Protein Tyrosine Phosphatase Nonreceptor Type 2 Expression Does Not Correlate with Viral Load or Response to Direct-Acting Antiviral Therapy in Hepatitis C Virus Infections-Infected Patients.

BACKGROUND/AIMS: The hepatitis C virus nonstructural 3/4A protease has been shown to cleave protein tyrosine phosphatase nonreceptor type 2 (PTPN2, also known as T cell protein tyrosine phosphatase), thereby inducing a shift from a Th1 toward a nonantiviral Th2 immunity. Ribavirin therapy reverses these effects and supports direct-acting antiviral (DAA) therapy as an immunomodulatory compound and ultimately improves the response to DAA therapy. Here we aimed to assess whether intrahepatic levels of PTPN2 might be used as a clinical prognostic marker for the response to DAA therapy. METHODS: Liver biopsies from hepatitis C virus-infected patients with and without cirrhosis were immunohistochemically stained for PTPN2 and scored for staining intensity as well as percentage of hepatocytes positive for nuclear PTPN2 localization. PTPN2 scores were correlated to sustained virologic response after DAA therapy, viral load, serum levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase (GGT), and the Model for End-Stage Liver Disease (MELD) score at the time of liver biopsy. RESULTS: We did not detect a difference in intrahepatic PTPN2 levels between responders with cirrhosis, responders without cirrhosis, and nonresponders to DAA therapy. There was no correlation between intrahepatic PTPN2 levels and viral load or clinical markers such as liver transaminases, GGT, or the MELD score. CONCLUSION: Intrahepatic PTPN2 levels assessed via IHC staining do not represent a clinical prognostic marker for the response to DAA therapy.

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.

Loss of PTPN22 Promotes Intestinal Inflammation by Compromising Granulocyte-mediated Antibacterial Defence.

BACKGROUND AND AIMS: A single nucleotide polymorphism in protein tyrosine phosphatase non-receptor type 22 [PTPN22] has been associated with the onset of autoimmune disorders, but protects from Crohn's disease. PTPN22 deficiency in mice promotes intestinal inflammation by modulating lymphocyte function. However, the impact of myeloid PTPN22 in colitis development remains unclear. The aim of this study was to investigate the role of PTPN2 in the IL-10 and the T cell transfer colitis models. METHODS: PTPN22-deficient mice were crossed with IL-10-/- and RAG2-/- mice. Naïve T cells were injected in RAG-/- mice to induce T-cell transfer colitis. Spontaneous colitis in IL-10-/- mice was monitored for up to 200 days. RESULTS: Here, we demonstrate that PTPN22 in non-lymphoid immune cells is required to protect against T cell transfer-mediated and IL-10 knock-out colitis. Analysis of the intestinal immune landscape demonstrated a marked reduction of granulocyte influx into the inflamed colon in PTPN22-deficient mice. On a molecular level, granulocytes were not only reduced by numbers, but also revealed a defective function. In particular, granulocyte activation and granulocyte-mediated bacteria killing was impaired upon loss of PTPN22, resulting in elevated bacterial burden and translocation beyond the intestinal epithelial barrier in PTPN22-deficient mice. Consistently, antibiotic-induced depletion of bacteria reverted the increased colitis susceptibility in PTPN22-deficient mice, whereas granulocyte depletion induced acolitis phenotype in wild-type mice similar to that observed in PTPN22-deficient mice. CONCLUSIONS: In conclusion, our data demonstrate that PTPN22 is essential for adequate granulocyte activation and antimicrobial defence to protect the inflamed intestine from bacterial invasion and exacerbated colitis.

Commensal Clostridiales strains mediate effective anti-cancer immune response against solid tumors.

Despite overall success, T cell checkpoint inhibitors for cancer treatment are still only efficient in a minority of patients. Recently, intestinal microbiota was found to critically modulate anti-cancer immunity and therapy response. Here, we identify Clostridiales members of the gut microbiota associated with a lower tumor burden in mouse models of colorectal cancer (CRC). Interestingly, these commensal species are also significantly reduced in CRC patients compared with healthy controls. Oral application of a mix of four Clostridiales strains (CC4) in mice prevented and even successfully treated CRC as stand-alone therapy. This effect depended on intratumoral infiltration and activation of CD8+ T cells. Single application of Roseburia intestinalis or Anaerostipes caccae was even more effective than CC4. In a direct comparison, the CC4 mix supplementation outperformed anti-PD-1 therapy in mouse models of CRC and melanoma. Our findings provide a strong preclinical foundation for exploring gut bacteria as novel stand-alone therapy against solid tumors.

Protein Tyrosine Phosphatase Non-Receptor Type 2 Function in Dendritic Cells Is Crucial to Maintain Tissue Tolerance.

Protein tyrosine phosphatase non-receptor type 2 (PTPN2) plays a pivotal role in immune homeostasis and has been associated with human autoimmune and chronic inflammatory diseases. Though PTPN2 is well-characterized in lymphocytes, little is known about its function in innate immune cells. Our findings demonstrate that dendritic cell (DC)-intrinsic PTPN2 might be the key to explain the central role for PTPN2 in the immune system to maintain immune tolerance. Partial genetic PTPN2 ablation in DCs resulted in spontaneous inflammation, particularly in skin, liver, lung and kidney 22 weeks post-birth. DC-specific PTPN2 controls steady-state immune cell composition and even incomplete PTPN2 deficiency in DCs resulted in enhanced organ infiltration of conventional type 2 DCs, accompanied by expansion of IFNγ-producing effector T-cells. Consequently, the phenotypic effects of DC-specific PTPN2 deficiency were abolished in T-cell deficient Rag knock-out mice. Our data add substantial knowledge about the molecular mechanisms to prevent inflammation and maintain tissue tolerance.

Presence of PTPN2 SNP rs1893217 Enhances the Anti-inflammatory Effect of Spermidine.

BACKGROUND: The single nucleotide polymorphism (SNP) rs1893217 within the gene locus encoding PTPN2 represents a risk factor for inflammatory bowel disease (IBD). Our previous work demonstrated reduced PTPN2 activity and subsequently increased inflammatory signaling upon presence of SNP rs1893217. The naturally occurring polyamine spermidine reduces pro-inflammatory signaling via induction of PTPN2 activity; however, the effect of SNP rs1893217 on the anti-inflammatory potential of spermidine is still unknown. Here, we investigated how presence of SNP rs1893217 affects treatment efficacy of spermidine and whether it might serve as a potential biomarker for spermidine treatment. METHODS: Human T84 (wild-type [WT] for PTPN2 SNP rs1893217) and HT29 (heterozygous for PTPN2 SNP rs1893217) intestinal epithelial cells (IECs) were treated with several polyamines from the putrescine-spermidine pathway. T84 and HT29 IECs, THP-1 monocytes (WT and transfected with a lentiviral vector expressing PTPN2 SNP rs1893217) and genotyped, patient-derived peripheral blood mononuclear cells were challenged with IFN-γ and/or spermidine. RESULTS: Among the analyzed polyamines, spermidine was the most efficient activator of PTPN2 phosphatase activity, regardless of the PTPN2 genotype. Spermidine suppressed IFN-γ-induced STAT1 and STAT3 phosphorylation, along with decreased mRNA expression of ICAM-1, NOD2, and IFNG in IECs and monocytes. Of note, these effects were clearly more pronounced when the disease-associated PTPN2 C-variant in SNP rs1893217 was present. CONCLUSIONS: Our data demonstrate that spermidine is the most potent polyamine in the putrescine-spermine axis for inducing PTPN2 enzymatic activity. The anti-inflammatory effect of spermidine is potentiated in the presence of SNP rs1893217, and this SNP might thus be a useful biomarker for possible spermidine-treatment in IBD patients.

Deletion of Protein Tyrosine Phosphatase Nonreceptor Type 2 in Intestinal Epithelial Cells Results in Upregulation of the Related Phosphatase Protein Tyrosine Phosphatase Nonreceptor Type 23.

BACKGROUND/AIMS: Knockdown of protein tyrosine phosphatase nonreceptor type 2 (PTPN2) exaggerates IFN-γ-induced intestinal barrier defects, but mice constitutively lacking PTPN2 in epithelial cells (PTPN2xVilCre mice) do not show changes in epithelial function or enhanced susceptibility to experimental colitis. Here, we investigated whether PTPN2 modulates the expression of related tyrosine phosphatases. METHODS: PTPN2 knockdown in HT-29 cells was induced using siRNA constructs. Acute colitis in PTPN2xVilCre mice was induced by 2% dextran sulfate sodium (DSS) in drinking water for 7 days. Colitis-associated tumors were induced by injection of azoxymethane prior to treatment with DSS for 3 consecutive cycles. RESULTS: In HT-29 cells, PTPN2 depletion resulted in enhanced mRNA expression of PTPN11 and PTPN23 and in parallel to upregulation of IL-18 mRNA upon treatment with TNF for 24 h. DSS treatment of PTPN2-deficient mice resulted in a strong induction of Ptpn23 mRNA in colon tissue in vivo. In the tumor model, Ptpn23 mRNA was again clearly upregulated in nontumor tissue from PTPN2-deficient mice; however, this was not observed in tumor tissue. CONCLUSIONS: Our experiments show that PTPN23 function might, at least partially, compensate lack of PTPN2 in epithelial cells. Upregulation of PTPN23 might therefore crucially contribute to the lack of a colitis phenotype in PTPN2-VilCre mice.

Loss of PTPN23 Promotes Proliferation and Epithelial-to-Mesenchymal Transition in Human Intestinal Cancer Cells.

BACKGROUND/OBJECTIVES: Protein tyrosine phosphatase nonreceptor type 23 (PTPN23) has recently been associated with several human epithelial cancers via regulation of growth factor signaling. Colorectal carcinoma (CRC) is a leading cause for cancer-related death worldwide and is associated with aberrant epidermal (EGF) and vascular endothelial growth factor signaling. Here, we investigated whether PTPN23 might play a role in CRC. METHODS: Expression of PTPN23 was analyzed in CRC tissue by immunohistochemistry. PTPN23 was silenced in HT-29 cells to address the role of PTPN23 in EGF signaling, gene expression, and cell migration. RESULTS: PTPN23 silencing in HT-29 and Caco-2 intestinal epithelial cancer cells significantly enhanced activation of pro-oncogenic signaling molecules and genes promoting epithelial-to-mesenchymal transition (EMT) upon EGF treatment, while genes encoding tight junction proteins were significantly reduced. CONCLUSIONS: Our data clearly indicate that loss of PTPN23 is associated with increased activation of pro-oncogenic signaling pathways and an enhanced ability of human intestinal cancer cells to undergo EMT. Taken together, these findings show that PTPN23 acts as a tumor suppressor gene in CRC.

Transplantation of Human Intestine Into the Mouse: A Novel Platform for Study of Inflammatory Enterocutaneous Fistulas.

BACKGROUND AND AIMS: Enteric fistulas represent a severe and medically challenging comorbidity commonly affecting Crohn's disease [CD] patients. Gut fistulas do not develop in animal models of the disease. We have used transplantation of the human fetal gut into mice as a novel platform for studying inflammatory enterocutaneous fistulas. METHODS: Human fetal gut segments were transplanted subcutaneously into mature SCID mice, where they grew and fully developed over the course of several months. We first analysed the resident immune cells and inflammatory response elicited by systemic lipopolysaccharide [LPS] in normal, fully developed human gut xenografts. Thereafter, we used immunostaining to analyse fully developed xenografts that spontaneously developed enterocutaneous fistulas. RESULTS: Resident human innate and adaptive immune cells were demonstrated in gut xenografts during steady state and inflammation. The expression of human IL-8, IL-1ß, IL-6, TNF-α, A20, and IkBα was significantly elevated in response to LPS, with no change in IL-10 gene expression. Approximately 17% [19/110] of fully developed subcutaneous human gut xenografts spontaneously developed enterocutaneous fistulas, revealing striking histopathological similarities with CD fistula specimens. Immunohistochemical analyses of fistulating xenografts revealed transmural lymphocytic enteritis associated with massive expansion of resident human CD4+ lymphocytes and their migration into the intraepithelial compartment. Regionally, mucosal epithelial cells assumed spindle-shaped mesenchymal morphology and formed fistulous tracts towards chronic non-healing wounds in the host mouse skin overlying the transplants. CONCLUSIONS: Inflammation and fistulas developed in human gut xenografts lacking IL-10 gene response. This novel model system will enable systematic studies of the inflamed and fistulating human gut in live animals.

Protein tyrosine phosphatase non-receptor type 22 modulates colitis in a microbiota-dependent manner.

The gut microbiota is crucial for our health, and well-balanced interactions between the host's immune system and the microbiota are essential to prevent chronic intestinal inflammation, as observed in inflammatory bowel diseases (IBD). A variant in protein tyrosine phosphatase non-receptor type 22 (PTPN22) is associated with reduced risk of developing IBD, but promotes the onset of autoimmune disorders. While the role of PTPN22 in modulating molecular pathways involved in IBD pathogenesis is well studied, its impact on shaping the intestinal microbiota has not been addressed in depth. Here, we demonstrate that mice carrying the PTPN22 variant (619W mice) were protected from acute dextran sulfate sodium (DSS) colitis, but suffered from pronounced inflammation upon chronic DSS treatment. The basal microbiota composition was distinct between genotypes, and DSS-induced dysbiosis was milder in 619W mice than in WT littermates. Transfer of microbiota from 619W mice after the first DSS cycle into treatment-naive 619W mice promoted colitis, indicating that changes in microbial composition enhanced chronic colitis in those animals. This indicates that presence of the PTPN22 variant affects intestinal inflammation by modulating the host's response to the intestinal microbiota.

Loss of PTPN22 abrogates the beneficial effect of cohousing-mediated fecal microbiota transfer in murine colitis.

Fecal microbiota transfer (FMT) is a very efficient approach for the treatment of severe and recurring C. difficile infections. However, the beneficial effect of FMT in other disorders such as ulcerative colitis (UC) or Crohn's disease remains unclear. Furthermore, it is currently unknown how disease-associated genetic variants in donors or recipients influence the effect of FMT. We found that bacteria-transfer from wild-type (WT) donors via cohousing was efficient in inducing recovery from colitis in WT mice, but not in mice deficient in protein-tyrosine phosphatase non-receptor type 22 (PTPN22), a known risk gene for several chronic inflammatory diseases. Also cohousing of PTPN22-deficient mice with diseased WT mice failed to induce faster recovery. Our data indicate that the genetic background of the donor and the recipient influences the outcome of microbiota transfer, and offers a potential explanation why transfer of fecal microbes from some, but not all donors is efficient in UC patients.

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-1ß-dependent manner. Elevated levels of mature IL-1ß 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-1ß production to suppress pro-inflammatory responses during colitis but promote intestinal tumor development.

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

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.

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.

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.