RESUMEN
Resveratrol (RES) is a nutraceutical with promising anti-inflammatory properties for the treatment of inflammatory bowel diseases (IBD). However, the clinical effectiveness of resveratrol as an oral anti-inflammatory agent is hindered by its extremely poor solubility and poor stability. In this study, we encapsulated resveratrol in ß-lactoglobulin (BLG) nanospheres and systematically analyzed their formulation parameters in vitro followed by a thorough in vivo anti-inflammatory testing in a highly specialized spontaneous murine UC model (Winnie mice model). Complexation of resveratrol with BLG increased the aqueous solubility of resveratrol by ≈1.7 times with 10% w/w loading. Additionally, the in vitro dissolution of resveratrol from the particles was found to be higher compared to resveratrol alone, resulting in >90% resveratrol dissolution in â¼8 h. The anti-inflammatory activity of resveratrol was examined for the first time in Winnie mice, a mouse model that closely represents the clinical signs of IBD. At a 50 mg/kg oral dose for 2 weeks, BLG-RES significantly improved both % body weight and disease activity index (DAI), compared to free resveratrol in Winnie mice. Importantly, histological evaluations revealed a similar trend with striking improvement in the pathology of the colon via an increase in goblet cell numbers and recovery of colonic epithelium. BLG-RES significantly increased the expression level of cytokine interleukin-10 (Il10), which confirms the reduction in inflammation potentially because of the increased dissolution and stability of resveratrol by complexation with BLG. This comprehensive study demonstrates the effectiveness of biocompatible nanomaterials such as BLG in oral delivery of poorly soluble anti-inflammatory molecules such as resveratrol in the treatment of IBD.
Asunto(s)
Antiinflamatorios/administración & dosificación , Colitis Ulcerosa/tratamiento farmacológico , Portadores de Fármacos/química , Resveratrol/administración & dosificación , Administración Oral , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacocinética , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Colon/efectos de los fármacos , Colon/inmunología , Colon/patología , Modelos Animales de Enfermedad , Composición de Medicamentos/métodos , Liberación de Fármacos , Femenino , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Lactoglobulinas/química , Masculino , Ratones , Nanosferas/química , Resveratrol/química , Resveratrol/farmacocinética , SolubilidadRESUMEN
Rationale: Non-cystic fibrosis bronchiectasis is characterized by airway mucus accumulation and sputum production, but the role of mucus concentration in the pathogenesis of these abnormalities has not been characterized.Objectives: This study was designed to: 1) measure mucus concentration and biophysical properties of bronchiectasis mucus; 2) identify the secreted mucins contained in bronchiectasis mucus; 3) relate mucus properties to airway epithelial mucin RNA/protein expression; and 4) explore relationships between mucus hyperconcentration and disease severity.Methods: Sputum samples were collected from subjects with bronchiectasis, with and without chronic erythromycin administration, and healthy control subjects. Sputum percent solid concentrations, total and individual mucin concentrations, osmotic pressures, rheological properties, and inflammatory mediators were measured. Intracellular mucins were measured in endobronchial biopsies by immunohistochemistry and gene expression. MUC5B (mucin 5B) polymorphisms were identified by quantitative PCR. In a replication bronchiectasis cohort, spontaneously expectorated and hypertonic saline-induced sputa were collected, and mucus/mucin concentrations were measured.Measurements and Main Results: Bronchiectasis sputum exhibited increased percent solids, total and individual (MUC5B and MUC5AC) mucin concentrations, osmotic pressure, and elastic and viscous moduli compared with healthy sputum. Within subjects with bronchiectasis, sputum percent solids correlated inversely with FEV1 and positively with bronchiectasis extent, as measured by high-resolution computed tomography, and inflammatory mediators. No difference was detected in MUC5B rs35705950 SNP allele frequency between bronchiectasis and healthy individuals. Hypertonic saline inhalation acutely reduced non-cystic fibrosis bronchiectasis mucus concentration by 5%.Conclusions: Hyperconcentrated airway mucus is characteristic of subjects with bronchiectasis, likely contributes to disease pathophysiology, and may be a target for pharmacotherapy.
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Bronquiectasia/tratamiento farmacológico , Bronquiectasia/fisiopatología , Eritromicina/uso terapéutico , Moco/química , Sistema Respiratorio/fisiopatología , Esputo/química , Anciano , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Moco/microbiología , Queensland , Esputo/microbiologíaRESUMEN
Mucins are heavily glycosylated proteins that give mucus its gel-like properties. Moreover, the glycans decorating the mucin protein core can alter the protective properties of the mucus barrier. To investigate whether these alterations could be parasite-induced we utilized the Trichuris muris (T. muris) infection model, using different infection doses and strains of mice that are resistant (high dose infection in BALB/c and C57BL6 mice) or susceptible (high dose infection in AKR and low dose infection in BALB/c mice) to chronic infection by T. muris. During chronicity, within the immediate vicinity of the T. muris helminth the goblet cell thecae contained mainly sialylated mucins. In contrast, the goblet cells within the epithelial crypts in the resistant models contained mainly sulphated mucins. Maintained mucin sulphation was promoted by TH2-immune responses, in particular IL-13, and contributed to the protective properties of the mucus layer, making it less vulnerable to degradation by T. muris excretory secretory products. Mucin sulphation was markedly reduced in the caecal goblet cells in the sulphate anion transporter-1 (Sat-1) deficient mice. We found that Sat-1 deficient mice were susceptible to chronic infection despite a strong TH2-immune response. Lower sulphation levels lead to decreased efficiency of establishment of T. muris infection, independent of egg hatching. This study highlights the complex process by which immune-regulated alterations in mucin glycosylation occur following T. muris infection, which contributes to clearance of parasitic infection.
Asunto(s)
Mucinas/química , Mucinas/inmunología , Tricuriasis/inmunología , Animales , Modelos Animales de Enfermedad , Glicosilación , Células Caliciformes/química , Células Caliciformes/inmunología , Humanos , Inmunohistoquímica , Mucosa Intestinal/química , Mucosa Intestinal/inmunología , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa , Trichuris/inmunologíaRESUMEN
Donor T-cell-derived interleukin-17A (IL-17A) can mediate late immunopathology in graft-versus-host disease (GVHD), however protective roles remain unclear. Using multiple cytokine and cytokine receptor subunit knockout mice, we demonstrate that stem cell transplant recipients lacking the ability to generate or signal IL-17 develop intestinal hyper-acute GVHD. This protective effect is restricted to the molecular interaction of IL-17A and/or IL-17F with the IL-17 receptor A/C (IL-17RA/C). The protection from GVHD afforded by IL-17A required secretion from, and signaling in, both hematopoietic and nonhematopoietic host tissue. Given the intestinal-specificity of the disease in these animals, we cohoused wild-type (WT) with IL-17RA and IL-17RC-deficient mice, which dramatically enhanced the susceptibility of WT mice to acute GVHD. Furthermore, the gut microbiome of WT mice shifted toward that of the IL-17RA/C mice during cohousing prior to transplant, confirming that an IL-17-sensitive gut microbiota controls susceptibility to acute GVHD. Finally, induced IL-17A depletion peritransplant also enhanced acute GVHD, consistent with an additional protective role for this cytokine independent of effects on dysbiosis.
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Microbioma Gastrointestinal/inmunología , Enfermedad Injerto contra Huésped , Interleucina-17/inmunología , Enfermedades Intestinales , Enfermedad Aguda , Animales , Modelos Animales de Enfermedad , Disbiosis/genética , Disbiosis/inmunología , Disbiosis/patología , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Interleucina-17/genética , Enfermedades Intestinales/genética , Enfermedades Intestinales/inmunología , Enfermedades Intestinales/patología , Transfusión de Linfocitos , Ratones , Ratones Noqueados , Receptores de Interleucina/genética , Receptores de Interleucina/inmunología , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/inmunologíaRESUMEN
The interleukin (IL)-20 subfamily of cytokines consists of IL-19, IL-20, IL-22, IL-24, and IL-26, and the expression of IL-20, IL-22, and IL-24 is reported to be higher in the colon of patients with ulcerative colitis. Although the receptors for these cytokines are highly expressed in the colon epithelium, their effects on epithelial renewal are not clearly understood. This study evaluated the effects of IL-20, IL-22, and IL-24 in epithelial renewal using the LS174T human colon cancer epithelial cell line. LS174T cells were treated with IL-20, IL-22, and IL-24 (25, 50, and 100 ng/mL) and a live-cell imaging system was used to evaluate the effects on cell proliferation. Following treatment, the signaling pathways contributing to cell proliferation were investigated through Western blotting in LS174T cells and downstream transcriptional changes through qRT-PCR in LS174T cells, and RNA-Seq in primary murine intestinal epithelial cells. Our results demonstrated that only IL-22 promoted LS174T cell proliferation, mediated via extracellular-signal-regulated kinase (ERK)1/2-mediated downstream regulation of p90RSK, c-Jun, and transcriptional changes of TRIM15 and STOM. IL-22 also promoted expression of ERK1/2-independent genes such as DDR2, LCN2, and LRG1, which are known to be involved in cell proliferation and migration. This study suggests that IL-22 induces cell proliferation in highly proliferative cells such as intestinal epithelial cells.
Asunto(s)
Proliferación Celular , Neoplasias del Colon/metabolismo , Enterocitos/metabolismo , Interleucinas/metabolismo , Sistema de Señalización de MAP Quinasas , Animales , Línea Celular Tumoral , Células Cultivadas , Neoplasias del Colon/patología , Enterocitos/fisiología , Humanos , Interleucinas/genética , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismoRESUMEN
AIMS/HYPOTHESIS: The aim of this study was to determine whether therapy with the cytokine IL-22 could be used to prevent the development of, or treat, autoimmune diabetes in the NOD mouse. METHODS: Six-week-old NOD mice were administered bi-weekly either recombinant mouse IL-22 (200 ng/g) or PBS (vehicle control) intraperitoneally until overt diabetes was diagnosed as two consecutive measurements of non-fasting blood glucose ≥ 11 mmol/l. At this time, NOD mice in the control arm were treated with LinBit insulin pellets and randomised to bi-weekly therapeutic injections of either PBS or IL-22 (200 ng/g) and followed until overt diabetes was diagnosed, as defined above. RESULTS: IL-22 therapy did not delay the onset of diabetes in comparison with the vehicle-treated mice. We did not observe an improvement in islet area, glycaemic control, beta cell residual function, endoplasmic reticulum stress, insulitis or macrophage and neutrophil infiltration as determined by non-fasting blood glucose, C-peptide and histological scoring. Therapeutic administration of IL-22 did not reduce circulating lipopolysaccharide, a marker of impaired gut mucosal integrity. CONCLUSIONS/INTERPRETATION: Our study suggests that, at this dosing regimen introduced either prior to overt diabetes or at diagnosis of diabetes, recombinant mouse IL-22 therapy cannot prevent autoimmune diabetes, or prolong the honeymoon period in the NOD mouse.
Asunto(s)
Diabetes Mellitus Tipo 1/tratamiento farmacológico , Interleucinas/uso terapéutico , Animales , Bioensayo , Diabetes Mellitus Tipo 1/inmunología , Femenino , Técnicas In Vitro , Ratones , Ratones Endogámicos NOD , Interleucina-22RESUMEN
Metastatic renal cell carcinoma is a largely incurable disease, and existing treatments targeting angiogenesis and tyrosine kinase receptors are only partially effective. Here we reveal that MUC13, a cell surface mucin glycoprotein, is aberrantly expressed by most renal cell carcinomas, with increasing expression positively correlating with tumor grade. Importantly, we demonstrated that high MUC13 expression was a statistically significant independent predictor of poor survival in two independent cohorts, particularly in stage 1 cancers. In cultured renal cell carcinoma cells MUC13 promoted proliferation and induced the cell cycle regulator, cyclin D1, and inhibited apoptosis by inducing the anti-apoptotic proteins, BCL-xL and survivin. Silencing of MUC13 expression inhibited migration and invasion, and sensitized renal cancer cells to killing by the multi-kinase inhibitors used clinically, sorafenib and sunitinib, and reversed acquired resistance to these drugs. Furthermore, we demonstrated that MUC13 promotion of renal cancer cell growth and survival is mediated by activation of nuclear factor κB, a transcription factor known to regulate the expression of genes that play key roles in the development and progression of cancer. These results show that MUC13 has potential as a prognostic marker for aggressive early stage renal cell cancer and is a plausible target to sensitize these tumors to therapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma de Células Renales/patología , Resistencia a Antineoplásicos , Neoplasias Renales/patología , Mucinas/metabolismo , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor , Western Blotting , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Técnicas para Inmunoenzimas , Indoles/administración & dosificación , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/metabolismo , Estadificación de Neoplasias , Niacinamida/administración & dosificación , Niacinamida/análogos & derivados , Compuestos de Fenilurea/administración & dosificación , Pronóstico , Pirroles/administración & dosificación , Sorafenib , Sunitinib , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
OBJECTIVE: To assess whether FUT2 (secretor) genotype affects disease severity and airway infection in patients with non-cystic fibrosis bronchiectasis. PARTICIPANTS: Induced sputum samples were obtained from 112 adult patients with high-resolution CT scan-proven bronchiectasis and at least two exacerbations in the previous year, as part of an unrelated randomised control trial. OUTCOME MEASURES: Presence of null FUT2 polymorphisms were determined by gene sequencing and verified by endobronchial biopsy histochemical staining. Outcome measures were FEV1% predicted, exacerbation frequency, and bacterial, fungal and viral components of the microbiota (measured by culture independent approaches). RESULTS: Patients were grouped by FUT2 loss-of-function genotype; categorised as non-secretors (n=27, sese), heterozygous secretors (n=54, Sese) or homozygous secretors (n=31, SeSe). FEV1% was significantly lower in SeSe patients compared with sese patients (mean 61.6 (SD 20.0) vs 74.5 (18.0); p=0.023). Exacerbation frequency was significantly higher in SeSe (mean count 5.77) compared with sese (4.07; p=0.004) and Sese (4.63; p=0.026) genotypes. The time until first exacerbation was significantly shorter in SeSe compared with Sese (HR=0.571 (95% CI 0.343 to 0.950); p=0.031), with a similar trend for sese patients (HR=0.577 (0.311 to 1.07); p=0.081). sese had a significantly reduced frequency of Pseudomonas aeruginosa-dominated airway infection (8.7%) compared with Sese (31%; p=0.042) and SeSe (36%; p=0.035). In contrast, fungal, viral and non-dominant bacterial components of the microbiome were not significantly different between FUT2 genotypes. CONCLUSIONS: FUT2 genotype in patients with non-cystic fibrosis bronchiectasis was significantly associated with disease outcomes, with homozygous secretors exhibiting lower lung function, higher exacerbation number and a higher frequency of P. aeruginosa-dominated infection. TRIAL REGISTRATION NUMBER: ACTRN12609000578202 (anzctr.org.au); Pre-results.
Asunto(s)
Bronquiectasia/genética , Bronquiectasia/microbiología , Bronquiectasia/fisiopatología , Fucosiltransferasas/genética , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Broncoscopía , Progresión de la Enfermedad , Femenino , Genotipo , Humanos , Masculino , Microbiota , Persona de Mediana Edad , Infecciones por Pseudomonas/diagnóstico , Infecciones por Pseudomonas/fisiopatología , Pseudomonas aeruginosa/aislamiento & purificación , Pruebas de Función Respiratoria , Índice de Severidad de la Enfermedad , Esputo/microbiología , Tomografía Computarizada por Rayos X , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
INTRODUCTION: Case control studies suggest an inverse association between Helicobacter pylori (H. pylori) and Crohn's disease (CD). It is possible this could be accounted for by confounders such as antibiotic therapy. Analyzing the geographic distribution of H. pylori and the links with the incidence and prevalence of CD would be an alternative approach to circumvent these confounders. METHODS: The literature was searched for studies published between 1990 and 2016 that reported incidence or prevalence data for CD in random population samples in developed countries (GDP per capita >20,000 USD/year). Corresponding prevalence studies for H. pylori in these same regions were then sought matched to the same time period (±6 years). The association between the incidence and prevalence of CD and H. pylori prevalence rates were assessed before and after adjusting for GDP and life expectancy. RESULTS: A total of 19 CD prevalence and 22 CD incidence studies from 10 European countries, Japan, USA, and Australia with date-matched H. pylori prevalence data were identified. The mean H. pylori prevalence rate was 43.4% (range 15.5-85%), and the mean rates for incidence and prevalence for CD were 6.9 and 91.0/100,000 respectively. The incidence (r = -0.469, p < 0.03) and prevalence (r = -0.527, p = 0.02) of CD was inversely and significantly associated with prevalence of H. pylori infection. CONCLUSIONS: Our data demonstrate a significant inverse association between geographic distribution of H. pylori and CD. Thus, it is highly unlikely that the findings of previous case control studies were simply due to confounding factors such as concomitant antibiotic use in CD patients.
Asunto(s)
Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/epidemiología , Infecciones por Helicobacter/diagnóstico , Infecciones por Helicobacter/epidemiología , Helicobacter pylori , Estudios de Casos y Controles , Helicobacter pylori/aislamiento & purificación , HumanosRESUMEN
OBJECTIVES: The mucin MUC1, best known for providing an epithelial barrier, is an important protective host factor in both humans and mice during Helicobacter pylori pathogenesis. This study aimed to identify the long-term consequences of MUC1 deficiency on H. pylori pathogenesis and the mechanism by which MUC1 protects against H. pylori gastritis. DESIGN: Wildtype and Muc1(-/-) mice were infected for up to 9â months, and the gastric pathology, immunological response and epigenetic changes assessed. The effects of MUC1 on the inflammasome, a potent inflammatory pathway, were examined in macrophages and H. pylori-infected mice deficient in both MUC1 and inflammasome components. RESULTS: Muc1(-/-) mice began to die 6â months after challenge, indicating Muc1 deficiency made H. pylori a lethal infection. Surprisingly, chimaeric mouse infections revealed MUC1 expression by haematopoietic-derived immune cells limits H. pylori-induced gastritis. Gastritis in infected Muc1(-/-) mice was associated with elevated interleukin (IL)-1ß and epigenetic changes in their gastric mucosa similar to those in transgenic mice overexpressing gastric IL-1ß, implicating MUC1 regulation of an inflammasome. In support of this, infected Muc1(-/-)Casp1(-/-) mice did not develop severe gastritis. Further, MUC1 regulated Nlrp3 expression via an nuclear factor (NF)-κB-dependent pathway and reduced NF-κB pathway activation via inhibition of IRAK4 phosphorylation. The importance of this regulation was proven using Muc1(-/-)Nlrp3(-/-) mice, which did not develop severe gastritis. CONCLUSIONS: MUC1 is an important, previously unidentified negative regulator of the NLRP3 inflammasome. H. pylori activation of the NLRP3 inflammasome is normally tightly regulated by MUC1, and loss of this critical regulation results in the development of severe pathology.
Asunto(s)
Gastritis/microbiología , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/patogenicidad , Inflamasomas/metabolismo , Mucina-1/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Animales , Caspasa 1/genética , Metilación de ADN , Femenino , Mucosa Gástrica/inmunología , Mucosa Gástrica/metabolismo , Gastritis/patología , Expresión Génica , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/inmunología , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/inmunología , Mucina-1/genética , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Transducción de Señal , Factores de Tiempo , Factor Trefoil-2/genéticaRESUMEN
BACKGROUND & AIMS: Protein misfolding and endoplasmic reticulum (ER) stress have been observed in intestinal secretory cells from patients with inflammatory bowel diseases and induce intestinal inflammation in mice. However, it is not clear how immune factors affect ER stress and therefore disease symptoms. METHODS: We analyzed the effects of interleukin (IL)-10 on ER stress in intestinal tissues in wild-type C57BL/6, Winnie, IL-10(-/-), and Winnie × IL-10(+/-) mice. In Winnie mice, misfolding of the intestinal mucin Muc2 initiates ER stress and inflammation. We also analyzed the effects of different inhibitors of IL-10 signaling and the N-glycosylation inhibitor tunicamycin in cultured human LS174T goblet cells. RESULTS: Administration of neutralizing antibodies against IL-10 or its receptor (IL-10R1) to Winnie mice rapidly exacerbated ER stress and intestinal inflammation compared with mice given vehicle (controls). Antibodies against IL-10 also increased accumulation of misfolded Muc2 in the ER of goblet cells of Winnie mice and increased T-cell production of inflammatory cytokines. Winnie × IL-10(+/-) mice and IL-10(-/-) mice with a single Winnie allele each developed more severe inflammation than Winnie mice or IL-10(-/-) mice. Administration of tunicamycin to wild-type mice caused intestinal ER stress, which increased when IL-10R1 was blocked. In LS174T cells, induction of ER stress with tunicamycin and misfolding of MUC2 were reduced by administration of IL-10; this reduction required STAT1 and STAT3. In LS174T cells incubated with tunicamycin, IL-10 up-regulated genes involved in MUC2 folding and in ER-associated degradation and maintained correct folding of MUC2, its transport from the ER, and its O-glycosylation and secretion. CONCLUSIONS: IL-10 prevents protein misfolding and ER stress by maintaining mucin production in goblet cells and helps the intestine preserve the mucus barrier.
Asunto(s)
Estrés del Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Células Caliciformes/metabolismo , Interleucina-10/farmacología , Mucosa Intestinal/metabolismo , Moco/metabolismo , Deficiencias en la Proteostasis/tratamiento farmacológico , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Retículo Endoplásmico/efectos de los fármacos , Células Caliciformes/efectos de los fármacos , Células Caliciformes/patología , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Moco/efectos de los fármacos , Deficiencias en la Proteostasis/metabolismo , Deficiencias en la Proteostasis/patologíaRESUMEN
The type IV secretion system (T4SS) of Helicobacter pylori triggers massive inflammatory responses during gastric infection by mechanisms that are poorly understood. Here we provide evidence for a novel pathway by which the T4SS structural component, CagL, induces secretion of interleukin-8 (IL-8) independently of CagA translocation and peptidoglycan-sensing nucleotide-binding oligomerization domain 1 (NOD1) signalling. Recombinant CagL was sufficient to trigger IL-8 secretion, requiring activation of α5 ß1 integrin and the arginine-glycine-aspartate (RGD) motif in CagL. Mutation of the encoded RGD motif to arginine-glycine-alanine (RGA) in the cagL gene of H. pylori abrogated its ability to induce IL-8. Comparison of IL-8 induction between H. pylori ΔvirD4 strains bearing wild-type or mutant cagL indicates that CagL-dependent IL-8 induction can occur independently of CagA translocation. In line with this notion, exogenous CagL complemented H. pylori ΔcagL mutant in activating NF-κB and inducing IL-8 without restoring CagA translocation. The CagA translocation-independent, CagL-dependent IL-8 induction involved host signalling via integrin α5 ß1 , Src kinase, the mitogen-activated protein kinase (MAPK) pathway and NF-κB but was independent of NOD1. Our findings reveal a novel pathway whereby CagL, via interaction with host integrins, can trigger pro-inflammatory responses independently of CagA translocation or NOD1 signalling.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos , Helicobacter pylori/inmunología , Interleucina-8/metabolismo , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteínas Bacterianas/genética , Línea Celular , Interacciones Huésped-Patógeno , Humanos , Integrina alfa5beta1/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Proteínas Mutantes/metabolismo , Mutación , FN-kappa B/metabolismo , Transducción de SeñalRESUMEN
OBJECTIVE: The anti-leukemic drugs, azathioprine and 6-mercaptopurine (6MP), are important in the treatment of inflammatory bowel disease but an alternative faster-acting, less-allergenic thiopurine, 6-thioguanine (6TG), can cause hepatic veno-occlusive disease/sinusoidal obstructive syndrome (SOS). Understanding of SOS has been hindered by inability to ethically perform serial liver biopsies on patients and the lack of an animal model. DESIGN: Normal and C57Bl/6 mice with specific genes altered to elucidate mechanisms responsible for 6TG-SOS, were gavaged daily for upto 28d with 6TG, 6MP or methylated metabolites. Animal survival was monitored and at sacrifice a histological score of SOS, haematology and liver biochemistry were measured. RESULTS: Only 6TG caused SOS, which was dose related. 6TG and to a lesser extent 6MP but not methylated metabolites were associated with dose-dependent haematopoietic toxicity. SOS was not detected with non-lethal doses of 6TG. SOS did not occur in hypoxanthine-phosphoribosyl transferase-deficient C57Bl/6 mice, demonstrating that 6TG-SOS requires thioguanine nucleotides. Hepatic inflammation was characteristic of SOS, and C57Bl/6 mice deficient in P- and E-selectins on the surface of vascular endothelial cells showed markedly reduced SOS, demonstrating a major role for leukocytes recruited from blood. Split dosing of 6TG markedly attenuated SOS but still effected immunosuppression and prevented spontaneous colitis in Winnie mice, which have a single nucleotide polymorphism mutation in Muc2. CONCLUSION: This novel model provides clinically relevant insights into how 6TG induces SOS, and how this dangerous adverse drug reaction may be avoided by either inhibition of endothelial activation or simple changes to dosing regimens of 6TG, while still being effective treatment for colitis.
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Modelos Animales de Enfermedad , Enfermedad Veno-Oclusiva Hepática/inducido químicamente , Tioguanina/toxicidad , Animales , Relación Dosis-Respuesta a Droga , Enfermedad Veno-Oclusiva Hepática/prevención & control , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Estadísticas no Paramétricas , Análisis de Supervivencia , Tioguanina/administración & dosificaciónRESUMEN
OBJECTIVE: Cathepsin K is a lysosomal cysteine protease that has pleiotropic roles in bone resorption, arthritis, atherosclerosis, blood pressure regulation, obesity and cancer. Recently, it was demonstrated that cathepsin K-deficient (Ctsk(-/-) ) mice are less susceptible to experimental autoimmune arthritis and encephalomyelitis, which implies a functional role for cathepsin K in chronic inflammatory responses. Here, the authors address the relevance of cathepsin K in the intestinal immune response during chronic intestinal inflammation. DESIGN: Chronic colitis was induced by administration of 2% dextran sodium sulphate (DSS) in distilled water. Mice were assessed for disease severity, histopathology and endoscopic appearance. Furthermore, DSS-exposed Ctsk(-/-) mice were treated by rectal administration of recombinant cathepsin K. Intestinal microflora was assessed by real-time PCR and 16srDNA molecular fingerprinting of ileal and colonic mucosal and faecal samples. RESULTS: Using Ctsk(-/-) mice, the authors demonstrate a protective role of cathepsin K against chronic DSS colitis. Dissecting the underlying mechanisms the authors found cathepsin K to be present in intestinal goblet cells and the mucin layer. Furthermore, a direct cathepsin K-mediated bactericidal activity against intestinal bacteria was demonstrated, which potentially explains the alteration of intestinal microbiota observed in Ctsk(-/-) mice. Rectal administration of recombinant cathepsin K in DSS-treated Ctsk(-/-) mice ameliorates the severity of intestinal inflammation. CONCLUSION: These data identify extracellular cathepsin K as an intestinal antibacterial factor with anti-inflammatory potential and suggest that topical administration of cathepsin K might provide a therapeutic option for patients with inflammatory bowel disease.
Asunto(s)
Catepsina K/farmacología , Colitis/tratamiento farmacológico , Colitis/microbiología , Animales , Western Blotting , Catepsina K/metabolismo , Colitis/inducido químicamente , Colitis/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Endoscopía Gastrointestinal , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The IL-22RA1 receptor is highly expressed in the pancreas, and exogenous IL-22 has been shown to reduce endoplasmic reticulum and oxidative stress in human pancreatic islets and promote secretion of high-quality insulin from beta-cells. However, the endogenous role of IL-22RA1 signaling on these cells remains unclear. Here, we show that antibody neutralisation of IL-22RA1 in cultured human islets leads to impaired insulin quality and increased cellular stress. Through the generation of mice lacking IL-22ra1 specifically on pancreatic alpha- or beta-cells, we demonstrate that ablation of murine beta-cell IL-22ra1 leads to similar decreases in insulin secretion, quality and islet regeneration, whilst increasing islet cellular stress, inflammation and MHC II expression. These changes in insulin secretion led to impaired glucose tolerance, a finding more pronounced in female animals compared to males. Our findings attribute a regulatory role for endogenous pancreatic beta-cell IL-22ra1 in insulin secretion, islet regeneration, inflammation/cellular stress and appropriate systemic metabolic regulation.
Asunto(s)
Glucosa , Homeostasis , Células Secretoras de Insulina , Insulina , Ratones Noqueados , Receptores de Interleucina , Animales , Células Secretoras de Insulina/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina/genética , Femenino , Humanos , Masculino , Insulina/metabolismo , Ratones , Glucosa/metabolismo , Secreción de Insulina , Ratones Endogámicos C57BL , Interleucina-22 , Intolerancia a la Glucosa/metabolismo , Interleucinas/metabolismo , Interleucinas/genética , Envejecimiento/metabolismoRESUMEN
Metabolic dysfunction-associated steatohepatitis (MASH) is the most prevalent cause of liver disease worldwide, with a single approved therapeutic. Previous research has shown that interleukin-22 (IL-22) can suppress ß-cell stress, reduce local islet inflammation, restore appropriate insulin production, reverse hyperglycemia, and ameliorate insulin resistance in preclinical models of diabetes. In clinical trials long-acting forms of IL-22 have led to increased proliferation in the skin and intestine, where the IL-22RA1 receptor is highly expressed. To maximise beneficial effects whilst reducing the risk of epithelial proliferation and cancer, we designed short-acting IL-22-bispecific biologic drugs that successfully targeted the liver and pancreas. Here we show 10-fold lower doses of these bispecific biologics exceed the beneficial effects of native IL-22 in multiple preclinical models of MASH, without off-target effects. Treatment restores glycemic control, markedly reduces hepatic steatosis, inflammation, and fibrogenesis. These short-acting IL-22-bispecific targeted biologics are a promising new therapeutic approach for MASH.
Asunto(s)
Hígado Graso , Interleucina-22 , Interleucinas , Hígado , Páncreas , Interleucinas/metabolismo , Animales , Hígado/metabolismo , Hígado/patología , Hígado/efectos de los fármacos , Páncreas/patología , Páncreas/metabolismo , Páncreas/efectos de los fármacos , Humanos , Ratones , Hígado Graso/tratamiento farmacológico , Hígado Graso/metabolismo , Masculino , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Resistencia a la Insulina , Receptores de Interleucina/metabolismoRESUMEN
Mucinous differentiation is associated with both CpG island methylator phenotype and microsatellite instability in colorectal cancer. The mucinous phenotype derives from abundant expression of the colonic goblet cell mucin, MUC2, and de novo expression of gastric foveolar mucin, MUC5AC. We, therefore, investigated the protein expression levels of MUC2 and MUC5AC, as well as MUC5B and MUC6, in molecular subtypes of colorectal cancer. Seven-hundred and twenty-two incident colorectal carcinomas occurring in 702 participants of the Melbourne Collaborative Cohort Study were characterized for methylator status, MLH1 methylation, somatic BRAF and KRAS mutations, microsatellite-instability status, MLH1, MSH2, MSH6, and PMS2 mismatch repair, and p53 protein expression, and their histopathology was reviewed. Protein expression levels of MUC2, MUC5AC, MUC5B, MUC6, and the putative mucin regulator CDX2 were compared with molecular and clinicopathological features of colorectal cancers using odds ratios and corresponding 95% confidence intervals. MUC2 overexpression (>25% positive tumor cells) was observed in 33% colorectal cancers, MUC5B expression in 53%, and de novo MUC5AC and MUC6 expression in 50% and 39%, respectively. Co-expression of two or more of the mucins was commonly observed. Expression of MUC2, MUC5AC and MUC6 was strongly associated with features associated with tumorigenesis via the serrated neoplasia pathway, including methylator positivity, somatic BRAF p.V600E mutation, and mismatch repair deficiency, as well as proximal location, poor differentiation, lymphocytic response, and increased T stage (all P<0.001). Overexpression was observed in tumors with and without mucinous differentiation. There were inverse associations between expression of all four mucins and p53 overexpression. CDX2 expression was inversely associated with MUC2, MUC5AC and MUC6 expression. Our results suggest that, in methylator-positive tumors, mucin genes on chromosome 11p15.5 region undergo increased expression via mechanisms other than direct regulation by CDX2.
Asunto(s)
Carcinoma/genética , Carcinoma/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Islas de CpG/genética , Mucinas/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Factor de Transcripción CDX2 , Metilación de ADN/genética , Femenino , Silenciador del Gen , Proteínas de Homeodominio/biosíntesis , Humanos , Inmunohistoquímica , Masculino , Inestabilidad de Microsatélites , Persona de Mediana Edad , Mucina 5AC/análisis , Mucina 5AC/biosíntesis , Mucina 2/análisis , Mucina 2/biosíntesis , Mucina 5B/análisis , Mucina 5B/biosíntesis , Mucina 6/análisis , Mucina 6/biosíntesis , Mucinas/análisis , Fenotipo , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
Mismatch repair-deficient breast cancers may be identified in Lynch syndrome mutation carriers, and have clinicopathological features in common with mismatch repair-deficient colorectal and endometrial cancers such as tumour-infiltrating lymphocytes and poor differentiation. Mismatch repair-deficient colorectal cancers frequently show mucinous differentiation associated with upregulation of chromosome 11 mucins. The aim of this study was to compare the protein expression of these mucins in mismatch repair-deficient and -proficient breast cancers. Cases of breast cancer (n=100) were identified from families where (1) both breast and colon cancer co-occurred and (2) families met either modified Amsterdam criteria or had at least one early-onset (<50 years) colorectal cancer. Tumour sections were stained for the epithelial mucins, MUC2, MUC5AC, MUC5B and MUC6, and the homeobox protein CDX2, a regulator of MUC2 expression. In all, 16 mismatch repair-deficient Lynch syndrome breast cancers and 84 non-Lynch breast cancers were assessed for altered mucin expression. No significant difference in the expression of MUC2, MUC5AC or MUC6 was observed between the mismatch repair-deficient and mismatch repair-proficient breast cancers; however, there was a trend for mismatch repair-deficient tumours to express high levels of MUC5B less frequently (P=0.07, OR=0.2 (0.0-1.0)). Co-expression of two or more gel-forming mucins was common. Ectopic expression of CDX2 was associated with expression of MUC2 (P=0.035, OR=8.7 (1.3-58.4)). Mismatch repair-deficient breast cancers do not show differential expression of the mucins genes on chromosome 11 when compared with mismatch repair-proficient breast cancers, in contrast with mismatch repair-deficient colorectal and endometrial cancers, which frequently have increased mucin protein expression when compared with their mismatch repair-proficient counterparts. In addition, ectopic CDX2 expression is positively associated with de novo MUC2 expression.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias Colorrectales Hereditarias sin Poliposis/metabolismo , Mucinas/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/complicaciones , Neoplasias de la Mama/genética , Cromosomas Humanos Par 11 , Neoplasias Colorrectales Hereditarias sin Poliposis/complicaciones , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mucinas/análisisRESUMEN
OBJECTIVE: The spondylarthritides (SpA), including ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, and arthritis associated with inflammatory bowel disease, cause chronic inflammation of the large peripheral and axial joints, eyes, skin, ileum, and colon. Genetic studies reveal common candidate genes for AS, PsA, and Crohn's disease, including IL23R, IL12B, STAT3, and CARD9, all of which are associated with interleukin-23 (IL-23) signaling downstream of the dectin 1 ß-glucan receptor. In autoimmune-prone SKG mice with mutated ZAP-70, which attenuates T cell receptor signaling and increases the autoreactivity of T cells in the peripheral repertoire, IL-17-dependent inflammatory arthritis developed after dectin 1-mediated fungal infection. This study was undertaken to determine whether SKG mice injected with 1,3-ß-glucan (curdlan) develop evidence of SpA, and the relationship of innate and adaptive autoimmunity to this process. METHODS: SKG mice and control BALB/c mice were injected once with curdlan or mannan. Arthritis was scored weekly, and organs were assessed for pathologic features. Anti-IL-23 monoclonal antibodies were injected into curdlan-treated SKG mice. CD4+ T cells were transferred from curdlan-treated mice to SCID mice, and sera were analyzed for autoantibodies. RESULTS: After systemic injection of curdlan, SKG mice developed enthesitis, wrist, ankle, and sacroiliac joint arthritis, dactylitis, plantar fasciitis, vertebral inflammation, ileitis resembling Crohn's disease, and unilateral uveitis. Mannan triggered spondylitis and arthritis. Arthritis and spondylitis were T cell- and IL-23-dependent and were transferable to SCID recipients with CD4+ T cells. SpA was associated with collagen- and proteoglycan-specific autoantibodies. CONCLUSION: Our findings indicate that the SKG ZAP-70W163C mutation predisposes BALB/c mice to SpA, resulting from innate and adaptive autoimmunity, after systemic ß-glucan or mannan exposure.
Asunto(s)
Artritis Experimental/patología , Artritis Reumatoide/patología , Ileítis/inducido químicamente , Espondiloartritis/inducido químicamente , beta-Glucanos , Animales , Artritis Experimental/inmunología , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Autoinmunidad/inmunología , Ileítis/inmunología , Ileítis/patología , Interleucina-17/inmunología , Articulaciones/inmunología , Articulaciones/patología , Ratones , Espondiloartritis/inmunología , Espondiloartritis/patología , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
Campylobacter jejuni is an important human food-borne intestinal pathogen, however relatively little is known about its mechanisms of pathogenesis or pathogen-host interactions. To monitor changes in gene expression and glycan binding of C. jejuni within a common avian host, an immunomagnetic separation technique (IMS) was utilised to directly isolate infecting C. jejuni 81116 from a chicken host. An average of 10(5) cells/g was re-isolated from chicken caecal samples by IMS technique. The in vivo passaged strains were used successfully in evaluation of carbohydrate binding through the use of a glycan array and were further suitable for transcriptome analysis. The glycan microarray analysis demonstrated differences in binding to negatively charged glycans of laboratory grown strains of C. jejuni compared with strains isolated after in vivo passage. The in vivo passaged strains showed marked up-regulation of chemotaxis receptors and toxin genes. The optimised Campylobacter IMS technique described in this study allowed isolation directly from an animal host. Changes in gene expression and glycan binding at an in vivo level can also be identified by using this method.