RÉSUMÉ
INTRODUCTION: An association between functional dyspepsia (FD) and wheat-containing foods has been reported in observational studies; however, an adaptive response has not been demonstrated. We examined whether antigens present in wheat could provoke a response from FD duodenal lymphocytes. METHODS: Lamina propria mononuclear cells (LPMCs) were isolated from duodenal biopsies from 50 patients with FD and 23 controls. LPMCs were exposed to gluten (0.2 mg/mL) or gliadin (0.2 mg/mL) for 24 hours. Flow cytometry was performed to phenotype lymphocytes. Quantitative PCR was used to measure the expression of gliadin-associated T-cell receptor alpha variant ( TRAV ) 26-2. RESULTS: In response to gliadin (but not gluten) stimulation, the effector Th2-like population was increased in FD LPMCs compared with that in controls and unstimulated FD LPMCs. Duodenal gene expression of TRAV26- 2 was decreased in patients with FD compared with that in controls. We identified a positive association between gene expression of this T-cell receptor variant and LPMC effector Th17-like cell populations in patients with FD, but not controls after exposure to gluten, but not gliadin. DISCUSSION: Our findings suggest that gliadin exposure provokes a duodenal effector Th2-like response in patients with FD, supporting the notion that food antigens drive responses in some patients. Furthermore, these findings suggest that altered lymphocyte responses to wheat proteins play a role in FD pathogenesis.
Sujet(s)
Dyspepsie , Humains , Dyspepsie/étiologie , Gliadine/métabolisme , Triticum/génétique , Lymphocytes/métabolisme , Lymphocytes/anatomopathologie , Glutens , Muqueuse intestinale/anatomopathologie , Récepteurs aux antigènes des cellules T/métabolismeRÉSUMÉ
Background: Functional dyspepsia is characterised by chronic symptoms of post-prandial distress or epigastric pain not associated with defined structural pathology. Increased peripheral gut-homing T cells have been previously identified in patients. To date, it is unknown if these T cells were antigen-experienced, or if a specific phenotype was associated with FD. Objective: This study aimed to characterise T cell populations in the blood and duodenal mucosa of FD patients that may be implicated in disease pathophysiology. Methods: We identified duodenal T cell populations from 23 controls and 49 Rome III FD patients by flow cytometry using a surface marker antibody panel. We also analysed T cell populations in peripheral blood from 37 controls and 61 patients. Where available, we examined the number of duodenal eosinophils in patients and controls. Results: There was a shift in the duodenal T helper cell balance in FD patients compared to controls. For example, patients had increased duodenal mucosal Th2 populations in the effector (13.03 ± 16.11, 19.84 ± 15.51, p=0.038), central memory (23.75 ± 18.97, 37.52 ± 17.51, p=0.007) and effector memory (9.80±10.50 vs 20.53±14.15, p=0.001) populations. Th17 populations were also increased in the effector (31.74±24.73 vs 45.57±23.75, p=0.03) and effector memory (11.95±8.42 vs 18.44±15.63, p=0.027) subsets. Peripheral T cell populations were unchanged between FD and control. Conclusion: Our findings identify an association between lymphocyte populations and FD, specifically a Th2 and Th17 signature in the duodenal mucosa. The presence of effector and memory cells suggest that the microinflammation in FD is antigen driven.
Sujet(s)
Dyspepsie , Humains , Dyspepsie/diagnostic , Dyspepsie/anatomopathologie , Duodénum , Douleur abdominale/métabolisme , Granulocytes éosinophiles/métabolisme , Muqueuse/métabolismeRÉSUMÉ
Intestinal epithelia are critical for maintaining gastrointestinal homeostasis. Epithelial barrier injury, causing inflammation and vascular damage, results in inflammatory hypoxia, and thus, healing occurs in an oxygen-restricted environment. The transcription factor hypoxia-inducible factor (HIF)-1 regulates genes important for cell survival and repair, including the cell adhesion protein ß1-integrin. Integrins function as αß-dimers, and α-integrin-matrix binding is critical for cell migration. We hypothesized that HIF-1 stabilization accelerates epithelial migration through integrin-dependent pathways. We aimed to examine functional and posttranslational activity of α-integrins during HIF-1-mediated intestinal epithelial healing. Wound healing was assessed in T84 monolayers over 24 h with/without prolyl-hydroxylase inhibitor (PHDi) (GB-004), which stabilizes HIF-1. Gene and protein expression were measured by RT-PCR and immunoblot, and α-integrin localization was assessed by immunofluorescence. α-integrin function was assessed by antibody-mediated blockade, and integrin α6 regulation was determined by HIF-1α chromatin immunoprecipitation. Models of mucosal wounding and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis were used to examine integrin expression and localization in vivo. PHDi treatment accelerated wound closure and migration within 12 h, associated with increased integrin α2 and α6 protein, but not α3. Functional blockade of integrins α2 and α6 inhibited PHDi-mediated accelerated wound closure. HIF-1 bound directly to the integrin α6 promoter. PHDi treatment accelerated mucosal healing, which was associated with increased α6 immunohistochemical staining in wound-associated epithelium and wound-adjacent tissue. PHDi treatment increased α6 protein levels in colonocytes of TNBS mice and induced α6 staining in regenerating crypts and reepithelialized inflammatory lesions. Together, these data demonstrate a role for HIF-1 in regulating both integrin α2 and α6 responses during intestinal epithelial healing.NEW & NOTEWORTHY HIF-1 plays an important role in epithelial restitution, selectively inducing integrins α6 and α2 to promote migration and proliferation, respectively. HIF-stabilizing prolyl-hydroxylase inhibitors accelerate intestinal mucosal healing by inducing epithelial integrin expression.
Sujet(s)
Colite/prévention et contrôle , Côlon/effets des médicaments et des substances chimiques , Sous-unité alpha du facteur-1 induit par l'hypoxie/métabolisme , Intégrines alpha/métabolisme , Muqueuse intestinale/effets des médicaments et des substances chimiques , Inhibiteurs de prolyle hydroxylases/pharmacologie , Cicatrisation de plaie/effets des médicaments et des substances chimiques , Animaux , Lignée cellulaire tumorale , Mouvement cellulaire/effets des médicaments et des substances chimiques , Prolifération cellulaire/effets des médicaments et des substances chimiques , Colite/induit chimiquement , Colite/métabolisme , Colite/anatomopathologie , Côlon/métabolisme , Côlon/anatomopathologie , Modèles animaux de maladie humaine , Femelle , Humains , Sous-unité alpha du facteur-1 induit par l'hypoxie/génétique , Intégrines alpha/génétique , Intégrine alpha2/métabolisme , Intégrine alpha6/métabolisme , Muqueuse intestinale/métabolisme , Muqueuse intestinale/anatomopathologie , Souris de lignée BALB C , Stabilité protéique , Transduction du signal , Acide 2,4,6-trinitro-benzènesulfoniqueRÉSUMÉ
Liver inflammation is a common extraintestinal manifestation in inflammatory bowel disease (IBD), yet, the mechanisms driving gut-liver axis inflammation remain poorly understood. IBD leads to a breakdown in the integrity of the intestinal barrier causing an increase in portal and systemic gut-derived antigens, which challenge the liver. Here, we examined the role of platelet activating factor receptor (PAFR) in colitis-associated liver damage using dextran sulfate sodium (DSS) and anti-CD40-induced colitis models. Both DSS and anti-CD40 models exhibited liver inflammation associated with colitis. Colitis reduced global PAFR protein expression in mouse livers causing an exclusive re-localization of PAFR to the portal triad. The global decrease in liver PAFR was associated with increased sirtuin 1 while relocalized PAFR expression was limited to Kupffer cells (KCs) and co-localized with toll-like receptor 4. DSS activated the NLRP3-inflammasome and increased interleukin (IL)-1ß in the liver. Antagonism of PAFR amplified the inflammasome response by increasing NLRP3, caspase-1, and IL-1ß protein levels in the liver. LPS also increased NLRP3 response in human hepatocytes, however, overexpression of PAFR restored the levels of NLPR3 and caspase-1 proteins. Interestingly, KCs depletion also increased IL-1ß protein in mouse liver after DSS challenge. These data suggest a protective role for PAFR-expressing KCs during colitis and that regulation of PAFR is important for gut-liver axis homeostasis.
Sujet(s)
Colite/métabolisme , Colite/anatomopathologie , Inflammation/métabolisme , Inflammation/anatomopathologie , Foie/métabolisme , Glycoprotéines de membrane plaquettaire/métabolisme , Récepteurs couplés aux protéines G/métabolisme , Animaux , Caspase-1/métabolisme , Cellules cultivées , Colite/induit chimiquement , Côlon/métabolisme , Côlon/anatomopathologie , Sulfate dextran/pharmacologie , Hépatocytes/effets des médicaments et des substances chimiques , Hépatocytes/métabolisme , Hépatocytes/anatomopathologie , Humains , Inflammasomes/métabolisme , Inflammation/induit chimiquement , Maladies inflammatoires intestinales/métabolisme , Maladies inflammatoires intestinales/anatomopathologie , Interleukine-1 bêta/métabolisme , Cellules de Küpffer/métabolisme , Cellules de Küpffer/anatomopathologie , Foie/effets des médicaments et des substances chimiques , Foie/anatomopathologie , Mâle , Souris , Souris de lignée C57BL , Protéine-3 de la famille des NLR contenant un domaine pyrine/métabolisme , Récepteur de type Toll-4/métabolismeRÉSUMÉ
Extra-intestinal manifestations (EIM) are common in inflammatory bowel disease (IBD). One such EIM is sub-clinical pulmonary inflammation, which occurs in up to 50% of IBD patients. In animal models of colitis, pulmonary inflammation is driven by neutrophilic infiltrations, primarily in response to the systemic bacteraemia and increased bacterial load in the lungs. Platelet activating factor receptor (PAFR) plays a critical role in regulating pulmonary responses to infection in conditions, such as chronic obstructive pulmonary disease and asthma. We investigated the role of PAFR in pulmonary EIMs of IBD, using dextran sulfate sodium (DSS) and anti-CD40 murine models of colitis. Both models induced neutrophilic inflammation, with increased TNF and IL-1ß levels, bacterial load and PAFR protein expression in mouse lungs. Antagonism of PAFR decreased lung neutrophilia, TNF, and IL-1ß in an NLRP3 inflammasome-dependent manner. Lipopolysaccharide from phosphorylcholine (ChoP)-positive bacteria induced NLRP3 and caspase-1 proteins in human alveolar epithelial cells, however antagonism of PAFR prevented NLRP3 activation by ChoP. Amoxicillin reduced bacterial populations in the lungs and reduced NLRP3 inflammasome protein levels, but did not reduce PAFR. These data suggest a role for PAFR in microbial pattern recognition and NLRP3 inflammasome signaling in the lung.
Sujet(s)
Colite/complications , Prédisposition aux maladies , Protéine-3 de la famille des NLR contenant un domaine pyrine/métabolisme , Glycoprotéines de membrane plaquettaire/métabolisme , Pneumopathie infectieuse/étiologie , Pneumopathie infectieuse/métabolisme , Récepteurs couplés aux protéines G/métabolisme , Animaux , Biopsie , Sulfate dextran/effets indésirables , Modèles animaux de maladie humaine , Endoscopie , Immunohistochimie , Inflammasomes/métabolisme , Maladies inflammatoires intestinales/complications , Souris , Infiltration par les neutrophiles/immunologie , Pneumopathie infectieuse/anatomopathologie , Transduction du signalRÉSUMÉ
BACKGROUND: Cell-free mitochondrial DNA (mtDNA) is proinflammatory and has been detected in high concentrations in trauma patients' plasma. Deoxyribonuclease (DNAse) is the free plasma enzyme responsible for the digestion of extracellular DNA. The relationship between mtDNA and DNAse after major trauma is unknown. We hypothesized that DNAse activity would be elevated after injury and trauma surgery and would be associated with high concentrations of extracellular DNA. METHODS: Two-year prospective study was performed on 103 consecutive trauma patients (male, 81%; age, 38 years [interquartile range, 30-59 years]; injury severity score, 18 [interquartile range, 12-26 years]) who underwent standardized major orthopedic trauma surgical interventions. Blood was collected at five perioperative time points (preoperative, postoperative, 7 hours, 24 hours, and 3 days postoperatively). Healthy control subjects (n = 20) were also sampled. Cell-free mtDNA and nuclear DNA (nDNA) were measured using quantitative polymerase chain reaction. Deoxyribonuclease was also assayed in the same plasma samples. RESULTS: Increased levels of mtDNA (from preoperative 163 ± 86 ng/mL to 3 days 282 ± 201 ng/mL, p < 0.0001) and nDNA (from preoperative 28 ± 20 ng/mL to 3 days 37 ± 27 ng/mL, p < 0.05) were present in trauma patients at all perioperative time points compared with healthy controls (mtDNA: 4 ± 2 ng/mL; nDNA: 10 ± 5 ng/mL). Deoxyribonuclease activity was lower in the trauma cohort (from preoperative 0.06 ± 0.04U/mL to 3 days 0.08 ± 0.04U/mL, p < 0.0001) compared with healthy controls (DNAse: 0.17 ± 0.03U/mL). There was no correlation between DNAse and perioperative DNA concentrations. Elevated mtDNA (but not nDNA) correlated with the development of systemic inflammatory response syndrome (SIRS) (p = 0.026) but not multiple organ failure. CONCLUSIONS: The significant perioperative elevation in plasma-free mtDNA concentration is associated with the development of SIRS. The fact that increased cell-free DNA concentrations present with significantly lower than healthy control DNAse activity suggests a potential therapeutic opportunity with DNAse administration to modulate postinjury severe SIRS. LEVEL OF EVIDENCE: Prognostic/Epidemiological, level II.
Sujet(s)
ADN mitochondrial/sang , Désoxyribonucléases/sang , Syndrome de réponse inflammatoire généralisée/sang , Adulte , Études cas-témoins , Acides nucléiques acellulaires/sang , Femelle , Humains , Score de gravité des lésions traumatiques , Mâle , Adulte d'âge moyen , Défaillance multiviscérale/sang , Études prospectivesRÉSUMÉ
Inflammatory bowel disease (IBD) is associated with several immune-mediated extraintestinal manifestations. More than half of all IBD patients have some form of respiratory pathology, most commonly neutrophil-mediated diseases, such as bronchiectasis and chronic bronchitis. Using murine models of colitis, we aimed to identify the immune mechanisms driving pulmonary manifestations of IBD. We found increased neutrophil numbers in lung tissue associated with the pulmonary vasculature in both trinitrobenzenesulfonic acid- and dextran sulfate sodium-induced models of colitis. Analysis of systemic inflammation identified that neutrophilia was associated with bacteremia and pyrexia in animal models of colitis. We further identified IL-6 as a systemic mediator of neutrophil recruitment from the bone marrow of dextran sulfate sodium animals. Functional inhibition of IL-6 led to reduced systemic and pulmonary neutrophilia, but it did not attenuate established colitis pathology. These data suggest that systemic bacteremia and pyrexia drive IL-6 secretion, which is a critical driver for pulmonary manifestation of IBD. Targeting IL-6 may reduce neutrophil-associated extraintestinal manifestations in IBD patients.
Sujet(s)
Bactériémie/anatomopathologie , Colite/complications , Modèles animaux de maladie humaine , Interleukine-6/toxicité , Granulocytes neutrophiles/immunologie , Pneumopathie infectieuse/anatomopathologie , Animaux , Bactériémie/étiologie , Bactériémie/métabolisme , Colite/induit chimiquement , Sulfate dextran/toxicité , Femelle , Souris , Souris de lignée BALB C , Souris de lignée C57BL , Granulocytes neutrophiles/effets des médicaments et des substances chimiques , Granulocytes neutrophiles/anatomopathologie , Pneumopathie infectieuse/étiologie , Pneumopathie infectieuse/métabolismeRÉSUMÉ
BACKGROUND: Pharmacological induction of hypoxia-inducible factor (HIF), a global transcriptional regulator of the hypoxic response, by prolyl hydroxylase inhibitors (PHDi) is protective in murine models of colitis, and epithelial cells are critical for the observed therapeutic efficacy. Because systemic HIF activation may lead to potentially negative off-target effects, we hypothesized that targeting epithelial HIF through oral delivery of PHDi would be sufficient to protect against colitis in a mouse model. METHODS: Using a chemically induced trinitrobenzene sulfonic acid murine model of colitis, we compared the efficacy of oral and intraperitoneal (i.p.) delivery of the PHDi; AKB-4924 in preventing colitis, as measured by endoscopy, histology, barrier integrity, and immune profiling. Furthermore, we measured potential off-target effects, examining HIF and HIF target genes in the heart and kidney, as well as erythropoietin and hematocrit levels. RESULTS: Oral administration of AKB-4924 exhibited mucosal protection comparable i.p. dosing. Oral delivery of PHDi led to reduced colonic epithelial HIF stabilization compared with i.p. delivery, but this was still sufficient to induce transcription of downstream HIF targets. Furthermore, oral delivery of PHDi led to reduced stabilization of HIF and activation of HIF targets in extraintestinal organs. CONCLUSIONS: Oral delivery of PHDi therapies to this intestinal mucosa protects against colitis in animal models and represents a potential therapeutic strategy for inflammatory bowel disease, which also precludes unwanted extraintestinal effects.
Sujet(s)
Colite/traitement médicamenteux , Modèles animaux de maladie humaine , Muqueuse/effets des médicaments et des substances chimiques , Pipérazines/administration et posologie , Inhibiteurs de prolyle hydroxylases/administration et posologie , Pyridones/administration et posologie , Cicatrisation de plaie/effets des médicaments et des substances chimiques , Administration par voie orale , Animaux , Colite/induit chimiquement , Colite/anatomopathologie , Femelle , Facteur-1 induit par l'hypoxie/agonistes , Facteur-1 induit par l'hypoxie/métabolisme , Techniques immunoenzymatiques , Muqueuse intestinale/cytologie , Muqueuse intestinale/effets des médicaments et des substances chimiques , Muqueuse intestinale/métabolisme , Souris , Souris de lignée BALB C , Muqueuse/métabolisme , Pipérazines/pharmacologie , Inhibiteurs de prolyle hydroxylases/pharmacologie , Pyridones/pharmacologie , Acide 2,4,6-trinitro-benzènesulfonique/toxicitéRÉSUMÉ
FZR1, an activator of the anaphase-promoting complex/cyclosome (APC/C), is recognized for its roles in the mitotic cell cycle. To examine its meiotic function in females we generated an oocyte-specific knockout of the Fzr1 gene (Fzr1(Δ/Δ)). The total number of fully grown oocytes enclosed in cumulus complexes was 35-40% lower in oocytes from Fzr1(Δ/Δ) mice and there was a commensurate rise in denuded, meiotically advanced and/or fragmented oocytes. The ability of Fzr1(Δ/Δ) oocytes to remain prophase I/germinal vesicle (GV) arrested in vitro was also compromised, despite the addition of the phosphodiesterase milrinone. Meiotic competency of smaller diameter oocytes was also accelerated by Fzr1 loss. Cyclin B1 levels were elevated ~5-fold in Fzr1(Δ/Δ) oocytes, whereas securin and CDC25B, two other APC/C(FZR1) substrates, were unchanged. Cyclin B1 overexpression can mimic the effects of Fzr1 loss on GV arrest and here we show that cyclin B1 knockdown in Fzr1(Δ/Δ) oocytes affects the timing of meiotic resumption. Therefore, the effects of Fzr1 loss are mediated, at least in part, by raised cyclin B1. Thus, APC/C(FZR1) activity is required to repress cyclin B1 levels in oocytes during prophase I arrest in the ovary, thereby maintaining meiotic quiescence until hormonal cues trigger resumption.
Sujet(s)
Protéines du cycle cellulaire/physiologie , Méiose , Prophase I de méiose , Ovocytes/cytologie , Ubiquitin-protein ligase complexes/physiologie , Complexe promoteur de l'anaphase , Animaux , Protéines Cdh1 , Cycline B1/génétique , Femelle , Souris , Souris knockout , Ovaire , Facteurs tempsRÉSUMÉ
Mature mammalian eggs are ovulated arrested at meiotic metaphase II. Sperm break this arrest by an oscillatory Ca(2+) signal that is necessary and sufficient for the two immediate events of egg activation: cell cycle resumption and cortical granule release. Previous work has suggested that cell cycle resumption, but not cortical granule release, is mediated by calmodulin-dependent protein kinase II (CamKII). Here we find that mouse eggs contain detectable levels of only one CamKII isoform, gamma 3. Antisense morpholino knockdown of CamKIIgamma3 during oocyte maturation produces metaphase II eggs that are insensitive to parthenogenetic activation by Ca(2+) stimulation and insemination. The effect is specific to this morpholino, as a 5-base-mismatch morpholino is without effect, and is rescued by CamKIIgamma3 or constitutively active CamKII cRNAs. Although CamKII-morpholino-treated eggs fail to exit metaphase II arrest, cortical granule exocytosis is not blocked. Therefore, CamKIIgamma3 plays a necessary and sufficient role in transducing the oscillatory Ca(2+) signal into cell cycle resumption, but not into cortical granule release.