Claudia Gottier

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.

Bilberry-Derived Anthocyanins Modulate Cytokine Expression in the Intestine of Patients with Ulcerative Colitis

Background/Aims We previously demonstrated that anthocyanin-rich bilberry extract (ARBE) inhibits IFN-γ-induced signalling and downstream effects in human monocytic cells and ameliorates disease activity in ulcerative colitis (UC) patients. Here, we studied the molecular mechanisms of ARBE-mediated effects in vitro and by analysing colonic tissue and serum samples of UC patients treated with an oral anthocyanin-rich bilberry preparation during an open label clinical trial. Methods Colon specimens obtained during an open pilot study using ARBE for the treatment of mild-to-moderate UC were analyzed by immunohistochemistry. Cytokine levels in patients’ serum were quantified by ELISA. Cell culture experiments were performed using THP-1 monocytic cells. Results ARBE treatment inhibited the expression of IFN-γ-receptor 2 in human THP-1 monocytic cells. Colon biopsies of UC patients who responded to the 6-week long ARBE treatment revealed reduced amounts of the pro-inflammatory cytokines IFN-γ and TNF-α. Levels of phosphorylated (activated) p65-NF-κB were reduced in these patients. Further, patients with successful ARBE treatment featured enhanced levels of Th17-cell specific cytokine IL-22 and immunoregulatory cytokine IL-10 as well as reduced serum levels of TNF-α and MCP-1, but enhanced levels of IL-17A, in contrast to patients that did not reach remission after ARBE treatment. Conclusions Our data suggest a molecular mechanism underlying the anti-inflammatory effects of ARBE treatment in UC patients by modulating T-cell cytokine signalling and inhibiting IFN-γ signal transduction. These data are of particular interest, since ARBE is a promising therapeutic approach for the treatment of IBD.

PTPN2 Regulates Inflammasome Activation and Controls Onset of Intestinal Inflammation and Colon Cancer

Variants in the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) are associated with inflammatory disorders, including inflammatory bowel diseases, rheumatoid arthritis, and type 1 diabetes. The anti-inflammatory role of PTPN2 is highlighted by the fact that PTPN2-deficient mice die a few weeks after birth because of systemic inflammation and severe colitis. However, the tissues, cells, and molecular mechanisms that contribute to this phenotype remain unclear. Here, we demonstrate that myeloid cell-specific deletion of PTPN2 in mice (PTPN2-LysMCre) promotes intestinal inflammation but protects from colitis-associated tumor formation in an IL-1β-dependent manner. Elevated levels of mature IL-1β production in PTPN2-LysMCre mice are a consequence of increased inflammasome assembly due to elevated phosphorylation of the inflammasome adaptor molecule ASC. Thus, we have identified a dual role for myeloid PTPN2 in directly regulating inflammasome activation and IL-1β production to suppress pro-inflammatory responses during colitis but promote intestinal tumor development.

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.

PTPN22 regulates NLRP3-mediated IL1B secretion in an autophagy-dependent manner

ABSTRACT A variant within the gene locus encoding PTPN22 (protein tyrosine phosphatase, non-receptor type 22) emerged as an important risk factor for auto-inflammatory disorders, including rheumatoid arthritis, systemic lupus erythematosus and type 1 diabetes, but at the same time protects from Crohn disease, one of the 2 main forms of inflammatory bowel diseases. We have previously shown that loss of PTPN22 results in decreased NLRP3 (NLR family pyrin domain containing 3) activation and that this effect is mediated via enhanced NLRP3 phosphorylation. However, it is unclear how phosphorylation of NLRP3 mediates its inhibition. Here, we demonstrate that loss of macroautophagy/autophagy abrogates the inhibitory effect on NLRP3 activation observed upon loss of PTPN22. Phosphorylated, but not nonphosphorylated NLRP3 is found in autophagosomes, indicating that NLRP3 phosphorylation mediates its inactivation via promoting sequestration into phagophores, the precursors to autophagosomes. This finding shows that autophagy and NLRP3 inflammasome activation are connected, and that PTPN22 plays a key role in the regulation of those 2 pathways. Given its role in inflammatory disorders, PTPN22 might be an attractive therapeutic target, and understanding the cellular mechanisms modulated by PTPN22 is of crucial importance.

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.