Protein Tyrosine Phosphatase Non-receptor Type Research Articles

Gene expression and Levels of Plasma Protein Tyrosine Phosphatase Non-Receptor Type 22 (PTPN22)in Pulmonary Tuberculosis Patients and their Household Contacts in Makassar, Indonesia

Gene expression and Levels of Plasma Protein Tyrosine Phosphatase Non-Receptor Type 22 (PTPN22)in Pulmonary Tuberculosis Patients and their Household Contacts in Makassar, Indonesia

PTPN2 Regulates Interactions Between Macrophages and Intestinal Epithelial Cells to Promote Intestinal Barrier Function

The mechanisms by which macrophages regulate intestinal epithelial cell (IEC) barrier properties are poorly understood. Protein tyrosine phosphatase non-receptor type 2 (PTPN2) protects the IEC barrier from inflammation-induced disruption and regulates macrophage functions. We investigated whether PTPN2 controls interactions between IECs and macrophages to maintain intestinal barrier function. Human IEC (Caco-2BBe/HT-29.cl19a cells) and mouse enteroid monolayers were cocultured with human macrophages (THP-1, U937, primary monocyte-derived macrophages from patients with inflammatory bowel disease [IBD]) or mouse macrophages, respectively. We assessed barrier function (transepithelial electrical resistance [TEER] and permeability to 4-kDa fluorescently labeled dextran or 70-kDa rhodamine B-dextran) and macrophage polarization. We analyzed intestinal tissues from mice with myeloid cell-specific deletion of PTPN2 (Ptpn2-LysMCre mice) and mice without disruption of Ptpn2 (controls); some mice were given injections of a neutralizing antibody against interleukin (IL) 6. Proteins were knocked down in macrophages and/or IECs with small hairpin RNAs. Knockdown of PTPN2 in either macrophages and/or IECs increased the permeability of IEC monolayers, had a synergistic effect when knocked down from both cell types, and increased the development of inflammatory macrophages in macrophage-IEC cocultures. Colon lamina propria from Ptpn2-LysMCre mice had significant increases in inflammatory macrophages; these mice had increased invivo and exvivo colon permeability to 4-kDa fluorescently labeled dextran and reduced exvivo colon TEER. Nanostring analysis showed significant increases in the expression of IL6 in colon macrophages from Ptpn2-LysMCre mice. An IL6-blocking antibody reversed the effects of PTPN2-deficient macrophages, reducing the permeability of IEC monolayers in culture and in Ptpn2-LysMCre mice. Macrophages from patients with IBD carrying a single-nucleotide polymorphism associated with the disease (PTPN2 rs1893217) had the same features of PTPN2-deficient macrophages from mice, including reduced TEER and increased permeability in cocultures with human IEC or mouse enteroid monolayers, which were restored by anti-IL6. PTPN2 is required for interactions between macrophages and IECs; loss of PTPN2 from either cell type results in intestinal barrier defects, and loss from both cell types has a synergistic effect. We provide a mechanism by which the PTPN2 gene variants compromise intestinal epithelial barrier function and increase the risk of inflammatory disorders such as IBD.

PTPN14 acts as a candidate tumor suppressor in prostate cancer and inhibits cell proliferation and invasion through modulating LATS1/YAP signaling

Protein tyrosine phosphatase, non-receptor type 14 (PTPN14) exerts a profound effect in the progression of multiple malignant tumors. However, whether PTPN14 plays a role in prostate cancer has not been well investigated. Herein, we evaluated the function and potential underlying mechanism of PTPN14 in prostate cancer. Decreased PTPN14 expression was detected in prostate cancer, and restoration of PTPN14 expression in prostate cancer cells inhibited the proliferative and invasive potential. Mechanistically, PTPN14 increased the phosphorylation of Yes-associated protein (YAP) by activation of large tumor suppressor 1 (LATS1), an action that resulted in a significant reduction in YAP-mediated transcriptional activity. Inactivation of YAP by its inhibitor markedly abrogated the PTPN14-knockdown-induced promotion effect on prostate cancer cell proliferation and invasion. Notably, PTPN14 up-regulation also exerted a remarkable suppressive impact on tumorigenesis of prostate cancer in vivo. Taken together, the study reveals a tumor-inhibition role of PTPN14 that represses the proliferation and invasion of prostate cancer by down-regulating YAP activation.

Linking protein to phenotype with Mendelian Randomization detects 38 proteins with causal roles in human diseases and traits.

To efficiently transform genetic associations into drug targets requires evidence that a particular gene, and its encoded protein, contribute causally to a disease. To achieve this, we employ a three-step proteome-by-phenome Mendelian Randomization (MR) approach. In step one, 154 protein quantitative trait loci (pQTLs) were identified and independently replicated. From these pQTLs, 64 replicated locally-acting variants were used as instrumental variables for proteome-by-phenome MR across 846 traits (step two). When its assumptions are met, proteome-by-phenome MR, is equivalent to simultaneously running many randomized controlled trials. Step 2 yielded 38 proteins that significantly predicted variation in traits and diseases in 509 instances. Step 3 revealed that amongst the 271 instances from GeneAtlas (UK Biobank), 77 showed little evidence of pleiotropy (HEIDI), and 92 evidence of colocalization (eCAVIAR). Results were wide ranging: including, for example, new evidence for a causal role of tyrosine-protein phosphatase non-receptor type substrate 1 (SHPS1; SIRPA) in schizophrenia, and a new finding that intestinal fatty acid binding protein (FABP2) abundance contributes to the pathogenesis of cardiovascular disease. We also demonstrated confirmatory evidence for the causal role of four further proteins (FGF5, IL6R, LPL, LTA) in cardiovascular disease risk.

PTPN2 Deficiency Enhances Programmed T Cell Expansion and Survival Capacity of Activated T Cells

SummaryManipulating molecules that impact T cell receptor (TCR) or cytokine signaling, such as the protein tyrosine phosphatase non-receptor type 2 (PTPN2), has significant potential for advancing T cell-based immunotherapies. Nonetheless, it remains unclear how PTPN2 impacts the activation, survival, and memory formation of T cells. We find that PTPN2 deficiency renders cells in vivo and in vitro less dependent on survival-promoting cytokines, such as interleukin (IL)-2 and IL-15. Remarkably, briefly ex vivo-activated PTPN2-deficient T cells accumulate in 3- to 11-fold higher numbers following transfer into unmanipulated, antigen-free mice. Moreover, the absence of PTPN2 augments the survival of short-lived effector T cells and allows them to robustly re-expand upon secondary challenge. Importantly, we find no evidence for impaired effector function or memory formation. Mechanistically, PTPN2 deficiency causes broad changes in the expression and phosphorylation of T cell expansion and survival-associated proteins. Altogether, our data underline the therapeutic potential of targeting PTPN2 in T cell-based therapies to augment the number and survival capacity of antigen-specific T cells.

Genetic susceptibility of increased intestinal permeability is associated with progressive liver disease and diabetes in patients with non-alcoholic fatty liver disease

Background and aimIncreased intestinal permeability plays a key role in the pathogenesis of fat deposition in the liver. The aim of our study was to assess whether a single nucleotide polymorphism of protein tyrosine phosphatase non-receptor type 2 (PTPN2) (rs2542151 T→G), involved in intestinal permeability, may be associated with non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM). Methods and resultsWe recruited a prospective cohort of NAFLD subjects and matched controls. Clinical data, PTPN2 genotype and laboratory data were collected for each patient. Results were stratified according to liver histology and diabetes. We enrolled 566 cases and 377 controls. PTPN2 genotype distribution did not significantly differ between patients and controls. In the entire population, patients with PTPN2 rs2542151 T→G (dominant model) have a higher prevalence of diabetes; 345 patients (60.9%) underwent liver biopsy: 198 (57.4%) had steatohepatitis and 75 (21.7%) had advanced fibrosis. At multiple logistic regression analysis PTPN2 rs2542151 T→G was associated with T2DM (OR 2.14, 95% CI 1.04–4.40, P = 0.03). Patients who underwent liver biopsy, rs2542151 T→G of PTPN2 was independently associated with severe steatosis (OR 2.00, 95% CI 1.17–3.43, p = 0.01) and severe fibrosis (OR 2.23, 95% CI 1.06–4.72, P = 0.03). ConclusionOur study shows that NAFLD patients with rs2542151 T→G of PTPN2 have a higher severity of fatty liver disease and a higher prevalence of T2DM. These results suggest that individual genetic susceptibility to intestinal permeability could play a role in liver disease progression.

The autoimmune susceptibility gene, PTPN2, restricts expansion of a novel mouse adherent-invasive E. coli

ABSTRACT 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.

Gene testing for osteonecrosis of the femoral head in systemic lupus erythematosus using targeted next-generation sequencing: A pilot study.

BACKGROUNDPrevious publications indicated that genetic predisposition might play important roles in the onset of osteonecrosis of the femoral head (ONFH) in systemic lupus erythematosus (SLE). Some gene loci such as complement C3d receptor 2 (CR2), nitric oxide synthase 3 (NOS3), collagen type II alpha 1 chain (COL2A1), protein tyrosine phosphatase non-receptor type 22 (PTPN22), and transient receptor potential cation channel subfamily V member 4 (TRPV4) were reported to be involved in this process.AIMTo investigate whether the risk of ONFH in SLE is associated with single nucleotide variations (SNVs) in these five genes.METHODSSNVs in the CR2, NOS3, COL2A1, PTPN22, and TRPV4 genes were examined by using FastTarget and Illumina Miseq sequencing technologies in 49 cases of SLE with ONFH. Burrows–wheeler aligner was used to align the sequencing reads to hg19, and GATK and Varscan programs were used to perform SNV calling. PolyPhen-2, SIFT, and MutationTaster were used to assess the functional effects of non-synonymous SNVs.RESULTSSix of the 49 patients were confirmed to have low frequency SNVs, including one patient with SNVs in NOS3 (exon 6: c.814G>A: p.E272K and exon 7: c.814G>A: p.E272K.), four in COL2A1 (rs41263847: exon 29: c.1913C>T: p.T638I, exon 28: c.1706C>T: p.T569I, and rs371445823: exon 8: c.580G>A: p.A194T, exon 7: c.373G>A: p.A125T), and one in CR2 (rs45573035: exon 2: c.200C>G: p.T67S).CONCLUSIONThe onset of ONFH in SLE might be associated with the identified SNVs in NOS3, COL2A1, and CR2.

Progress in the correlation between PTPN12 gene expression and human tumors.

Background:The global morbidity of cancer is rising rapidly. Despite advances in molecular biology, immunology, and cytotoxic and immune-anticancer therapies, cancer remains a major cause of death worldwide. Protein tyrosine phosphatase non-receptor type 12 (PTPN12) is a new member of the cytoplasmic protein tyrosine phosphatase family, isolated from a cDNA library of adult colon tissue. Thus far, no studies have reviewed the correlation between PTPN12 gene expression and human tumors.Methods:This article summarizes the latest domestic and international research developments on how the expression of PTPN12 relates to human tumors. The extensive search in Web of Science and PubMed with the keywords including PTPN12, tumor, renal cell carcinoma, proto-oncogenes, tumor suppressor genes was undertaken.Results:More and more studies have shown that a tumor is essentially a genetic disease, arising from a broken antagonistic function between proto-oncogenes and tumor suppressor genes. When their antagonistic effect is out of balance, it may cause uncontrolled growth of cells and lead to the occurrence of tumors. PTPN12 is a tumor suppressor gene, so inhibiting its activity will lead directly or indirectly to the occurrence of tumors.Conclusion:The etiology, prevention, and treatment of tumors have become the focus of research around the world. PTPN12 is a tumor suppressor gene. In the future, PTPN12 might serve as a novel molecular marker to benefit patients, and even the development of tumor suppressor gene activation agents can form a practical research direction.

PTPN3 Inhibits the Growth and Metastasis of Clear Cell Renal Cell Carcinoma via Inhibition of PI3K/AKT Signaling.

The underlying molecular mechanism driving clear cell renal cell carcinoma (ccRCC) progression is still to be explored. The significant downregulation of protein tyrosine phosphatase nonreceptor type 3 (PTPN3) expression in the tumor tissues suggested its protective role in ccRCC progression. IHC analysis of PTPN3 protein in 172 ccRCC tissue revealed that PTPN3 was an independently favorable prognostic factor for progression-free survival (P = 0.0166) and overall survival (P = 0.0343) of patients. The ccRCC cell lines SN12C, 1932, ACHN, and Caki-1 were used to evaluate, both in vitro and in vivo, the biological roles of PTPN3. We observed that overexpression of PTPN3 significantly inhibited the proliferation, migration, and invasion of ccRCC cells. In contrast, the knocking down of PTPN3 elicited opposite effects. Overexpressing PTPN3 inhibited xenograft tumor growth and lung metastasis displayed by the in vivo mice models. PTPN3 inhibited tumor cell motility by suppressing the phosphorylation of AKT, and subsequently inactivating the PI3K/AKT signaling pathway of renal cell carcinoma cells. Furthermore, the inhibition of phospho-AKTThr308 and phospho-AKTSer473 reversed PTPN3-induced silencing in tumor cell migration. Our work revealed that the overexpression of PTPN3 could suppress kidney cancer progression by negatively regulating the AKT signaling pathway, and served as a favorable prognostic factor in patients with ccRCC. Our findings provided insight that PTPN3 could be a potential target for therapy aiming to inhibit the malignant behaviors of ccRCC. IMPLICATIONS: PTPN3 is an independent favorable prognostic factor for patients with ccRCC and could be a potential target for therapy aiming to inhibit the malignant behaviors of ccRCC.

The Analysis of PTPN6 for Bladder Cancer: An Exploratory Study Based on TCGA.

PTPN6 (protein tyrosine phosphatase nonreceptor type 6), a tyrosine phosphatase, is known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. Previous studies have demonstrated that PTPN6 expression is relatively elevated in several malignancies. However, the role of PTPN6 in bladder cancer (BC) remains unclear. The purpose of this study was to explore the prognostic value of PTPN6 in BC. RNA-seq data from The Cancer Genome Atlas (TCGA) was used to identify the expression level of PTPN6 in BC. The relationship between clinical pathologic features and PTPN6 were analyzed with the Wilcoxon signed-rank test. The prognostic and predictive value of PTPN6 was evaluated by survival analysis and nomogram. Gene Set Enrichment Analysis (GSEA) was conducted to explore the potential molecular mechanisms of PTPN6 in BC. Finally, Tumor Immune Estimation Resource (TIMER) was applied to investigate the relationship between PTPN6 and immune cell infiltration in the tumor microenvironment. Results indicated that PTPN6 was overexpressed in BC tissues compared with normal bladder tissues and was significantly correlated with grade, stage, T, and N. Survival analysis showed that low expression of PTPN6 was significantly related to the poor overall survival (OS) in BC patients. Coexpression analysis showed that PTPN6 and TNFRSF14 (Tumor necrosis factor receptor superfamily member 14) have a close correlation in BC. GSEA showed that multiple cancer-associated signaling pathways are differentially enriched in the PTPN6 high expression phenotype. Moreover, the expression level of PTPN6 was positively associated with the infiltration of B cells, CD4+T cells, dendritic cells, and neutrophils and negatively associated with CD8+ T cells and macrophages in BC. In conclusion, we identified that PTPN6 may be a novel prognostic biomarker in BC based on the TCGA database. Further clinical trials are needed to confirm our observations and mechanisms underlying the prognostic value of PTPN6 in BC also deserve further experimental exploration.

Phospholipase D2 restores endothelial barrier function by promoting PTPN14-mediated VE-cadherin dephosphorylation

Increased permeability of vascular lung tissues is a hallmark of acute lung injury and is often caused by edemagenic insults resulting in inflammation. Vascular endothelial (VE)-cadherin undergoes internalization in response to inflammatory stimuli and is recycled at cell adhesion junctions during endothelial barrier re-establishment. Here, we hypothesized that phospholipase D (PLD)-generated phosphatidic acid (PA) signaling regulates VE-cadherin recycling and promotes endothelial barrier recovery by dephosphorylating VE-cadherin. Genetic deletion of PLD2 impaired recovery from protease-activated receptor-1-activating peptide (PAR-1-AP)-induced lung vascular permeability and potentiated inflammation in vivo In human lung microvascular endothelial cells (HLMVECs), inhibition or deletion of PLD2, but not of PLD1, delayed endothelial barrier recovery after thrombin stimulation. Thrombin stimulation of HLMVECs increased co-localization of PLD2-generated PA and VE-cadherin at cell-cell adhesion junctions. Inhibition of PLD2 activity resulted in prolonged phosphorylation of Tyr-658 in VE-cadherin during the recovery phase 3 h post-thrombin challenge. Immunoprecipitation experiments revealed that after HLMVECs are thrombin stimulated, PLD2, VE-cadherin, and protein-tyrosine phosphatase nonreceptor type 14 (PTPN14), a PLD2-dependent protein-tyrosine phosphatase, strongly associate with each other. PTPN14 depletion delayed VE-cadherin dephosphorylation, reannealing of adherens junctions, and barrier function recovery. PLD2 inhibition attenuated PTPN14 activity and reversed PTPN14-dependent VE-cadherin dephosphorylation after thrombin stimulation. Our findings indicate that PLD2 promotes PTPN14-mediated dephosphorylation of VE-cadherin and that redistribution of VE-cadherin at adherens junctions is essential for recovery of endothelial barrier function after an edemagenic insult.

Protein tyrosine phosphatase nonreceptor type 22 gene polymorphism in alopecia areata: Does it have an association with disease severity?

Alopecia areata is a condition involving hair loss from certain or all areas of the body. It has been considered as an immune-mediated disease, characterized by the infiltration of CD4+ and CD8+ lymphocytes in the hair follicles. The study aimed to assess whether protein tyrosine phosphatase nonreceptor type 22 gene single nucleotide polymorphism 1858C/T has any relationship with alopecia areata in Egyptian patients and whether it is associated with disease severity or not. Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) C1858T gene polymorphism was identified using polymerase chain reaction-restriction fragment length polymorphism analysis technique in 60 patients suffering from alopecia areata and 30 age- and sex-matched healthy controls. Disease severity was assessed using SALT score. CT and TT genotypes were significantly higher in the patients' group (P=.005), OR=4.38 95% CI [1.48-12.96], with significant statistical predominance of T allele in patients, P=.003, OR=3.86, 95% CI [1.52-9.77]. There was also a positive significant relationship between protein tyrosine phosphatase nonreceptor type 22 genotype CT and SALT score. PTPN22 1858T allele is associated with the development and severity of alopecia areata in Egyptians.

T‐cell Protein Tyrosine Phosphatase is a Key Regulator of Paneth Cell Function

Loss‐of‐function single nucleotide polymorphisms (SNPs) in the Protein Tyrosine Phosphatase non‐receptor Type 2 (PTPN2) gene, are associated with increased susceptibility to Inflammatory Bowel Disease (IBD). PTPN2 encodes the T Cell Protein Tyrosine Phosphatase (TCPTP) which is a negative regulator of intracellular signaling pathways including JAK‐STAT. We previously presented that Paneth and goblet cell numbers are reduced in Ptpn2 knockout (KO) mice, consistent with altered differentiation of these intestinal epithelial cell (IEC) types in IBD. Alterations in IEC populations are detrimental for intestinal homeostasis and increase susceptibility to microbial infection.GoalTo identify whether Ptpn2‐KO mice display altered IEC differentiation and Paneth cell antimicrobial peptide (AMP) production.MethodsIntestinal tissues (ileum, cecum, proximal and distal colon) from 21 day old constitutive Ptpn2‐KO mice and control littermates, and from adult tamoxifen‐inducible intestinal epithelial‐specific Ptpn2ΔIEC mice (Vil‐Cre/Ptpn2), were harvested and processed for imaging, western blot, flow cytometry and qPCR.ResultsConstitutive Ptpn2‐KO mice display reduced numbers of Paneth cells and loss of the AMP lysozyme (p=0.0158; n=5). This was confirmed by Western blots of isolated IECs (~67% reduction; n=4). Expression of Paneth cell markers and differentiation factors (Mmp7, Defa5 and Sox9) were not altered in KO mice (n=10). Interestingly, Reg3‐γ, which like lysozyme also targets gram‐(+) bacteria and is secreted by Paneth cells and enterocytes, was increased in KO mice vs. control littermates (p=0.0066; n=10). Next, we identified by immunofluorescence that loss of Ptpn2 only in intestinal epithelium (Ptpn2ΔIEC) dramatically reduced colocalization of lysozyme and another Paneth cell marker, UEA‐1 (n=6). Decreased lysozyme expression in Ptpn2ΔIEC ileum was confirmed by western blot (p=0.0315; n=6). Since Paneth cells reside in the stem cell compartment, we investigated whether Ptpn2 deficiency affects the intestinal stem cells. Gene expression of Lgr5, an intestinal stem cell marker, Ephb3, a marker of the stem cell compartment, and Ascl2, a gene that controls stem cell renewal in the crypts, was unchanged in Ptpn2‐KO mice (n=10). In addition, expression of early IEC differentiation factors, Hes1 and Math1, were not altered (n=10). Il‐22 and IFN‐γ are known to stimulate secretion of AMPs by Paneth cells. Interestingly, flow cytometry showed increased abundance of Il‐22+ T cells (p=0.0187; n=5) and IFN‐γ+ T cells (p=0.0277; n=5) in the Ileum of constitutive KO mice, indicating that the defect in AMP production is likely not due to a lack of immune cell‐derived stimuli.ConclusionPtpn2‐deficiency decreases Paneth cell number and has opposing effects on their production of specific AMPs. These effects on Paneth cell number and function may contribute to the increased susceptibility to infection and the dysbiotic microbiota in Ptpn2‐deficient mice.Support or Funding InformationSupported by NIH 2R01DK091281 (DFM) and the Crohn’s and Colitis Foundation (DFM)

PTPN2 Dysfunction Exacerbates C. rodentium Infection and Prevents Bacterial Clearance in a Cell‐Type Specific Manner

Background/AimLoss‐of‐function variants within the gene locus encoding protein tyrosine phosphatase non‐receptor type 2 (PTPN2) are linked with increased susceptibility to inflammatory diseases, including gastrointestinal disorders such as inflammatory bowel disease. Deletion of PTPN2 in myeloid cells promotes susceptibility to experimental colitis, and its loss in intestinal epithelial cells results in intestinal barrier defects, but it is not known how this affects susceptibility to bacterial infections. The aim of this project was to investigate the role of PTPN2 during infection with enteropathogenic bacteria.Methods8–12 week old, male and female mice (i) constitutively heterozygous for PTPN2 (PTPN2‐Het mice), (ii) lacking PTPN2 specifically in myeloid immune cells (PTPN2‐LysMCre mice), or (iii) with tamoxifen‐induced deletion in intestinal epithelial cells (PTPN2‐VilCre mice) and their respective wild‐type (WT) littermates were infected with Citrobacter rodentium (C. rodentium) and disease monitored over 21 days.ResultsWT mice suffered from moderate disease activity (moderate diarrhea and weight loss, reduced activity/grooming) upon infection with C. rodentium and recovered from disease by day 20. PTPN2‐Het mice showed significantly enhanced disease activity (pronounced weight loss (p<0.01), severe diarrhea, reduced activity and lack of grooming), enhanced C. rodentium load in feces and translocation (p<0.01, each) and were not able to clear infection. In contrast, PTPN2‐LysMCre mice showed severe early‐onset disease (drastic weight loss (p<0.05), severe diarrhea, reduced activity and lack of grooming) and clearly enhanced C. rodentium fecal load and translocation (p<0.01, each) between day 5 and 10 of the infection, but recovered faster from disease (p<0.05) and completely cleared the infection by day 16. PTPN2‐VilCre mice showed more severe disease than floxed control mice (p<0.05), but were able to recover/clear the infection by day 21. PTPN2‐LysMCre and PTPN2‐Het, but not PTPN2‐VilCre mice, mounted a more pronounced type 1 immune response (increased numbers of pro‐inflammatory macrophages and T helper (Th)1 and Th17 cells, p<0.05, each) and enhanced IL‐22 expression (LysMCre: p<0.05; Het: p<0.01). Furthermore, C. rodentium infection induced expression of anti‐microbial peptides in IECs from WT mice, an effect potentiated in PTPN2‐LysMCre mice, which might account for the faster bacterial clearance/recovery. In contrast, IECs from PTPN2‐Het and PTPN2VilCre mice were unable to upregulate anti‐microbial peptides.ConclusionPTPN2 protects from bacterial infections by restricting worsens disease burden and defects in bacterial clearance. This highlights the importance of PTPN2 for intestinal health and might partially explain the increased disease risk in individuals carrying PTPN2 variants with respect to host responses to bacterial triggers of inflammatory disease.Support or Funding InformationNIH 2R01DK091281 to DFM and a Postdoctoral Fellowship from the Swiss National Science Foundation to MRS.

Blood Serum Cytokines Induce Changes in Blood‐Brain Barrier Permeability and Gene Expression in an In Vitro Blood‐Brain Barrier Model

The blood‐brain barrier (BBB), a semi‐permeable interface between the brain and the blood, is a key player in the of development and progression of brain‐related illnesses such as traumatic brain injury (TBI), Alzheimer’s disease (AD), multiple sclerosis (MS), and schizophrenia (SCZ). With these disorders, pathologic BBB leakiness is caused by changes in the cellular milieu on the brain‐side as well as on the blood‐side of the BBB. On the brain‐side, injury to neurons, astrocytes, microglia, and pericytes can lead to increased BBB leakiness, while on the blood‐side, pathophysiologic levels of circulating factors such as hormones, inflammatory cytokines, and immune cells can significantly alter BBB leakiness.The objective of this study was to discover novel biomarkers of BBB leakiness using the in vitro three‐dimensional Neurovascular Unit (3D NVU; Research Triangle Institute International, Durham, NC) to examine the impact of human blood serum on transcriptional regulation and function of the BBB. The 3D NVU integrates endothelial, neuronal, astrocytic, and microglial cells into a three‐dimensional platform, and allows for the assessment of BBB permeability (transendothelial electrical resistance (TEER, Ω cm2)). Blood serum of secondary progressive MS patients (two men, two women) and four healthy individuals (two men, two women) were purchased from PrecisionMed, Inc. (Solana Beach, CA), and diluted to 40% with cell culture media for testing. TEER of the 3D NVU was recorded immediately prior to serum exposure (baseline), and again at 24 hours after serum treatment. At the end of the experiment, cells of the 3D NVU were collected, and expression of 84 MS‐related genes were measured by quantitative RT‐PCR. BBB permeability was calculated as TEERSerum – TEERBaseline. The protein levels of 100 serum cytokines were measured in undiluted serum samples by membrane‐based sandwich immunoassay. Multiple regression analysis was used to determine the relationship among TEER, serum cytokine levels, and BBB gene expression.We found that no single serum cytokine was a strong predictor of BBB permeability, however, serum‐induced changes in expression of chemokine ligand 12 (Ccl12/Mcp‐5), chemokine ligand 7 (Ccl7), hexosaminidase subunit beta (Hexb), interleukin 13 (Il13), interleukin 1 beta (Il1b), protein tyrosine phosphatase non‐receptor type 11 (Ptpn11), and vascular adhesion molecule 1 (Vcam1) were strong and significant predictors of BBB permeability (R2’s ≥ 0.76, P’s ≤ 0.004). Some of these genes (Il1b, Hexb and Vcam1) have been shown previously to play a role in regulation of BBB function, however, genes such as Ptpn11 have not yet been implicated in BBB permeability. This preliminary study demonstrates that the 3D NVU can be used to elucidate novel, physiologically relevant biomarkers of BBB‐related neurological and psychological disorders. In the future, we plan to perform RNA‐sequencing to examine a broader range of differential gene expression in our study.Support or Funding InformationFunded by TATRC AMEDD Advanced Medical Technology Initiative grant. The views expressed in this abstract are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the U.S. Government.

A Novel IBD‐Associated Mouse Pathobiont E. coli Causes Disease and Prevents Recovery from Colitis

IntroductionExpansion of intestinal pathobionts, such as adherent‐invasive E. coli (AIEC), are strongly implicated in the pathogenesis of Inflammatory Bowel Disease (IBD) as AIEC increases pro‐inflammatory cytokine production and alters tight junction protein regulation, suggesting a potential mechanism of pathogen‐induced barrier dysfunction and inflammation. In mice lacking the IBD risk gene, protein tyrosine phosphatase non‐receptor Type 2 (PTPN2), we previously showed increased abundance of a novel mouse AIEC (mAIEC) which had sequence match identity of a 250bp segment to the human IBD‐associated AIEC, LF82. We functionally validated the ability of mAIEC to adhere to and invade intestinal epithelial cells in vitro. The aim of this study was to determine if mAIEC can cause disease in vivo and whether it requires other bacteria to support its pathogenicity in mice.Methods(1) Germ‐free ~12‐week old C57Bl/6 male and female mice; or (2) wild‐type 10‐week old confirmed segmented filamentous bacteria (SFB)‐free C57Bl/6 female mice (JAX labs, Stock# 000664) ± 3% dextran‐sodium sulfate (DSS) for 7 days housed in a specific pathogen‐free (SPF) vivarium; were orally gavaged with PBS or gavaged with 109 CFU of either pure mAIEC or K12 at exponential‐phase growth. Germ‐free mice received fecal microbiome transplant (FMT) by oral gavage of fresh caecal content from a Ptpn2‐KO (KO‐FMT) mouse at day 22 post‐mAIEC colonization. Disease activity index (DAI) was assessed by measuring weight loss, physical activity, general appearance, and stool consistency.ResultsmAIEC infected SFB‐free mice had a significant decrease in body weight development (P = 0.002, n = 5) but no apparent increase in DAI compared with the control E. coli K12 or PBS conditions. Infection of germ‐free mice with mAIEC did not alter body weight development compared with K12 infected mice. However, recolonization with KO‐FMT post‐mAIEC infection caused sustained weight loss in male but not female mice compared with K12 mice (P = 0.02, n = 3). To identify the effects of mAIEC infection on inflammation, SFB‐free mice were orally gavaged with mAIEC on days 0–3 of DSS administration. mAIEC+DSS treated mice showed a more severe drop in body weight development, increased disease severity (P < 0.001, n = 4–5), and a worse barrier defect associated with translocation of bacteria to the spleen and liver (P ≤ 0.002, n = 3–5) compared with DSS+PBS, DSS+K12 or non‐DSS treated PBS, mAIEC or K12 alone control groups. When administered to mice with established DSS colitis, mAIEC prevented recovery from DSS‐induced colitis compared with K12 and PBS conditions (P ≤ 0.009, n = 5).ConclusionsWe describe for the first time a novel IBD‐associated mouse AIEC that can cause or worsen disease in a host, but requires other bacteria to support its pathogenicity. This study generates new insights into environment‐microbiome interactions in IBD.Support or Funding InformationCrohn’s and Colitis Foundation Senior Research Award (D.F.M.); NIH‐2R01‐DK091281 (D.F.M.); American Gastroenterological Association IBD Research Award (D.F.M.). UCR Office of Research & Economic Development Pilot Award (D.F.M. & J.B.).

Histone Deacetylation 10 Alleviates Inflammation After Intracerebral Hemorrhage via the PTPN22/NLRP3 Pathway in Rats

The NOD-like receptor family Pyrin domain-containing 3 (NLRP3) inflammasome has a crucial role in the inflammatory process that occurs during intracerebral hemorrhage (ICH)-induced injury. Histone deacetylase 10 (HDAC10) is a newly identified class II histone deacetylase involved in immune responses. However, how HDAC10 affects the inflammatory response after ICH remains unknown. In this study, we investigated whether HDAC10 relieves ICH injury by suppressing NLRP3 inflammasome activation through the protein tyrosine phosphatase, nonreceptor type 22 (PTPN22) pathway. We induced ICH in Sprague-Dawley rats (healthy, male adult) with a single infusion of autologous blood. To knockdown HDAC10, we injected siRNA into the rats. To further explore the mechanisms underlying the role of HDAC10 in ICH injury, PTPN22 was silenced. HDAC10 levels were upregulated after ICH in humans and rats, and reached peak levels 24 h after ICH induction in rats. HDAC10 silencing aggravated ICH injury, as demonstrated by increased modified neurological severity scores, brain water content, Evans blue extravasation, and number of myeloperoxidase (MPO) cells, and the results of Nissl and H&E staining. Furthermore, HDAC10 knockdown increased the expression of PTPN22 and accentuated inflammatory responses mediated by the NLRP3 inflammasome. HDAC10 silencing increased NLRP3 inflammasome activation, and this was effectively reversed by PTPN22 knockdown using siRNA. Furthermore, HDAC10 silencing also promoted the interaction of PTPN22 and NLRP3. Our study demonstrated that HDAC10 silencing aggravated NLRP3-mediated inflammatory responses after ICH in rats via the PTPN22 pathway. These results suggest that regulating the NLRP3 inflammasome may be a novel method to ameliorate ICH injury.

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.

Molecular and environmental characterization of Noonan syndrome in Morocco reveals a significant association with consanguinity and advanced parental age

BackgroundNoonan syndrome (NS) is one of the most common RASopathies, with an autosomal dominant inheritance. This disorder is caused by a range of genes belonging to the RAS-MAP kinase (rat sarcoma viral oncogene homolog/mitogen-activated protein kinases) pathway, with PTPN11 (protein-tyrosine phosphatase, non-receptor type 11) being the most involved genetic factor.The aim of this study is to report PTPN11 mutations found in a cohort of Moroccans with Noonan syndrome, compare the mutation rate with various studies, and statistically assess involvement of prominent risk factors in manifestation of this disorder.Thirty-one NS patients were screened for PTPN11 mutations using PCR-Sanger sequencing method. Pathogenic effect prediction, for detected variants, was carried out using PROVEAN, MutationTaster2, and HSF programs. Statistical tests were performed with R software. Chi-square and Fisher’s exact tests were used in percentage comparisons, while Student’s test was used in average comparisons.ResultsWe detected five pathogenic mutations, one synonymous variant with a potential altering effect on splicing function, and three novel intronic duplications. PTPN11 mutation rate in our cohort is around 16.13%. Comparison of this rate with the corresponding rates in various populations shows notably significant differences across continents.ConclusionsBesides genetic factors, the present study suggests involvement of additional environmental factors. Statistical assessment of clinical data confirms particularly the association of NS manifestation with consanguinity and advanced paternal age, and suggests an eventual implication of advanced maternal age as well.