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.

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.

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.

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.

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.

Neutralization of CD95 ligand promotes regeneration and functional recovery after spinal cord injury.

The clinical outcome of spinal cord injury (SCI) depends in part on the extent of secondary damage, to which apoptosis contributes. The CD95 and tumor necrosis factor (TNF) ligand/receptor systems play an essential role in various apoptotic mechanisms. To determine the involvement of these ligands in SCI-induced damage, we neutralized the activity of CD95 ligand (CD95L) and/or TNF in spinal cord-injured mice. Therapeutic neutralization of CD95L, but not of TNF, significantly decreased apoptotic cell death after SCI. Mice treated with CD95L-specific antibodies were capable of initiating active hind-limb movements several weeks after injury. The improvement in locomotor performance was mirrored by an increase in regenerating fibers and upregulation of growth-associated protein-43 (GAP-43). Thus, neutralization of CD95L promoted axonal regeneration and functional improvement in injured adult animals. This therapeutic strategy may constitute a potent future treatment for human spinal injury.

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 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. 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.

CpG motifs of bacterial DNA exert protective effects in mouse models of IBD by antigen-independent tolerance induction.

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 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.

Caspase 3 inactivates biologically active full length interleukin-33 as a classical cytokine but does not prohibit nuclear translocation.

IL-33 is a member of the IL-1 family of cytokines with dual function which either activates cells via the IL-33 receptor in a paracrine fashion or translocates to the nucleus to regulate gene transcription in an intracrine manner. We show that full length murine IL-33 is active as a cytokine and that it is not processed by caspase 1 to mature IL-33 but instead cleaved by caspase 3 at aa175 to yield two products which are both unable to bind to the IL-33 receptor. Full length IL-33 and its N-terminal caspase 3 breakdown product, however, translocate to the nucleus. Finally, bioactive IL-33 is not released by cells constitutively or after activation. This suggests that IL-33 is not a classical cytokine but exerts its function in the nucleus of intact cells and only activates others cells via its receptor as an alarm mediator after destruction of the producing cell.

IL-1 receptor accessory protein is essential for IL-33-induced activation of T lymphocytes and mast cells.

Lack of the IL-1 receptor accessory protein (IL-1RAcP) abrogates responses to IL-33 and IL-1 in the mouse thymoma clone EL-4 D6/76 cells. Reconstitution with full-length IL-1RAcP is sufficient to restore responsiveness to IL-33 and IL-1. IL-33 activates IL-1 receptor-associated kinase-1, cJun-N-terminal kinase, and the NF-kappaB pathway in an IL-1RAcP-dependent manner and results in IL-2 release. IL-33 is able to induce the release of proinflammatory cytokines in bone marrow-derived (BMD) mast cells, indicating that IL-33 may have a proinflammatory potential like its relatives IL-1 and IL-18, in addition to its Th2-skewing properties in the adaptive response described previously. Blocking of murine IL-1RAcP with the neutralizing antibody 4C5 inhibits response of mouse thymoma cells and BMD mast cells to IL-33. The interaction of either membrane-bound or soluble forms of IL-1RAcP and IL-33Ralpha-chain depends on the presence of IL-33, as demonstrated by coimmunoprecipitation assays. These data demonstrate that IL-1RAcP is indispensable for IL-33 signaling. Furthermore, they suggest that IL-1RAcP is used by more than one alpha-chain of the IL-1 receptor family and thus may resemble a common beta-chain of that family.

Lipopolysaccharide-trap-Fc, a multifunctional agent to battle gram-negative bacteria.

The family of Toll-like receptors (TLRs) plays a pivotal role in host defense against pathogens. However, overstimulation of these receptors may lead to uncontrolled general inflammation and eventually to systemic organ dysfunction or failure. With the intent to control overwhelming inflammation during gram-negative bacterial sepsis, we constructed soluble fusion proteins of the lipopolysaccharide (LPS)-receptor complex to modulate TLR signaling in multiple ways. The extracellular domain of mouse TLR4 and mouse myeloid differentiation factor 2 (MD-2) fusions (LPS-Trap) were linked to human immunoglobulin G Fc domains (LPS-Trap-Fc). In addition to the ability to bind LPS or gram-negative bacteria and to inhibit interleukin-6 secretion of monocytic cells after LPS treatment, LPS-Trap-Fc was able to opsonize fluorescent Escherichia coli particles. This led to enhancement of phagocytosis by monocytic cells which was strictly dependent on the presence of the Fc region. Moreover, only LPS-Trap-Fc- and not LPS-Trap-coated bacteria were sensitized to complement killing. Therefore, LPS-Trap-Fc not only neutralizes LPS but also, after binding to bacteria, enhances phagocytosis and complement-mediated killing and could thus act as a multifunctional agent to fight gram-negative bacteria in vivo.

Fatty acid-induced induction of Toll-like receptor-4/nuclear factor-kappaB pathway in adipocytes links nutritional signalling with innate immunity.

To study the effects of fatty acids and the involvement of the Toll-like receptor-4/nuclear factor-kappaB (TLR-4/NF-kappaB) pathway with respect to the secretion of adipokines from adipocytes 3T3-L1 adipocytes were stimulated with increasing doses of fatty acids. The secretion of adiponectin, resistin and monocyte chemoattractant protein-1 (MCP-1) was measured by enzyme-linked immunosorbent assay. The NF-kappaB p65 nuclear translocation and TLR-4 expression were investigated by Western blot. The effects mediated by NF-kappaB were tested using a specific NF-kappaB-inhibitor and TLR-4-induced effects were analysed with a neutralizing TLR-4 antibody. Binding of (14)C-labelled fatty acids to TLR-4/MD-2 was investigated using a FLAG-tagged extracellular part of TLR-4 fused to full-length MD-2 via a linker (lipopolysaccharide-Trap). The messenger RNA (mRNA) expression of adipokines in abdominal adipose tissue of rats fed a standard chow or a high-fat diet was investigated by reverse transcription-polymerase chain reaction. The TLR-4 is induced during adipocyte differentiation and its expression is enhanced following fatty acid stimulation. The stimulatory effects of stearic and palmitic acids on MCP-1 secretion and of palmitoleic acid on resistin secretion are mediated via NF-kappaB. The stimulatory effects of stearic, palmitic and palmitoleic acids on resistin secretion and the stimulatory effect of stearic acid on MCP-1 secretion are mediated via TLR-4. Fatty acid-mediated effects are caused by an endogenous ligand because fatty acids were shown not to bind directly to TLR-4/MD-2. Adipose tissue mRNA expression and serum levels of adipokines did not differ in rats fed a high-fat diet. These data provide a new molecular mechanism by which fatty acids can link nutrition with innate immunity.

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.

A TLR4/MD2 fusion protein inhibits LPS-induced pro-inflammatory signaling in hepatic stellate cells.

Activated hepatic stellate cells (HSCs) play a key role in hepatic fibrogenesis. In injured liver they are the main extracellular matrix protein producing cell type and further perpetuate hepatic injury by secretion of pro-inflammatory mediators. Since LPS-mediated signaling through toll-like receptor 4 (TLR4) has been identified as key fibrogenic signal in HSCs we aimed to test TLR4 as potential target of therapy via ligand-binding soluble receptors. Incubation of human HSCs with a fusion protein between the extracellular domain of TLR4 and MD2 which binds LPS inhibited LPS-induced NFkappaB and JNK activation. TLR4/MD2 abolished LPS-induced secretion of IL-6, IL-8, MCP1, and RANTES in HSCs. In addition, TLR4/MD2 fused to human IgG-Fc neutralized LPS activity. Since TLR4 mutant mice are resistant to liver fibrosis, the TLR4/MD2 soluble receptor might represent a new therapeutic molecule for liver fibrogenesis in vivo.

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.

13-Oxo-ODE is an endogenous ligand for PPARgamma in human colonic epithelial cells.

BACKGROUND: The ligand activated nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma) induces transcriptional repression of pro-inflammatory factors. Activation of PPARgamma is followed by amelioration of colitis in animal models of inflammatory bowel disease (IBD). A reduced expression of PPARgamma was found in epithelial cells of patients with ulcerative colitis. The eicosanoids 13-HODE and 15-HETE are products of 12/15-lipoxygenase (LOX) and endogenous ligands for PPARgamma. Dehydrogenation of 13-HODE by 13-HODE dehydrogenase results in formation of the 13-Oxo-ODE. Highest activity of 13-HODE dehydrogenase is found in colonic epithelial cells (CECs). We therefore investigated whether 13-Oxo-ODE is a new endogenous ligand of PPARgamma in CECs. METHODS: LOX activity and 13-HODE dehydrogenase in CECs were investigated after stimulation with arachidonic or linoleic acid. LOX metabolites were identified by RP-18 reversed-phase HPLC. Binding of (14)C-labelled 13-Oxo-ODE was demonstrated using a His-tagged PPARgamma. RESULTS: Stimulation of HT-29 and primary CECs homogenates with and without Ca-ionophor was followed by the formation of high amounts of the linoleic acid metabolite 13-Oxo-ODE (155 and 85 ng/ml). The decrease of IL-8 secretion from IEC was more pronounced after pre-incubation with 13-Oxo-ODE compared to the PPARgamma agonist troglitazone and higher as with the known PPARgamma ligands 13-HODE and 15-HETE. Binding assays with (14)C-labelled 13-Oxo-ODE clearly demonstrated a direct interaction. CONCLUSION: High amounts of 13-Oxo-ODE can be induced in CECs by stimulation of linoleic acid metabolism. 13-Oxo-ODE binds to PPARgamma and has anti-inflammatory effects. 13-HODE dehydrogenase might be a therapeutic target in IBD.

Interleukin-10 does not affect IL-1-induced interleukin-6 and metalloproteinase production in human chondrosarcoma cells, SW1353.

Cartilage repair by transplantation of autologous chondrocytes is an option when restoring functional joints. Control of chondrocyte function is thus required. Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine affecting the expression of a wide range of immune mediators in hematopoietic and non-hematopoietic cells. Previous studies indicated that IL-10 has therapeutic potential in the treatment of chronic inflammatory joint disorders such as rheumatoid arthritis and osteoarthritis. IL-10 has been found to be chondroprotective by down-regulating metalloproteinase expression and by inhibiting the synthesis of pro-inflammatory cytokines, such as IL-6, in immune cells. In contrast, the effects of IL-10 on chondrocytes are poorly understood and have to be identified with regard to their future clinical use. In this study, we investigated the effects of IL-10 on the expression of cartilage-degrading mediators in the human chondrosarcoma cell line, SW1353, after exposure to IL-1, a key mediator in cartilage and bone destruction. We found a strong induction of the pro-inflammatory cytokine, IL-6, in IL-1-exposed SW1353 cells. Surprisingly, IL-10 had no effect on IL-1-induced IL-6, pro-MMP1, and pro-MMP13 secretion. Although RT-PCR analyses demonstrated the expression of both receptor chains of the IL-10 receptor complex (IL-10R1 and IL-10R2), exposure of SW1353 to IL-10 did not lead to phosphorylation of STAT3, the major transcription factor induced by IL-10. This was not due to a defect in STAT3, because stimulation with IL-6 resulted in its phosphorylation. Failure of SW1353 cells to respond to IL-10 was consistent with a deficient surface expression of IL-10R1. From these results we conclude that IL-10 does not exert its chondroprotective character on chondrocytes directly. Furthermore, the unresponsiveness of chondrocytes towards IL-10 might explain the vulnerability of joint cartilage to inflammation.

p38MAPK mediates IL-1-induced down-regulation of aggrecan gene expression in human chondrocytes.

The pleiotropic cytokine interleukin 1 (IL-1) is considered to be the principal inducer of mediators of cartilage degradation in both, osteoarthritis (OA) and rheumatoid arthritis (RA). IL-1 activates numerous signaling pathways involved in cartilage destruction and dedifferentiation of chondrocytes. In this study, we analyzed expression and functional effects of IL-1 in human chondrocytes. We found an IL-1-induced reduction in the expression of the cartilage specific proteoglycan aggrecan as an indicator for the IL-1-mediated dedifferentiation of chondrocytes. To block the IL-1-induced signaling pathways specifically, we incubated human chondrocytes and cartilage explants with IL-1 in the presence of different signal transduction inhibitors and analyzed their effect on aggrecan mRNA expression and IL-6 secretion. IL-6 has been found to act synergistically in the IL-1-induced suppression of the proteoglycan synthesis in chondrocytes. Our results led to the identification of p38MAPK and/or PI3K/JNK as being crucial for IL-1-induced IL-6 secretion by chondrocytes. IL-1-induced down-regulation of aggrecan expression was found to be mediated by p38MAPK and/or ERK1/2. The identification and characterization of these signaling pathways will enable us to develop new modulation strategies for therapeutic use in inflammatory joint diseases.

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.