RESUMO
BACKGROUND & AIMS: It is not clear how rapid ascent to a high altitude causes the gastrointestinal symptoms of acute mountain sickness (AMS). We assessed the incidence of endoscopic lesions in the upper gastrointestinal tract in healthy mountaineers after a rapid ascent to high altitude, their association with symptoms, and their pathogenic mechanisms. METHODS: In a prospective study, 25 mountaineers (10 women; mean age, 43.8 ± 9.5 y) underwent unsedated, transnasal esophagogastroduodenoscopy in Zurich (490 m) and then on 2 test days (days 2 and 4) at a high altitude laboratory in the Alps (Capanna Regina Margherita, 4559 m). Symptoms were assessed using validated instruments for AMS (the acute mountain sickness score and the Lake Louise scoring system) and visual analogue scales (scale, 0-100). Levels of messenger RNAs (mRNAs) in duodenal biopsy specimens were measured by quantitative polymerase chain rection. RESULTS: The follow-up endoscopy at high altitude was performed in 19 of 25 patients on day 2 and in 23 of 25 patients on day 4. The frequency of endoscopic lesions increased from 12% at baseline to 26.3% on day 2 and to 60.9% on day 4 (P < .001). The incidence of ulcer disease increased from 0 at baseline to 10.5% on day 2 and to 21.7% on day 4 (P = .014). Mucosal lesions were associated with lower hunger scores (37.3 vs 67.4 in patients without lesions; P = .012). Subjects with peptic lesions had higher levels of HIF2A mRNA, which encodes a hypoxia-induced transcription factor, and ICAM1 mRNA, which encodes an adhesion molecule, compared with subjects without lesions (fold changes, 1.38 vs 0.63; P = .001; and 1.37 vs 0.66; P = .011, respectively). CONCLUSIONS: In a prospective study of 25 mountaineers, fast ascent to a high altitude resulted in rapid onset of clinically meaningful mucosal lesions and ulcer disease. Duodenal biopsy specimens from these subjects had increased levels of HIF2A mRNA and ICAM1 mRNA, which might contribute to the formation of hypoxia-induced peptic lesions. Further studies are needed of the mechanisms of this process.
Assuntos
Doença da Altitude , Doença Aguda , Adulto , Altitude , Doença da Altitude/diagnóstico , Endoscopia do Sistema Digestório , Feminino , Humanos , Hipóxia , Estudos ProspectivosRESUMO
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
Solute carrier (SLC) transporters mediate the uptake of biologically active compounds in the intestine. Reduced oxygenation (hypoxia) is an important factor influencing intestinal homeostasis. The aim of this study was to investigate the pathophysiological consequences of hypoxia on the expression and function of SLCs in human intestine. Hypoxia was induced in human intestinal epithelial cells (IECs) in vitro (0.2; 1% O2 or CoCl2). For human in vivo studies, duodenal biopsies and serum samples were obtained from individuals (n = 16) acutely exposed to 4,554 meters above sea levels. Expression of relevant targets was analyzed by quantitative PCR, Western blotting, or immunofluorescence. Serum levels of inflammatory mediators and nucleosides were determined by ELISA and LC/MS-MS, respectively. In the duodenum of volunteers exposed to high altitude we observed decreased mRNA levels of apical sodium-dependent bile acid transporter (ASBT), concentrative nucleoside transporters 1/2 (CNT1/2), organic anion transporting polypeptide 2B1 (OATP2B1), organic cation transporter 2 (OCTN2), peptide transporter 1 (PEPT1), serotonin transporter (SERT), and higher levels of IFN-γ, IL-6, and IL-17A. Serum levels of IL-10, IFN-γ, matrix metalloproteinase-2 (MMP-2), and serotonin were elevated, whereas the levels of uridine decreased upon exposure to hypoxia. Hypoxic IECs showed reduced levels of equilibrative nucleoside transporter 2 (ENT2), OCTN2, and SERT mRNAs in vitro, which was confirmed on the protein level and was accompanied by activation of ERK1/2, increase of hypoxia-inducible factor (HIF) proteins, and production of IL-8 mRNA. Costimulation with IFN-γ and IL-6 during hypoxia further decreased the expression of SERT, ENT2, and CNT2 in vitro. Reduced oxygen supply affects the expression pattern of duodenal SLCs that is accompanied by changes in serum levels of proinflammatory cytokines and biologically active compounds demonstrating that intestinal transport is affected during systemic exposure to hypoxia in humans.
Assuntos
Aclimatação , Altitude , Citocinas/sangue , Duodeno/metabolismo , Hipóxia/metabolismo , Mediadores da Inflamação/sangue , Proteínas de Membrana Transportadoras/metabolismo , Transdução de Sinais , Biomarcadores/sangue , Hipóxia Celular , Linhagem Celular , Citocinas/genética , Regulação para Baixo , Duodeno/fisiopatologia , Humanos , Hipóxia/sangue , Hipóxia/genética , Hipóxia/fisiopatologia , Absorção Intestinal , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiopatologia , Proteínas de Membrana Transportadoras/genética , Oxigênio/metabolismo , RNA Mensageiro/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
UNLABELLED: Human iron homeostasis is regulated by intestinal iron transport, hepatic hepcidin release, and signals from pathways that consume or supply iron. The aim of this study was to characterize the adaptation of iron homeostasis under hypoxia in mountaineers at the levels of (1) hepatic hepcidin release, (2) intestinal iron transport, and (3) systemic inflammatory and erythropoietic responses. Twenty-five healthy mountaineers were studied. Blood samples and duodenal biopsies were taken at baseline of 446 m as well as on day 2 (MG2) and 4 (MG4) after rapid ascent to 4559 m. Divalent metal-ion transporter 1 (DMT-1), ferroportin 1 (FP-1) messenger RNA (mRNA), and protein expression were analyzed in biopsy specimens by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. Serum hepcidin levels were analyzed by mass spectrometry. Serum iron, ferritin, transferrin, interleukin (IL)-6, and C-reactive protein (CRP) were quantified by standard techniques. Serum erythropoietin and growth differentiation factor 15 (GDF15) levels were measured by enzyme-linked immunosorbent assay (ELISA). Under hypoxia, erythropoietin peaked at MG2 (P < 0.001) paralleled by increased GDF15 on MG2 (P < 0.001). Serum iron and ferritin levels declined rapidly on MG2 and MG4 (P < 0.001). Duodenal DMT-1 and FP-1 mRNA expression increased up to 10-fold from baseline on MG2 and MG4 (P < 0.001). Plasma CRP increased on MG2 and MG4, while IL-6 only increased on MG2 (P < 0.001). Serum hepcidin levels decreased at high altitude on MG2 and MG4 (P < 0.001). CONCLUSION: This study in healthy volunteers showed that under hypoxemic conditions hepcidin is repressed and duodenal iron transport is rapidly up-regulated. These changes may increase dietary iron uptake and allow release of stored iron to ensure a sufficient iron supply for hypoxia-induced compensatory erythropoiesis.
Assuntos
Adaptação Fisiológica , Altitude , Hipóxia/metabolismo , Ferro/metabolismo , Adulto , Doença da Altitude/tratamento farmacológico , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Proteína C-Reativa/metabolismo , Proteínas de Transporte de Cátions/sangue , Dexametasona/uso terapêutico , Duodeno/metabolismo , Feminino , Ferritinas/sangue , Fator 15 de Diferenciação de Crescimento/sangue , Hepcidinas/sangue , Humanos , Interleucina-6/sangue , Fígado , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Transferrina/metabolismoRESUMO
Signaling via cAMP plays an important role in apical cell surface dynamics in epithelial cells. In hepatocytes, elevated levels of cAMP as well as extracellular oncostatin M stimulate apical lumen development in a manner that depends on protein kinase A (PKA) activity. However, neither the identity of PKA isoforms involved nor the mechanisms of the cross-talk between oncostatin M and cAMP/PKA signaling pathways have been elucidated. Here we demonstrate that oncostatin M and PKA signaling converge at the level of the PKA holoenzyme downstream of oncostatin M-stimulated MAPK activation. Experiments were performed with chemically modified cAMP analogues that preferentially target regulatory subunit (R) I or RII holoenzymes, respectively, in hepatocytes. The data suggest that the dissociation of RI- but not RII-containing holoenzymes, as well as catalytic activity of PKA, is required for apical lumen development in response to elevated levels of cAMP and oncostatin M. However, oncostatin M signaling does not stimulate PKA holoenzyme dissociation in living cells. Based on pharmacological and cell biological studies, it is concluded that RI-controlled PKA activity is essential for cAMP- and oncostatin M-stimulated development of apical bile canalicular lumens.
Assuntos
Canalículos Biliares/citologia , Canalículos Biliares/enzimologia , Polaridade Celular , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Hepatócitos/citologia , Hepatócitos/enzimologia , Inibidores de Adenilil Ciclases , Canalículos Biliares/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Polaridade Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , AMP Cíclico/farmacologia , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Transferência de Energia/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Hepatócitos/efeitos dos fármacos , Holoenzimas/metabolismo , Humanos , Isoenzimas/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Oncostatina M/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Oncostatin M and cAMP signaling stimulate apical surface-directed membrane trafficking and apical lumen development in hepatocytes, both in a protein kinase A (PKA)-dependent manner. Here, we show that oncostatin M, but not cAMP, promotes the A-kinase anchoring protein (AKAP)-dependent anchoring of the PKA regulatory subunit (R)IIalpha to subapical centrosomes and that this requires extracellular signal-regulated kinase 2 activation. Stable expression of the RII-displacing peptide AKAP-IS, but not a scrambled peptide, inhibits the association of RIIalpha with centrosomal AKAPs and results in the repositioning of the centrosome from a subapical to a perinuclear location. Concomitantly, common endosomes, but not apical recycling endosomes, are repositioned from a subapical to a perinuclear location, without significant effects on constitutive or oncostatin M-stimulated basolateral-to-apical transcytosis. Importantly, however, the expression of the AKAP-IS peptide completely blocks oncostatin M-, but not cAMP-stimulated apical lumen development. Together, the data suggest that centrosomal anchoring of RIIalpha and the interrelated subapical positioning of these centrosomes is required for oncostatin M-, but not cAMP-mediated, bile canalicular lumen development in a manner that is uncoupled from oncostatin M-stimulated apical lumen-directed membrane trafficking. The results also imply that multiple PKA-mediated signaling pathways control apical lumen development and that subapical centrosome positioning is important in some of these pathways.
Assuntos
Canalículos Biliares/enzimologia , Bucladesina/farmacologia , Polaridade Celular/efeitos dos fármacos , Centrossomo/efeitos dos fármacos , Centrossomo/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Oncostatina M/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/farmacologia , Canalículos Biliares/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Humanos , Ligação Proteica/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacosRESUMO
Inflammatory bowel disease (IBD) is an inflammatory condition that affects the gastrointestinal tract. The solute carrier (SLC) superfamily of transporters comprise proteins involved in the uptake of drugs, hormones, and other biologically active compounds. The purpose of this study was to determine the mRNA expression levels of 15 solute carrier transporters in two regions of the intestine in IBD patients. Endoscopic biopsy specimens were taken from two locations (terminal ileum and colon) for histological examination and RNA extraction. We quantitatively measured the mRNA expression of 15 SLC transporters in 107 IBD patients (53 with Crohn's disease and 54 with ulcerative colitis) and 23 control subjects. mRNA expression was evaluated using the quantitative reverse transcription-polymerase chain reaction technique. We observed that in the ileum of IBD patients, mRNA levels for serotonin transporter, equilibrative nucleoside transporter (ENT) 1, ENT2, and organic anion-transporting polypeptide (OATP) 2B1 were significantly elevated, whereas levels for apical sodium-dependent bile acid transporter (ASBT) and organic zwitterion/cation transporter (OCTN) 2 were significantly lower. In colon, mRNA levels for ENT1, ENT2, concentrative nucleoside transporter (CNT) 2, OATP2B1, and OATP4A1 were significantly higher, whereas mRNA levels for OCTN2 were significantly decreased. In inflamed colon of IBD patients the mRNA expression levels of ENT1, ENT2, CNT2, OATP2B1, OATP4A1, and peptide transporter 1 were significantly higher. We conclude that intestinal SLC mRNA levels are dysregulated in IBD patients, which may be linked to the inflammation of the tissue and provides an indication about the role of inflammatory signaling in regulation of SLC expression.
Assuntos
Proteínas de Transporte/biossíntese , Doenças Inflamatórias Intestinais/metabolismo , RNA Mensageiro/biossíntese , Anti-Inflamatórios/uso terapêutico , Biópsia , Colo/metabolismo , Colonoscopia , DNA Complementar/biossíntese , DNA Complementar/genética , Humanos , Íleo/metabolismo , Doenças Inflamatórias Intestinais/tratamento farmacológico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteroides/uso terapêuticoRESUMO
In hepatocytes, cAMP/PKA activity stimulates the exocytic insertion of apical proteins and lipids and the biogenesis of bile canalicular plasma membranes. Here, we show that the displacement of PKA-RIIalpha from the Golgi apparatus severely delays the trafficking of the bile canalicular protein MDR1 (P-glycoprotein), but not that of MRP2 (cMOAT), DPP IV and 5'NT, to newly formed apical surfaces. In addition, the direct trafficking of de novo synthesized glycosphingolipid analogues from the Golgi apparatus to the apical surface is inhibited. Instead, newly synthesized glucosylceramide analogues are rerouted to the basolateral surface via a vesicular pathway, from where they are subsequently endocytosed and delivered to the apical surface via transcytosis. Treatment of HepG2 cells with the glucosylceramide synthase inhibitor PDMP delays the appearance of MDR1, but not MRP2, DPP IV, and 5'NT at newly formed apical surfaces, implicating glucosylceramide synthesis as an important parameter for the efficient Golgi-to-apical surface transport of MDR1. Neither PKA-RIIalpha displacement nor PDMP inhibited (cAMP-stimulated) apical plasma membrane biogenesis per se, suggesting that other cAMP effectors may play a role in canalicular development. Taken together, our data implicate the involvement of PKA-RIIalpha anchoring in the efficient direct apical targeting of distinct proteins and glycosphingolipids to newly formed apical plasma membrane domains and suggest that rerouting of Golgi-derived glycosphingolipids may underlie the delayed Golgi-to-apical surface transport of MDR1.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Canalículos Biliares/metabolismo , Membrana Celular/metabolismo , Polaridade Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glucosilceramidas/metabolismo , Hepatócitos/citologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Canalículos Biliares/citologia , Canalículos Biliares/efeitos dos fármacos , Bucladesina/farmacologia , Membrana Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico , Glucosilceramidas/biossíntese , Complexo de Golgi/metabolismo , Hepatócitos/metabolismo , Humanos , Transporte Proteico/efeitos dos fármacosRESUMO
Oncostatin M regulates membrane traffic and stimulates apicalization of the cell surface in hepatoma cells in a protein kinase A-dependent manner. Here, we show that oncostatin M enhances the expression of the cyclin-dependent kinase (cdk)2 inhibitor p27(Kip1), which inhibits G(1)-S phase progression. Forced G(1)-S-phase transition effectively renders presynchronized cells insensitive to the apicalization-stimulating effect of oncostatin M. G(1)-S-phase transition prevents oncostatin M-mediated recruitment of protein kinase A to the centrosomal region and precludes the oncostatin M-mediated activation of a protein kinase A-dependent transport route to the apical surface, which exits the subapical compartment (SAC). This transport route has previously been shown to be crucial for apical plasma membrane biogenesis. Together, our data indicate that oncostatin M-stimulated apicalization of the cell surface is critically dependent on the ability of oncostatin M to control p27(Kip1)/cdk2-mediated G(1)-S-phase progression and suggest that the regulation of apical plasma membrane-directed traffic from SAC is coupled to centrosome-associated signaling pathways.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Peptídeos/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Compartimento Celular , Linhagem Celular , Polaridade Celular , Centrossomo/metabolismo , Meios de Cultura , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidor de Quinase Dependente de Ciclina p27 , Fase G1 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Lipídeos de Membrana/metabolismo , Modelos Biológicos , Oncostatina M , Fase S , Transdução de SinaisRESUMO
BACKGROUND AND AIMS: An imbalance between cellular antioxidant defence system[s] and reactive oxygen species [ROS]-driven oxidative stress has been implicated in the pathogenesis of inflammatory bowel disease. Peroxiredoxin [PRDX] 6 contributes to an appropriate redox balance by clearing ROS and reducing peroxidized membrane phospholipids. We here studied the role of PRDX6 in acute and chronic dextran sodium sulphate [DSS]-induced colitis. METHODS: To investigate the impact of PRDX6 on intestinal inflammation, we used wild type [WT], Prdx6 knock-out mice [Prdx6-/-] and transgenic mice [Prdx6tg/tg], overexpressing Prdx6. Acute and chronic colitis was induced by DSS in WT, Prdx6-/- and Prdx6tg/tg mice. Colitis was evaluated by endoscopy, colon length, histopathological assessment and myeloperoxidase [MPO] activity. Changes in mRNA and protein expression of pro-inflammatory cytokines and antioxidant enzymes were evaluated by real-time quantitative polymerase chain reaction [RT-qPCR] and western blot. Total glutathione [GSH] levels in colon samples were determined. RESULTS: Prdx6-/- mice exposed to acute and chronic DSS showed a significant decrease in the clinical parameters and in colonic expression of pro-inflammatory cytokines compared with WT mice. mRNA expression of antioxidant enzymes in colon samples was significantly increased in Prdx6-/- compared with WT mice exposed to acute and chronic DSS. In addition, total GSH levels were increased in Prdx6-/- mice treated with DSS in comparison with WT. Overexpression of Prdx6 did not significantly influence acute and chronic colitis. CONCLUSIONS: Our data indicate that a lack of the antioxidant enzyme PRDX6 protects against the development of acute and chronic experimental colitis and is associated with increased expression and function of other antioxidant enzymes, suggesting effective compensatory mechanisms.
Assuntos
Colite/enzimologia , Colite/genética , Citocinas/metabolismo , Peroxirredoxina VI/genética , Doença Aguda , Animais , Doença Crônica , Colite/induzido quimicamente , Colite/patologia , Colo/metabolismo , Sulfato de Dextrana , Endoscopia Gastrointestinal , Células Epiteliais/metabolismo , Feminino , Glutamato-Cisteína Ligase/genética , Glutationa/metabolismo , Glutationa Sintase/genética , Humanos , Doenças Inflamatórias Intestinais/genética , Interferon gama/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fator 2 Relacionado a NF-E2/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Peroxidase/metabolismo , Peroxirredoxina III/genética , Peroxirredoxina VI/metabolismo , Peroxirredoxinas/genética , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND AND AIMS: Hypoxia can induce inflammation in the gastrointestinal tract. However, the impact of hypoxia on the course of inflammatory bowel disease (IBD) is poorly understood. We aimed to evaluate whether flights and/or journeys to regions lying at an altitude of >2000 m above the sea level are associated with flare-ups within 4 weeks of the trip. METHODS: IBD patients with at least one flare-up during a 12-month observation period were compared to a group of patients in remission. Both groups completed a questionnaire. RESULTS: A total of 103 IBD patients were included (43 with Crohn's disease (CD): mean age 39.3 ± 14.6 years; 60 with ulcerative colitis (UC): mean age 40.4 ± 15.1 years). Fifty-two patients with flare-ups were matched to 51 patients in remission. IBD patients experiencing flare-ups had more frequently undertaken flights and/or journeys to regions >2000 m above sea level within four weeks of the flare-up when compared to patients in remission (21/52 [40.4%] vs. 8/51 [15.7%], p=0.005). CONCLUSIONS: Journeys to high altitude regions and/or flights are a risk factor for IBD flare-ups occurring within 4 weeks of travel.
Assuntos
Viagem Aérea , Altitude , Colite Ulcerativa/etiologia , Doença de Crohn/etiologia , Hipóxia/complicações , Adulto , Medicina Aeroespacial , Colite Ulcerativa/fisiopatologia , Doença de Crohn/fisiopatologia , Progressão da Doença , Humanos , Pessoa de Meia-Idade , Fatores de Risco , Inquéritos e Questionários , Fatores de Tempo , Adulto JovemRESUMO
Retinoid treatment is suggested to promote development of inflammatory bowel disease, although preclinical studies are not supportive. We evaluated the effect of retinoids on cytokine response in in vitro-differentiated human dendritic cells (ivDCs) and macrophages (ivMACs) derived from healthy human donors and in cultured human THP-1 cells. Effect on human intestinal epithelial cell integrity was also assessed. Each cell type was incubated (±lipopolysaccharide [LPS]) with all-trans retinoic acid (ATRA), 13-cis-RA (isotretinoin) and 4-oxo-13-cis-RA. Cytokine analysis was performed by array analysis. Cultured human endothelial colorectal adenocarcinoma (Caco-2) cells were incubated with these retinoids and media analyzed for leakage by spectrofluorometric analysis. ATRA consistently and significantly inhibited LPS-induced release of the pro-inflammatory cytokines tumor necrosis factor, interleukin (IL)-6, macrophage inflammatory protein (MIP)-1α and MIP-1ß. All retinoids tested stimulated release of the anti-inflammatory cytokines granulocyte-macrophage colony-stimulating factor and IL-10, and also monocyte chemotactic protein-1, vascular endothelial growth factor and eotaxin-1. Incubation with retinoids did not significantly alter the permeability of Caco-2 monolayers. Pre-treatment of each cell type with retinoids promoted an anti-inflammatory cytokine profile with only minimal effect on intestinal epithelial cell permeability; consistent with in vivo studies.
Assuntos
Células Dendríticas/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Retinoides/farmacologia , Adulto , Células CACO-2 , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/metabolismo , Feminino , Humanos , Imunidade Inata/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Lipopolissacarídeos , Macrófagos/metabolismo , Masculino , PermeabilidadeRESUMO
The chaperone function of the ER-residing heat shock protein gp96 plays an important role in protein physiology and has additionally important immunological functions due to its peptide-binding capacity. Low amounts of gp96 stimulate immunity; high quantities induce tolerance by mechanisms not fully understood. A lack of gp96 protein in intestinal macrophages (IMACs) from Crohn`s disease (CD) patients correlates with loss of tolerance against the host gut flora, leading to chronic inflammation. Since gp96 shows dose-dependent direction of immunological reactions, we studied primary IMACs and developed cell models to understand the regulation of gp96 expression. Induction of gp96-expression was higher in in vitro differentiated dendritic cells (i.v.DCs) than in in vitro differentiated macrophages (i.v.MACs), whereas monocytes (MOs) expressed only low gp96 levels. The highest levels of expression were found in IMACs. Lipopolysaccharide (LPS), muramyl dipeptide (MDP), tumour necrosis factor (TNF), and Interleukin (IL)-4 induced gp96-expression, while IL12, IL-17, IL-23 and interferon (IFN)-γ were not effective indicating that Th1 and Th17 cells are probably not involved in the induction of gp96. Furthermore, gp96 was able to induce its own expression. The ER-stress inducer tunicamycin increased gp96-expression in a concentration- and time-dependent manner. Both ulcerative colitis (UC) and CD patients showed significantly elevated gp96 mRNA levels in intestinal biopsies which correlated positively with the degree of inflammation of the tissue. Since gp96 is highly expressed on the one hand upon stress induction as during inflammation and on the other hand possibly mediating tolerance, these results will help to understand the whether gp96 plays a role in the pathophysiology of inflammatory bowel disease (IBD).
Assuntos
Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Biópsia , Estudos de Casos e Controles , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Íleo/efeitos dos fármacos , Íleo/metabolismo , Íleo/patologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Doenças Inflamatórias Intestinais/patologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Glicoproteínas de Membrana/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
BACKGROUND: Tumor necrosis factor (TNF) is an important cytokine in the pathogenesis of inflammatory bowel disease (IBD). Anti-TNF antibodies have been successfully implemented in IBD therapy, however their efficacies differ among IBD patients. Here we investigate the influence of CD64 Fc receptor on the inhibitory activity of anti-TNFs in cells of intestinal wall. METHODS: Intestinal cell lines, monocytes/macrophages and peripheral blood mononuclear cells (PBMCs) were used as models. The efficacies of adalimumab, infliximab and certolizumab-pegol were assessed by RT-PCR for target genes. Protein levels and localizations were examined by Western blotting and immunofluorescence. Antibody fragments were obtained by proteolytic digestion, immunoprecipitation and protein chip analysis. Knock-down of specific gene expression was performed using siRNAs. RESULTS: Infliximab had limited efficacy towards soluble TNF in cell types expressing Fc gamma receptor CD64. Both adalimumab and infliximab had lower efficacies in PBMCs of IBD patients, which express elevated levels of CD64. Infliximab-TNF complexes were more potent in activating CD64 in THP-1 cells than adalimumab, which was accompanied by distinct phospho-tyrosine signals. Blocking Fc parts and isolation of Fab fragments of infliximab improved its efficacy. IFN-γ-induced expression of CD64 correlated with a loss of efficacy of infliximab, whereas reduction of CD64 expression by either siRNA or PMA treatment improved inhibitory activity of this drug. Colonic mRNA expression levels of CD64 and other Fc gamma receptors were significantly increased in the inflamed tissues of infliximab non-responders. CONCLUSIONS: CD64 modulates the efficacy of infliximab both in vitro and ex vivo, whereas the presence of this receptor has no impact on the inhibitory activity of certolizumab-pegol, which lacks Fc fragment. These data could be helpful in both predicting and evaluating the outcome of anti-TNF therapy in IBD patients with elevated systemic and local levels of Fc receptors.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Receptores de IgG/metabolismo , Adalimumab , Adulto , Linhagem Celular , Células Cultivadas , Certolizumab Pegol , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Doenças Inflamatórias Intestinais/metabolismo , Infliximab , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Polietilenoglicóis/farmacologia , Fator de Necrose Tumoral alfa/imunologia , Adulto JovemRESUMO
BACKGROUND: Autophagy is a process of central importance for maintaining cell homeostasis, survival, and the regulation of inflammation. Recent studies associated variants within the gene loci, encoding protein tyrosine phosphatase nonreceptor type 2 (PTPN2), and autophagy genes, such as autophagy-related 16-like 1 (ATG16L1), with chronic inflammatory disorders, such as Crohn's disease (CD). We show that PTPN2 regulates autophagy in human intestinal epithelial cells (IEC) and primary colonic lamina propria fibroblasts (CLPF). METHODS: Protein analysis in IEC and CLPF was performed by western blotting. Autophagososme formation was assessed by LC3B immunofluorescence or immunohistochemistry. Human intestinal tissue samples were obtained from noninflammatory bowel disease (IBD) control or from CD patients and genotyped for disease-associated PTPN2 or ATG16L1 variations. RESULTS: Knockdown of PTPN2 causes impaired autophagosome formation and dysfunctional autophagy resulted in increased levels of intracellular Listeria monocytogenes (LM) and elevated IEC apoptosis in response to tumor necrosis factor (TNF) and interferon gamma (IFN-γ). Similar findings were observed in primary CLPF derived from CD patients carrying the CD-associated PTPN2 variant. Presence of the ATG16L1 variant prevented the cytokine-induced rise in PTPN2 protein, finally resulting in impaired LC3B-II levels in IEC. Actively inflamed intestinal biopsies from CD patients carrying either ATG16L1 or PTPN2 genetic variants revealed aberrant LC3B expression patterns when compared with samples from non-IBD control patients. CONCLUSIONS: Our results demonstrate that PTPN2 regulates autophagosome formation in human intestinal cells. We provide a model of how a dysfunction of the CD susceptibility genes, PTPN2 and/or ATG16L1, may contribute to the onset and perpetuation of chronic intestinal inflammation.
Assuntos
Autofagia , Proteínas de Transporte/metabolismo , Doença de Crohn/patologia , Fibroblastos/patologia , Intestinos/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Adulto , Idoso , Proteínas Relacionadas à Autofagia , Estudos de Casos e Controles , Comunicação Celular , Células Cultivadas , Colo/metabolismo , Colo/patologia , Doença de Crohn/metabolismo , Doença de Crohn/microbiologia , Citocinas/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Técnicas Imunoenzimáticas , Mucosa Intestinal/metabolismo , Listeria monocytogenes/patogenicidade , Listeriose/metabolismo , Listeriose/microbiologia , Listeriose/patologia , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Mucosa/metabolismo , Mucosa/patologia , Fosforilação , Prognóstico , Estudos Prospectivos , Proteína Tirosina Fosfatase não Receptora Tipo 2/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , RNA Interferente Pequeno/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Nod1 and Nod2 are members of the Nod-like receptor family that detect intracellular bacterial peptidoglycan-derived muramyl peptides. The biological effects of muramyl peptides have been described for over three decades, but the mechanism underlying their internalization to the cytosol remains unclear. Using the human epithelial cell line HEK293T as a model system, we demonstrate here that Nod1-activating ligands entered cells through endocytosis, most likely by the clathrin-coated pit pathway, as internalization was dynamin-dependent but not inhibited by methyl-beta-cyclodextrin. In the endocytic pathway, the cytosolic internalization of Nod1 ligands was pH-dependent, occurred prior to the acidification mediated by the vacuolar ATPase, and was optimal at pH ranging from 5.5 to 6. Similarly, the Nod2 ligand MDP was internalized into host cytosol through a similar pathway with optimal pH for internalization ranging from 5.5 to 6.5. Moreover, Nod1-activating muramyl peptides likely required processing by endosomal enzymes, prior to transport into the cytosol, suggesting the existence of a sterically gated endosomal transporter for Nod1 ligands. In support for this, we identified a role for SLC15A4, an oligopeptide transporter expressed in early endosomes, in Nod1-dependent NF-kappaB signaling. Interestingly, SLC15A4 expression was also up-regulated in colonic biopsies from patients with inflammatory bowel disease, a disorder associated with mutations in Nod1 and Nod2. Together, our results shed light on the mechanisms by which muramyl peptides get access to the host cytosol, where they are detected by Nod1 and Nod2, and might have implications for the understanding of human diseases, such as inflammatory bowel disease.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/metabolismo , Endocitose , Endossomos/fisiologia , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Ácido Diaminopimélico/análogos & derivados , Ácido Diaminopimélico/metabolismo , Endossomos/química , Endossomos/genética , Humanos , Concentração de Íons de Hidrogênio , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Ligantes , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação ProteicaRESUMO
Cyclic adenosine monophosphate (cAMP) and cAMP-dependent protein kinase A (PKA) are evolutionary conserved molecules with a well-established position in the complex network of signal transduction pathways. cAMP/PKA-mediated signaling pathways are implicated in many biological processes that cooperate in organ development including the motility, survival, proliferation and differentiation of epithelial cells. Cell surface polarity, here defined as the anisotropic organisation of cellular membranes, is a critical parameter for most of these processes. Changes in the activity of cAMP/PKA elicit a variety of effects on intracellular membrane dynamics, including membrane sorting and trafficking. One of the most intriguing aspects of cAMP/PKA signaling is its evolutionary conserved abundance on the one hand and its precise spatial-temporal actions on the other. Here, we review recent developments with regard to the role of cAMP/PKA in the regulation of intracellular membrane trafficking in relation to the dynamics of epithelial surface domains.