RESUMEN

Inflammation of the ileum, or ileitis, is commonly caused by Crohn's disease (CD) but can also accompany ulcerative colitis (backwash ileitis), infections or drug-related damage. Oxidative tissue injury triggered by reactive oxygen species (ROS) is considered part of the ileitis etiology. However, not only elevated ROS but also permanently decreased ROS are associated with inflammatory bowel disease (IBD). While very early onset IBD (VEO-IBD) is associated with a spectrum of NOX1 variants, how NOX1 inactivation contributes to disease development remains ill-defined. Besides propagating signaling responses, NOX1 provides superoxide for peroxynitrite formation in the epithelial barrier. Here we report that NOX4, an H2O2-generating NADPH oxidase with documented tissue protective effects in the intestine and other tissues, limits the generation of ileal peroxynitrite by NOX1/NOS2. Deletion of NOX4 leads to persistent peroxynitrite excess, hyperpermeability, villus blunting, muscular hypertrophy, chemokine/cytokine upregulation and dysbiosis. Conversely, SAMP1/YitFc mice, a CD-like ileitis model, showed age-dependent NOX1/NOS2 downregulation preventing ileal peroxynitrite formation in homeostasis and LPS-induced acute inflammation. Deficiency in NOX1 correlated with the upregulation of antimicrobial peptides, suggesting that ileal peroxynitrite acts as chemical barrier and microbiota modifier in the ileum.

RESUMEN

Loss of appetite and negative energy balance are common features of endotoxemia in all animals and are thought to have protective roles by reducing nutrient availability to host and pathogen metabolism. Accordingly, fasting and caloric restriction have well-established anti-inflammatory properties. However, in response to reduced nutrient availability at the cellular and organ levels, negative energy balance also recruits distinct energy-sensing brain circuits, but it is not known whether these neuronal systems have a role in its anti-inflammatory effects. Here, we report that hypothalamic AgRP neurons-a critical neuronal population for the central representation of negative energy balance-have parallel immunoregulatory functions. We found that when endotoxemia occurs in fasted mice, the activity of AgRP neurons remains sustained, but this activity does not influence feeding behavior and endotoxemic anorexia. Furthermore, we found that endotoxemia acutely desensitizes AgRP neurons, which also become refractory to inhibitory signals. Mimicking this sustained AgRP neuron activity in fed mice by chemogenetic activation-a manipulation known to recapitulate core behavioral features of fasting-results in reduced acute tumor necrosis factor alpha (TNF-α) release during endotoxemia. Mechanistically, we found that endogenous glucocorticoids play an important role: glucocorticoid receptor deletion from AgRP neurons prevents their endotoxemia-induced desensitization, and importantly, it counteracts the fasting-induced suppression of TNF-α release, resulting in prolonged sickness. Together, these findings provide evidence directly linking AgRP neuron activity to the acute response during endotoxemia, suggesting that these neurons are a functional component of the immunoregulatory effects associated with negative energy balance and catabolic metabolism.

Asunto(s)

Endotoxemia , Factor de Necrosis Tumoral alfa , Ratones , Animales , Proteína Relacionada con Agouti/genética , Proteína Relacionada con Agouti/metabolismo , Factor de Necrosis Tumoral alfa/genética , Endotoxemia/metabolismo , Endotoxemia/patología , Hipotálamo/metabolismo , Neuronas/fisiología , Metabolismo Energético

RESUMEN

Kaposi's sarcoma (KS) is a rare inflammation- based vascular cancer involving the skin. The viral aetiology of KS is the Human Herpesvirus 8. KS may be frequently diagnosed in immunosuppressed kidneytransplanted patients, while is less common in patients with dialysis. It is known that various immunological abnormalities can lead to impaired immune status in uremic patients. It is noteworthy that despite the incidence of KS in patients with renal impairment, only few cases have reported efficacy and safety profile of KS targeting anti-cancer drugs in this kidney disease population. Herein, we report the first case of a symptomatic KS patient with renal disease in haemodialysis and focus on its therapeutic management. We also review the main data available from literature regarding the safety of KS therapy in dialysis patients.

RESUMEN

OBJECTIVE: Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are effective medications to reduce appetite and body weight. These actions are centrally mediated; however, the neuronal substrates involved are poorly understood. METHODS: We employed a combination of neuroanatomical, genetic, and behavioral approaches in the mouse to investigate the involvement of caudal brainstem cholecystokinin-expressing neurons in the effect of the GLP-1RA exendin-4. We further confirmed key neuroanatomical findings in the non-human primate brain. RESULTS: We found that cholecystokinin-expressing neurons in the caudal brainstem are required for the anorectic and body weight-lowering effects of GLP-1RAs and for the induction of GLP-1RA-induced conditioned taste avoidance. We further show that, while cholecystokinin-expressing neurons are not a direct target for glucose-dependent insulinotropic peptide (GIP), GIP receptor activation results in a reduced recruitment of these GLP-1RA-responsive neurons and a selective reduction of conditioned taste avoidance. CONCLUSIONS: In addition to disclosing a neuronal population required for the full appetite- and body weight-lowering effect of GLP-1RAs, our data also provide a novel framework for understanding and ameliorating GLP-1RA-induced nausea - a major factor for withdrawal from treatment.

Asunto(s)

Colecistoquinina/farmacología , Polipéptido Inhibidor Gástrico/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Animales , Apetito/efectos de los fármacos , Depresores del Apetito/farmacología , Glucemia/efectos de los fármacos , Exenatida/farmacología , Femenino , Glucagón/metabolismo , Péptido 1 Similar al Glucagón/farmacología , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/fisiología , Hipoglucemiantes/farmacología , Insulina/farmacología , Liraglutida/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo

RESUMEN

Given the high-energy requirements to sustain immune responses and healing processes, it is intriguing that lack of appetite (i.e., anorexia) is a cardinal feature of sickness behaviour. While our understanding of the brain mechanisms that control appetite is rapidly growing, how inflammation affects these mechanisms is not fully understood. Here, we discuss advances in our understanding of discrete appetite controlling mechanisms and how inflammation influences their function. We further discuss the pathophysiological significance of anorexia and negative energy balance during the immune regulatory response. LINKED ARTICLES: This article is part of a themed issue on Cellular metabolism and diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.10/issuetoc.

Asunto(s)

Apetito , Inflamación , Encéfalo , Humanos , Inmunidad

RESUMEN

Dysregulated redox signaling and oxidative injury are associated with inflammatory processes and fibrosis. H2O2 generation by NOX4 has been suggested as a key driver in the development of fibrosis and a small molecule drug is under evaluation in clinical trials for idiopathic pulmonary fibrosis and primary biliary cholangitis. Fibrosis is a common complication in Crohn's disease (CD) leading to stricture formation in 35-40% of patients, who require surgical interventions in the absence of therapeutic options. Here we assess NOX4 expression in CD patients with inflammatory or stricturing disease and examine whether loss of NOX4 is beneficial in acute and fibrotic intestinal disease. NOX4 was upregulated in inflamed mucosal tissue of CD and ulcerative colitis (UC) patients, in CD ileal strictures, and in mice with intestinal inflammation. Nox4 deficiency in mice promoted pathogen colonization and exacerbated tissue injury in acute bacterial and chemical colitis. In contrast, in two chronic injury models aberrant tissue remodeling and fibrosis-related gene expression did not differ substantially between Nox4-/- mice and wildtype mice, suggesting that Nox4 is dispensable in TGF-ß1-driven intestinal fibrogenesis. While animal models do not recapitulate all the hallmarks of CD fibrosis, the tissue-protective role of Nox4 warrants a cautious approach to pharmacological inhibitors.

Asunto(s)

Miofibroblastos , NADPH Oxidasas , Animales , Fibrosis , Humanos , Peróxido de Hidrógeno , Inflamación/genética , Inflamación/patología , Ratones , Miofibroblastos/patología , NADPH Oxidasa 4/genética

RESUMEN

Pain conditions, such as neuropathic pain (NP) and persistent inflammatory pain are therapeutically difficult to manage. Previous studies have shown the involvement of glutamate receptor in pain modulation and in particular same of these showed the key role of the AMPA ionotropic glutamate receptor subtype. Antiseizure medications (ASMs) are often used to treat this symptom, however the effect of perampanel (PER), an ASM acting as selective, non-competitive inhibitor of the AMPA receptor on the management of pain has not well been investigated yet. Here we tested the potential analgesic and anti-inflammatory effects of PER, in acute and chronic pain models. PER was given orally either in acute (5 mg/kg) or repeated administration (3 mg/kg/d for 4 days). Pain response was assessed using models of nociceptive sensitivity, visceral and inflammatory pain, and mechanical allodynia and hyperalgesia induced by chronic constriction injury to the sciatic nerve. PER significantly reduced pain perception in all behavioral tests as well as CCI-induced mechanical allodynia and hyperalgesia in acute regimen (5 mg/kg). This effect was also observed after repeated treatment using the dose of 3 mg/kg/d. The antinociceptive, antiallodynic and antihyperalgesic effects of PER were attenuated when the CB1 antagonist AM251 (1 mg/kg/i.p.) was administered before PER treatment, suggesting the involvement of the cannabinergic system. Moreover, Ex vivo analyses showed that PER significantly increased CB1 receptor expression and reduced inflammatory cytokines (i.e. TNFα, IL-1ß, and IL-6) in the spinal cord. In conclusion, these results extend our knowledge on PER antinociceptive and antiallodynic effects and support the involvement of cannabinergic system on its mode of action.

RESUMEN

Reactive oxygen species (ROS) generated by NADPH oxidases (NOX/DUOX) provide antimicrobial defense, redox signaling, and gut barrier maintenance. Inactivating NOX variants are associated with comorbid intestinal inflammation in chronic granulomatous disease (CGD; NOX2) and pediatric inflammatory bowel disease (IBD; NOX1); however Nox-deficient mice do not reflect human disease susceptibility. Here we assessed if a hypomorphic patient-relevant CGD mutation will increase the risk for intestinal inflammation in mice. Cyba (p22phox) mutant mice generated low intestinal ROS, while maintaining Nox4 function. The Cyba variant caused profound mucus layer disruption with bacterial penetration into crypts, dysbiosis, and a compromised innate immune response to invading microbes, leading to mortality. Approaches used in treatment-resistant CGD or pediatric IBD such as bone marrow transplantation or oral antibiotic treatment ameliorated or prevented disease in mice. The Cyba mutant mouse phenotype implicates loss of both mucus barrier and efficient innate immune defense in the pathogenesis of intestinal inflammation due to ROS deficiency, supporting a combined-hit model where a single disease variant compromises different cellular functions in interdependent compartments.

Asunto(s)

Colitis/enzimología , Colon/enzimología , Grupo Citocromo b/metabolismo , Mucosa Intestinal/enzimología , Moco/enzimología , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Antibacterianos/farmacología , Colitis/inmunología , Colitis/microbiología , Colitis/prevención & control , Colon/efectos de los fármacos , Colon/inmunología , Colon/microbiología , Grupo Citocromo b/deficiencia , Grupo Citocromo b/genética , Modelos Animales de Enfermedad , Disbiosis , Femenino , Microbioma Gastrointestinal , Inmunidad Innata , Inmunidad Mucosa , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Moco/inmunología , Moco/microbiología , Mutación Missense , NADPH Oxidasas/deficiencia , NADPH Oxidasas/genética , Transducción de Señal

RESUMEN

Reactive oxygen species (ROS) are critical redox regulators of cellular dynamics controlling homeostasis. Although numerous fluorescent probes are currently available to measure ROS in cell-based assays, the short-lived nature of these molecules renders their detection challenging in more complex biological systems, such as the gastrointestinal tract in vivo. However, in the past decade, significant progress has been made in the development of novel imaging technologies and probes, facilitating ROS quantification with high sensitivity, selectivity, and temporal resolution. The IVIS Spectrum (PerkinElmer) is an optical imaging system for small animal imaging allowing precise and noninvasive visualization of fluorescent or bioluminescent signals. Here, we describe a reproducible and comprehensive method for the measurement of physiological intestinal NADPH oxidase-derived ROS by using the chemiluminescent probe L-012. Using transgenic mice deficient in Nox isoforms expressed in the intestinal mucosa, we delineate the contribution of gut epithelial versus immune cell NADPH oxidase activity in homeostatic conditions. We also discuss L-012 probe specificity and potential alternatives for in vivo studies.

Asunto(s)

Homeostasis , Intestinos/fisiología , Luminol/análogos & derivados , Imagen Molecular , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Animales , Biomarcadores , Análisis de Datos , Procesamiento de Imagen Asistido por Computador , Mucosa Intestinal/metabolismo , Mediciones Luminiscentes/métodos , Luminol/química , Luminol/metabolismo , Ratones , Imagen Molecular/métodos , Sondas Moleculares , Estructura Molecular , NADPH Oxidasas/metabolismo

RESUMEN

Infection with parasite helminths induces potent modulation of the immune system of the host. Epidemiological and animal studies have shown that helminth infections can suppress or exacerbate unrelated autoimmune, allergic, and other inflammatory disorders. There is growing evidence that helminth infection-mediated suppression of bystander inflammatory responses is influenced by alterations in the intestinal microbiome modulating metabolic and immune functions of the infected host. We analyzed the fecal microbiota of mice infected with adult male Schistosoma mansoni worms, which are less susceptible to experimental colitis, and male- and female-worm-infected mice, which are highly sensitive to colitis. While both groups of infected mice developed a disrupted microbiota, there were marked alterations in mice with male and female worm infections. Antibiotic-treated recipients that were cohoused with both types of S. mansoni worm-infected mice acquired a colitogenic microbiome, leading to increased susceptibility to experimental colitis. Following anthelmintic treatment to remove worms from worm-only-infected mice, the mice developed exacerbated colitis. This study provides evidence that adult male S. mansoni worm infection modulates the host's immune system and suppresses bystander colitis while limiting dysbiosis of the host's intestinal microbiome during infection.

Asunto(s)

Colitis/prevención & control , Microbioma Gastrointestinal , Esquistosomiasis mansoni/inmunología , Animales , Susceptibilidad a Enfermedades , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Esquistosomiasis mansoni/microbiología

RESUMEN

The A20-binding inhibitor of NF-κB 2 (ABIN2) interacts with Met1-linked ubiquitin chains and is an integral component of the tumor progression locus 2 (Tpl2) kinase complex. We generated a knock-in mouse expressing the ubiquitin-binding-defective mutant ABIN2[D310N]. The expression of Tpl2 and its activation by TLR agonists in macrophages or by IL-1ß in fibroblasts from these mice was unimpaired, indicating that the interaction of ABIN2 with ubiquitin oligomers is not required for the stability or activation of Tpl2. The ABIN2[D310N] mice displayed intestinal inflammation and hypersensitivity to dextran sodium sulfate-induced colitis, an effect that was mediated by radiation-resistant cells rather than by hematopioetic cells. The IL-1ß-dependent induction of cyclooxygenase 2 (COX2) and the secretion of PGE2 was reduced in mouse embryonic fibroblasts and intestinal myofibroblasts (IMFs) from ABIN2[D310N] mice. These observations are similar to those reported for the Tpl2 knockout (KO) mice (Roulis et al. 2014. Proc. Natl. Acad. Sci. USA 111: E4658-E4667), but the IL-1ß-dependent production of COX2 and PGE2 in mouse embryonic fibroblasts or IMFs was unaffected by pharmacological inhibition of Tpl2 in wild-type mice. The expression of ABIN2 is decreased drastically in Tpl2 KO mice. These and other lines of evidence suggest that the hypersensitivity of Tpl2 KO mice to dextran sodium sulfate-induced colitis is not caused by the loss of Tpl2 catalytic activity but by the loss of ABIN2, which impairs COX2 and PGE2 production in IMFs by a Tpl2 kinase-independent pathway.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Colitis/inmunología , Quinasas Quinasa Quinasa PAM/metabolismo , Macrófagos/inmunología , Miofibroblastos/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células Cultivadas , Colitis/inducido químicamente , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Sulfato de Dextran , Dinoprostona/metabolismo , Técnicas de Sustitución del Gen , Interleucina-1beta/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutación/genética , Unión Proteica/genética , Proteínas Proto-Oncogénicas/genética , Ribonucleasa Pancreática/metabolismo , Transducción de Señal , Ubiquitinas/metabolismo

RESUMEN

Hypothalamic inflammation is thought to contribute to obesity. One potential mechanism is via gut microbiota derived bacterial lipopolysaccharide (LPS) entering into the circulation and activation of Toll-like receptor-4. This is called metabolic endotoxemia. Another potential mechanism is systemic inflammation arising from sustained exposure to high-fat diet (HFD) over more than 12 weeks. In this study we show that mice fed HFD over 8 weeks become obese and show elevated plasma LPS binding protein, yet body weight gain and adiposity is not attenuated in mice lacking Tlr4 or its co-receptor Cd14. In addition, caecal microbiota composition remained unchanged by diet. Exposure of mice to HFD over a more prolonged period (20 weeks) to drive systemic inflammation also caused obesity. RNAseq used to assess hypothalamic inflammation in these mice showed increased hypothalamic expression of Serpina3n and Socs3 in response to HFD, with few other genes altered. In situ hybridisation confirmed increased Serpina3n and Socs3 expression in the ARC and DMH at 20-weeks, but also at 8-weeks and increased SerpinA3N protein could be detected as early as 1 week on HFD. Overall these data show lack of hypothalamic inflammation in response to HFD and that metabolic endotoxemia does not link HFD to obesity.

Asunto(s)

Proteínas de Fase Aguda/genética , Dieta Alta en Grasa/efectos adversos , Endotoxemia/complicaciones , Obesidad/etiología , Serpinas/genética , Receptor Toll-Like 4/inmunología , Regulación hacia Arriba , Animales , Endotoxemia/genética , Endotoxemia/inmunología , Endotoxemia/patología , Microbioma Gastrointestinal , Regulación de la Expresión Génica , Genotipo , Hipotálamo/inmunología , Hipotálamo/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/genética , Obesidad/inmunología , Obesidad/patología , Transducción de Señal , Receptor Toll-Like 4/genética

RESUMEN

Reactive oxygen species (ROS), initially categorized as toxic by-products of aerobic metabolism, have often been called a double-edged sword. ROS are considered indispensable when host defense and redox signaling is concerned and a threat in inflammatory or degenerative diseases. This generalization does not take in account the diversity of oxygen metabolites being generated, their physicochemical characteristics and their production by distinct enzymes in space and time. NOX/DUOX NADPH oxidases are the only enzymes solely dedicated to ROS production and the prime ROS producer for intracellular and intercellular communication due to their widespread expression and intricate regulation. Here we discuss new insights of how NADPH oxidases act via ROS as multifaceted regulators of the intestinal barrier in homeostasis, infectious disease and intestinal inflammation. A closer look at monogenic VEOIBD and commensals as ROS source supports the view of H2O2 as key beneficial messenger in the barrier ecosystem.

Asunto(s)

Tracto Gastrointestinal/fisiología , Inflamación/metabolismo , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Comunicación Celular , Homeostasis , Humanos , Peróxido de Hidrógeno/metabolismo , Oxidación-Reducción , Transducción de Señal

RESUMEN

Colorectal cancer (CRC) is a major health problem in Western countries. The endocannabinoid 2-arachidonoyl-glycerol (2-AG) exerts antiproliferative actions in a number of tumoral cell lines, including CRC cells. Monoacylglycerol lipase (MAGL), a serine hydrolase that inactivates 2-AG, is highly expressed in aggressive human cancer cells. Here, we investigated the role of MAGL in experimental colon carcinogenesis. The role of MAGL was assessed in vivo by using the xenograft and the azoxymethane models of colon carcinogenesis; MAGL expression was evaluated by RT-PCR and immunohistochemistry; 2-AG levels were measured by liquid chromatography mass spectrometry; angiogenesis was evaluated in tumor tissues [by microvessel counting and by investigating the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) proteins] as well as in human umbilical vein endothelial cells (HUVEC); cyclin D1 was evaluated by RT-PCR. MAGL and 2-AG were strongly expressed in tumor tissues. The MAGL inhibitor URB602 reduced xenograft tumor volume, this effect being associated to down-regulation of VEGF and FGF-2, reduction in the number of vessels and down-regulation of cyclin D1. In HUVEC, URB602 exerted a direct antiangiogenic effect by inhibiting FGF-2 induced proliferation and migration, and by modulating pro/anti-angiogenic agents. In experiments aiming at investigating the role of MAGL in chemoprevention, URB602 attenuated azoxymethane-induced preneoplastic lesions, polyps and tumors. MAGL, possibly through modulation of angiogenesis, plays a pivotal role in experimental colon carcinogenesis. Pharmacological inhibition of MAGL could represent an innovative therapeutic approach to reduce colorectal tumor progression.

Asunto(s)

Antineoplásicos/uso terapéutico , Compuestos de Bifenilo/uso terapéutico , Colon/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Monoacilglicerol Lipasas/antagonistas & inhibidores , Recto/efectos de los fármacos , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Ácidos Araquidónicos/metabolismo , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Colon/irrigación sanguínea , Colon/metabolismo , Colon/patología , Neoplasias Colorrectales/irrigación sanguínea , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Regulación hacia Abajo/efectos de los fármacos , Endocannabinoides/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glicéridos/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones Endogámicos ICR , Ratones Desnudos , Monoacilglicerol Lipasas/genética , Monoacilglicerol Lipasas/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Recto/irrigación sanguínea , Recto/metabolismo , Recto/patología

RESUMEN

BACKGROUND: Hydrocyanines are widely used as fluorogenic probes to monitor reactive oxygen species (ROS) generation in cells. Their brightness, stability to autoxidation and photobleaching, large signal change upon oxidation, pH independence and red/near infrared emission are particularly attractive for imaging ROS in live tissue. METHODS: Using confocal fluorescence microscopy we have examined an interference of mitochondrial membrane potential (ΔΨm) with fluorescence intensity and localisation of a commercial hydro-Cy3 probe in respiring and non-respiring colon carcinoma HCT116 cells. RESULTS: We found that the oxidised (fluorescent) form of hydro-Cy3 is highly homologous to the common ΔΨm-sensitive probe JC-1, which accumulates and aggregates only in 'energised' negatively charged mitochondrial matrix. Therefore, hydro-Cy3 oxidised by hydroxyl and superoxide radicals tends to accumulate in mitochondrial matrix, but dissipates and loses brightness as soon as ΔΨm is compromised. Experiments with mitochondrial inhibitor oligomycin and uncoupler FCCP, as well as a common ROS producer paraquat demonstrated that signals of the oxidised hydro-Cy3 probe rapidly and strongly decrease upon mitochondrial depolarisation, regardless of the rate of cellular ROS production. CONCLUSIONS: While analysing ROS-derived fluorescence of commercial hydrocyanine probes, an accurate control of ΔΨm is required. GENERAL SIGNIFICANCE: If not accounted for, non-specific effect of mitochondrial polarisation state on the behaviour of oxidised hydrocyanines can cause artefacts and data misinterpretation in ROS studies.

Asunto(s)

Carbocianinas/metabolismo , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/metabolismo , Mitocondrias/fisiología , Especies Reactivas de Oxígeno/metabolismo , Animales , Línea Celular Tumoral , Fluorescencia , Células HCT116 , Humanos , Ratones , Ratones Endogámicos C57BL , Oligomicinas/metabolismo , Oxidación-Reducción , Superóxidos/metabolismo

RESUMEN

NOX/DUOX family of NADPH oxidases are expressed in diverse tissues and are the primary enzymes for the generation of reactive oxygen species (ROS). The intestinal epithelium expresses NOX1, NOX4, and DUOX2, whose functions are not well understood. To address this, we generated mice with complete or epithelium-restricted deficiency in the obligatory NOX dimerization partner Cyba (p22(phox)). We discovered that NOX1 regulates DUOX2 expression in the intestinal epithelium, which magnified the epithelial ROS-deficiency. Unexpectedly, epithelial deficiency of Cyba resulted in protection from C. rodentium and L. monocytogenes infection. Microbiota analysis linked epithelial Cyba deficiency to an enrichment of H2O2-producing bacterial strains in the gut. In particular, elevated levels of lactobacilli physically displaced and attenuated C. rodentium virulence by H2O2-mediated suppression of the virulence-associated LEE pathogenicity island. This transmissible compensatory adaptation relied on environmental factors, an important consideration for prevention and therapy of enteric disease.

Asunto(s)

Citrobacter rodentium/metabolismo , Infecciones por Enterobacteriaceae/enzimología , Microbioma Gastrointestinal/inmunología , Mucosa Intestinal/enzimología , Mucosa Intestinal/microbiología , NADPH Oxidasas/metabolismo , Animales , Disbiosis , Infecciones por Enterobacteriaceae/microbiología , Enfermedad Granulomatosa Crónica/microbiología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Lactobacillus , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasas/deficiencia , Especies Reactivas de Oxígeno/metabolismo , Simbiosis

RESUMEN

BACKGROUND & AIMS: Defects in intestinal innate defense systems predispose patients to inflammatory bowel disease (IBD). Reactive oxygen species (ROS) generated by nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases in the mucosal barrier maintain gut homeostasis and defend against pathogenic attack. We hypothesized that molecular genetic defects in intestinal NADPH oxidases might be present in children with IBD. METHODS: After targeted exome sequencing of epithelial NADPH oxidases NOX1 and DUOX2 on 209 children with very early onset inflammatory bowel disease (VEOIBD), the identified mutations were validated using Sanger Sequencing. A structural analysis of NOX1 and DUOX2 variants was performed by homology in silico modeling. The functional characterization included ROS generation in model cell lines and in in vivo transduced murine crypts, protein expression, intracellular localization, and cell-based infection studies with the enteric pathogens Campylobacter jejuni and enteropathogenic Escherichia coli. RESULTS: We identified missense mutations in NOX1 (c.988G>A, p.Pro330Ser; c.967G>A, p.Asp360Asn) and DUOX2 (c.4474G>A, p.Arg1211Cys; c.3631C>T, p.Arg1492Cys) in 5 of 209 VEOIBD patients. The NOX1 p.Asp360Asn variant was replicated in a male Ashkenazi Jewish ulcerative colitis cohort. All NOX1 and DUOX2 variants showed reduced ROS production compared with wild-type enzymes. Despite appropriate cellular localization and comparable pathogen-stimulated translocation of altered oxidases, cells harboring NOX1 or DUOX2 variants had defective host resistance to infection with C. jejuni. CONCLUSIONS: This study identifies the first inactivating missense variants in NOX1 and DUOX2 associated with VEOIBD. Defective ROS production from intestinal epithelial cells constitutes a risk factor for developing VEOIBD.

RESUMEN

Activation of the noncanonical inflammasome, mediated by caspase-11, serves as an additional pathway for the production of the proinflammatory cytokines IL-1ß and IL-18. Noncanonical inflammasome activity occurs during host defense against Gram-negative bacteria and in models of acute septic shock. We propose that the noncanonical inflammasome is activated in mice during acute intestinal inflammation elicited by dextran sodium sulfate (DSS), a model of experimental colitis. We find that caspase-11(-/-) mice display enhanced susceptibility to DSS, because of impaired IL-18 production. The impaired IL-18 levels observed are shown to result in reduced intestinal epithelial cell proliferation and increased cell death. We also suggest that a novel type II IFN-dependent, type I IFN-TRIF-independent signaling pathway is required for in vivo caspase-11 production in intestinal epithelial cells during DSS colitis. Collectively, these data suggest that IFN-γ-mediated caspase-11 expression has a key role maintaining intestinal epithelial barrier integrity in vivo during experimentally induced acute colitis.

Asunto(s)

Caspasas/metabolismo , Colitis/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Caspasas/genética , Caspasas Iniciadoras , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Citocinas/metabolismo , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Expresión Génica , Predisposición Genética a la Enfermedad , Inmunohistoquímica , Interferón gamma/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Noqueados , Fenotipo , Transducción de Señal