Search

ObjectiveInflammatory bowel disease (IBD) is a multifactorial condition driven by genetic and environmental risk factors. A genetic variation in the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene has been associated with autoimmune disorders while protecting from the IBD subtype Crohn’s disease. Mice expressing the murine orthologous PTPN22-R619W variant are protected from intestinal inflammation in the model of acute dextran sodium sulfate (DSS)-induced colitis. We previously identified food-grade titanium dioxide (TiO2, E171) as a neglected IBD risk factor. Here, we investigate the interplay of the PTPN22 variant and TiO2-mediated effects during IBD pathogenesis.DesignAcute DSS colitis was induced in wild-type and PTPN22 variant mice (PTPN22-R619W) and animals were treated with TiO2nanoparticles during colitis induction. Disease-triggering mechanisms were investigated using bulk and single-cell RNA sequencing.ResultsIn mice, administration of TiO2nanoparticles abrogated the protective effect of the variant, rendering PTPN22-R619W mice susceptible to DSS colitis. In early disease, cytotoxic CD8+T-cells were found to be reduced in the lamina propria of PTPN22-R619W mice, an effect reversed by TiO2administration. Normalisation of T-cell populations correlated with increasedIfngexpression and, at a later stage of disease, the promoted prevalence of proinflammatory macrophages that triggered severe intestinal inflammation.ConclusionOur findings indicate that the consumption of TiO2nanoparticles might have adverse effects on the gastrointestinal health of individuals carrying the PTPN22 variant. This demonstrates that environmental factors interact with genetic risk variants and can reverse a protective mechanism into a disease-promoting effect.

Background and Aims Exacerbated immune activation, intestinal dysbiosis and a disrupted intestinal barrier are common features among inflammatory bowel disease [IBD] patients. The polyamine spermidine, which is naturally present in all living organisms, is an integral component of the human diet, and exerts beneficial effects in human diseases. Here, we investigated whether spermidine treatment ameliorates intestinal inflammation and offers therapeutic potential for IBD treatment. Methods We assessed the effect of oral spermidine administration on colitis severity in the T cell transfer colitis model in Rag2−/− mice by endoscopy, histology and analysis of markers of molecular inflammation. The effects on the intestinal microbiome were determined by 16S rDNA sequencing of mouse faeces. The impact on intestinal barrier integrity was evaluated in co-cultures of patient-derived macrophages with intestinal epithelial cells. Results Spermidine administration protected mice from intestinal inflammation in a dose-dependent manner. While T helper cell subsets remained unaffected, spermidine promoted anti-inflammatory macrophages and prevented the microbiome shift from Firmicutes and Bacteroides to Proteobacteria, maintaining a healthy gut microbiome. Consistent with spermidine as a potent activator of the anti-inflammatory molecule protein tyrosine phosphatase non-receptor type 2 [PTPN2], its colitis-protective effect was dependent on PTPN2 in intestinal epithelial cells and in myeloid cells. The loss of PTPN2 in epithelial and myeloid cells, but not in T cells, abrogated the barrier-protective, anti-inflammatory effect of spermidine and prevented the anti-inflammatory polarization of macrophages. Conclusion Spermidine reduces intestinal inflammation by promoting anti-inflammatory macrophages, maintaining a healthy microbiome and preserving epithelial barrier integrity in a PTPN2-dependent manner.

Macrophages are a heterogeneous population of innate immune cells that are often divided into two major subsets: classically activated, typically pro-inflammatory (M1) macrophages that mediate host defense, and alternatively activated, tolerance-inducing (M2) macrophages that exert homeostatic and tissue-regenerative functions. Disturbed macrophage function/differentiation results either in inadequate, excessive immune activation or in a failure to induce efficient protective immune responses against pathogens. Loss-of-function variants in protein tyrosine phosphatase non-receptor type 2 (PTPN2) are associated with chronic inflammatory disorders, but the effect of macrophage-intrinsic PTPN2 loss is still poorly understood. Here we report that PTPN2-deficient macrophages fail to acquire an alternatively activated/M2 phenotype. This was the consequence of reduced IL-6 receptor expression and a failure to induce IL-4 receptor in response to IL-6, resulting in an inability to respond to the key M2-inducing cytokine IL-4. Ultimately, failure to adequately respond to IL-6 and IL-4 resulted in increased levels of M1 macrophage marker expression in vitro and exacerbated lung inflammation upon infection with Nippostrongylus brasiliensis in vivo. These results demonstrate that PTPN2 loss interferes with the ability of macrophages to adequately respond to inflammatory stimuli and might explain the increased susceptibility of PTPN2 loss-of-function carriers to developing inflammatory diseases.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease affecting the colonic mucosa. There is no cure for UC and its chronic relapsing/remitting nature strongly affects patient quality of life. Current treatment options frequently have significant side effects and remission rates are limited raising a demand for new treatment strategies. Novel therapeutic approaches that could maximize the drug concentration at the site of inflammation with minimal systemic exposure, like topical applications, would address this unmet clinical need. To date, few drug delivery systems (DDSs) have been designed to topically convey small molecules to the rectum and left-sided colon. Here, we developed and tested a drug delivery platform for topical treatment of UC based on a temperature-triggered in situ forming adhesive lipid gel (TIF-Gel). Due to its soft, gel-like consistency, its high encapsulation efficacy, and its drug-controlled release, TIF-Gel suggests a more patient-friendly and effective application with respect to the rectal formulations currently available.Capitalizing on the biocompatible and biodegradable self-assembled structure of lipid mesophases (LMPs), we loaded TIF-Gel with tofacitinib (TOFA; a hydrophilic inhibitor of the enzymes Janus kinase 1 and 3) or TAC (a hydrophobic immunosuppressive drug), both of which are indicated in the treatment of UC. We designed and fully characterized our biocompatible lipid formulation in vitro and tested it in vivo using two different murine models of inflammatory bowel disease: chemically-induced and T cell transfer-mediated. Both approaches (TIF-Gel-TOFA and TIF-Gel-TAC) led to reductions in colitis disease severity and intestinal inflammation compared to vehicles, therefore showing therapeutic efficacy.Overall, our findings show that TIF-Gel can deliver drugs locally to the colonic mucosa to mitigate intestinal inflammatory disease in a pre-clinical model. They also suggest that, in a clinical setting, TIF-Gel might provide a patient-friendly approach to improve colitis while allowing for a reduction of the adverse effects associated with a systemic therapy.

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.

Background Vedolizumab is a widely used and safe therapy in inflammatory bowel disease, particularly in ulcerative colitis (UC), making it a promising candidate for enhanced efficacy by combining it with additional immunomodulatory medications. In this study, we studied the impact of vedolizumab monotreatment vs vedolizumab coadministration with other immunomodulatory drugs on intestinal inflammation and intestinal immune cells in vivo. Methods Colon tissue from human patients with UC with active disease or in remission with or without vedolizumab treatment was stained by immunohistochemistry. We reconstituted NOD-SCID-SGM3 mice with human CD34+ cells and treated them with dextran sodium sulfate to induce acute colitis. Mice were treated with vedolizumab alone, or in combination with tacrolimus, ozanimid, or tofacitinib. Results Vedolizumab reduced the number of CD3+ T cells and CD68+ monocytes/macrophages in the colon of patients with UC with active disease. Vedolizumab moderately decreased immune cell numbers in acute dextran sodium sulfate–induced colitis. The combination of vedolizumab with tacrolimus further reduced the number of infiltrating CD3+ T cells and CD68+ monocytes/macrophages and was superior in ameliorating intestinal inflammation when compared to vedolizumab monotreatment. In contrast, cotreatment using vedolizumab with ozanimod or tofacitinib had no additive effect. Conclusions Our data show that vedolizumab reduces the number of innate and adaptive immune cells in the mucosa of patients with UC. Further, the combination of vedolizumab with tacrolimus was more efficient to reduce immune cell numbers and to increase therapeutic efficacy than vedolizumab monotreatment. This finding indicates that combination treatment using these two drugs may be beneficial for patients who do not respond to vedolizumab monotherapy.

Inflammatory bowel disease (IBD) pathogenesis involves significant contributions from genetic and environmental factors. Loss-of-function single-nucleotide polymorphisms (SNPs) in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene increase IBD risk and are associated with altered microbiome population dynamics in IBD. Expansion of intestinal pathobionts, such as adherent-invasive E. coli (AIEC), is strongly implicated in IBD pathogenesis as AIEC increases pro-inflammatory cytokine production and alters tight junction protein regulation - suggesting a potential mechanism of pathogen-induced barrier dysfunction and inflammation. We aimed to determine if PTPN2 deficiency alters intestinal microbiome composition to promote expansion of specific bacteria with pathogenic properties. In mice constitutively lacking Ptpn2, we identified increased abundance of a novel mouse AIEC (mAIEC) that showed similar adherence and invasion of intestinal epithelial cells, but greater survival in macrophages, to the IBD-associated AIEC, LF82. Furthermore, mAIEC caused disease when administered to mice lacking segmented-filamentous bacteria (SFB), and in germ-free mice but only when reconstituted with a microbiome, thus supporting its classification as a pathobiont, not a pathogen. Moreover, mAIEC infection increased the severity of, and prevented recovery from, induced colitis. Although mAIEC genome sequence analysis showed >90% similarity to LF82, mAIEC contained putative virulence genes with >50% difference in gene/protein identities from LF82 indicating potentially distinct genetic features of mAIEC. We show for the first time that an IBD susceptibility gene, PTPN2, modulates the gut microbiome to protect against a novel pathobiont. This study generates new insights into gene-environment-microbiome interactions in IBD and identifies a new model to study AIEC-host interactions.

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.

ObjectivesAlterations in the intestinal microbiota are linked with a wide range of autoimmune and inflammatory conditions, including inflammatory bowel diseases (IBD), where pathobionts penetrate the intestinal barrier and promote inflammatory reactions. In patients with IBD, the ability of intestinal macrophages to efficiently clear invading pathogens is compromised resulting in increased bacterial translocation and excessive immune reactions. Here, we investigated how an IBD-associated loss-of-function variant in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene, or loss of PTPN2 expression affected the ability of macrophages to respond to invading bacteria.DesignIBD patient-derived macrophages with wild-type (WT) PTPN2 or carrying the IBD-associated PTPN2 SNP, peritoneal macrophages from WT and constitutive PTPN2-knockout mice, as well as mice specifically lacking PTPN2 in macrophages were infected with non-invasive K12 Escherichia coli, the human adherent-invasive E. coli (AIEC) LF82, or a novel mouse AIEC (mAIEC) strain.ResultsLoss of PTPN2 severely compromises the ability of macrophages to clear invading bacteria. Specifically, loss of functional PTPN2 promoted pathobiont invasion/uptake into macrophages and intracellular survival/proliferation by three distinct mechanisms: Increased bacterial uptake was mediated by enhanced expression of carcinoembryonic antigen cellular adhesion molecule (CEACAM)1 and CEACAM6 in PTPN2-deficient cells, while reduced bacterial clearance resulted from defects in autophagy coupled with compromised lysosomal acidification. In vivo, mice lacking PTPN2 in macrophages were more susceptible to mAIEC infection and mAIEC-induced disease.ConclusionsOur findings reveal a tripartite regulatory mechanism by which PTPN2 preserves macrophage antibacterial function, thus crucially contributing to host defence against invading bacteria.

Plastic pollution is a major global challenge of our times, baring a potential threat for the environment and the human health. The increasing abundance of nanoplastic (NP) and microplastic (MP) particles in the human diet might negatively affect human health since they - particularly in patients suffering from inflammatory bowel disease (IBD) - might surpass the intestinal barrier. To investigate whether ingested plastic particles cross the intestinal epithelium and promote bowel inflammation, mice were supplemented with NP or MP polystyrene (PS) particles for 24 or 12 weeks before inducing acute or chronic dextran sodium sulfate (DSS) colitis with continuous plastic administration. Although ingested PS particles accumulated in the small intestine and organs distant from the gastrointestinal tract, PS ingestion did not affect intestinal health nor did it promote colitis severity. Although the lack of colitis-promoting effects of small PS particles might be a relief for IBD patients, potential accumulative effects of ingested plastic particles on the gastrointestinal health cannot be excluded.

Medullary thymic epithelial cells (mTECs) are key antigen-presenting cells mediating T cell tolerance to prevent harmful autoimmunity. mTECs both negatively select self-reactive T cells and promote...

Medullary thymic epithelial cells (mTECs) are key antigen-presenting cells mediating T cell tolerance to prevent harmful autoimmunity. mTECs both negatively select self-reactive T cells and promote the development of thymic regulatory T cells (tT

The successes of fecal microbiota transplants (FMT) have provided the necessary proof-of-concept for microbiome therapeutics. Because of the many risks and uncertainties associated with feces-based therapies, defined microbial consortia that modify the microbiome in a targeted manner have emerged as a promising safer alternative to FMT. The development of such live biotherapeutic products has important challenges, including the selection of appropriate strains and the production of the consortia at scale. Here, we report on an ecology and biotechnology-based approach to microbial consortium design that overcomes these issues. We designed a nine-strain consortium that emulates the central metabolic pathways of carbohydrate fermentation in the healthy human gut microbiota. We show that continuous co-culturing the bacteria produce a stable consortium whose activity is distinct from an equivalent mix of individually cultured strains. Further, we showed that our function-based consortium is as effective as FMT in counteracting dysbiosis in a dextran sodium sulfate mouse model of acute colitis. We propose that combining a bottom-up functional design with continuous co-cultivation is a powerful strategy to produce robust, functionally designed synthetic consortia for therapeutic use.

Genome-wide association studies revealed that loss-of-function mutations in protein tyrosine phosphatase non-receptor type 2 (PTPN2) increase the risk of developing chronic immune diseases, such as inflammatory bowel disease (IBD) and celiac disease. These conditions are associated with increased intestinal permeability as an early etiological event. The aim of this study was to examine the consequences of deficient activity of the PTPN2 gene product, T cell protein tyrosine phosphatase (TCPTP), on intestinal barrier function and tight junction organization in vivo and in vitro. Here, we demonstrate that TCPTP protected against intestinal barrier dysfunction induced by the inflammatory cytokine IFN-γ by 2 mechanisms: it maintained localization of zonula occludens 1 and occludin at apical tight junctions and restricted both expression and insertion of the cation pore-forming transmembrane protein, claudin-2, at tight junctions through upregulation of the inhibitory cysteine protease, matriptase. We also confirmed that the loss-of-function PTPN2 rs1893217 SNP was associated with increased intestinal claudin-2 expression in patients with IBD. Moreover, elevated claudin-2 levels and paracellular electrolyte flux in TCPTP-deficient intestinal epithelial cells were normalized by recombinant matriptase. Our findings uncover distinct and critical roles for epithelial TCPTP in preserving intestinal barrier integrity, thereby proposing a mechanism by which PTPN2 mutations contribute to IBD.

Inflammatory bowel disease (IBD) is a multifactorial condition driven by genetic and environmental risk factors. A genetic variation in the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene has been associated with autoimmune disorders while protecting from the IBD subtype Crohn's disease. Mice expressing the murine orthologous PTPN22-R619W variant are protected from intestinal inflammation in the model of acute dextran sodium sulfate (DSS)-induced colitis. We previously identified food-grade titanium dioxide (TiOAcute DSS colitis was induced in wild-type and PTPN22 variant mice (PTPN22-R619W) and animals were treated with TiOIn mice, administration of TiOOur findings indicate that the consumption of TiO

Background and Aims: Local extracellular acidification is associated with several conditions, such as ischemia, cancer, metabolic disease, respiratory diseases, and inflammatory bowel disease (IBD). Several recent studies reported a link between IBD and a family of pH-sensing G protein-coupled receptors. Our previous studies point to an essential role for OGR1 (GPR68) in the modulation of intestinal inflammation and fibrosis. In the current study, we evaluated the effects of a novel OGR1 inhibitor in murine models of colitis. Methods: The effects of a novel small-molecule OGR1 inhibitor were assessed in the acute and chronic dextran sulfate sodium (DSS) murine models of colitis. Macroscopic disease indicators of intestinal inflammation were evaluated, and epithelial damage and immune cell infiltration and proliferation were assessed by immunohistochemistry. Results: The OGR1 inhibitor ameliorated clinical parameters in acute and chronic DSS-induced colitis. In mice treated with the OGR1 inhibitor, endoscopy showed no thickening and normal vascularity, while fibrin was not detected. Histopathological findings revealed a decrease in severity of colonic inflammation in the OGR1 inhibitor group when compared to vehicle-DSS controls. In OGR1 inhibitor-treated mice, staining for the macrophage marker F4/80 and cellular proliferation marker Ki-67 revealed a reduction of infiltrating macrophages and slightly enhanced cell proliferation, respectively. This was accompanied by a reduction in pro-inflammatory cytokines, TNF and IL-6, and the fibrosis marker TGF-β1. Conclusion: This is the first report providing evidence that a pharmacological inhibition of OGR1 has a therapeutic effect in murine colitis models. Our data suggest that targeting proton-sensing OGR1 using specific small-molecule inhibitors may be a novel therapeutic approach for the treatment of IBD.

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.

Intestinal fibrosis and stenosis are common complications of Crohn’s disease [CD], frequently requiring surgery. Anti-inflammatory strategies can only partially prevent fibrosis; hence, anti-fibrotic therapies remain an unmet clinical need. Oxysterols are oxidised cholesterol derivatives with important roles in various biological processes. The enzyme cholesterol 25-hydroxylase [CH25H] converts cholesterol to 25-hydroxycholesterol [25-HC], which modulates immune responses and oxidative stress. In human intestinal samples from CD patients, we found a strong correlation of CH25H mRNA expression with the expression of fibrosis markers. We demonstrate reduced intestinal fibrosis in mice deficient for the CH25H enzyme, using the sodium dextran sulphate [DSS]-induced chronic colitis model. Additionally, using a heterotopic transplantation model of intestinal fibrosis, we demonstrate reduced collagen deposition and lower concentrations of hydroxyproline in CH25H knockouts. In the heterotopic transplant model, CH25H was expressed in fibroblasts. Taken together, our findings indicate an involvement of oxysterol synthesis in the pathogenesis of intestinal fibrosis.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide and the need for novel biomarkers and therapeutic strategies to improve diagnosis and surveillance is obvious. This study aims to identify β6 -integrin (ITGB6) as a novel serum tumor marker for diagnosis, prognosis, and surveillance of CRC. ITGB6 serum levels were validated in retro- and prospective CRC patient cohorts. ITGB6 serum levels were analyzed by ELISA. Using an initial cohort of 60 CRC patients, we found that ITGB6 is present in the serum of CRC, but not in non-CRC control patients. A cut-off of ≥2 ng/mL ITGB6 reveals 100% specificity for the presence of metastatic CRC. In an enlarged study cohort of 269 CRC patients, ITGB6 predicted the onset of metastatic disease and was associated with poor prognosis. Those data were confirmed in an independent, prospective cohort consisting of 40 CRC patients. To investigate whether ITGB6 can also be used for tumor surveillance, serum ITGB6-levels were assessed in 26 CRC patients, pre- and post-surgery, as well as during follow-up visits. After complete tumor resection, ITGB6 serum levels declined completely. During follow-up, a new rise in ITGB6 serum levels indicated tumor recurrence or the onset of new metastasis as confirmed by CT scan. ITGB6 was more accurate for prognosis of advanced CRC and for tumor surveillance as the established marker carcinoembryonic antigen (CEA). Our findings identify ITGB6 as a novel serum marker for diagnosis, prognosis, and surveillance of advanced CRC. This might essentially contribute to an optimized patient care.

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.

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.

Only a fraction of patients benefits from immune checkpoint inhibitors (ICI). Tumor cells exhibit a distinct glycosylation pattern and supplementation of glycans has the potential to promote response and overcome resistance to ICI, thus addressing an urgent clinical need. Using a subcutaneous tumor mouse model, we investigated whether two glycopeptide-based test items reduce tumor burden when orally administered alone or in combination with ICI. C57/BL6 mice were supplemented with 3% GNU-201 (n=10) or 3% GNU-101 (n=10) in the drinking water starting 14 days prior to tumor cell injection (d-14), while control mice (n=10) received normal drinking water. On day 0, all mice were injected subcutaneously with YUMM1.7 melanoma cells in each flank. On days 6, 9 and 12, mice in each group received anti-PD1 antibody or an isotype control. Tumor size was measured every 3rd day. On day 15, the mice were sacrificed to analyze tumor weight and tumor infiltrating immune cells. 16S rDNA sequencing of fecal samples was performed on days -14, 0 and 14. GNU-201 and GNU-101 supplementation was well tolerated, no difference in water consumption and no abnormal behavior were detected that would indicate adverse effects of the products. While anti-PD1 on its own had no effect on tumor growth, we observed reduced tumor growth in the GNU-201+anti-PD1, GNU-101 and GNU-101+anti-PD1-treated groups, resulting in a significantly reduced tumor volume in these three groups on day 15. Administration of the two test items had a clear effect on anti-tumor T cell responses. In combination with anti-PD1, GNU-201 promoted the overall abundance of T cells, and among those the proportion of CD8+ T cells and IFNγ producing CD4+ T cells. Even in absence of anti-PD1, GNU-201 promoted the number of IFNγ+ CD8+ T cells, indicating that GNU-201 did not only promote tumor infiltration but also activation of CD8+ T cells, which are known to directly kill tumor cells. Finally, GNU-201+anti-PD1 resulted in significantly increased levels of Perforin and TNFα producing CD8+ T cells. With GNU-101 there was a trend towards elevated numbers of T cells and TNFα+ CD4 T cells. In mice that received GNU-101 with anti-PD1, we observed significantly elevated levels of TNFα+ CD8+ T cells and a trend towards higher numbers of IFNγ+ CD8+ T cells. GNU-201 and GNU-101 induced a significant modulation of the gut microbiota. In particular, the products promoted the growth of Akkermansia spp., a genus previously associated with anti-tumor immunity. In conclusion, GNU-201 and GNU-101 are both able to boost an anti-tumor immune response upon PD1 inhibition and GNU-101 shows moderate anti-tumor effects on its own. The response is linked with tumor-infiltration and activation of T cells and seems to be associated with changes in the gut microbiome. This study highlights a potential therapeutic role for glycopeptides in boosting anti-tumor immunity in response to ICI therapy. Citation Format: Marianne R. Spalinger, Romain Wyss, Sara Vidal, Yong Miao, Michael Scharl, Yemi Adesokan. Glycopeptides promote anti-cancer immune response against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5600.

Background Fistulas represent a frequent and severe complication in patients with Crohn disease (CD). Tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta, and interleukin (IL)-13 are known to trigger epithelial-mesenchymal transition (EMT), promoting fistula formation. Here, we investigated the role of T-lymphocytes (T cells) in fistula pathogenesis. Methods CD3+CD8-, CD3+CD8+, or CD45+CD3- cells from healthy volunteers, patients with CD, and patients with CD with perianal fistula were co-cultured with HT-29 cells. The EMT, cytokine production, and mRNA expression were analyzed. Perianal CD fistula specimens were immunohistochemically stained for cytokines and their receptors. The effect of cytokines on EMT induction was investigated using an EMT spheroid model. Results Patients with CD with fistula revealed more CD3+CD8- and less CD3+CD8+ T cells in blood than healthy control patients and patients with CD without fistula. In perianal fistula specimens, CD4+ cells—and to a lesser extent CD8+ cells—were highly present around fistula tracts. When co-cultured with HT-29 cells, both cell subsets promoted EMT-related gene expression and TNF-α production in a time-dependent manner. The CD3+CD8- T cells from patients with CD with fistula also produced higher amounts of IL-13 than cells from healthy control patients or patients with CD without a fistula. We found that IL-22 and IL-22Rα1 were highly expressed in perianal CD fistula specimens and that IL-22 cotreatment potentiated TNF-α-induced EMT in HT-29 spheroids. Conclusions Our data indicate that both CD3+CD8- and CD3+CD8+ T cells play an important role in the pathogenesis of perianal CD fistulas by the secretion of TNF-α. Our data support clinical evidence indicating that anti-TNF-α therapy is effective in fistula treatment and identify IL-13 and IL-22 as possible novel therapeutic targets for fistula therapy.

Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) recently emerged as a promising cancer immunotherapy target. We set out to investigate the functional role of PTPN2 in the pathogenesis of human colorectal carcinoma (CRC), as its role in immune-silent solid tumors is poorly understood. We demonstrate that in human CRC, increased PTPN2 expression and activity correlated with disease progression and decreased immune responses in tumor tissues. In particular, stage II and III tumors displayed enhanced PTPN2 protein expression in tumor-infiltrating T cells, and increased PTPN2 levels negatively correlated with expression of PD-1, CTLA4, STAT1, and granzyme A. In vivo, T cell– and DC-specific PTPN2 deletion reduced tumor burden in several CRC models by promoting CD44(+) effector/memory T cells, as well as CD8(+) T cell infiltration and cytotoxicity in the tumor. In direct relevance to CRC treatment, T cell–specific PTPN2 deletion potentiated anti–PD-1 efficacy and induced antitumor memory formation upon tumor rechallenge in vivo. Our data suggest a role for PTPN2 in suppressing antitumor immunity and promoting tumor development in patients with CRC. Our in vivo results identify PTPN2 as a key player in controlling the immunogenicity of CRC, with the strong potential to be exploited for cancer immunotherapy.

Many patients suffering from inflammatory bowel disease (IBD) follow restrictive diets, as many respective recommendations circulate. Efforts are made to evaluate and summarize the published information, for example, in a recent consensus manuscript by the International Organization for the Study of IBD (IOIBD). However, the standards that should be applied to make claims about dietary effects are poorly defined. In this manuscript, the scientific basis of recommendations for nutritional interventions in IBD is analyzed. Epidemiological evidence on diet in IBD is always biased by numerous factors, and the number of robust dietary intervention studies is limited due to methodological difficulties. Therefore, animal models are used to test hypotheses with respect to dietary factors and intestinal inflammation. Naturally, animal models have limitations, and knowledge of key characteristics of colitis animal models is crucial to understand their advantages and disadvantages. In recent years the important role of the microbiota for IBD and dietary factors has been discovered. Microbiota data are added to many publications on IBD and nutrition. The quality of those data varies largely. Subsequently, quality standards for microbiota analyses also are discussed. Finally, quality requirements to be applied on recommendations for dietary changes in patients with IBD are suggested.

Coronavirus disease (COVID-19), caused by SARS-CoV-2, has affected over 65 million individuals and killed over 1.5 million persons (December 8, 2020;www.who.int)1. While fatality rates are higher among the elderly and those with underlying comorbidities2, host factors that promote susceptibility to SARS-CoV-2 infection and severe disease are poorly understood. Although individuals with certain autoimmune/inflammatory disorders show increased susceptibility to viral infections, there is incomplete knowledge of SARS-CoV-2 susceptibility in these diseases.3–7We report that the autoimmunePTPN2risk variant rs1893217 promotes expression of the SARS-CoV-2 receptor, ACE2, and increases cellular entry mediated by SARS-CoV-2 spike protein. Elevated ACE2 expression and viral entry were mediated by increased JAK-STAT signalling, and were reversed by the JAK inhibitor, tofacitinib. Collectively, our findings uncover a novel risk biomarker for increased expression of the SARS-CoV-2 receptor and viral entry, and identify a clinically approved therapeutic agent to mitigate this risk.

BackgroundThe rise in the prevalence of inflammatory bowel diseases in the past decades coincides with changes in nutritional habits, such as adaptation of a Western diet. However, it is largely unknown how certain nutritional habits, such as energy drink consumption, affect intestinal inflammation. Here, we assessed the effect of energy drink supplementation on the development of intestinal inflammation in vitro and in vivo.MethodsHT-29 and T84 intestinal epithelial cells and THP-1 monocytic cells were treated with IFNγ in presence or absence of different concentrations of an energy drink. Colitis was induced in C57BL/6 mice by addition of dextran sodium sulfate (DSS) to drinking water with or without supplementation of the energy drink.ResultsEnergy drink supplementation caused a dose-dependent decrease in IFNγ-induced epithelial barrier permeability, which was accompanied by upregulation of the pore-forming protein claudin-2. Administration of the energy drink reduced secretion of the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-α from HT-29, T84, and THP-1 cells. In vivo, energy drink administration reduced clinical symptoms of DSS-induced colitis and epithelial barrier permeability. Endoscopic and histologic colitis scores and expression of pro-inflammatory cytokines were significantly reduced by energy drink co-administration.ConclusionEnergy drink consumption seems to exert an unexpected anti-inflammatory effect in vitro and in vivo in our experimental setting. However, our experimental approach focuses on intestinal inflammation and neglects additional effects of energy drink consumption on the body (eg, on metabolism or sleep). Therefore, the translation of our findings into the human situation must be taken with caution.

Background and Aims Loss-of-function variants in protein tyrosine phosphatase non-receptor type-2 [PTPN2] promote susceptibility to inflammatory bowel diseases [IBD]. PTPN2 regulates Janus-kinase [JAK] and signal transducer and activator of transcription [STAT] signalling, while protecting the intestinal epithelium from inflammation-induced barrier disruption. The pan-JAK inhibitor tofacitinib is approved to treat ulcerative colitis, but its effects on intestinal epithelial cell-macrophage interactions and on barrier properties are unknown. We aimed to determine if tofacitinib can rescue disrupted epithelial-macrophage interaction and barrier function upon loss of PTPN2. Methods Human Caco-2BBe intestinal epithelial cells [IECs] and THP-1 macrophages expressing control or PTPN2-specific shRNA were co-cultured with tofacitinib or vehicle. Transepithelial electrical resistance and 4 kDa fluorescein-dextran flux were measured to assess barrier function. Ptpn2fl/fl and Ptpn2-LysMCre mice, which lack Ptpn2 in myeloid cells, were treated orally with tofacitinib citrate twice daily to assess the in vivo effect on the intestinal epithelial barrier. Colitis was induced via administration of 1.5% dextran sulphate sodium [DSS] in drinking water. Results Tofacitinib corrected compromised barrier function upon PTPN2 loss in macrophages and/or IECs via normalisation of: [i] tight junction protein expression; [ii] excessive STAT3 signalling; and [iii] IL-6 and IL-22 secretion. In Ptpn2-LysMCre mice, tofacitinib reduced colonic pro-inflammatory macrophages, corrected underlying permeability defects, and prevented the increased susceptibility to DSS colitis. Conclusions PTPN2 loss in IECs or macrophages compromises IEC-macrophage interactions and reduces epithelial barrier integrity. Both of these events were corrected by tofacitinib in vitro and in vivo. Tofacitinib may have greater therapeutic efficacy in IBD patients harbouring PTPN2 loss-of-function mutations.

Activity of the NLRP3 inflammasome, a critical mediator of inflammation, is controlled by accessory proteins, posttranslational modifications, cellular localization, and oligomerization. How these factors relate is unclear. We show that a well-established drug target, Bruton’s tyrosine kinase (BTK), affects several levels of NLRP3 regulation. BTK directly interacts with NLRP3 in immune cells and phosphorylates four conserved tyrosine residues upon inflammasome activation, in vitro and in vivo. Furthermore, BTK promotes NLRP3 relocalization, oligomerization, ASC polymerization, and full inflammasome assembly, probably by charge neutralization, upon modification of a polybasic linker known to direct NLRP3 Golgi association and inflammasome nucleation. As NLRP3 tyrosine modification by BTK also positively regulates IL-1β release, we propose BTK as a multifunctional positive regulator of NLRP3 regulation and BTK phosphorylation of NLRP3 as a novel and therapeutically tractable step in the control of inflammation.

Inflammasomes are multi-protein complexes that mediate the activation and secretion of the inflammatory cytokines IL-1β and IL-18. More than half a decade ago, it has been shown that the inflammasome adaptor molecule, ASC requires tyrosine phosphorylation to allow effective inflammasome assembly and sustained IL-1β/IL-18 release. This finding provided evidence that the tyrosine phosphorylation status of inflammasome components affects inflammasome assembly and that inflammasomes are subjected to regulation via kinases and phosphatases. In the subsequent years, it was reported that activation of the inflammasome receptor molecule, NLRP3, is modulated via tyrosine phosphorylation as well, and that NLRP3 de-phosphorylation at specific tyrosine residues was required for inflammasome assembly and sustained IL-1β/IL-18 release. These findings demonstrated the importance of tyrosine phosphorylation as a key modulator of inflammasome activity. Following these initial reports, additional work elucidated that the activity of several inflammasome components is dictated via their phosphorylation status. Particularly, the action of specific tyrosine kinases and phosphatases are of critical importance for the regulation of inflammasome assembly and activity. By summarizing the currently available literature on the interaction of tyrosine phosphatases with inflammasome components we here provide an overview how tyrosine phosphatases affect the activation status of inflammasomes.

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.

Background Tissue inflammation in inflammatory bowel diseases [IBD] is associated with local acidification. Genetic variants in the pH-sensing G protein-coupled receptor 65, also known as T cell death-associated gene 8 [TDAG8], have been implicated in IBD and other autoimmune diseases. Since the role of TDAG8 in intestinal inflammation remains unclear, we investigated the function of TDAG8 using murine colitis models. Methods The effects of TDAG8 deficiency were assessed in dextran sodium sulphate [DSS], IL-10-/-, and T cell transfer colitis murine models. RNA sequencing of acidosis-activated TDAG8-/- and wild-type [WT] peritoneal macrophages [MΦs] was performed. Results mRNA expression of IFN-γ, TNF, IL-6, and iNOS in TDAG8-/- mice increased significantly in colonic lymphoid patches and in colonic tissue in acute and chronic DSS colitis, respectively. In transfer colitis, there was a trend towards increased IFN-γ, iNOS, and IL-6 expression in mice receiving TDAG8-/- T cells. However, absence of TDAG8 did not lead to changes in clinical scores in the models tested. Increased numbers of infiltrating MΦs and neutrophils, but not CD3+ T cells, were observed in DSS-treated TDAG8-/- mice. No differences in infiltrating CD3+ T cells were observed between mice receiving TDAG8-/- or WT naive T cells in transfer colitis. RNA sequencing showed that acidosis activation of TDAG8 in MΦs modulated the expression of immune response genes. Conclusions TDAG8 deficiency triggers colonic MΦ and neutrophil infiltration, and expression of pro-inflammatory mediators in DSS colitis models. In transfer colitis, mice receiving TDAG8-/- T cells presented a significantly higher spleen weight and a tendency towards increased expression of pro-inflammatory markers of monocyte/MΦ activity.

Summary: 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.