RESUMO
OBJECTIVE: 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 (TiO2, E171) as a neglected IBD risk factor. Here, we investigate the interplay of the PTPN22 variant and TiO2-mediated effects during IBD pathogenesis. DESIGN: Acute DSS colitis was induced in wild-type and PTPN22 variant mice (PTPN22-R619W) and animals were treated with TiO2 nanoparticles during colitis induction. Disease-triggering mechanisms were investigated using bulk and single-cell RNA sequencing. RESULTS: In mice, administration of TiO2 nanoparticles 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 TiO2 administration. Normalisation of T-cell populations correlated with increased Ifng expression and, at a later stage of disease, the promoted prevalence of proinflammatory macrophages that triggered severe intestinal inflammation. CONCLUSION: Our findings indicate that the consumption of TiO2 nanoparticles 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.
Assuntos
Colite , Doença de Crohn , Doenças Inflamatórias Intestinais , Nanopartículas , Camundongos , Animais , Doença de Crohn/genética , Doença de Crohn/complicações , Linfócitos T CD8-Positivos/metabolismo , Colite/induzido quimicamente , Colite/genética , Colite/prevenção & controle , Inflamação/complicações , Sulfato de Dextrana , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Proteína Tirosina Fosfatase não Receptora Tipo 22/genéticaRESUMO
BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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 in vivo and ex vivo colon permeability to 4-kDa fluorescently labeled dextran and reduced ex vivo 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. CONCLUSIONS: 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.
Assuntos
Comunicação Celular , Células Epiteliais/enzimologia , Doenças Inflamatórias Intestinais/enzimologia , Absorção Intestinal , Mucosa Intestinal/enzimologia , Macrófagos/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Adulto , Células CACO-2 , Técnicas de Cocultura , Células Epiteliais/imunologia , Feminino , Humanos , Imunidade Inata , Imunidade nas Mucosas , Mediadores da Inflamação/metabolismo , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Permeabilidade , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Transdução de Sinais , Células THP-1 , Células U937RESUMO
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.
Assuntos
Doença Hepática Terminal , Hepatite C Crônica , Antivirais/uso terapêutico , Doença Hepática Terminal/tratamento farmacológico , Hepacivirus/genética , Hepatite C Crônica/tratamento farmacológico , Humanos , Índice de Gravidade de Doença , Carga ViralRESUMO
Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) plays a critical role in the pathogenesis of inflammatory bowel diseases (IBD). Mice lacking PTPN2 in dendritic cells (DCs) develop skin and liver inflammation by the age of 22 weeks due to a generalized loss of tolerance leading to uncontrolled immune responses. The effect of DC-specific PTPN2 loss on intestinal health, however, is unknown. The aim of this study was to investigate the DC-specific role of PTPN2 in the intestine during colitis development. PTPN2fl/flxCD11cCre mice were subjected to acute and chronic DSS colitis as well as T cell transfer colitis. Lamina propria immune cell populations were analyzed using flow cytometry. DC-specific PTPN2 deletion promoted infiltration of B and T lymphocytes, macrophages, and DCs into the lamina propria of unchallenged mice and elevated Th1 abundance during acute DSS colitis, suggesting an important role for PTPN2 in DCs in maintaining intestinal immune cell homeostasis. Surprisingly, those immune cell alterations did not translate into increased colitis susceptibility in acute and chronic DSS-induced colitis or T cell transfer colitis models. However, macrophage depletion by clodronate caused enhanced colitis severity in mice with a DC-specific loss of PTPN2. Loss of PTPN2 in DCs affects the composition of lamina propria lymphocytes, resulting in increased infiltration of innate and adaptive immune cells. However, this did not result in an elevated colitis phenotype, likely because increased infiltration of macrophages in the intestine upon loss of PTPN2 loss in DCs can compensate for the inflammatory effect of PTPN2-deficient DCs.
Assuntos
Colite/etiologia , Colite/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/deficiência , Animais , Colite/patologia , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Suscetibilidade a Doenças , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Transgênicos , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Fator de Transcrição STAT1/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologiaRESUMO
Inflammasomes are multi-protein complexes that mediate the activation and secretion of the inflammatory cytokines IL-1ß 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-1ß/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-1ß/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.
Assuntos
Inflamassomos/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Animais , Humanos , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , FosforilaçãoRESUMO
Nonalcoholic steatohepatitis (NASH), a primary cause of liver disease, leads to complications such as fibrosis, cirrhosis, and carcinoma, but the pathophysiology of NASH is incompletely understood. Epstein-Barr virus-induced G protein-coupled receptor 2 (EBI2) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-diHC) are recently discovered immune regulators. Several lines of evidence suggest a role of oxysterols in NASH pathogenesis, but rigorous testing has not been performed. We measured oxysterol levels in the livers of NASH patients by LC-MS and tested the role of the EBI2-7α,25-diHC system in a murine feeding model of NASH. Free oxysterol profiling in livers from NASH patients revealed a pronounced increase in 24- and 7-hydroxylated oxysterols in NASH compared with controls. Levels of 24- and 7-hydroxylated oxysterols correlated with histological NASH activity. Histological analysis of murine liver samples demonstrated ballooning and liver inflammation. No significant genotype-related differences were observed in Ebi2-/- mice and mice with defects in the 7α,25-diHC synthesizing enzymes CH25H and CYP7B1 compared with wild-type littermate controls, arguing against an essential role of these genes in NASH pathogenesis. Elevated 24- and 7-hydroxylated oxysterol levels were confirmed in murine NASH liver samples. Our results suggest increased bile acid synthesis in NASH samples, as judged by the enhanced level of 7α-hydroxycholest-4-en-3-one and impaired 24S-hydroxycholesterol metabolism as characteristic biochemical changes in livers affected by NASH.
Assuntos
Fígado/metabolismo , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Oxisteróis/metabolismo , Adulto , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Colesterol/sangue , Cromatografia Líquida , Citometria de Fluxo , Humanos , Hidroxicolesteróis/sangue , Hidroxicolesteróis/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Hepatopatia Gordurosa não Alcoólica/sangue , Oxisteróis/sangue , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismoRESUMO
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 ß6 -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.
Assuntos
Neoplasias Colorretais/sangue , Cadeias beta de Integrinas/sangue , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Neoplasias Colorretais/genética , Humanos , Cadeias beta de Integrinas/biossíntese , Cadeias beta de Integrinas/genética , Prognóstico , Estudo de Prova de Conceito , Modelos de Riscos Proporcionais , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: Western lifestyle and diet are major environmental factors playing a role in the development of IBD. Titanium dioxide (TiO2) nanoparticles are widely used as food additives or in pharmaceutical formulations and are consumed by millions of people on a daily basis. We investigated the effects of TiO2 in the development of colitis and the role of the nucleotide-binding oligomerisation domain receptor, pyrin domain containing (NLRP)3 inflammasome. DESIGN: Wild-type and NLRP3-deficient mice with dextran sodium sulfate-induced colitis were orally administered with TiO2 nanoparticles. The proinflammatory effects of TiO2 particles in cultured human intestinal epithelial cells (IECs) and macrophages were also studied, as well as the ability of TiO2 crystals to traverse IEC monolayers and accumulate in the blood of patients with IBD using inductively coupled plasma mass spectrometry. RESULTS: Oral administration of TiO2 nanoparticles worsened acute colitis through a mechanism involving the NLRP3 inflammasome. Importantly, crystals were found to accumulate in spleen of TiO2-administered mice. In vitro, TiO2 particles were taken up by IECs and macrophages and triggered NLRP3-ASC-caspase-1 assembly, caspase-1 cleavage and the release of NLRP3-associated interleukin (IL)-1ß and IL-18. TiO2 also induced reactive oxygen species generation and increased epithelial permeability in IEC monolayers. Increased levels of titanium were found in blood of patients with UC having active disease. CONCLUSION: These findings indicate that individuals with a defective intestinal barrier function and pre-existing inflammatory condition, such as IBD, might be negatively impacted by the use of TiO2 nanoparticles.
Assuntos
Colite/imunologia , Corantes/efeitos adversos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nanopartículas/efeitos adversos , Titânio/efeitos adversos , Animais , Caspase 1/metabolismo , Colite/induzido quimicamente , Colite/metabolismo , Corantes/administração & dosagem , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Humanos , Interleucina-18/biossíntese , Interleucina-1beta/metabolismo , Intestinos/citologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nanopartículas/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Baço/patologia , Titânio/administração & dosagem , Titânio/sangueRESUMO
BACKGROUND/AIMS: The protein tyrosine phosphatase non-receptor type 2 (PTPN2) is known to mediate susceptibility to inflammatory bowel diseases. Cell culture experiments suggest that PTPN2 influences barrier function, autophagy and secretion of pro-inflammatory cytokines. PTPN2 knockout mice die a few weeks after birth due to systemic inflammation, emphasizing the importance of this phosphatase in inflammatory processes. The aim of this study was to investigate the role of PTPN2 in colon epithelial cells by performing dextran sulphate sodium (DSS)-induced colitis in PTPN2xVilCre mice. METHODS: Acute colitis was induced by administering 2.5 or 2% DSS for 7 days and chronic colitis by 4 cycles of treatment using 1% DSS. Body weight of mice was measured regularly and colonoscopy was done at the end of the experiments. Mice were sacrificed afterwards and colon specimens were obtained for H&E staining. For analysis of wound healing, mechanical wounds were introduced during endoscopy and wound closure assessed by daily colonoscopy. RESULTS: Although colonoscopy and weight development suggested changes in colitis severity, the lack of any influence of PTPN2 deficiency on histological scoring for inflammation severity after acute or chronic DSS colitis indicates that colitis severity is not influenced by epithelial-specific loss of PTPN2. Chronic colitis induced the development of aberrant crypt foci more frequently in PTPN2xVilCre mice compared to their wild type littermates. On the other hand, loss of PTPN2-induced enhanced epithelial cell proliferation and promoted wound closure. CONCLUSIONS: Loss of PTPN2 in intestinal epithelial cells (IECs) has no significant influence on inflammation in DSS colitis. Obviously, loss of PTPN2 in IECs can be compensated in vivo, thereby suppressing a phenotype. This lack of a colitis-phenotype might be due to enhanced epithelial cell proliferation and subsequent increased wound-healing capacity of the epithelial layer.
Assuntos
Colite/genética , Doenças Inflamatórias Intestinais/genética , Mucosa Intestinal/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 2/fisiologia , Cicatrização/genética , Animais , Proliferação de Células/genética , Doença Crônica , Colite/induzido quimicamente , Colite/patologia , Colo/patologia , Colonoscopia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Predisposição Genética para Doença , Humanos , Doenças Inflamatórias Intestinais/induzido quimicamente , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Tirosina Fosfatase não Receptora Tipo 2/genéticaRESUMO
BACKGROUND/AIMS: The single nucleotide polymorphism (SNP) rs1893217 within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) results in a dysfunctional PTPN2 protein is associated with Crohn's disease (CD) and exists in perfect linkage disequilibrium with the CD- and ulcerative colitis (UC)-associated PTPN2 SNP rs2542151. We investigated associations of PTPN2 SNP rs1893217 and clinical characteristics of inflammatory bowel disease (IBD) patients. METHODS: One thousand seventy three patients with CD and 734 patients with UC from the Swiss IBD Cohort Study (SIBDCS) were included. Epidemiologic, disease and treatment characteristics were analysed for an association with the presence of one of the rs1893217 genotypes 'homozygous wild-type' (TT), 'heterozygous' (CT) and 'homozygous variant' (CC). RESULTS: About 2.88% of IBD patients were identified with CC, 26.8% with CT and 70.4% with TT genotype. The CC-genotype was associated with the existence of gallstones in CD and pancolitis in UC patients. The presence of the C-allele (i.e. either CC or CT genotype) was associated with the onset of uveitis, but protected from aphthous oral ulcers in CD patients. UC patients carrying a C-allele were diagnosed at an older age but required intestinal surgery more often. The presence of the C-allele was associated with a successful treatment with anti-TNF antibodies in both CD and UC patients. CONCLUSION: IBD patients carrying the C-allele of PTPN2 SNP rs1893217 are at greater risk for developing a severe disease course but are more likely to respond to treatment with anti-TNF antibodies. These findings demonstrate a clinical relevance of this PTPN2 risk variant in IBD patients.
Assuntos
Predisposição Genética para Doença/genética , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/terapia , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adalimumab/uso terapêutico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Certolizumab Pegol/uso terapêutico , Criança , Pré-Escolar , Feminino , Fármacos Gastrointestinais/uso terapêutico , Genótipo , Humanos , Lactente , Doenças Inflamatórias Intestinais/epidemiologia , Infliximab/uso terapêutico , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Estudos Prospectivos , Fatores de Risco , Suíça/epidemiologia , Adulto JovemRESUMO
BACKGROUND/AIMS: Genetic polymorphisms within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) have been associated with inflammatory bowel disease (IBD). A recent study demonstrated that PTPN2 regulates ER stress signalling in pancreatic ß-cells. Therefore, we investigated whether PTPN2 regulates ER stress pathways, apoptosis and cytokine secretion in human intestinal epithelial cells (IECs) and monocytes. METHODS: THP-1 and HT-29 IECs were stimulated with 2 µg/ml tunicamycin (TNM) for the indicated periods of time. For knockdown experiments, cells were transfected using a mixture of three different PTPN2-specific siRNA oligonucleotides. Cell lysates were analysed by Western blot and real-time PCR. Cytokine secretion was studied by ELISA measurements of cell culture supernatant. RESULTS: TNM treatment reduced PTPN2 protein levels in HT-29 IECs and THP-1 monocytes. Knockdown of PTPN2 in THP-1 monocytes led to an exaggerated induction of phospho-eIF2α, enhanced PARP cleavage, indicative of apoptosis, and attenuated IL-8 and TNF secretion upon TNM stimulation. In HT-29 cells PTPN2 deficiency caused reduced phosphorylation of eIF2α and PARP cleavage under ER stress conditions. CONCLUSIONS: Whereas the knockdown of PTPN2 made THP-1 cells more susceptible to ER stress, PTPN2 deficiency reduced ER stress responses in HT-29 IECs. This suggests that PTPN2 regulates adaptation to ER stress in a cell type-specific manner.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Citocinas/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Técnicas de Silenciamento de Genes , Células HT29 , Humanos , Interleucina-8/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Fator de Necrose Tumoral alfa/metabolismo , Tunicamicina/farmacologiaRESUMO
BACKGROUND & AIMS: A gain-of-function variation within the locus that encodes protein tyrosine phosphatase nonreceptor type (PTPN)22 is associated with a reduced risk for Crohn's disease (CD), whereas a loss-of-function variant seems to promote autoimmune disorders. We investigated how loss of PTPN22 could contribute to chronic inflammation of the intestine. METHODS: Intestinal tissue samples from patients with or without inflammatory bowel disease (controls) were analyzed for levels of PTPN22 messenger RNA (mRNA) and protein. In human THP-1 monocytes, protein levels were analyzed by immunoblotting, mRNA levels by real-time polymerase chain reaction, and cytokine release by enzyme-linked immunosorbent assay. RESULTS: Intestinal tissue samples from patients with CD had reduced levels of PTPN22 mRNA and protein, compared with samples from controls. In human THP-1 monocytes, interferon-γ (IFN-γ) induced expression and activity of PTPN22. Loss of PTPN22 increased levels of p38-mitogen-activated protein kinase, but reduced phosphorylation of nuclear factor-κB subunits. Increased activity of suppressor of cytokine signaling-1 was accompanied by reduced phosphorylation of signal-transducer and activator of transcription protein 1 and signal-transducer and activator of transcription protein 3 in PTPN22-deficient cells incubated with cytokines. PTPN22 knockdown increased secretion of the inflammatory cytokines interleukin (IL)-6 and IL-17, but reduced expression or secretion of T-bet, intercellular adhesion molecule-1, nucleotide-binding oligomerization domain containing-2, monocyte chemoattractant protein-1, IL-2, and IL-12p40 in response to IFN-γ. CONCLUSIONS: PTPN22 expression is reduced in intestinal tissues of patients with active CD. PTPN22 regulates intracellular signaling events and is induced by IFN-γ in human monocytes. Knockdown of PTPN22 alters activation of inflammatory signal transducers, increasing secretion of T-helper 17-related inflammatory mediators. Genetic variants that reduce PTPN22 activity might contribute to the pathogenesis of CD by these mechanisms.
Assuntos
Doença de Crohn/genética , Regulação da Expressão Gênica , Interferon gama/farmacologia , Intestino Delgado/metabolismo , Monócitos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , RNA Mensageiro/genética , Adolescente , Adulto , Idoso , Comunicação Celular , Doença de Crohn/metabolismo , Doença de Crohn/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Immunoblotting , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/patologia , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 22/metabolismo , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/farmacologia , Transdução de Sinais/genética , Adulto JovemRESUMO
Crohn's disease (CD) is characterized by a breakdown of the intestinal epithelial barrier function leading to an uncontrolled immune response to bacterial antigens. Available data demonstrate that appropriate response and early host defense against invading bacteria are crucial to maintain tolerance towards commensal bacteria. When the mechanisms of early removal of invading bacteria are disturbed, a loss of tolerance and a full-blown adaptive immune reaction, which is mounted against the usually harmless commensal flora, are induced. Dysfunction of autophagy caused by genetic variations within CD susceptibility genes, such as ATG16L1 and IRGM, results in defective handling of intracellular and invading bacteria and causes prolonged survival and defective clearance of those microbes. Dysfunction of ATG16L1 and IRGM has also been shown to cause aberrant Paneth cell function and uncontrolled secretion of proinflammatory cytokines finally resulting in increased susceptibility to bacterial infection and the onset of colitis. Interestingly, autophagy can also be regulated by other CD susceptibility genes, such as NOD2 (nucleotide oligomerization domain 2) or PTPN2 (protein tyrosine phosphatase nonreceptor type 2) and the presence of the CD-associated variations within these genes results in similar effects. Taken together, more and more evidence suggests a close functional correlation between loss of tolerance and defective autophagy in CD patients. Therefore, most likely, the onset of CD is triggered by both a loss of tolerance as well as a dysfunction of autophagy, which finally results in the onset of chronic intestinal inflammation.
Assuntos
Doença de Crohn/imunologia , Doença de Crohn/patologia , Tolerância Imunológica , Autofagia , Proteínas de Transporte/metabolismo , Humanos , Celulas de Paneth/patologia , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
BACKGROUND/AIMS: Anthocyanins are plant-derived dietary components that are highly abundant, for example, in bilberries. We have previously demonstrated that anthocyanins exert anti-inflammatory properties in mouse colitis models and ameliorate disease activity in ulcerative colitis patients. Here, we studied the molecular mechanisms through which anthocyanin-containing bilberry extract (BE) exerts anti-inflammatory effects in human monocytic THP-1 cells. METHODS: THP-1 cells were pre-incubated with BE 20 min prior to TNF-α or IFN-γ (100 ng/ml each) stimulation. Signalling protein activation was studied by Western blotting, mRNA expression by quantitative PCR and cytokine secretion by ELISA. RESULTS: IFN-γ-induced phosphorylation of STAT1 and STAT3 was significantly reduced by BE co-treatment. Consequently, levels of mRNA expression and/or cytokine secretion of MCP-1, IL-6, TNF-α, ICAM-1, and T-bet were lower with BE co-treatment. In contrast, BE enhanced TNF-α-mediated p65-NF-κB phosphorylation but reduced ERK1/2 phosphorylation. BE co-treatment further increased TNF-α-induced mRNA expression and secretion of NF-κB target genes, such as IL-6, IL-8, and MCP-1, while mRNA levels of ICAM-1 were reduced. CONCLUSIONS: BE co-treatment reduced IFN-γ-induced signal protein activation, pro-inflammatory gene expression, and cytokine secretion, whereas it enhanced TNF-α-induced responses. These findings suggest a distinct role for anthocyanins in modulating inflammatory responses that need to be further studied to fully understand anthocyanin-mediated effects.
Assuntos
Antocianinas/farmacologia , Citocinas/metabolismo , Interferon gama/antagonistas & inibidores , Monócitos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Vaccinium myrtillus/química , Animais , Antocianinas/isolamento & purificação , Anti-Inflamatórios/farmacologia , Linhagem Celular , Sinergismo Farmacológico , Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/farmacologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/química , Monócitos/imunologia , NF-kappa B/química , Fosforilação/efeitos dos fármacos , Extratos Vegetais , Coelhos , Fator de Transcrição STAT1/química , Fator de Transcrição STAT3/química , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: The increasing presence of plastics in the human diet is raising public concern about the potential risks posed by nanoplastic (NP) particles, which can emerge from the degradation of plastic debris. NP ingestion poses particular risks to individuals with inflammatory bowel disease (IBD), as compromised epithelial barriers may facilitate NP translocation. METHODS: In vitro, bone-marrow-derived macrophages (BMDMs) were exposed to 25 nm polymethacrylate (PMMA) or 50 nm polystyrene (PS) particles to assess morphological changes and alterations in pro- and anti-inflammatory gene expression. In vivo, mice received PMMA NP particles for 6 months before acute dextran sodium sulfate (DSS) colitis was induced to investigate NP impacts on intestinal health and inflammation. RESULTS: PMMA and PS NP exposure in BMDMs induced morphological changes indicative of a proinflammatory phenotype characterized by enlarged amoeboid cell shapes. It also triggered an inflammatory response, indicated by increased expression of proinflammatory cytokines such as Tnfa and Il6. Unexpectedly, long-term PMMA NP administration did not affect the intestinal epithelial barrier or exacerbate acute DSS-induced colitis in mice. Colonoscopy and histological analysis revealed no NP-related changes, suggesting adverse effects on intestinal health or inflammation. CONCLUSION: Our findings from animal models offer some reassurance to IBD patients regarding the effects of NP ingestion. However, variations in lifestyle and dietary habits may lead to significantly higher plastic intake in certain individuals, raising concerns about potential long-term gastrointestinal effects of lifelong plastic consumption.
RESUMO
Ulcerative colitis is a chronic inflammatory bowel disease that strongly affects patient quality of life. Side effects of current therapies necessitate new treatment strategies that maximise the drug concentration at the site of inflammation, while minimizing systemic exposure. Capitalizing on the biocompatible and biodegradable structure of lipid mesophases, we present a temperature-triggered in situ forming lipid gel for topical treatment of colitis. We show that the gel is versatile and can host and release drugs of different polarities, including tofacitinib and tacrolimus, in a sustained manner. Further, we demonstrate its adherence to the colonic wall for at least 6 h, thus preventing leakage and improving drug bioavailability. Importantly, we find that loading known colitis treatment drugs into the temperature-triggered gel improves animal health in two mouse models of acute colitis. Overall, our temperature-triggered gel may prove beneficial in ameliorating colitis and decreasing adverse effects associated with systemic application of immunosuppressive treatments.
Assuntos
Colite Ulcerativa , Colite , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Animais , Camundongos , Colite Ulcerativa/tratamento farmacológico , Qualidade de Vida , Temperatura , Colite/induzido quimicamente , Colite/tratamento farmacológico , LipídeosRESUMO
Macrophages intimately interact with intestinal epithelial cells, but the consequences of defective macrophage-epithelial cell interactions for protection against enteric pathogens are poorly understood. Here, we show that in mice with a deletion in protein tyrosine phosphatase nonreceptor type 2 (PTPN2) in macrophages, infection with Citrobacter rodentium, a model of enteropathogenic and enterohemorrhagic E. coli infection in humans, promoted a strong type 1/IL-22-driven immune response, culminating in accelerated disease but also faster clearance of the pathogen. In contrast, deletion of PTPN2 specifically in epithelial cells rendered the epithelium unable to upregulate antimicrobial peptides and consequently resulted in a failure to eliminate the infection. The ability of PTPN2-deficient macrophages to induce faster recovery from C. rodentium was dependent on macrophage-intrinsic IL-22 production, which was highly increased in macrophages deficient in PTPN2. Our findings demonstrate the importance of macrophage-mediated factors, and especially macrophage-derived IL-22, for the induction of protective immune responses in the intestinal epithelium, and show that normal PTPN2 expression in the epithelium is crucial to allow for protection against enterohemorrhagic E. coli and other intestinal pathogens.
Assuntos
Infecções por Enterobacteriaceae , Escherichia coli Êntero-Hemorrágica , Infecções por Escherichia coli , Proteína Tirosina Fosfatase não Receptora Tipo 2 , Animais , Humanos , Camundongos , Células Epiteliais/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismoRESUMO
BACKGROUND & AIMS: Glycoprotein (GP)96 is an endoplasmic reticulum-resident master chaperone for cell surface receptors including the Wnt co-receptors low-density lipoprotein-receptor-related protein 5/6. Intestinal epithelial cell (IEC)-specific deletion of Gp96 is embryonically lethal. However, the role of GP96 in adult intestinal tissue and especially within the intestinal stem cell (ISC) niche is unknown. Here, we investigated how GP96 loss interferes with intestinal homeostasis by compromising viability, proliferation, and differentiation of IECs. METHODS: Tamoxifen was used to induce Cre-mediated deletion of Gp96 in GP96-VillincreERT2 (Cre recombinase-Estrogen-Receptor Transgene 2) mice and intestinal organoids. With H&E and immunofluorescence staining we assessed alterations in intestinal morphology and the presence and localization of IEC types. Real-time polymerase chain reaction and Western blot analysis were performed to explore the molecular mechanisms underlying the severe phenotype of Gp96 KO mice and organoids. RESULTS: IEC-specific deletion of Gp96 in adult mice resulted in a rapid degeneration of the stem cell niche, followed by complete eradication of the epithelial layer and death within a few days. These effects were owing to severe defects in ISC renewal and premature ISC differentiation, which resulted from defective Wnt and Notch signaling. Furthermore, depletion of GP96 led to massive induction of endoplasmic reticulum stress. Although effects on ISC renewal and adequate differentiation were partly reversed upon activation of Wnt/Notch signaling, viability could not be restored, indicating that reduced viability was mediated by other mechanisms. CONCLUSIONS: Our work shows that GP96 plays a fundamental role in regulating ISC fate and epithelial regeneration and therefore is indispensable for maintaining intestinal epithelial homeostasis.
Assuntos
Células Epiteliais , Intestinos , Glicoproteínas de Membrana , Animais , Camundongos , Proliferação de Células , Células Epiteliais/metabolismo , Glicoproteínas/metabolismo , Intestinos/citologia , Via de Sinalização Wnt/genética , Glicoproteínas de Membrana/metabolismoRESUMO
BACKGROUND & AIMS: Loss-of-function variants in the PTPN2 gene are associated with increased risk of inflammatory bowel disease. We recently showed that Ptpn2 is critical for intestinal epithelial cell (IEC) barrier maintenance, IEC-macrophage communication, and modulation of the gut microbiome in mice, restricting expansion of a small intestinal pathobiont associated with inflammatory bowel disease. Here, we aimed to identify how Ptpn2 loss affects ileal IEC subtypes and their function in vivo. METHODS: Constitutive Ptpn2 wild-type, heterozygous, and knockout (KO) mice, as well as mice with inducible deletion of Ptpn2 in IECs, were used in the study. Investigation was performed using imaging techniques, flow cytometry, enteroid culture, and analysis of gene and protein levels of IEC markers. RESULTS: Partial transcriptome analysis showed that expression of Paneth cell-associated antimicrobial peptides Lyz1, Pla2g2a, and Defa6 was down-regulated markedly in Ptpn2-KO mice compared with wild-type and heterozygous. In parallel, Paneth cell numbers were reduced, their endoplasmic reticulum architecture was disrupted, and the endoplasmic reticulum stress protein, C/EBP-homologous protein (CHOP), was increased in Ptpn2-KO mice. Despite reduced Paneth cell number, flow cytometry showed increased expression of the Paneth cell-stimulatory cytokines interleukin 22 and interferon γ+ in CD4+ T cells isolated from Ptpn2-KO ileum. Key findings in constitutive Ptpn2-KO mice were confirmed in epithelium-specific Ptpn2ΔIEC mice, which also showed impaired lysozyme protein levels in Paneth cells compared with Ptpn2fl/fl control mice. CONCLUSIONS: Constitutive Ptpn2 deficiency affects Paneth cell viability and compromises Paneth cell-specific antimicrobial peptide production. The observed effects may contribute to the increased susceptibility to intestinal infection and dysbiosis in these mice.
Assuntos
Doenças Inflamatórias Intestinais , Celulas de Paneth , Camundongos , Animais , Celulas de Paneth/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Sobrevivência Celular , Doenças Inflamatórias Intestinais/genética , Íleo/metabolismo , Camundongos KnockoutRESUMO
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.