Stephanie Kasper

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-1β, which influences a variety of inflammatory responses. Excessive inflammasome activation results in severe inflammatory conditions, but physiological IL-1β 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-1β release. In murine models, PTPN22 deficiency resulted in pronounced colitis, increased NLRP3 phosphorylation, but reduced levels of mature IL-1β. Conversely, patients with inflammatory bowel disease (IBD) that carried an autoimmunity-associated PTPN22 variant had increased IL-1β levels. Together, our results identify tyrosine phosphorylation as an important regulatory mechanism for NLRP3 that prevents aberrant inflammasome activation.

Activation of protein tyrosine phosphatase non-receptor type 2 by spermidine exerts anti-inflammatory effects in human THP-1 monocytes and in a mouse model of acute colitis.

Background Spermidine is a dietary polyamine that is able to activate protein tyrosine phosphatase non-receptor type 2 (PTPN2). As PTPN2 is known to be a negative regulator of interferon-gamma (IFN-γ)-induced responses, and IFN-γ stimulation of immune cells is a critical process in the immunopathology of inflammatory bowel disease (IBD), we wished to explore the potential of spermidine for reducing pro-inflammatory effects in vitro and in vivo. Methods Human THP-1 monocytes were treated with IFN-γ and/or spermidine. Protein expression and phosphorylation were analyzed by Western blot, cytokine expression by quantitative-PCR, and cytokine secretion by ELISA. Colitis was induced in mice by dextran sodium sulfate (DSS) administration. Disease severity was assessed by recording body weight, colonoscopy and histology. Results Spermidine increased expression and activity of PTPN2 in THP-1 monocytes and reduced IFN-γ-induced phosphorylation of signal transducer and activator of transcription (STAT) 1 and 3, as well as p38 mitogen-activated protein kinase (MAPK) in a PTPN2 dependent manner. Subsequently, IFN-γ-induced expression/secretion of intracellular cell adhesion molecule (ICAM)-1 mRNA, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 was reduced in spermidine-treated cells. The latter effects were absent in PTPN2-knockdown cells. In mice with DSS-induced colitis, spermidine treatment resulted in ameliorated weight loss and decreased mucosal damage indicating reduced disease severity. Conclusions Activation of PTPN2 by spermidine ameliorates IFN-γ-induced inflammatory responses in THP-1 cells. Furthermore, spermidine treatment significantly reduces disease severity in mice with DSS-induced colitis; hence, spermidine supplementation and subsequent PTPN2 activation may be helpful in the treatment of chronic intestinal inflammation such as IBD.

Deficiency of Protein Tyrosine Phosphatase Non-Receptor Type 2 in Intestinal Epithelial Cells Has No Appreciable Impact on Dextran Sulphate Sodium Colitis Severity But Promotes Wound Healing.

Background/Aims: The protein tyrosine phosphatase non-receptor type 2 (PTPN2) is known to mediate susceptibility to inflammatory bowel diseases. Cell culture experiments suggest that PTPN2 influences barrier function, autophagy and secretion of pro-inflammatory cytokines. PTPN2 knockout mice die a few weeks after birth due to systemic inflammation, emphasizing the importance of this phosphatase in inflammatory processes. The aim of this study was to investigate the role of PTPN2 in colon epithelial cells by performing dextran sulphate sodium (DSS)-induced colitis in PTPN2xVilCre mice. Methods: Acute colitis was induced by administering 2.5 or 2% DSS for 7 days and chronic colitis by 4 cycles of treatment using 1% DSS. Body weight of mice was measured regularly and colonoscopy was done at the end of the experiments. Mice were sacrificed afterwards and colon specimens were obtained for H&E staining. For analysis of wound healing, mechanical wounds were introduced during endoscopy and wound closure assessed by daily colonoscopy. Results: Although colonoscopy and weight development suggested changes in colitis severity, the lack of any influence of PTPN2 deficiency on histological scoring for inflammation severity after acute or chronic DSS colitis indicates that colitis severity is not influenced by epithelial-specific loss of PTPN2. Chronic colitis induced the development of aberrant crypt foci more frequently in PTPN2xVilCre mice compared to their wild type littermates. On the other hand, loss of PTPN2-induced enhanced epithelial cell proliferation and promoted wound closure. Conclusions: Loss of PTPN2 in intestinal epithelial cells (IECs) has no significant influence on inflammation in DSS colitis. Obviously, loss of PTPN2 in IECs can be compensated in vivo, thereby suppressing a phenotype. This lack of a colitis-phenotype might be due to enhanced epithelial cell proliferation and subsequent increased wound-healing capacity of the epithelial layer.

A cell type-specific role of protein tyrosine phosphatase non-receptor type 2 in regulating ER stress signalling.

Background/Aims: Genetic polymorphisms within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) have been associated with inflammatory bowel disease (IBD). A recent study demonstrated that PTPN2 regulates ER stress signalling in pancreatic β-cells. Therefore, we investigated whether PTPN2 regulates ER stress pathways, apoptosis and cytokine secretion in human intestinal epithelial cells (IECs) and monocytes. Methods: THP-1 and HT-29 IECs were stimulated with 2 µg/ml tunicamycin (TNM) for the indicated periods of time. For knockdown experiments, cells were transfected using a mixture of three different PTPN2-specific siRNA oligonucleotides. Cell lysates were analysed by Western blot and real-time PCR. Cytokine secretion was studied by ELISA measurements of cell culture supernatant. Results: TNM treatment reduced PTPN2 protein levels in HT-29 IECs and THP-1 monocytes. Knockdown of PTPN2 in THP-1 monocytes led to an exaggerated induction of phospho-eIF2α, enhanced PARP cleavage, indicative of apoptosis, and attenuated IL-8 and TNF secretion upon TNM stimulation. In HT-29 cells PTPN2 deficiency caused reduced phosphorylation of eIF2α and PARP cleavage under ER stress conditions. Conclusions: Whereas the knockdown of PTPN2 made THP-1 cells more susceptible to ER stress, PTPN2 deficiency reduced ER stress responses in HT-29 IECs. This suggests that PTPN2 regulates adaptation to ER stress in a cell type-specific manner.

Tu1985 Loss of Integrin β6 (ITGB6) Protects From Colon Cancer Development In Vivo

Background: Colorectal carcinoma (CRC) represents the 3rd most frequent malignancy in industrialised countries and the 2nd most common cause of cancer-related deaths. Nearly each 4th of CRC patients develop metastatic disease that can only hardly be treated. A central process in CRC progression and metastasis is epithelial-mesenchymal-transition (EMT), which is partially mediated by certain integrins. An important molecule contributing to EMT in epithelial cells is integrin β6 (ITGB6), which has been associated with proliferation and invasion of colon cancer cells. We have recently shown that ITGB6 can be detected in the serum of CRC patients. ITGB6 serum levels above the threshold of 2/ml are associated with 100% metastatic disease and correlate with poor survival. Here, we investigated the relevance of ITGB6 in the development of colon cancer in mouse models in vivo. Methods: We performed the azoxymethane (AOM)/dextran sodium sulphate (DSS) colon cancer model in female weight-matched ITGB6-/and C57BL/6J wild-type (WT) littermates. ITGB6-/and WT mice were randomly assigned to either a saline/water group (n=6-9) or an AOM/DSS group (n=11-14). Mice were treated 3 cycles, each for 7 day with 1% DSS followed by 14 days recovery period with drinking water. At the 1st day of each DSS cycle and the 2nd day of each water cycle, mice were treated interperitoneally with AOM (10 /kg body weight). Control animals were treated with saline at the same time points. All mice were weighed and monitored throughout the experiment and killed 15 weeks after the treatment start. Results: AOM/DSS-treated ITGB6-/mice (n=14) suffered from aggravated DSS-induced colitis, when compared to AOM/DSS-treated WT mice (n=10). Clinical signs of colitis such as enhanced weight loss (p<.01), increased number of mice with rectal bleeding and severe diarrhoea were clearly more observed in ITGB6-/mice during DSS-treatment. Interestingly, by mouse endoscopy, we detected no colon tumours at all in AOM/DSS-treated ITGB6-/mice, whereas AOM/DSS-treated WT mice displayed up to 4 tumours per animal (p<.001). These findings could be fully confirmed by histologic analysis of the mouse colon. Colon tumours of WT mice featured also an invasive pattern infiltrating into the lamina muscularis mucosae of the intestinal epithelium. DSS/AOM-treated WT mice only revealed hyper proliferative epithelium, but no signs of malignancy in the histopathologic assessment. Conclusions: In summary, we demonstrate that ITGB6-/completely protects mice from AOM/ DSS-induced colon tumour formation. In contrast, WT mice receiving AOM/DSS regularly developed colon tumours. Furthermore, we also found an aggravation of colitis symptoms in ITGB6-/mice, whereas WT mice showed only mild colitis symptoms. Taken together our results suggest that ITGB6 might play a crucial role for colon cancer development.

125 Protein Tyrosine Phosphatase Non-Receptor Type 22 Dephosphorylates NLRP3 to Enable Efficient Inflammasome Activation

Background and aim: Inflammasomes are large, multi-protein complexes in the cytosol, which form upon intracellular presence of danger-associated molecular patterns. Inflammasome assembly results in auto-cleavage and activation of the protease caspase-1, which in turn cleaves pro-IL-1β into its active form, and mediates its secretion. Excessive inflammasome activation results in severe inflammatory conditions, but in the intestine physiological IL1β secretion is important for immune homeostasis. Loss of function variants in the gene encoding the inflammasome receptor Nod-like receptor protein (NLRP)3 are associated with increased risk for Crohns disease (CD). Here, we addressed if loss of protein tyrosine phosphatase non-receptor 22 (PTPN22) which is associated with several inflammatory disorders, including CD influences inflammasome activation. Methods: human cell lines, as wella as murine and human primary cells were treated with different inflammasome inducing agents and analysed by ELISA, qPCR and Western blot. Colitis in mice was induced by application of 2% DSS in the drinking water for 7 days. Intestinal biopsies and serum from CD and control patients were analysed by qPCR and ELISA. Results: Knockdown of PTPN22 in human cell lines as well as loss of PTPN22 in primary murine dendritic cells resulted in markedly reduced caspase-1 cleavage and IL-1 β secretion upon activation of the NLRP3 inflammasome. In contrast, IL-1 β secretion upon activation of AIM2 or NLRC4 inflammasomes was not affected. Immunoprecipitation of NLRP3 revealed tyrosine phosphorylation of NLRP3 and a direct interaction between NLRP3 and PTPN22. Loss of PTPN22 enhanced NLRP3 tyrosine phosphorylation, while a targeted mutation of tyrosine 861 in NLRP3 completely abolished its phosphorylation, and resulted in increased caspase-1 cleavage and IL-1β secretion. In acute DDS-induced colitis, PTPN22 knockout resulted in pronounced weight loss and aggravated macroscopic and microscopic colitis scores. Loss of PTPN22 enhanced NLRP3 phosphorylation together with reduced levels of mature IL-1 β in lamina propria mononuclear cells, but not in epithelial cells. In IBD patients, presence of a disease-associated gain-of-function variant within the gene locus encoding PTPN22 was accompanied with increased IL-1β mRNA and serum levels. Summary: we describe a novel and important regulatory mechanism of NLRP3 by tyrosine phosphorylation, which prevents aberrant inflammasome activation. We demonstrate that PTPN22 dephosphorylates NLRP3 to allow efficient inflammasome activation upon inflammatory insults. This helps to explain, how variants in PTPN22 contribute to the pathogenesis of CD as well as other inflammatory disorders.

A9.13 TNF-Induced- Protein Tyrosine Phosphatase Nonreceptor Type 2 (PTPN2) as a Negative Regulator of Inflammation in Rheumatoid Arthritis

Background and Objectives Protein Tyrosine Phosphatase Nonreceptor Type 2 (PTPN2) is a protein tyrosine phosphatase that plays a role in the development of autoimmune diseases. PTPN2 function has not been studied in rheumatoid arthritis (RA), although single nucleotide polymorphisms within the gene have been described to be associated with RA in genome wide association studies. Considering the involvement of PTPN2 in the regulation of key inflammatory pathways, our aim was to analyse the expression and function of PTPN2 in RA synovial fibroblasts (RASF). Materials and Methods The expression of PTPN2 was assessed in synovial tissue and fibroblasts (passage 4–10) from patients with RA and osteoarthritis (OA) using immunohistochemistry, real-time PCR (w/o tumour necrosis factor α (TNFα), IL1β, LPS and hypoxia) and Western blotting. PTPN2 was silenced with silencing RNA. Levels of IL-6 and IL-8 expression were measured by commercially available ELISA in cell culture supernatants after silencing PTPN2 in RASF w/o stimulation with tumour necrosis factor α (TNFα). Apoptosis of RASF was evaluated by AnnexinV staining using flow cytometry after stimulation with TNF-related apoptosis-inducing ligand (TRAIL, 20 ng/ml) for 24 hours. Results In RA synovial tissue, compared with OA, we observed a stronger staining of ptpn2 in both the lining and the sublining layer by immunohistochemistry. On mRNA level we confirmed this overexpression in RA synovial tissue (2.0 fold, n = 4–5). In isolated RASF the constitutive mRNA level of PTPN2 was higher than in OASF (1.6 fold, p < 0.01, n = 10–16). Levels of PTPN2 were further upregulated in RASF after stimulation with inflammatory cytokines such as TNFα (10 ng/ml, 24 hours, 3.1 fold, p < 0.05, n = 4), TNFα and IL-1β (1 ng/ml, 2.3 fold, n = 5), LPS (100 µg/ml, 24 hours, 1.9 fold, n = 5) and by 1% hypoxia (1.3 fold, n = 3). Accordingly, basal PTPN2 protein expression was 2.0 fold higher in RASF than in OASF (n = 4) and TNFα upregulated levels of PTPN2 (1.7 fold). ptpn2-deficient RASF produced 2.4 times more IL-6 than scrambled siRNA transfected cells (mean ± SD pg/ml 11412 ± 6313 versus 28133 ± 12734, n = 3). On the other hand, levels of IL-8 were not affected (35800 pg/ml versus 24330 pg/ml, n = 3). Furthermore, after silencing, 34% increase in TRAIL-induced apoptosis was detected in RASF (n = 5) compared to scrambled controls. Conclusions Our findings indicate that PTPN2, known to be involved in the pathogenesis of several autoimmune diseases, could be an important negative regulator of inflammation in RASF. Acknowledgement This work was supported by IMI BTCure, IAR and Masterswitch-FP7.

THU0071 Protein Tyrosine Phosphatase Nonreceptor Type 2 (PTPN2) is an Important Regulator of Inflammation and Autophagy in Rheumatoid Arthritis

Background Recently we found that Protein Tyrosine Phosphatase Nonreceptor Type 2 (PTPN2) is involved in the apoptosis resistance of rheumatoid arthritis synovial fibroblasts (RASF). Objectives Autophagy is strongly connected to apoptosis, and is activated via a series of phosphorylation events. We wanted to investigate whether PTPN2 plays a role in autophagy and inflammation in RASF. Methods PTPN2 expression was analysed in synovial tissues by immunohistochemistry and real-time PCR. Transcription levels of PTPN2 were detected with or without tumor necrosis factor α (TNFα 10 ng/ml, 24 h), IL1β (1 ng/ml), LPS (100 µg/ml) and hypoxia (1%). PTPN2 protein levels were assessed by Western blotting. After silencing PTPN2 in RASF, commercially available ELISA was used to measure IL-6 and IL-8 in culture supernatants. TNF-related apoptosis-inducing ligand (TRAIL, 20 ng/ml, 24 h) induced apoptosis was measured after staining with AnnexinV using flow cytometry. Autophagy was induced by Thapsigargin (5 µM, 24h). Western blot was used to measure LC3B-I and LC3B-II. Results In the lining and the sublining layer of RA synovial tissue, a stronger PTPN2 staining was detected by immunohistochemistry compared to OA (n=7). That overexpression could also be confirmed on mRNA level (2.0 fold, RA tissue n=4, OA tissue n=5). Moreover, in RASF, the constitutive mRNA level of PTPN2 was higher than in OA synovial fibroblasts (OASF) (1.6 fold, p<0.01, n=10-16). Stimulation with inflammatory cytokines such as TNFα (3.1 fold, p<0.05, n=4), TNFα and IL-1β (2.3 fold, n=5), LPS (1.9 fold, n=5) and hypoxia (1.3 fold, n=3) resulted in a further increase in PTPN2 mRNA levels. We found a 2.0 fold higher PTPN2 protein expression in RASF compared to OASF (n=4), and a 1.7±0.4 fold induction of PTPN2 in RASF after TNFα stimulation (n=4) by densitometric analysis of Western blot signals. IL-6 production increased 2.1 times in PTPN2 deficient cells (mean±SD pg/ml 11159±5179 vs. 24400±12801, n=4) compared to scrambled control. We could not see this effect concerning IL-8 production (35450±20619 pg/ml vs. 24148±24450 pg/ml, n=4). TRAIL-induced apoptosis increased by 34% after PTPN2 silencing (n=5). The level of autophagy (LC3B-II) did not differ basally and after TNFα stimulation in scrambled control and PTPN2 silenced cells. However, in scrambled control transfected cells, the addition of Thapsigargin induced the expression of LC3B-II protein (2.07±0.97 fold, n=5), indicative for autophagy. PTPN2 silencing reduced the induction of autophagy to 0.8±0.2 fold (n=5) compared to scrambled control. Conclusions Since levels of autophagy are lower in PTPN2 silenced RASF compared to scrambled control, it is indicated that increased PTPN2 protein expression in RASF induces inflammation and elevated autophagy in RASF. Acknowledgements This work was supported by IMI BTCure, IAR, Masterswitch-FP7 and ZIHP. Disclosure of Interest None Declared