RESUMEN
Platelets are specialized cells essential for hemostasis that also function as crucial effectors capable of mediating inflammatory and immune responses. These sentinels continually survey their environment and discriminate between homeostatic and danger signals such as modified components of the extracellular matrix. The glycosaminoglycan hyaluronan (HA) is a major extracellular matrix component that coats the vascular lumen and, under normal conditions, restricts access of inflammatory cells. In response to tissue damage, the endothelial HA matrix enhances leukocyte recruitment and regulates the early stages of the inflammatory response. We have shown that platelets can degrade HA from the surface of activated endothelial cells via the enzyme hyaluronidase-2 (HYAL2) and that HYAL2 is deficient in platelets isolated from patients with inflammatory bowel disease (IBD). Platelets are known to be involved in the pathogenesis of several chronic disease states, including IBD, but they have been largely overlooked in the context of intestinal inflammation. We therefore wanted to define the mechanism by which platelet HYAL2 regulates the inflammatory response during colitis. In this study, we provide evidence that HA catabolism is disrupted in human intestinal microvascular endothelial cells isolated from patients with IBD. Furthermore, mice deficient in HYAL2 are more susceptible to an acute model of colitis, and this increased susceptibility is abrogated by transfusion of HYAL2-competent platelets. Finally, we show that platelets, via HYAL2-dependent degradation of endothelial HA, regulate the early stages of inflammation in colitis by limiting leukocyte extravasation.
Asunto(s)
Plaquetas/inmunología , Colitis/inmunología , Hialuronoglucosaminidasa/inmunología , Animales , Plaquetas/patología , Células Cultivadas , Colitis/patología , Células Endoteliales/inmunología , Células Endoteliales/patología , Proteínas Ligadas a GPI/inmunología , Humanos , Ácido Hialurónico/inmunología , Inflamación/inmunología , Inflamación/patología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Ratones , Ratones NoqueadosRESUMEN
BACKGROUND: Specific-sized species of the carbohydrate hyaluronan elicit a variety of cellular responses mediating tissue integrity and repair, as well as regulating inflammatory responses. Orally provided hyaluronan with an average molecular weight of 35 kDa (HA35) protects mice from short-term ethanol (EtOH)-induced liver injury. This protection was associated with maintenance of the colocalization of zonula occludens-1 (ZO-1) and occludin at tight junctions in the proximal colon. However, it is not known whether HA35 also protects other regions of the intestine or whether protection is due to a direct and/or indirect interaction of HA35 with the intestinal epithelium. METHODS: Female C57BL/6J mice were fed an EtOH containing diet or pair-fed control diet (4 days) and treated with or without HA35 via daily gavage during the last 3 days of EtOH feeding. Intestinal morphology and tight junction integrity were assessed. Differentiated Caco-2 cells were transfected or not with scrambled siRNA or siRNA targeting layilin, a hyaluronan receptor. Caco-2 cells were treated with or without HA35 prior to challenge with EtOH. Localization of tight junction proteins, fluorescein isothiocyanate (FITC)-dextran permeability, and transepithelial electrical resistance (TEER) were evaluated. RESULTS: While short-term EtOH did not result in any apparent changes in the gross morphology of the intestine, colocalization of ZO-1 and occludin at tight junctions was decreased in the proximal and distal colon. HA35 prevented these effects of EtOH. In differentiated Caco-2 cells, EtOH decreased the localization of ZO-1 and occludin at tight junctions and increased permeability of FITC-dextran. At higher concentrations, EtOH also decreased TEER. Pretreatment with HA35 prevented these changes. When the hyaluronan receptor layilin was knocked down in Caco-2 cells, HA35 no longer protected cells from EtOH-induced loss of tight junctions. CONCLUSIONS: Taken together, these data indicate that HA35 interacts with layilin on intestinal epithelial cells and maintains intestinal tight junction integrity during short-term EtOH exposure.
Asunto(s)
Ácido Hialurónico/uso terapéutico , Mucosa Intestinal/efectos de los fármacos , Hepatopatías Alcohólicas/prevención & control , Uniones Estrechas/efectos de los fármacos , Viscosuplementos/uso terapéutico , Animales , Células CACO-2 , Depresores del Sistema Nervioso Central/efectos adversos , Evaluación Preclínica de Medicamentos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Etanol/efectos adversos , Femenino , Humanos , Ácido Hialurónico/farmacología , Lectinas Tipo C/metabolismo , Ratones Endogámicos C57BL , Viscosuplementos/farmacologíaRESUMEN
Dynamic alterations of the extracellular matrix in response to injury directly modulate inflammation and consequently the promotion and resolution of disease. During inflammation, hyaluronan (HA) is increased at sites of inflammation where it may be covalently modified with the heavy chains (HC) of inter-α-trypsin inhibitor. Deposition of this unique, pathological form of HA (HC-HA) leads to the formation of cable-like structures that promote adhesion of leukocytes. Naive mononuclear leukocytes bind specifically to inflammation-associated HA matrices but do not adhere to HA constitutively expressed under homeostatic conditions. In this study, we have directly investigated a role for the blood-coagulation protease thrombin in regulating the adhesion of monocytic cells to smooth muscle cells producing an inflammatory matrix. Our data demonstrate that the proteolytic activity of thrombin negatively regulates the adhesion of monocytes to an inflammatory HC-HA complex. This effect is independent of protease-activated receptor activation but requires proteolytic activity toward a novel substrate. Components of HC-HA complexes were predicted to contain conserved thrombin-susceptible cleavage sites based on sequence analysis, and heavy chain 1 (HC1) was confirmed to be a substrate of thrombin. Thrombin treatment is sufficient to cleave HC1 associated with either cell-surface HA or serum inter-α-trypsin inhibitor. Furthermore, thrombin treatment of the inflammatory matrix leads to dissolution of HC-HA cable structures and abolishes leukocyte adhesion. These data establish a novel mechanism whereby thrombin cleavage of HC1 regulates the adhesive properties of an inflammatory HA matrix.
Asunto(s)
alfa-Globulinas/metabolismo , Matriz Extracelular/metabolismo , Ácido Hialurónico/metabolismo , Leucocitos/metabolismo , Trombina/metabolismo , Adhesión Celular , Humanos , Leucocitos/citologíaRESUMEN
Hyaluronan is the predominant glycosaminoglycan component of the extracellular matrix with an emerging role in hematopoiesis. Modulation of hyaluronan polymer size is responsible for its control over cellular functions, and the balance of hyaluronan synthesis and degradation determines its molecular size. Although two active somatic hyaluronidases are expressed in mammals, only deficiency in hyaluronidase-2 (Hyal-2) results in thrombocytopenia of unknown mechanism. Our results reveal that Hyal-2 knockout mice accumulate hyaluronan within their bone marrow and within megakaryocytes, the cells responsible for platelet generation. Proplatelet formation by Hyal-2 knockout megakaryocytes was disrupted because of abnormal formation of the demarcation membrane system, which was dilated and poorly developed. Importantly, peptide-mediated delivery of exogenous hyaluronidase rescued deficient proplatelet formation in murine and human megakaryocytes lacking Hyal-2. Together, our data uncover a previously unsuspected mechanism of how hyaluronan and Hyal-2 control platelet generation.
Asunto(s)
Ácido Hialurónico/metabolismo , Hialuronoglucosaminidasa/metabolismo , Megacariocitos/metabolismo , Trombopoyesis/fisiología , Animales , Apoptosis/fisiología , Humanos , Immunoblotting , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Following injury, platelets rapidly interact with the exposed extracellular matrix (ECM) of the vessel wall and the surrounding tissues. Hyaluronan (HA) is a major glycosaminoglycan component of the ECM and plays a significant role in regulating inflammation. We have recently reported that human platelets degrade HA from the surfaces of activated endothelial cells into fragments capable of inducing immune responses by monocytes. We also showed that human platelets contain the enzyme hyaluronidase-2 (HYAL2), one of two major hyaluronidases that digest HA in somatic tissues. The deposition of HA increases in inflamed tissues in several inflammatory diseases, including inflammatory bowel disease (IBD). We therefore wanted to define the mechanism by which platelets degrade HA in the inflamed tissues. In this study, we show that human platelets degrade the proinflammatory matrix HA through the activity of HYAL2 and that platelet activation causes the immediate translocation of HYAL2 from a distinct population of α-granules to platelet surfaces where it exerts its catalytic activity. Finally, we show that patients with IBD have lower platelet HYAL2 levels and activity than healthy controls.
Asunto(s)
Plaquetas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Gránulos Citoplasmáticos/metabolismo , Matriz Extracelular/metabolismo , Ácido Hialurónico/metabolismo , Hialuronoglucosaminidasa/metabolismo , Enfermedades Inflamatorias del Intestino/enzimología , Estudios de Casos y Controles , Células Cultivadas , Técnicas de Cocultivo , Citometría de Flujo , Proteínas Ligadas a GPI/metabolismo , Humanos , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Músculo Liso/citología , Músculo Liso/metabolismo , Activación Plaquetaria , Pronóstico , Transporte de ProteínasRESUMEN
PURPOSE OF REVIEW: The extracellular matrix (ECM) is a frequently overlooked component of the pathogenesis of inflammatory bowel disease (IBD). However, the functional and clinically significant interactions between immune as well as nonimmune cells with the ECM have important implications for disease pathogenesis. In this review, we discuss how the ECM participates in process associated with IBD that involves diverse cell types of the intestine. RECENT FINDINGS: Remodeling of the ECM is a consistent feature of IBD, and studies have implicated key ECM molecules in IBD pathogenesis. While the majority of prior studies have focused on ECM degradation by proteases, more recent studies have uncovered additional degrading enzymes, identified fragments of ECM components as potential biomarkers, and revealed that ECM synthesis is increased in IBD. These new studies support the notion that the ECM, rather than acting as a passive element, is an active participant in promoting inflammation. SUMMARY: New studies have offered exciting clues about the function of the ECM during IBD pathogenesis. The balance of ECM synthesis and turnover is altered in IBD, and the molecules involved exhibit discreet biological functions that regulate inflammation on the basis of specific cell type and matrix molecule.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/fisiología , Inmunidad Innata/inmunología , Inflamación/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/metabolismo , Proliferación Celular , Células Epiteliales/metabolismo , Humanos , Inflamación/fisiopatología , Enfermedades Inflamatorias del Intestino/fisiopatologíaRESUMEN
Hyaluronan (HA) in human milk mediates host responses to microbial infection via TLR4- and CD44-dependent signaling. Signaling by HA is generally size specific. Because pure HA with average molecular mass (M) of 35 kDa can elicit a protective response in intestinal epithelial cells, it has been proposed that human milk HA may have a bioactive low-M component. Here we report the size distribution of HA in human milk samples from 20 unique donors. A new method for HA analysis, employing ion exchange (IEX) chromatography to fractionate HA by size and specific quantification of each size fraction by competitive enzyme-linked sorbent assay (ELSA), was developed. When separated into four fractions, milk HA with M⩽20 kDa, Mâ¼20 to 60 kDa, and Mâ¼60 to 110 kDa comprised averages of 1.5, 1.4, and 2.0% of the total HA, respectively. The remaining 95% was HA with M⩾110 kDa. Electrophoretic analysis of the higher M HA from 13 samples showed nearly identical M distributions, with an average M of approximately 440 kDa. This higher M HA component in human milk is proposed to bind to CD44 and to enhance human beta defensin 2 (HBD2) induction by the low-M HA components.
Asunto(s)
Ácido Hialurónico/química , Leche Humana/química , Calibración , Fraccionamiento Químico , Densitometría , Electroforesis , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Ácido Hialurónico/aislamiento & purificación , Intercambio Iónico , Peso MolecularRESUMEN
Breast-feeding is associated with enhanced protection from gastrointestinal disease in infants, mediated in part by an array of bioactive glycan components in milk that act through molecular mechanisms to inhibit enteric pathogen infection. Human milk contains hyaluronan (HA), a glycosaminoglycan polymer found in virtually all mammalian tissues. We have shown that synthetic HA of a specific size range promotes expression of antimicrobial peptides in intestinal epithelium. We hypothesize that hyaluronan from human milk also enhances innate antimicrobial defense. Here we define the concentration of HA in human milk during the first 6 months postpartum. Importantly, HA isolated from milk has a biological function. Treatment of HT-29 colonic epithelial cells with human milk HA at physiologic concentrations results in time- and dose-dependent induction of the antimicrobial peptide human ß-defensin 2 and is abrogated by digestion of milk HA with a specific hyaluronidase. Milk HA induction of human ß-defensin 2 expression is also reduced in the presence of a CD44-blocking antibody and is associated with a specific increase in ERK1/2 phosphorylation, suggesting a role for the HA receptor CD44. Furthermore, oral administration of human milk-derived HA to adult, wild-type mice results in induction of the murine Hß D2 ortholog in intestinal mucosa and is dependent upon both TLR4 and CD44 in vivo. Finally, treatment of cultured colonic epithelial cells with human milk HA enhances resistance to infection by the enteric pathogen Salmonella typhimurium. Together, our observations suggest that maternally provided HA stimulates protective antimicrobial defense in the newborn.
Asunto(s)
Ácido Hialurónico/farmacología , Inmunidad Innata/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Leche Humana/química , Administración Oral , Animales , Anticuerpos/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Recuento de Colonia Microbiana , Resistencia a la Enfermedad/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Células HT29 , Humanos , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/administración & dosificación , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Lactancia/efectos de los fármacos , Ratones , Microscopía Fluorescente , Fosforilación/efectos de los fármacos , Periodo Posparto , Transporte de Proteínas/efectos de los fármacos , Salmonelosis Animal/inmunología , Salmonelosis Animal/patología , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/fisiología , Homología de Secuencia de Aminoácido , Receptor Toll-Like 4/metabolismo , beta-Defensinas/metabolismoRESUMEN
OBJECTIVE: Intestinal microflora and inflammatory cell infiltrates play critical roles in the pathogenesis of acute colitis. Ceruloplasmin is an acute-phase plasma protein produced by hepatocytes and activated macrophages, and has ferroxidase with bactericidal activities. The goal is to understand the role of ceruloplasmin in colitis progression in a genetically modified murine model. DESIGN: Experimental colitis was induced in ceruloplasmin null (Cp(-/-)) and wild-type (WT) mice by dextran sulphate sodium administration. The role of ceruloplasmin was further evaluated by transplantation of WT macrophages into Cp(-/-) mice. RESULTS: Cp(-/-) mice rapidly lost weight and were moribund by day 14, while WT mice survived at least 30 days. Colon culture supernatants from Cp(-/-) mice exhibited elevated levels of TNFα, KC and MCP-1, indicative of increased inflammation and neutrophil and macrophage infiltration. Elevated leucocytes and severe histopathology were observed in Cp(-/-) mice. Elevated protein carbonyl content was detected in colons from Cp(-/-) mice suggesting ceruloplasmin antioxidant activity might contribute to its protective function. Unexpectedly, intraperitoneal administration of human ceruloplasmin into Cp(-/-) mice did not afford protection. Bone marrow transplantation from WT mice or injection of isolated peripheral blood monocytes markedly reduced severity of colitis and morbidity in Cp(-/-) mice. CONCLUSION: Macrophage-derived ceruloplasmin contributes importantly to protection against inflammation and tissue injury in acute and chronic experimental colitis. The findings suggest that defects in ceruloplasmin expression or processing may influence the onset or progression of inflammatory bowel disease in patients.
Asunto(s)
Ceruloplasmina/fisiología , Colitis/prevención & control , Macrófagos Peritoneales/metabolismo , Animales , Trasplante de Médula Ósea , Quimiocinas/metabolismo , Colitis/inducido químicamente , Colitis/metabolismo , Cartilla de ADN/química , Sulfato de Dextran , Progresión de la Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BL , Carbonilación Proteica , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Hyaluronan (HA) is a glycosaminoglycan polymer found in the extracellular matrix of virtually all mammalian tissues. Recent work has suggested a role for small, fragmented HA polymers in initiating innate defense responses in immune cells, endothelium, and epidermis through interaction with innate molecular pattern recognition receptors, such as TLR4. Despite these advances, little is known regarding the effect of fragmented HA at the intestinal epithelium, where numerous pattern recognition receptors act as sentinels of an innate defense response that maintains epithelial barrier integrity in the presence of abundant and diverse microbial challenges. Here we report that HA fragments promote expression of the innate antimicrobial peptide human ß-defensin 2 (HßD2) in intestinal epithelial cells. Treatment of HT-29 colonic epithelial cells with HA fragment preparations resulted in time- and dose-dependent up-regulated expression of HßD2 protein in a fragment size-specific manner, with 35-kDa HA fragment preparations emerging as the most potent inducers of intracellular HßD2. Furthermore, oral administration of specific-sized HA fragments promotes the expression of an HßD2 ortholog in the colonic epithelium of both wild-type and CD44-deficient mice but not in TLR4-deficient mice. Together, our observations suggest that a highly size-specific, TLR4-dependent, innate defense response to fragmented HA contributes to intestinal epithelium barrier defense through the induction of intracellular HßD2 protein.
Asunto(s)
Colon/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Ácido Hialurónico/farmacología , Mucosa Intestinal/metabolismo , beta-Defensinas/biosíntesis , Animales , Línea Celular Tumoral , Colon/inmunología , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/inmunología , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/inmunología , Ácido Hialurónico/metabolismo , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Mucosa Intestinal/inmunología , Ratones , Ratones Mutantes , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismo , beta-Defensinas/genética , beta-Defensinas/inmunologíaRESUMEN
Hyaluronan (HA) is widely detected in biological samples and its concentration is most commonly determined by the use of a labeled specific HA binding protein (aggrecan G1-IGD-G2, HABP), employing membrane blotting and sandwich enzyme-linked immunosorbent assay (ELISA)-like methods. However, the detected signal intensity or the quantified value obtained by using these surface-based methods is related to the molecular mass (M) of HA, especially for HA in the low M range below ~150 kDa. At the same mass or mass concentration, higher M HA gives a higher signal than lower M HA. We have experimentally determined the quantitative relationship between the M of HA (in the range 20-150 kDa) and the relative signal intensity in comparison with a standard HA, in a sandwich ELISA-like assay. An M-dependent signal correction factor (SCF) was calculated and used to correct the signal intensity, so that the corrected concentration value would more accurately reflect the true HA concentration in solution. The SCF for polydisperse low M HA was also calculated and compared with experimental results. When the molecular mass distribution of an HA sample is determined by a method such as gel electrophoresis, then its appropriately averaged SCF can be calculated and used to correct the signal in sandwich ELISA to obtain a more accurate concentration estimation. The correction method works for HA with M between ~150 and 20 kDa, but lower M HA is too poorly detected for useful analysis. The physical basis of the M-dependent detection is proposed to be the increase in detector-accessible fraction of each surface-bound molecule as M increases.
Asunto(s)
Receptores de Hialuranos/química , Ácido Hialurónico/química , Biotinilación , Densitometría , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Immunoblotting , Peso MolecularRESUMEN
The mechanisms underlying synovial joint development remain poorly understood. Here we use complete and cell-specific gene inactivation to identify the roles of the redundant chondrogenic transcription factors Sox5 and Sox6 in this process. We show that joint development aborts early in complete mutants (Sox5(-/-)6(-/-)). Gdf5 and Wnt9a expression is punctual in articular progenitor cells, but Sox9 downregulation and cell condensation in joint interzones are late. Joint cell differentiation is unsuccessful, regardless of lineage, and cavitation fails. Sox5 and Sox6 restricted expression to chondrocytes in wild-type embryos and continued Erg expression and weak Ihh expression in Sox5(-/-)6(-/-) growth plates suggest that growth plate failure contribute to this Sox5(-/-)6(-/-) joint morphogenesis block. Sox5/6 inactivation in specified joint cells and chondrocytes (Sox5(fl/fl)6(fl/fl)Col2Cre) also results in a joint morphogenesis block, whereas Sox5/6 inactivation in specified joint cells only (Sox5(fl/fl)6(fl/fl)Gdf5Cre) results in milder joint defects and normal growth plates. Sox5(fl/fl)6(fl/fl)Gdf5Cre articular chondrocytes remain undifferentiated, as shown by continued Gdf5 expression and pancartilaginous gene downregulation. Along with Prg4 downregulation, these defects likely account for joint tissue overgrowth and incomplete cavitation in adult mice. Together, these data suggest that synovial joint morphogenesis relies on essential roles for Sox5/6 in promoting both growth plate and articular chondrocyte differentiation.
Asunto(s)
Cartílago Articular/crecimiento & desarrollo , Placa de Crecimiento , Articulaciones/crecimiento & desarrollo , Morfogénesis , Factores de Transcripción SOXD/metabolismo , Animales , Cartílago Articular/metabolismo , Diferenciación Celular , Condrocitos/metabolismo , Condrogénesis , Femenino , Ácido Hialurónico/metabolismo , Masculino , Proteínas Oncogénicas/metabolismo , Factor de Transcripción SOX9/metabolismo , Células Madre/metabolismo , Factores de Transcripción , Regulador Transcripcional ERGRESUMEN
The causes of fibrosis, or the inappropriate wound healing, that follows chronic intestinal inflammation are not well defined and likely involve the contributions of multiple cellular mechanisms. As other articles in this series confirm, inflammatory cytokines clearly play a role in driving cell differentiation to the myofibroblast phenotype, promoting proliferation and extracellular matrix deposition that are characteristic of fibrotic tissue. However, controlling the balance of cytokines produced and process of myofibroblast differentiation appears to be more complex. This review considers ways in which hyaluronan, an extracellular matrix component that is remodeled during the progression of colitis, may provide indirect as well as direct cues that influence the balancing act of intestinal wound healing.
Asunto(s)
Enterocolitis/inmunología , Enterocolitis/patología , Ácido Hialurónico/metabolismo , Inflamación/inmunología , Inflamación/patología , Animales , Fibrosis , Homeostasis/inmunología , HumanosRESUMEN
Intelectins are ancient carbohydrate binding proteins, spanning chordate evolution and implicated in multiple human diseases. Previous GWAS have linked SNPs in ITLN1 (also known as omentin) with susceptibility to Crohn's disease (CD); however, analysis of possible functional significance of SNPs at this locus is lacking. Using the Ensembl database, pairwise linkage disequilibrium (LD) analyses indicated that several disease-associated SNPs at the ITLN1 locus, including SNPs in CD244 and Ly9, were in LD. The alleles comprising the risk haplotype are the major alleles in European (67%), but minor alleles in African superpopulations. Neither ITLN1 mRNA nor protein abundance in intestinal tissue, which we confirm as goblet-cell derived, was altered in the CD samples overall nor when samples were analyzed according to genotype. Moreover, the missense variant V109D does not influence ITLN1 glycan binding to the glycan ß-D-galactofuranose or protein-protein oligomerization. Taken together, our data are an important step in defining the role(s) of the CD-risk haplotype by determining that risk is unlikely to be due to changes in ITLN1 carbohydrate recognition, protein oligomerization, or expression levels in intestinal mucosa. Our findings suggest that the relationship between the genomic data and disease arises from changes in CD244 or Ly9 biology, differences in ITLN1 expression in other tissues, or an alteration in ITLN1 interaction with other proteins.
Asunto(s)
Citocinas/genética , Tracto Gastrointestinal/metabolismo , Regulación de la Expresión Génica , Variación Genética , Lectinas/genética , Alelos , Enfermedad de Crohn/genética , Citocinas/química , Susceptibilidad a Enfermedades , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Sitios Genéticos , Humanos , Lectinas/química , Especificidad de Órganos/genéticaRESUMEN
Hyaluronan (HA) is best known as an abundantly present extracellular matrix component found throughout the body of all vertebrates, including humans. Recent evidence, however, has demonstrated benefits of providing HA exogenously as a therapeutic modality for several medical conditions. Here we discuss the effects of providing HA treatment to increase innate host defense of the intestine, elucidate the size specific effects of HA, and discuss the role of various HA receptors as potential mediators of the HA effects in the intestine. This review especially focuses on HA interaction with the epithelium because it is the primary cellular barrier of the intestine and these cells play a critical balancing role between allowing water and nutrient absorption while excluding microbes and harmful dietary metabolites that are constantly in that organ's environment.
Asunto(s)
Ácido Hialurónico/farmacología , Inmunidad Innata/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Animales , Humanos , Inmunidad Innata/inmunología , Intestinos/efectos de los fármacos , Intestinos/inmunologíaRESUMEN
Inflammatory bowel disease is a chronic inflammatory condition of the intestinal mucosa whose etiology is unclear but is likely to be multifactorial. We have shown previously that an increased amount of hyaluronan (HA) is present both in the inflamed mucosa of inflammatory bowel disease patients and in isolated human cells after polyI:C treatment. The signal transducer and activator of transcription (STAT)1 protein plays an important role in many signaling pathways that are associated with inflammation. We therefore investigated the role of STAT1 in adhesive interactions that occur between leukocytes and polyI:C-induced mucosal smooth muscle cells (M-SMCs). Activation of STAT1 was observed after the polyI:C treatment of M-SMCs. Specific phosphorylation of tyrosine and serine residues of STAT1 was observed in polyI:C-treated, but not untreated, M-SMC cultures. To evaluate further the role of STAT1, a corresponding STAT-1-null mouse was used. PolyI:C-induced, HA-mediated leukocyte adhesion to colon SMCs from STAT1-null mice was significantly decreased compared with that from wild-type control mice. In vivo, using the dextran sulfate sodium-induced model of colon inflammation, both tissue damage and HA deposition were attenuated in STAT1-null mice compared with that in wild-type control mice. Additionally, the inter-alpha-trypsin inhibitor (IalphaI), a proteoglycan essential for facilitating leukocyte binding to the HA matrix, was reduced in STAT1-null mice. Together, these results demonstrate that STAT1 plays an important role in HA-mediated inflammatory processes.
Asunto(s)
Adhesión Celular , Colitis/patología , Colitis/prevención & control , Ácido Hialurónico/metabolismo , Leucocitos/citología , Factor de Transcripción STAT1/deficiencia , Animales , Adhesión Celular/efectos de los fármacos , Colitis/inducido químicamente , Colitis/metabolismo , Sulfato de Dextran , Humanos , Mucosa Intestinal/citología , Leucocitos/efectos de los fármacos , Ratones , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Fosfotirosina/metabolismo , Poli I-C/farmacología , Factor de Transcripción STAT1/metabolismo , Células U937RESUMEN
Hyaluronan, a major extracellular matrix component, is an active participant in many disease states, including inflammatory bowel disease (IBD). The synthesis of this dynamic polymer is increased at sites of inflammation. Hyaluronan together with the enzymes responsible for its synthesis, degradation, and its binding proteins, directly modulates the promotion and resolution of disease by controlling recruitment of immune cells, by release of inflammatory cytokines, and by balancing hemostasis. This review discusses the functional significance of hyaluronan in the cells and tissues involved in inflammatory bowel disease pathobiology.
Asunto(s)
Ácido Hialurónico/metabolismo , Enfermedades Inflamatorias del Intestino/inmunología , Citocinas/metabolismo , Matriz Extracelular/metabolismo , Humanos , Hialuronano Sintasas/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismoRESUMEN
Intestinal epithelium plays a critical role in host defense against orally acquired pathogens. Dysregulation of this protective barrier is a primary driver of inflammatory bowel diseases (Crohn's and ulcerative colitis) and also infant gastrointestinal infections. Previously, our lab reported that hyaluronan (HA) isolated from human milk induces the expression of the antimicrobial peptide ß-defensin in vivo and protects against Salmonella Typhimurium infection of epithelial cells in vitro. In addition, we demonstrated that commercially available 35 kDa size HA induces the expression of ß-defensin, upregulates the expression of tight junction protein zonula occludens-1 (ZO-1), and attenuates murine Citrobacter rodentium infection in vivo. In this current study, we report that HA35 remains largely intact and biologically active during transit through the digestive tract where it directly induces ß-defensin expression upon epithelial cell contact. We also demonstrate HA35 abrogation of murine Salmonella Typhimurium infection as well as downregulation of leaky tight junction protein claudin-2 expression. Taken together, we propose a dual role for HA in host innate immune defense at the epithelial cell surface, acting to induce antimicrobial peptide production and also block pathogen-induced leaky gut. HA35 is therefore a promising therapeutic in the defense against bacterially induced colitis in compromised adults and vulnerable newborns.
Asunto(s)
Antibacterianos/uso terapéutico , Ácido Hialurónico/uso terapéutico , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/microbiología , Infecciones por Salmonella/tratamiento farmacológico , Salmonella typhimurium/efectos de los fármacos , Animales , Antibacterianos/farmacocinética , Claudina-2/análisis , Colon/microbiología , Colon/patología , Tránsito Gastrointestinal , Humanos , Ácido Hialurónico/farmacocinética , Inmunidad Innata/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Masculino , Ratones Endogámicos C57BL , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/patología , Salmonella typhimurium/inmunología , beta-Defensinas/análisisRESUMEN
Tight junction proteins are critical in maintaining homeostatic intestinal permeability. Multiple intestinal inflammatory diseases are correlated with reduced expression of tight junction proteins. We have recently reported that oral treatment of mice with Hyaluronan 35kDa (HA35) increases colonic expression of tight junction protein zonula occludens-1 (ZO-1). Here, we investigate whether HA35 treatment enhances ZO-1 expression by direct interaction with intestinal epithelium in vitro and have identified the HA receptor responsible for HA35-mediated ZO-1 induction in colonic epithelium in vitro and in vivo. Our results reveal that HA35 treatment increases ZO-1 expression in mouse intestinal epithelial organoids, while large HA 2000kDa is not internalized into the cells. Our immunofluorescence data indicate that layilin, but neither toll-like receptor-4 (TLR-4) nor CD44, mediate the HA35-induced ZO-1 expression in colonic epithelium in vitro and in vivo. Additionally, using layilin null mice we have determined that layilin mediates HA35 induction of ZO-1 in healthy mice and during dextran sulfate sodium (DSS)-induced colitis. Furthermore, we find that while ZO-1 expression levels are reduced, layilin expression levels are equivalent in inflammatory bowel disease (IBD) patients and non-IBD controls. Together, our data suggest that layilin is an important HA receptor, that mediates the effect of oral HA35 treatment on intestinal epithelium. HA35 holds promise as a simple dietary supplement to strengthen gut barrier defense.
Asunto(s)
Proteínas Portadoras/metabolismo , Colitis/metabolismo , Ácido Hialurónico/farmacología , Mucosa Intestinal/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , Animales , Proteínas Portadoras/genética , Células Cultivadas , Colitis/inducido químicamente , Colitis/genética , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Glicoproteínas de Membrana/genética , Ratones , Organoides/citología , Organoides/efectos de los fármacos , Organoides/metabolismoRESUMEN
Maintaining a healthy intestinal barrier, the primary physical barrier between intestinal microbiota and the underlying lamina propria, is critical for optimal health. Epithelial integrity is essential for the prevention of the entrance of luminal contents, such as bacteria and their products, through the large intestinal barrier. In this study, we investigated the protective functions of biosynthetic, specific sized, hyaluronan around 35kDa (HA35) on intestinal epithelium in healthy mice, as well as mice infected Citrobacter rodentium, an established model that mimics infection with a serious human pathogen, enteropathogenic E. coli (EPEC). Our results reveal that treatment with HA35 protects mice from Citrobacter infection and enhances the epithelial barrier function. In particular, we have found that HA35 induces the expression of tight junction protein zonula occludens (ZO)-1 in both healthy and Citrobacter infected mice, as demonstrated by immunoflurorescence and Western blot analyses. Furthermore, we determined that HA35 treatment enhances ZO-1 expression and reduces intestinal permeability at the early stages of dextran sulfate sodium (DSS)-induced colitis in mice. Together, our data demonstrate that the expression and functionality of tight junctions, are increased by HA35 treatment, suggesting a novel mechanism for the protection from Citrobacter infection.