treatment in different concentrations intraperitoneally. The distal third of the colon was used for histologic analysis. Colonic cytokine expression was determined by quantitative RT-PCR. RESULTS: In vitro, gal-3 induced IL-8 secretion was significantly reduced by co-incubation with IL-10 (5 ng/ml) and IL-4 (10 ng/ml). Acute DSS-induced colitis was ameliorated by gal-3 treatment as indicated by increased colonic length and reduced weight loss compared to that of controls. In acute and chronic colitis, gal-3 treatment resulted in a significant suppression of colonic IL-6. CONCLUSION: Gal-3 significantly reduces inflammation in acute and chronic DSS colitis in mice indicating a potential role in intestinal inflammation.

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 Crohn's disease. METHODS: To study immuno-modulating effects of gp96 in T-cell transfer colitis BALB/c donor mice were injected with 2 × 100 µg gp96. After 1 week, 2.5 × 10(5) 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 µg 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 CD4CD62L 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 Crohn's disease might provoke loss of this tolerance mediating mechanism.

Influence of natural killer cells and perforin­mediated cytolysis on the development of chemically induced lung cancer in A/J mice.

One alternative approach for the treatment of lung cancer might be the activation of the immune system using vaccination strategies. However, most of clinical vaccination trials for lung cancer did not reach their primary end points, suggesting that lung cancer is of low immunogenicity. To provide additional experimental information about this important issue, we investigated which type of immune cells contributes to the protection from lung cancer development. Therefore, A/J mice induced for lung adenomas/ adenocarcinomas by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were depleted of CD4+ or CD8+ T cells, CD11b+ macrophages, Gr-1+ neutrophils and asialo GM1+ natural killer (NK) cells. Subsequent analysis of tumour growth showed an increase in tumour number only in mice depleted of NK cells. Further asking by which mechanism NK cells suppressed tumour development, we neutralized several death ligands of the tumour necrosis factor (TNF) family known to be involved in NK cell-mediated cytotoxicity. However neither depletion of TNF-α, TNF-related apoptosis-inducing ligand, TNF-like weak inducer of apoptosis or FasL alone nor in combination induced an augmentation of tumour burden. To show whether an alternative cell death pathway is involved, we next generated A/J mice deficient for perforin. After challenging with NNK, mice deficient for perforin showed an increase in tumour number and volume compared to wild-type A/J mice. In summary, our data suggest that NK cells and perforin-mediated cytolysis are critically involved in the protection from lung cancer giving promise for further immunotherapeutic strategies for this disease.

Physiologic TLR9-CpG-DNA interaction is essential for the homeostasis of the intestinal immune system.

BACKGROUND: Cytosine-guanosine dinucleotide (CpG) motifs are immunostimulatory components of bacterial DNA and activators of innate immunity through Toll-like receptor 9 (TLR9). Administration of CpG oligodeoxynucleotides before the onset of experimental colitis prevents intestinal inflammation by enforcement of regulatory mechanisms. It was investigated whether physiologic CpG/TLR9 interactions are critical for the homeostasis of the intestinal immune system. METHODS: Mesenteric lymph node cell and lamina propria mononuclear cell (LPMC) populations from BALB/c wild-type (wt) or TLR9 mice were assessed by flow cytometry and proteome profiling. Cytokine secretion was determined and nuclear extracts were analyzed for nuclear factor kappa B (NF-κB) and cAMP response-element binding protein activity. To assess the colitogenic potential of intestinal T cells, CD4-enriched cells from LPMC of wt or TLR9 donor mice were injected intraperitoneally in recipient CB-17 SCID mice. RESULTS: TLR9 deficiency was accompanied by slight changes in cellular composition and phosphorylation of signaling proteins of mesenteric lymph node cell and LPMC. LPMC from TLR9 mice displayed an increased proinflammatory phenotype compared with wt LPMC. NF-κB activity in cells from TLR9 mice was enhanced, whereas cAMP response-element binding activity was reduced compared with wt. Transfer of lamina propria CD4-enriched T cells from TLR9 mice induced severe colitis, whereas wt lamina propria CD4-enriched T cells displayed an attenuated phenotype. CONCLUSIONS: Lack of physiologic CpG/TLR9 interaction impairs the function of the intestinal immune system indicated by enhanced proinflammatory properties. Thus, physiologic CpG/TLR interaction is essential for homeostasis of the intestinal immune system as it is required for the induction of counterregulating anti-inflammatory mechanisms.

Yip1 domain family, member 6 (Yipf6) mutation induces spontaneous intestinal inflammation in mice.

Using an environmentally sensitized genetic screen we identified mutations that cause inflammatory colitis in mice. The X-linked Klein-Zschocher (KLZ) mutation created a null allele of Yipf6, a member of a gene family believed to regulate vesicular transport in yeast, but without known functions in mammals. Yipf6 is a five transmembrane-spanning protein associated with Golgi compartments. Klein-Zschocher mutants were extremely sensitive to colitis induced by dextran sodium sulfate (DSS) and developed spontaneous ileitis and colitis after 16 mo of age in specific pathogen-free housing conditions. Electron microscopy, gene expression, and immunocytochemistry analyses provided evidence that impaired intestinal homeostasis stemmed from defective formation and secretion of large secretory granules from Paneth and goblet cells. These studies support a tissue- and organ-specific function for Yipf6 in the maintenance of intestinal homeostasis and implicate the orthologous human gene as a disease susceptibility locus.

(-)-Epigallocatechin-3-gallate, a green tea-derived catechin, synergizes with celecoxib to inhibit IL-1-induced tumorigenic mediators by human pancreatic adenocarcinoma cells Colo357.

Despite their toxic side effects prostaglandin H(2) synthase-2 (PGHS-2) inhibitors hold promise for cancer chemoprevention. In order to overcome adverse effects lower doses of PGHS-2 inhibitors could be applied in combination with other agents exhibiting complementary effects. Herein, the effects of the PGHS-2-specific inhibitor celecoxib either alone or in combination with the green tea-derived catechin (-)-epigallocatechin-3-gallate (EGCG) were studied on the expression of interleukin (IL)-1-induced tumorigenic factors in Colo357 human pancreatic adenocarcinoma cells. This approach mimics tumor-associated pancreatic inflammation which is considered as a key player in pancreatic malignancy. We found that co-incubation of Colo357 with celecoxib and EGCG synergistically diminished metabolic activity via apoptosis induction and down-regulated release of pro-angiogenic vascular endothelial growth factor (VEGF) and invasiveness-promoting matrix metalloproteinase (MMP)-2 to a maximum of 30%. Celecoxib and EGCG synergistically reduced IL-1-induced production of pro-inflammatory IL-6 and pro-angiogenic IL-8 to 23-50%. Celecoxib dose-dependently increased PGHS-2 levels. Whereas EGCG was able to compensate for celecoxib-mediated increase of PGHS-2, it failed to potentiate celecoxib-mediated suppression of prostaglandin E(2) (PGE(2)) release. Thus, in Colo357, EGCG synergistically boosts celecoxib-mediated effects and reduces the levels of celecoxib required to elicit beneficial effects on tumorigenic mediators by a factor of ten.

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

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 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.

Intracellular survival of Staphylococcus aureus in adipocyte-like differentiated 3T3-L1 cells is glucose dependent and alters cytokine, chemokine, and adipokine secretion.

Although obesity and type 2 diabetes mellitus are associated with Gram-positive infections and a worse clinical outcome, it is unknown whether adipocytes can be infected by Gram-positive bacteria. Adipocyte-like differentiated 3T3-L1 cells and Staphylococcus aureus were used for infection experiments under normoglycemic (100 mg/dl) and hyperglycemic (450 mg/dl) conditions in the presence/absence of insulin (1 µm). Intracellular presence and survival of S. aureus was investigated quantitatively. Supernatant cytokines, chemokines, and adipokines were measured by ELISA. Lipid metabolism and cellular morphology of infected adipocytes were investigated by different techniques. The present study provides the proof of principle that adipocyte-like cells can be infected by S. aureus dose dependently for up to 5 d. Importantly, low bacterial inocula did not affect cell viability. Intracellular survival of S. aureus was glucose dependent but not insulin dependent, and insulin receptor expression and insulin receptor signaling were not altered. Infection increased macrophage chemoattractant protein-1, visfatin, and IL-6 secretion, whereas resistin and adiponectin were decreased. Infected adipocytes had higher intracellular triacylglycerol concentrations and larger lipid droplets because of a decreased lipolysis. Taken together, infection of adipocytes by S. aureus is glucose dependent, inhibits cellular lipolysis, and affects the secretion of immunomodulating adipokines differentially. Because cell viability is not affected during infection, adipose tissue might function as a host for chronic infection by bacteria-causing metabolic, proinflammatory, and prodiabetic disturbances.

Interleukin-33 prolongs allograft survival during chronic cardiac rejection.

Interleukin-33 (IL-33) stimulates the generation of cells and cytokines characteristic of a Th2 immune response. We examined the effects of IL-33 on allografted heart tissue in a chronic cardiac rejection model, including analysis of the peripheral myeloid and lymphoid compartments. B6.C-H2bm12/KhEg hearts were transplanted into MHC class II-mismatched C57Bl/6J mice; IL-33 was administered daily. Cells from allografts and spleens were isolated for flow cytometry and cultured for cytokine production; some tissues were used for immunohistochemistry. Animals treated with IL-33 showed significantly longer allograft survival, which was associated with a distinct cytokine profile produced by graft-infiltrating cells. Proinflammatory IL-17A production was decreased with IL-33 treatment, while increased levels of IL-5, IL-10, and IL-13 were observed. After IL-33 therapy, flow cytometry showed a direct induction of CD4(+) Foxp3(+) Treg, whereas the number of B220(+) CD19(+) B cells, and circulating, as well as allograft deposited, alloantibodies was reduced. Following IL-33 treatment, a significant decrease in graft-infiltrating CD11b(high) Gr1(high) granulocytes coincided with a significant increase in CD11b(high) Gr1(intermediate) myeloid-derived suppressor cells (MDSC). In conclusion, IL-33 treatment in the setting of chronic rejection promotes the development of a Th2-type immune response that favors MDSC and Treg expansion, reduces antibody-mediated rejection (AMR), and ultimately, prolongs allograft survival.

EGCG downregulates IL-1RI expression and suppresses IL-1-induced tumorigenic factors in human pancreatic adenocarcinoma cells.

Human pancreatic cancer is currently one of the fifth-leading causes of cancer-related mortality with a 5-year survival rate of less than 5%. Since pancreatic carcinoma is largely refractory to conventional therapies, there is a strong medical need for the development of novel and innovative therapeutic strategies. Increasing evidence suggests an association of carcinogenesis and chronic inflammation. Because IL-1 plays a crucial role in inflammation-associated carcinogenesis, we analyzed the biological effects of IL-1 and its modulation by the chemopreventive green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) in the human pancreatic adenocarcinoma cell line Colo357. Proinflammatory IL-6 and PGHS-2 as well as proangiogenic IL-8 and VEGF were induced by IL-1, whereas the secretion of invasion-promoting MMP-2 remained unaffected. IL-1 responsiveness and constitutive MMP-2 release in Colo357 were downregulated by EGCG in a dose- and time-dependent manner. Moreover, EGCG reduced cell viability via induction of apoptosis in Colo357. Since EGCG effects on cytokine production precede reduction in cell viability, we hypothesize that these findings are not only a result of cell death but also depend on alterations in the IL-1 signaling cascade. In this context, we found for the first time an EGCG-induced downregulation of the IL-1RI expression possibly being caused by NF-κB inhibition and causative for its inhibitory action on the production of tumorigenic factors. Thus, our data might have future clinical implications with respect to the development of novel approaches as an adjuvant therapy in high-risk patients with human pancreatic carcinoma.

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.

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 Crohn's 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-ß 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-ß-CTGF-collagen I pathway.

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

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 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-ß (GSK3-ß) 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.

Glycogen synthase kinase 3-ß: 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-ß (GSK3-ß) 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-ß 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-ß 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. RESULTS: GSK3-ß blockade significantly reduced chronic intestinal inflammation and even abolished the colitis-intensifying effects of CpG-ODN treatment. In vitro inhibition of GSK3-ß reduced the proinflammatory phenotype of both murine and human intestinal immune cells from chronic inflamed tissue. In vivo blockade of GSK3-ß resulted in a shift from NF-κB activity toward CREB activity in murine MLC and LPMC. CONCLUSIONS: Blockade of GSK3-ß attenuates excessive proinflammatory TLR-mediated immune responses. GSK3-ß inhibition therefore constitutes a promising therapeutic option for selectively reducing exaggerated intestinal immune reactions toward the luminal flora in inflammatory bowel disease.

C1q/TNF-related protein-3 represents a novel and endogenous lipopolysaccharide antagonist of the adipose tissue.

Proteins secreted by adipocytes (adipokines) play an important role in the pathophysiology of type 2 diabetes mellitus and the associated chronic and low-grade state of inflammation. It was the aim to characterize the antiinflammatory potential of the new adipocytokine, C1q/TNF-related protein-3 (CTRP-3), which shows structural homologies to the pleiotropic adipocytokine adiponectin. mRNA and protein expression of CTRP-3 was analyzed by RT-PCR and Western blot. Recombinant CTRP-3 and small interfering RNA-based strategies were used to investigate the effect of CTRP-3 on toll-like receptor (TLR) ligand, lipopolysaccharide (LPS)-, and lauric acid-induced chemokine release of monocytes and adipocytes. Together with complex ELISA-based techniques, a designed TLR4/myeloid differentiation protein-2 fusion molecule shown to bind LPS was used to prove the ability of CTRP-3 to act as endogenous LPS antagonist. CTRP-3 is synthesized in monocytes and adipocytes. The recombinant protein dose-dependently inhibits the release of chemokines in monocytes and adipocytes that were induced by lauric acid, LPS, and other TLR ligands in vitro and ex vivo. CTRP-3 inhibits monocyte chemoattractant protein-1 release in adipocytes, whereas small interfering RNA-mediated knockdown of CTRP-3 up-regulates monocyte chemoattractant protein-1 release, reduces lipid droplet size, and decreases intracellular triglyceride concentration in adipocytes, causing a dedifferentiation into a more proinflammatory and immature phenotype. By using a designed TLR4/MD-2 fusion molecule, it is shown by different techniques that CTRP-3 specifically and effectively inhibits the binding of LPS to its receptor, TLR4/MD-2. CTRP-3 inhibits three basic and common proinflammatory pathways involved in obesity and type 2 diabetes mellitus (adipo-inflammation) by acting as an endogenous LPS antagonist of the adipose tissue.