Changes in phosphate metabolism in thymoma cells suggest mechanisms for resistance to dexamethasone-induced apoptosis. A 31P NMR spectroscopic study of cell extracts

Treatment of the mouse thymoma-derived WEHI7.2 cell line with dexamethasone, a synthetic glucocorticoid, causes the cells to undergo apoptosis. Previous studies have shown that WEHI7.2 cell variants with an increased antioxidant defense exhibit increased resistance to dexamethasone-induced apoptosis, suggesting that oxidative stress may play a role in glucocorticoid-induced apoptosis. In this work we compared metabolic profiles of WEHI7.2 parental cells with those of WEHI7.2 variants with an increased antioxidant defense or overexpressing bcl-2, to determine whether bolstering the antioxidant defense results in altered metabolic parameters that could translate into increased resistance to dexamethasone-induced apoptosis. WEHI7.2 parental cells and cells overexpressing catalase, thioredoxin or bcl-2, or selected for resistance to 200 micro M H(2)O(2) were cultured in low-glucose DMEM medium supplemented with 10% calf serum, and extracted using chloroform-methanol-water (1:1:1). Metabolites contained in the aqueous and organic phases of the extracts were processed separately and subjected to high-resolution (31)P NMR spectroscopy. In most of the steroid-resistant variants, ATP levels and energetic status were decreased compared with the steroid-sensitive parental cell line, while the concentrations of hexose and triose phosphates were increased. Furthermore, the ratio of choline-containing phospholipids to ethanolamine-containing phospholipids was generally reduced in steroid-resistant cells. Phosphatidylethanolamine and its derivatives contain a higher amount of polyunsaturated fatty acids (PUFA) than the choline-containing analogs, and PUFA are readily oxidized by reactive oxygen species. Therefore, an increased initial amount of phosphatidylethanolamine may increase the 'buffering capacity' of this antioxidant and may thus contribute to the steroid resistance of WEHI7.2 variants.     

Glucose 6-phosphate dehydrogenase overexpression models glucose deprivation and sensitizes lymphoma cells to apoptosis

Glucocorticoids are one component of combined treatment regimens for many types of lymphoma due to their ability to induce apoptosis in lymphoid cells. In WEHI7.2 murine thymic lymphoma cells, altering catalase and glutathione peroxidase activity by transfection or the use of chemical agents modulates the ability of glucocorticoids to induce apoptosis. This suggests that the oxidative stress response is important in determining the glucocorticoid sensitivity of the cells. For glutathione peroxidase and catalase to detoxify reactive oxygen species (ROS), reducing equivalents in the form of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) are ultimately required. The major source of NADPH in the cell is glucose 6-phosphate dehydrogenase (G6PDH). Therefore, we created G6PDH-overexpressing WEHI7.2 variants to test whether G6PDH activity is a key determinant of glucocorticoid sensitivity in WEHI7.2 cells. G6PDH-overexpressing WEHI7.2 cells were more sensitive to oxidative stress and glucocorticoids. The G6PDH-overexpressing WEHI7.2 variants appeared similar to cells undergoing glucose deprivation with decreased adenosine triphosphate (ATP) synthesis by the mitochondria and increased basal levels of ROS. Overexpression of G6PDH also sensitized the cells to other standard lymphoma chemotherapeutics including cyclophosphamide, doxorubicin, and vincristine. The decreased ATP and elevated ROS due to G6PDH overexpression may be key factors in increasing the sensitivity of the WEHI7.2 cells to lymphoma chemotherapeutics.     

Obesity and hepatocellular carcinoma: targeting obesity-related inflammation for chemoprevention of liver carcinogenesis

Obesity and related metabolic abnormalities, including a state of chronic inflammation, increase the risk of hepatocellular carcinoma (HCC). Adipose tissue constitutively expresses the proinflammatory cytokine tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which are important tumor promoters in inflammation-related carcinogenesis. Dysregulation of TNF-α and IL-6 is associated with the development of steatosis and inflammation within the liver. These cytokines also lie at the core of the association between obesity and insulin resistance, which is a key factor in the development of obesity-related HCC. Here we present a detailed review of the relationship between metabolic abnormalities and the development of HCC, focusing on the role played by inflammation. Drawing from our basic and clinical research, the present report also reviews evidence that targeting metabolic abnormalities, such as attenuation of chronic inflammation and improvement of insulin resistance by either pharmaceutical or nutritional intervention, may be an effective strategy in preventing the development of HCC in obese individuals.     

Tumor necrosis factor production by human granulocytes.

Human polymorphonuclear leukocytes kill WEHI 164 cells in an 18-hour 51Cr release assay. Antibody to human tumor necrosis factor (TNF) blocks the lysis of targets mediated by human granulocytes. Resting granulocytes produce an undetectable amount of TNF, if any. Granulocytes stimulated with Staphylococcus aureus release 250-500 U/ml TNF alpha. The specificity of the released TNF in the WEHI 164 cytotoxicity assay was confirmed by using neutralizing anti-TNF alpha monoclonal antibodies. The thymidine uptake of endothelial cells was inhibited by granulocyte-derived TNF. The identity of TNF alpha was further confirmed by molecular weight determination, by gel filtration on Sephacryl S-200, with a result of approximately 44,000. Besides their antimicrobial capacity, therefore, granulocytes may contribute to tumor rejection, inflammation and septic infections by releasing TNF.     

Involvement of tumor necrosis factor in human granulocyte-mediated killing of WEHI 164 cells

Human polymorphonuclear leukocytes (PMNLs) kill WEHI 164 clone 13 cells in an 18-hour 51Cr release assay. Antibody to human tumor necrosis factor (TNF) blocks the lysis of targets mediated by human granulocytes. PMNLs triggered by sensitive targets not only displayed cytotoxic activity, but also released a soluble factor capable of selectively lysing WEHI 164 cells. The killing of these cells by supernatants of triggered granulocytes was totally inhibited by anti-TNF antibody. These experiments suggest that the killing of WEHI 164 sarcoma cells by human PMNLs involves TNF or TNF-like molecules.     

Tumor necrosis factor-related apoptosis-inducing ligand mediates the resolution of allergic airway inflammation induced by chronic allergen inhalation

Allergic asthma can vanish over time either spontaneously or induced by allergen-specific immunotherapy. In mice with established airway allergic inflammation, chronic intranasal (IN) allergen challenges decreases progressively airway allergic inflammation. Here we compared the contribution of different regulatory pathways that could be associated with this phenomenon, known as local inhalational tolerance. We found that inhalational tolerance was not associated with increased number of regulatory T cells or suppressive cytokines. Instead, it was associated with increased apoptosis of airway inflammatory leukocytes revealed by annexin-V staining and the expression of apical caspase 8 and effector caspase 3. Also, the transition from acute to chronic phase was associated with a shift in the expression of pro-allergic to pro-apoptotic molecules. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was found to be a key molecule in mediating resolution of allergic inflammation because anti-TRAIL treatment blocked apoptosis and increased the infiltration of T helper type 2 (Th2) cells and eosinophils. Notably, repeated IN treatment with recombinant TRAIL in established airway allergic inflammation augmented leukocyte apoptosis and decreased the frequency of interleukin-5-producing Th2 cells and eosinophils to airways. Our data indicate that TRAIL signaling is sufficient for downmodulation of allergic airway disease, suggesting a potential therapeutic use of TRAIL for asthma treatment.     

Immunogold analysis of antioxidant enzymes in human renal cell carcinoma

Analysis of activities of the antioxidant enzyme manganese superoxide dismutase in human renal cell carcinomas often showed greatly altered enzyme levels (either elevated or depressed) compared to the cell of origin, the kidney proximal tubule. In order to better understand the variability observed, immunogold studies were performed on human renal cell carcinomas using a polyclonal antibody to human kidney manganese superoxide dismutase. For comparison, studies were also performed using antibodies to other antioxidant enzymes. For histologic studies, renal cell carcinomas were subclassified on the basis of light microscopy and ultrastructural analysis into clear cell, granular cell, or mixed clear and granular cell variants. In all three types of tumor, immunogold studies showed little staining using antibodies to copper, zinc superoxide dismutase or glutathione-dependent enzymes. However, intensity of labeling for manganese superoxide dismutase and catalase depended on the cell type(s) in the tumor. Clear cell variants demonstrated trace staining for manganese superoxide dismutase and catalase, while granular cell variants exhibited heavy staining for both of these enzymes. Mixed types of tumors showed clear cells with trace staining for all antioxidant enzymes examined, while granular cells again showed intense labeling for manganese superoxide dismutase and catalse. Using normal kidney proximal tubule as a comparison, immunogold ultrastructural analysis using antibody to manganese superoxide dismutase demonstrated infrequent small lightly labeled mitochondria in clear cell variants, while granular cell variants exhibited numerous medium-sized heavily labeled mitochondria. These data suggest that: 1) the variability in activity values for manganese superoxide dismutase may be due to heterogeneity of cell types in these tumors and 2) manganese superoxide dismutase immunoreactive protein was elevated in granular cells both because of an increase in number of mitochondria and because the labeling density in mitochondria was increased compared to mitochondria in clear cell types or in normal proximal tubular cells.     

Guinea pig kurloff (NK-like) cells mediate TNF-dependent cytotoxic activity: Analogy with NC effector cells

Kurloff cells are mononuclear cells possessing a large cytoplasmic inclusion body specific to the guinea pig. In this report, we present strong evidence that Kurloff cells can mediate NC activity against tumor cells in addition to their previously reported NK activity. Using an 18 h⁵¹Cr-release assay we have shown that Kurloff cells were highly effective in killing the TNF-sensitive WEHI 164 target cell line. Lower but significant cytotoxic activity was also observed after only 4 h. However, our results suggest a different mechanism of lysis in the 4 h and 18 h assay. Lysis of WEHI 164 target cells by Kurloff cells in the 4 h assay could be strongly increased in the presence of TPA alone or in combination with ionomycin whereas ionomycin alone was uneffective. In contrast, stimulation of Kurloff cells for 18 h with ionomycin alone or in combination with TPA could induce the release of TNF-like factor(s) as observed by the TNF bioassay using L-929 TNF-sensitive target cells. Release of TNF-like factor(s) could also be induced by stimulation with WEHI 164 target cells. Supernatants of Kurloff cells stimulated for 18 h with TPA + ionomycin were also highly cytotoxic against WEHI 164 target cells, but not against the TNF-resistant P815 target cell line. Pretreatment of these supernatants with antimurine TNF antibodies could almost completely inhibit their cytotoxic activity against WEHI 164 target cells. In contrast, supernatants of Kurloff cells stimulated for only 4 h did not show any TNF-like activity against the L-929 target cell line and were not cytotoxic against WEHI 164 target cells even after 18 h. Taken together, these results suggest that Kurloff cells can mediate NC activity against tumor cells in addition to their previously reported NK activity. By using multiple lytic pathways, these cells may play a crucial role in anti-tumor surveillance and defenses.Supported by grants from the National Cancer Institute and the Medical Research Council of Canada.Recipient of a studentship from the F.C.A.R.     

A role for microchimerism in obesity and evolution?

Cells exchanged between individuals, such as those passing the placenta from the mother to the child and vice versa, may survive in the fetal or maternal circulation and tissues for decades and result in microchimerism. Microchimeric cells may play a role in tissue repair, but they have also been implicated as inducers of chronic inflammation, leading to autoimmunity or even cancer. Here we propose that microchimerism may play a more fundamental role in health and evolution by setting a limit to genomic variability within populations. This means that microchimerism allows immune recognition of genomic differences between donor and host which may, depending on the level of variability, cause chronic inflammation. Since chronic inflammation has been experimentally linked to metabolic syndrome, we propose that genomic variability could affect the individual's weight. Thus, metabolic syndrome, which is a growing health problem, may not only result from our lifestyle, but in part be caused by global migration and the increasingly diverse origin of the present human population. Moreover, since in nature weight gain is associated with an increased risk of predation, we discuss the possibility that immunological incompatibility normally promotes the continuous development of new species.     

The relationship between the metabolic syndrome and energy-utilization deficit in the pathogenesis of obesity-induced osteoarthritis

We propose that the pathogenesis of obesity-induced osteoarthritis may be explained by the metabolic changes in the striated muscle induced by the interaction of insulin resistance and systemic inflammation in obese individuals with metabolic syndrome being osteoarthritis the latest consequence by the physiological changes seen in the metabolic syndrome. Increased levels of TH1 cytokines are produced by activated macrophages in the presence of an acute or chronic infectious disease and suppress the sensitivity of insulin receptors on the membrane of muscle cell and adipocytes. Both cells are activated by inflammatory cytokines and contribute to enhance acute inflammation and to maintain a state of chronic, low-grade inflammation in apparently healthy obese individuals. The increased number of macrophage in the adipose tissue of obese individuals acts as an amplifier of inflammation. Patients with osteoarthritis and metabolic syndrome frequently are complaining about hotness and recurrent edema of feet and hands. It is probable that hyperinsulinemia in the presence of insulin resistance and inflammation, induce vasodilation through the TNF mediated-iNOS overexpression. Patients with metabolic syndrome express clinically the consequence of a poor uptake, storage and energy expenditure by the muscle and any other insulin dependent tissue and the consequence of high insulin plasma levels are vasodilation and increased protein synthesis. The fatigue and muscle weakness induced by insulin resistance and inflammation in obese patients with metabolic syndrome increase the frequency and the intensity of traumatic events of peripheral or axial joints that result in stretch and breaking of tenoperiosteal junction and abrasive damage of cartilage and therefore in these patients with metabolic syndrome and pro-inflammatory state the reparative process of cartilage and periarticular tissues would be severely modified by the growth factor activity in presence of high levels of insulin.     

Mast cells in the promotion and limitation of chronic inflammation

Summary:  Observations of increased numbers of mast cells at sites of chronic inflammation have been reported for over a hundred years. Light and electron microscopic evidence of mast cell activation at such sites, taken together with the known functions of the diverse mediators, cytokines, and growth factors that can be secreted by appropriately activated mast cells, have suggested a wide range of possible functions for mast cells in promoting (or suppressing) many features of chronic inflammation. Similarly, these and other lines of evidence have implicated mast cells in a variety of adaptive or pathological responses that are associated with persistent inflammation at the affected sites. Definitively characterizing the importance of mast cells in chronic inflammation in humans is difficult. However, mice that genetically lack mast cells, especially those which can undergo engraftment with wildtype or genetically altered mast cells, provide a means to investigate the importance of mast cells and specific mast cell functions or products in diverse models of chronic inflammation. Such work has confirmed that mast cells can significantly influence multiple features of chronic inflammatory responses, through diverse effects that can either promote or, perhaps more surprisingly, suppress aspects of these responses.     

Antigen receptor signalling in apoptosis-resistant mutants of WEHI 231 cells

Ligation of membrane immunoglobulin M (mIgM) induces cell cycle arrest and apoptosis in the WEHI 231 B-lymphoma cell line. The molecular mechanisms which link receptor ligation and the nuclear events that underlie this response, have yet to be fully elucidated. Here we have examined the signals induced following mIgM cross-linking in variants of WEHI 231 that no longer undergo apoptosis in response to this stimulus. Tyrosine phosphorylation of cellular substrates in two of the variants is identical to that seen in wild-type cells but in one of the mutants, VS2.12, a restricted set of substrates becomes tyrosine phosphorylated. In a second variant (E8), mIgM cross-linking does not induce elevation of intracellular Ca2+, although tyrosine phosphorylation of PLCgamma2 is induced to an equivalent extent to that seen in WEHI 231 cells. A third variant, 2E10.F9, is resistant to apoptosis despite the fact that all signals analysed appear to be similar to those induced in wild-type cells. Our findings show that resistance to apoptosis can arise as a result of mutations affecting discrete stages of the mIgM signalling pathway. The mutant lines reported here show defects that have not yet been identified in previous studies and are likely to be useful tools in dissecting the signalling of cell death in B lymphocytes.     

Cytokines increase proliferation of human intestinal smooth muscle cells: Possible role in inflammation-induced stricture formation

Crohn's disease is an idiopathic, chronic inflammation of the gastrointestinal tract that causes narrowing and stricturing of primarily the small and large intestine. Although the mechanism(s) by which chronic inflammation promotes stricture formation remain to be defined, it does appear to be associated histologically with a hyperplasia of smooth muscle cells and an increased deposition of collagen within the bowel wall. The objective of this study was to assess the effect of two proinflammatory cytokines, tumor necrosis factor and interleukin-1, on the proliferation of human intestinal smooth muscle cells in vitro. Human intestinal smooth muscle cells were seeded at subconfluent densities into 24-well plates in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Human recombinant tumor necrosis factor (0.1–100 ng/ml), interleukin-1 (0.1–500 ng/ml), or control medium (without cytokines) was then added to the cells and incubation continued for 48 or 72 h. Proliferation was determined by the incorporation of tritiated thymidine, added during the final 18 h, into the cellular DNA of the smooth muscle cells. Both cytokines caused a significant dose-dependent increase in intestinal smooth muscle cell proliferation relative to control. These results suggest that the interleukin-1 and tumor necrosis factor produced during chronic inflammation in vivo may enhance the proliferation of smooth muscle cells within the intestinal bowel wall and hence potentially contribute to the narrowing and stricturing of the intestine that is observed in Crohn's disease.This work was supported by a Merit Review Grant from the Veterans Affairs Medical Research Service (M.W.O.) and by a grant from the NIH (DK43785, Project 6; M.B.G.).     

Common Regularities in Changes of the Neutrophil Functional Activity during <Emphasis Type="Italic">In Vivo</Emphasis> Growth of Tumors of Different Immunogenic Activity

Studies on BALB/c mice with tumors of different immunogenic activity (nonimmunogenic J774, WEHI 164 and immunogenic NS0) have showed that the development of a tumor is associated with changes in the neutrophil morphology and functions: the counts and size of the cells migrating to the focus increase and their capacity to produce active oxygen species is changed.     

Characterisation of functional biotinylated TNF-alpha targeted to the membrane of apoptotic melanoma cells

The physiologic clearance of apoptotic cells prevents inflammation at the site of cell death and limits the immunogenicity of tumors. In this study we report the functional characterisation of biotinylated tumor necrosis factor-alpha (TNF-alpha) after anchorage to apoptotic melanoma cells via a biotin-avidin-biotin bridge. Flow cytometric and morphological analysis showed that biotinylated TNF-alpha efficiently bound to apoptotic membrane blebs of dying cells. Membrane-bound TNF-alpha (12 fg/cell) killed sensitive WEHI164 cells 250-fold more effectively than equivalent amounts of the soluble cytokine. Furthermore, macrophages engulfing apoptotic cells with membrane-bound TNF-alpha secreted significantly higher amounts of soluble TNF-alpha and lower amounts of interleukin-10 (IL-10). Therefore the bridging of TNF-alpha potentiates its biological function and influences the outcome of the phagocytic clearance of apoptotic tumor cells.     

Development and maintenance of cancer stem cells under chronic inflammation

In many human cancers, tumorigenic potential is not equally shared by all cells but is restricted to phenotypically distinct subpopulations termed cancer stem cells. Cancer stem cells are also capable of both self-renewal and differentiation, and these functional properties have been suggested to play major roles in tumor initiation and progression. The factors responsible for the development of cancer stem cells and their subsequent regulation are unclear, but several chronic inflammatory states have been associated with an increased risk of malignancy. Therefore, it is possible that specific processes associated with chronic inflammation, as well as the adaptation to cellular stress, regulate cancer stem cells. Several factors associated with chronic inflammation, including cytokines, oxidative stress, and hypoxia, induce the activation of specific cellular response programs that can affect the survival, proliferation, metabolism, and differentiation of cancer cells, as well as the self-renewal and quiescence of normal stem cells. In this review, we discuss how these adaptive processes potentially become subverted to enhance the development and function of cancer stem cells.     

The effect of maternal Inflammation on foetal programming of metabolic disease

Maternal obesity during pregnancy increases the child's risk of developing obesity and obesity‐related diseases later in life. Key components in foetal programming of metabolic risk remain to be identified; however, chronic low‐grade inflammation associated with obesity might be responsible for metabolic imprinting in the offspring. We have therefore surveyed the literature to evaluate the role of maternal obesity‐induced inflammation in foetal programming of obesity and related diseases. The literature on this topic is limited, so this review also includes animal models where maternal inflammation is mimicked by single injections with lipopolysaccharide (LPS). An LPS challenge results in an immunological response that resembles the obesity‐induced immune profile, although LPS injections provoke a stronger response than the subclinical obesity‐associated response. Maternal LPS or cytokine exposures result in increased adiposity and impaired metabolic homeostasis in the offspring, similar to the phenotype observed after exposure to maternal obesity. The cytokine levels might be specifically important for the metabolic imprinting, as cytokines are both transferable from maternal to foetal circulation and have the capability to modulate placental nutrient transfer. However, the immune response associated with obesity is moderate and therefore potentially weakened by the pregnancy‐driven immune modulation, dominated by anti‐inflammatory Treg and Th2 cells. We know from other low‐grade inflammatory diseases, such as rheumatoid arthritis, that pregnancy can improve disease state. If pregnancy is also capable of suppressing the obesity‐associated inflammation, the immunological markers might be less likely to affect metabolic programming in the developing foetus than otherwise implied.     

The vitamin B5/coenzyme A axis: A target for immunomodulation?

Coenzyme A (CoA) serves as a vital cofactor in numerous enzymatic reactions involved in energy production, lipid metabolism, and synthesis of essential molecules. Dysregulation of CoA-dependent metabolic pathways can contribute to chronic diseases, such as inflammatory diseases, obesity, diabetes, cancer, and cardiovascular disorders. Additionally, CoA influences immune cell activation by modulating the metabolism of these cells, thereby affecting their proliferation, differentiation, and effector functions. Targeting CoA metabolism presents a promising avenue for therapeutic intervention, as it can potentially restore metabolic balance, mitigate chronic inflammation, and enhance immune cell function. This might ultimately improve the management and outcomes for these diseases. This review will more specifically focus on the contribution of pathways regulating the availability of the CoA precursor Vitamin B5/pantothenate in vivo and modulating the development of Th17-mediated inflammation, CD8-dependent anti-tumor immunity but also tissue repair processes in chronic inflammatory or degenerative diseases.     

Rapid killing of actinomycin D-treated tumor cells by mononuclear phagocytes: characterization of effector cells in mice

Pretreatment with Actinomycin D (Act D, 1 microgram/ml for 3 hr) rendered WEHI 164 tumor cells susceptible to killing by mouse resident or peptone-induced peritoneal exudate cells (PEC) in a 6-hr 51Cr release assay. Cytotoxicity was attributed to cells of the monocyte macrophage lineage on the basis of tissue distribution, separation by adherence on plastic and carbonyl iron, membrane antigens, and expression in mice with defective T cell- or NK cell-mediated immunity. Macrophages from four strains of mice (C3H/HeJ, A/J, P/J, C57B1/10 ScCR) previously shown to have defective "classical" nonspecific tumoricidal activity were examined for killing of Act-D-treated WEHI 164 cells. C3H/HeJ peritoneal macrophages had little or no DDCC, whereas cells from A/J, P/J, and C57B1/10 ScCR mice had normal levels of this reactivity. Tumor cells exposed to ActD were heterogenous in their susceptibility to killing by PEC, with five lines showing significant, though variable, lysis, whereas 12 tumor lines, normal fibroblasts, and lymphoblasts were not appreciably killed under the same conditions. Macrophage-mediated DDCC was also detectable in a colony assay. DDCC could explain how macrophages contribute to the antitumor activity of selected chemotherapeutic agents in murine tumor models.     

PTPN2 controls differentiation of CD4+ T cells and limits intestinal inflammation and intestinal dysbiosis

Loss-of-function variants within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) are associated with increased risk for Crohn's disease (CD). A disturbed regulation of T helper (Th) cell responses causing loss of tolerance against self- or commensal-derived antigens and an altered intestinal microbiota plays a pivotal role in CD pathogenesis. Loss of PTPN2 in the T-cell compartment causes enhanced induction of Th1 and Th17 cells, but impaired induction of regulatory T cells (Tregs) in several mouse colitis models, namely acute and chronic dextran sodium sulfate colitis, and T-cell transfer colitis models. This results in increased susceptibility to intestinal inflammation and intestinal dysbiosis which is comparable with that observed in CD patients. We detected inflammatory infiltrates in liver, kidney, and skin and elevated autoantibody levels indicating systemic loss of tolerance in PTPN2-deficient animals. CD patients featuring a loss-of-function PTPN2 variant exhibit enhanced Th1 and Th17 cell, but reduced Treg markers when compared with PTPN2 wild-type patients in serum and intestinal tissue samples. Our data demonstrate that dysfunction of PTPN2 results in aberrant T-cell differentiation and intestinal dysbiosis similar to those observed in human CD. Our findings indicate a novel and crucial role for PTPN2 in chronic intestinal inflammation.