RESUMEN
Porcine reproductive and respiratory syndrome (PRRS) induces respiratory disease and reproductive failure accompanied by gastroenteritis-like symptoms. The mechanism of intestinal barrier injury caused by PRRSV infection in piglets has yet to be investigated. An in vivo PRRSV-induced model was established in 30-day-old piglets by the intramuscular injection of 2 mL of 104 TCID50/mL PRRSV for 15 days. Observations of PRRSV replication and histology were conducted in the lungs and intestine, and goblet cell counts, relative MUC2 mRNA expression, and tight junction protein, proinflammatory cytokine, TLR4, MyD88, IκB and p-IκB expression were measured. PRRSV replicated in the lungs and small intestine, as demonstrated by absolute RT-qPCR quantification, and the PRRSV N protein was detected in the lung interstitium and jejunal mucosa. PRRSV infection induced both lung and gut injury, markedly decreased villus height and the villus to crypt ratio in the small intestine, and obviously increased the number of goblet cells and the relative expression of MUC2 mRNA in the jejunum. PRRSV infection aggravated the morphological depletion of tight junction proteins and increased IL-1ß, IL-6, IL-8 and TNF-α expression by activating the NF-κB signalling pathway in the jejunum. PRRSV infection impaired intestinal integrity by damaging physical and immune barriers in the intestine by inducing inflammation, which may be related to the regulation of the gut-lung axis. This study also provides a new hypothesis regarding the pathogenesis of PRRSV-induced diarrhoea.
Asunto(s)
Expresión Génica , Células Caliciformes/virología , Yeyuno/virología , Síndrome Respiratorio y de la Reproducción Porcina/fisiopatología , Proteínas de Uniones Estrechas/genética , Replicación Viral , Animales , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Sus scrofa , Porcinos , Proteínas de Uniones Estrechas/metabolismoRESUMEN
Although they globally cause viral gastroenteritis in children, astroviruses are understudied due to the lack of well-defined animal models. While murine astroviruses (muAstVs) chronically infect immunodeficient mice, a culture system and understanding of their pathogenesis is lacking. Here, we describe a platform to cultivate muAstV using air-liquid interface (ALI) cultures derived from mouse enteroids, which support apical infection and release. Chronic muAstV infection occurs predominantly in the small intestine and correlates with higher interferon-lambda (IFN-λ) expression. MuAstV stimulates IFN-λ production in ALI, recapitulating our in vivo findings. We demonstrate that goblet cells and enterocytes are targets for chronic muAstV infection in vivo, and that infection is enhanced by parasite co-infection or type 2 cytokine signaling. Depletion of goblet cells from ALI limits muAstV infection in vitro. During chronic infection, muAstV stimulates IFN-λ production in infected cells and induces ISGs throughout the intestinal epithelium in an IFN-λ-receptor-dependent manner. Collectively, our study provides insights into the cellular tropism and innate immune responses to muAstV and establishes an enteroid-based culture system to propagate muAstV in vitro.
Asunto(s)
Infecciones por Astroviridae/inmunología , Astroviridae/fisiología , Citocinas/metabolismo , Enterocitos/virología , Gastroenteritis/inmunología , Células Caliciformes/virología , Células Th2/inmunología , Animales , Células Cultivadas , Coinfección , Enterocitos/inmunología , Células Caliciformes/inmunología , Humanos , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de Órganos , Tropismo ViralRESUMEN
Goblet cells are specialized epithelial cells that are essential to the formation of the mucus barriers in the airways and intestines. Armed with an arsenal of defenses, goblet cells can rapidly respond to infection but must balance this response with maintaining homeostasis. Whereas goblet cell defenses against bacterial and parasitic infections have been characterized, we are just beginning to understand their responses to viral infections. Here, we outline what is known about the enteric and respiratory viruses that target goblet cells, the direct and bystander effects caused by viral infection and how viral interactions with the mucus barrier can alter the course of infection. Together, these factors can play a significant role in driving viral pathogenesis and disease outcomes.
Asunto(s)
Células Caliciformes/patología , Virosis/patología , Animales , Células Caliciformes/virología , Humanos , Virosis/virologíaRESUMEN
Astroviruses are a global cause of pediatric diarrhea, but they are largely understudied, and it is unclear how and where they replicate in the gut. Using an in vivo model, here we report that murine astrovirus preferentially infects actively secreting small intestinal goblet cells, specialized epithelial cells that maintain the mucus barrier. Consequently, virus infection alters mucus production, leading to an increase in mucus-associated bacteria and resistance to enteropathogenic E. coli colonization. These studies establish the main target cell type and region of the gut for productive murine astrovirus infection. They further define a mechanism by which an enteric virus can regulate the mucus barrier, induce functional changes to commensal microbial communities, and alter host susceptibility to pathogenic bacteria.
Asunto(s)
Infecciones por Astroviridae/patología , Infecciones por Astroviridae/virología , Astroviridae/fisiología , Tracto Gastrointestinal/patología , Tracto Gastrointestinal/virología , Células Caliciformes/virología , Moco/virología , Animales , Células Epiteliales/patología , Células Epiteliales/virología , Escherichia coli/fisiología , Femenino , Tracto Gastrointestinal/microbiología , Tracto Gastrointestinal/ultraestructura , Masculino , Ratones Endogámicos C57BL , Moco/microbiología , Transcriptoma/genética , Replicación Viral/fisiología , Esparcimiento de Virus/fisiologíaRESUMEN
The respiratory epithelium comprises polarized cells at the interface between the environment and airway tissues. Polarized apical and basolateral protein secretions are a feature of airway epithelium homeostasis. Human respiratory syncytial virus (hRSV) is a major human pathogen that primarily targets the respiratory epithelium. However, the consequences of hRSV infection on epithelium secretome polarity and content remain poorly understood. To investigate the hRSV-associated apical and basolateral secretomes, a proteomics approach was combined with an ex vivo pediatric human airway epithelial (HAE) model of hRSV infection (data are available via ProteomeXchange and can be accessed at https://www.ebi.ac.uk/pride/ with identifier PXD013661). Following infection, a skewing of apical/basolateral abundance ratios was identified for several individual proteins. Novel modulators of neutrophil and lymphocyte activation (CXCL6, CSF3, SECTM1 or CXCL16), and antiviral proteins (BST2 or CEACAM1) were detected in infected, but not in uninfected cultures. Importantly, CXCL6, CXCL16, CSF3 were also detected in nasopharyngeal aspirates (NPA) from hRSV-infected infants but not healthy controls. Furthermore, the antiviral activity of CEACAM1 against RSV was confirmed in vitro using BEAS-2B cells. hRSV infection disrupted the polarity of the pediatric respiratory epithelial secretome and was associated with immune modulating proteins (CXCL6, CXCL16, CSF3) never linked with this virus before. In addition, the antiviral activity of CEACAM1 against hRSV had also never been previously characterized. This study, therefore, provides novel insights into RSV pathogenesis and endogenous antiviral responses in pediatric airway epithelium.
Asunto(s)
Antivirales/metabolismo , Quimiocinas/metabolismo , Proteoma/metabolismo , Mucosa Respiratoria/virología , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/fisiología , Bronquios/patología , Línea Celular , Niño , Células Epiteliales/patología , Células Epiteliales/virología , Células Caliciformes/metabolismo , Células Caliciformes/virología , Homeostasis , Humanos , Lactante , Cinética , Nasofaringe/virología , Mucosa Respiratoria/metabolismo , Virus Sincitial Respiratorio Humano/crecimiento & desarrollo , Tropismo , Proteínas Virales/metabolismoRESUMEN
Ferrets represent an invaluable animal model to study influenza virus pathogenesis and transmission. To further characterize this model, we developed a differentiated primary ferret nasal epithelial cell (FNEC) culture model for investigation of influenza A virus infection and virus-host interactions. This well-differentiated culture consists of various cell types, a mucociliary clearance system, and tight junctions, representing the nasal ciliated pseudostratified respiratory epithelium. Both α2,6-linked and α2,3-linked sialic acid (SA) receptors, which preferentially bind the hemagglutinin (HA) of human and avian influenza viruses, respectively, were detected on the apical surface of the culture with different cellular tropisms. In accordance with the distribution of SA receptors, we observed that a pre-2009 seasonal A(H1N1) virus infected both ciliated and nonciliated cells, whereas a highly pathogenic avian influenza (HPAI) A(H5N1) virus primarily infected nonciliated cells. Transmission electron microscopy revealed that virions were released from or associated with the apical membranes of ciliated, nonciliated, and mucin-secretory goblet cells. Upon infection, the HPAI A(H5N1) virus replicated to titers higher than those of the human A(H1N1) virus at 37°C; however, replication of the A(H5N1) virus was significantly attenuated at 33°C. Furthermore, we found that infection with the A(H5N1) virus induced higher expression levels of immune mediator genes and resulted in more cell damage/loss than with the human A(H1N1) virus. This primary differentiated FNEC culture model, recapitulating the structure of the nasal epithelium, provides a useful model to bridge in vivo and in vitro studies of cellular tropism, infectivity, and pathogenesis of influenza viruses during the initial stages of infection.IMPORTANCE Although ferrets serve as an important model of influenza virus infection, much remains unknown about virus-host interactions in this species at the cellular level. The development of differentiated primary cultures of ferret nasal epithelial cells is an important step toward understanding cellular tropism and the mechanisms of influenza virus infection and replication in the airway milieu of this model. Using lectin staining and microscopy techniques, we characterized the sialic acid receptor distribution and the cellular composition of the culture model. We then evaluated the replication of and immune response to human and avian influenza viruses at relevant physiological temperatures. Our findings offer significant insight into this first line of defense against influenza virus infection and provide a model for the evaluation of emerging influenza viruses in a well-controlled in vitro environmental setting.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/genética , Tropismo Viral/genética , Animales , Bronquios/virología , Técnicas de Cultivo de Célula/métodos , Cilios/virología , Modelos Animales de Enfermedad , Células Epiteliales/virología , Hurones/virología , Células Caliciformes/metabolismo , Células Caliciformes/virología , Humanos , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Virus de la Influenza A/fisiología , Gripe Humana/virología , Mucosa Nasal/metabolismo , Mucosa Nasal/virología , Cultivo Primario de Células , Receptores de Superficie Celular/metabolismo , Receptores Virales/metabolismo , Mucosa Respiratoria/virología , Tráquea/virología , Virosis/genéticaRESUMEN
Recent worldwide outbreaks of enterovirus 71 (EV71) have caused major epidemics of hand, foot, and mouth disease with severe neurological complications, including acute flaccid paralysis. EV71 is transmitted by the enteral route, but little is known about the mechanisms it uses to cross the human gastrointestinal tract. Using primary human intestinal epithelial monolayers, we show that EV71 infects the epithelium from the apical surface, where it preferentially infects goblet cells. We found that EV71 infection did not alter epithelial barrier function but did reduce the expression of goblet cell-derived mucins, suggesting that it alters goblet cell function. We also show that the intestinal epithelium responds to EV71 infection through the selective induction of type III interferons (IFNs), which restrict EV71 replication. Collectively, these findings define the early events associated with EV71 infections of the human intestinal epithelium and show that host IFN signaling controls replication in an IFN-specific manner.
Asunto(s)
Enterovirus Humano A/fisiología , Infecciones por Enterovirus/metabolismo , Infecciones por Enterovirus/virología , Células Caliciformes/metabolismo , Células Caliciformes/virología , Interacciones Huésped-Patógeno , Interferones/metabolismo , Transducción de Señal , Permeabilidad de la Membrana Celular , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/virología , Interferón lambdaRESUMEN
Human adenoviruses (HAdV) are significant human pathogens. Although only a subset of HAdV serotypes commonly cause gastroenteritis in humans, most HAdV species replicate in the gastrointestinal tract. Knowledge of the complex interaction between HAdVs and the human intestinal epithelium has been limited by the lack of a suitable cell culture system containing relevant cell types. Recently, this need has been met by the stable and prolonged cultivation of primary intestinal epithelial cells as enteroids. Human enteroids have been used to reveal novel and interesting aspects of rotavirus, norovirus, and enterovirus replication, prompting us to explore their suitability for HAdV culture. We found that both prototype strains and clinical isolates of enteric and nonenteric HAdVs productively replicate in human enteroids. HAdV-5p, a respiratory pathogen, and HAdV-41p, an enteric pathogen, are both sensitive to type I and III interferons in human enteroid monolayers but not A549 cells. Interestingly, HAdV-5p, but not HAdV-41p, preferentially infected goblet cells. And, HAdV-5p but not HAdV-41p was potently neutralized by the enteric human alpha-defensin HD5. These studies highlight new facets of HAdV biology that are uniquely revealed by primary intestinal epithelial cell culture.IMPORTANCE Enteric adenoviruses are a significant cause of childhood gastroenteritis worldwide, yet our understanding of their unique biology is limited. Here we report robust replication of both prototype and clinical isolates of enteric and respiratory human adenoviruses in enteroids, a primary intestinal cell culture system. Recent studies have shown that other fastidious enteric viruses replicate in human enteroids. Therefore, human enteroids may provide a unified platform for culturing enteric viruses, potentially enabling isolation of a greater diversity of viruses from patients. Moreover, both the ability of interferon to restrict respiratory and enteric adenoviruses and a surprising preference of a respiratory serotype for goblet cells demonstrate the power of this culture system to uncover aspects of adenovirus biology that were previously unattainable with standard cell lines.
Asunto(s)
Infecciones por Adenovirus Humanos/virología , Adenovirus Humanos/aislamiento & purificación , Células Caliciformes/virología , Intestino Delgado/virología , Replicación Viral , Infecciones por Adenovirus Humanos/tratamiento farmacológico , Infecciones por Adenovirus Humanos/inmunología , Adulto , Antivirales/farmacología , Células Caliciformes/efectos de los fármacos , Células Caliciformes/inmunología , Humanos , Interferones/farmacología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/inmunologíaRESUMEN
Intestinal goblet cells secret mucins to form mucus layers critical for maintaining the integrity of the intestinal epithelium. Porcine epidemic diarrhea virus (PEDV) causes watery diarrhea and high mortality of suckling pigs. PEDV mainly infects villous epithelial cells of the small intestine, and infected cells undergo acute, massive necrosis, followed by severe villous atrophy. Conventional 9-day-old nursing pigs [PEDV-inoculated (n=9); Mock (n=11)] and 26-day-old weaned [PEDV-inoculated (n=11); Mock (n=9)] were inoculated orally [8.9 log10 genomic equivalents/pig] with PEDV strain PC21A or mock. We used alcian blue or Periodic-Acid-Schiff staining for the detection of acidic or neutral mucin-secreting goblet cells in the small intestine. We demonstrated that PEDV infection of the nursing pigs at post-inoculation days (PIDs) 1-5 and weaned pigs at PIDs 3-5 led to depletion or significant reduction in the number of goblet cells (and also the number of villous goblet cells normalized by jejunal villous crypt height to crypt depth ratios) in the villi or crypts. These findings coincided with the development of intestinal villous atrophy. By immunohistochemistry, a few PEDV antigen-positive goblet cells were identified in the jejunal or ileal villous epithelium of the infected nursing or weaned pigs. During the early stages of PEDV infection, goblet cell mucins in the small intestine may be decreased, possibly leading to an impaired mucus layer and increased susceptibility to secondary enteric bacterial infection.
Asunto(s)
Infecciones por Coronavirus/veterinaria , Células Caliciformes/virología , Intestino Delgado/virología , Enfermedades de los Porcinos/fisiopatología , Destete , Animales , Infecciones por Coronavirus/fisiopatología , Infecciones por Coronavirus/virología , Epitelio/virología , Virus de la Diarrea Epidémica Porcina/fisiología , Porcinos , Enfermedades de los Porcinos/virologíaRESUMEN
The authors report a case where undifferentiated (classic) penile intraepithelial neoplasia was associated with the presence of goblet cells throughout the full epithelial thickness and which later progressed into an invasive carcinoma. The lesion evolved in three consecutive biopsies from only surface epithelium occupying numerous goblet cells in the first to variably sized solid nodules in the dermis composed of atypical squamous and/or basaloid cells intermixed with numerous goblet cells in the third biopsy. Both cellular components expressed CK7 and p16 protein. Human Papillomavirus (HPV) genotyping revealed high risk HPV type 16. To the best of our knowledge, this is the first description of such a lesion occurring on the penis, which can be considered the penile analogue of cervical stratified mucin-producing intraepithelial lesion (SMILE). The correct diagnosis was rendered retrospectively, after recognition of the existence of a vulvar lesion resembling cervical SMILE. The initial biopsy was misinterpreted as extramammary Paget disease, which also constitutes the main pitfall in the differential diagnosis. Another important differential diagnosis is penile/vulvar mucinous metaplasia. The finding of atypical squamous epithelial cells positive for p16 associated with mucinous cells present throughout the full epithelial thickness is a clue to the diagnosis of penile SMILE.
Asunto(s)
Carcinoma in Situ/patología , Carcinoma/patología , Células Caliciformes/patología , Neoplasias Quísticas, Mucinosas y Serosas/patología , Enfermedad de Paget Extramamaria/patología , Neoplasias del Pene/patología , Displasia del Cuello del Útero/patología , Neoplasias del Cuello Uterino/patología , Biomarcadores de Tumor/análisis , Biopsia , Carcinoma/química , Carcinoma/virología , Carcinoma in Situ/química , Carcinoma in Situ/virología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/análisis , Errores Diagnósticos , Progresión de la Enfermedad , Femenino , Células Caliciformes/química , Células Caliciformes/virología , Papillomavirus Humano 16/aislamiento & purificación , Humanos , Inmunohistoquímica , Masculino , Metaplasia , Persona de Mediana Edad , Invasividad Neoplásica , Neoplasias Quísticas, Mucinosas y Serosas/química , Neoplasias Quísticas, Mucinosas y Serosas/virología , Neoplasias del Pene/química , Neoplasias del Pene/virología , Valor Predictivo de las Pruebas , Neoplasias del Cuello Uterino/química , Displasia del Cuello del Útero/químicaAsunto(s)
Células Caliciformes/patología , Células Caliciformes/virología , Mucinas/biosíntesis , Infecciones por Papillomavirus/patología , Integración Viral , Adulto , Femenino , Células Caliciformes/metabolismo , Humanos , Hibridación in Situ , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/patología , Displasia del Cuello del Útero/virologíaRESUMEN
Cyprinid herpesvirus-3 (CyHV-3) is recognised as a pathogen which causes mass mortality in populations of carp, Cyprinus carpio. One of the characteristic symptoms of the disease associated with CyHV-3 infection is the occurrence of skin lesions, sloughing off the epithelium and a lack of mucus. Furthermore, fish then seem to be more susceptible to secondary infections by bacterial, parasitic or fungal pathogens which may cause further mortality within the population. The observed pathological alterations lead to the assumption that the carp skin barrier is strongly challenged during CyHV-3 associated disease. Therefore we examined mRNA expression of genes encoding inflammatory mediators, type I interferons, and the following skin defence molecules: antimicrobial peptides, claudins, and mucin. In addition, we monitored changes in the bacterial flora of the skin during disease conditions. Our results show that CyHV-3 associated disease in the skin of common carp leads to a reduction in mRNA expression of genes encoding several important components of the mucosal barrier, in particular mucin 5B, beta defensin 1 and 2, and the tight junction proteins claudin 23 and 30. This caused changes in the bacterial flora and the development of secondary bacterial infection among some individual fish. To our knowledge this is the first report showing that under disease conditions associated with virus infection, the mucosal barrier of fish skin is disrupted resulting in a higher susceptibility to secondary infections. The reported clinical signs of CyHV-3 skin infection can now be explained by our results at the molecular level, although the mechanism of a probable virus induced immunomodulation has to be investigated further.
Asunto(s)
Carpas , Enfermedades de los Peces/virología , Infecciones por Herpesviridae/veterinaria , Herpesviridae/fisiología , Enfermedades Cutáneas Virales/veterinaria , Piel/patología , Piel/virología , Animales , Péptidos Catiónicos Antimicrobianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/metabolismo , Claudinas/biosíntesis , Claudinas/genética , Claudinas/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/virología , Enfermedades de los Peces/genética , Enfermedades de los Peces/metabolismo , Enfermedades de los Peces/patología , Células Caliciformes/citología , Células Caliciformes/virología , Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Interferón Tipo I/biosíntesis , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Mucina 5B/biosíntesis , Mucina 5B/genética , Mucina 5B/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , ARN Bacteriano/biosíntesis , ARN Bacteriano/genética , ARN Ribosómico 16S/biosíntesis , ARN Ribosómico 16S/genética , Piel/metabolismo , Piel/microbiología , Enfermedades Cutáneas Virales/metabolismo , Enfermedades Cutáneas Virales/patología , Enfermedades Cutáneas Virales/virología , Regulación hacia Arriba , Replicación ViralRESUMEN
Adenosine triphosphate (ATP) and its metabolite adenosine regulate airway mucociliary clearance via activation of purinoceptors. In this study, we investigated the contribution of goblet cells to airway epithelial ATP release. Primary human bronchial epithelial (HBE) cultures, typically dominated by ciliated cells, were induced to develop goblet cell metaplasia by infection with respiratory syncytial virus (RSV) or treatment with IL-13. Under resting conditions, goblet-cell metaplastic cultures displayed enhanced mucin secretion accompanied by increased rates of ATP release and mucosal surface adenosine accumulation as compared with nonmetaplastic control HBE cultures. Intracellular calcium chelation [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester] or disruption of the secretory pathways (nocodazole, brefeldin A, and N-ethylmaleimide) decreased mucin secretion and ATP release in goblet-cell metaplastic HBE cultures. Conversely, stimuli that triggered calcium-regulated mucin secretion (e.g., ionomycin or UTP) increased luminal ATP release and adenyl purine accumulation in control and goblet-cell metaplastic HBE cultures. Goblet cell-associated ATP release was not blocked by the connexin/pannexin hemichannel inhibitor carbenoxolone, suggesting direct nucleotide release from goblet cell vesicles rather than the hemichannel insertion. Collectively, our data demonstrate that nucleotide release is increased by goblet cell metaplasia, reflecting, at least in part, a mechanism tightly associated with goblet cell mucin secretion. Increased goblet cell nucleotide release and resultant adenosine accumulation provide compensatory mechanisms to hydrate mucins by paracrine stimulation of ciliated cell ion and water secretion and maintain mucociliary clearance, and to modulate inflammatory responses.
Asunto(s)
Adenosina Trifosfato/metabolismo , Bronquios/metabolismo , Epitelio/metabolismo , Células Caliciformes/metabolismo , Células Caliciformes/patología , Metaplasia/metabolismo , Mucinas/metabolismo , Western Blotting , Bronquios/citología , Bronquios/efectos de los fármacos , Bronquios/virología , Calcio/metabolismo , Células Cultivadas , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Ensayo de Inmunoadsorción Enzimática , Epitelio/efectos de los fármacos , Epitelio/virología , Etilmaleimida/farmacología , Exocitosis , Células Caliciformes/virología , Humanos , Técnicas para Inmunoenzimas , Interleucina-13/farmacología , Metaplasia/patología , Metaplasia/virología , ARN Mensajero/genética , Receptores Purinérgicos P2Y2/metabolismo , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/patogenicidad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Infection of airway epithelium by rhinovirus is the most common cause of asthma exacerbations. Even in mild asthma, airway epithelium exhibits mucous metaplasia, which increases with increasing severity of the disease. We previously showed that squamous cultures of human airway epithelium manifest rhinoviral infection at levels many times higher than in well-differentiated cultures of a mucociliary phenotype. Here we tested the hypothesis that mucous metaplasia is also associated with increased levels of rhinoviral infection. Mucous metaplasia was induced with IL-13, which doubled the numbers of goblet cells. In both control (mucociliary) and IL-13- treated (mucous metaplastic) cultures, goblet cells were preferentially infected by rhinovirus. IL-13 doubled the numbers of infected cells by increasing the numbers of infected goblet cells. Furthermore, IL-13 increased both the maturity of goblet cells and the probability that a goblet cell would be infected. The infection of cells other than goblet cells was unaltered by IL-13. Treatment with IL-13 did not alter the levels of rhinovirus receptor ICAM-1, nor did the proliferative effects of IL-13 enhance infection, because rhinovirus did not colocalize with dividing cells. However, the induction of mucous metaplasia caused changes in the apical membrane structure, notably a marked decrease in overall ciliation, and an increase in the overall flatness of the apical surface. We conclude that mucous metaplasia in asthma increases the susceptibility of airway epithelium to infection by rhinovirus because of changes in the overall architecture of the apical surface.
Asunto(s)
Epitelio/patología , Epitelio/virología , Interleucina-13/farmacología , Moco/efectos de los fármacos , Moco/virología , Infecciones por Picornaviridae/virología , Rhinovirus/fisiología , Recuento de Células , Células Cultivadas , Cilios/efectos de los fármacos , Cilios/metabolismo , Cilios/ultraestructura , Susceptibilidad a Enfermedades , Células Epiteliales/inmunología , Células Epiteliales/patología , Células Epiteliales/ultraestructura , Células Epiteliales/virología , Epitelio/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Células Caliciformes/efectos de los fármacos , Células Caliciformes/inmunología , Células Caliciformes/patología , Células Caliciformes/virología , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno Ki-67/metabolismo , Metaplasia/inducido químicamente , Metaplasia/inmunología , Moco/inmunología , Infecciones por Picornaviridae/patología , Rhinovirus/efectos de los fármacos , Factores de TiempoRESUMEN
Patients with mutations in the pulmonary surfactant protein C (SP-C) gene develop interstitial lung disease and pulmonary exacerbations associated with viral infections including respiratory syncytial virus (RSV). Pulmonary infection with RSV caused more severe interstitial thickening, air space consolidation, and goblet cell hyperplasia in SP-C-deficient (Sftpc(-/-)) mice compared with SP-C replete mice. The RSV-induced pathology resolved more slowly in Sftpc(-/-) mice with lung inflammation persistent up to 30 days postinfection. Polymorphonuclear leukocyte and macrophage counts were increased in the bronchoalveolar lavage (BAL) fluid of Sftpc(-/-) mice. Viral titers and viral F and G protein mRNA were significantly increased in both Sftpc(-/-) and heterozygous Sftpc(+/-) mice compared with controls. Expression of Toll-like receptor 3 (TLR3) mRNA was increased in the lungs of Sftpc(-/-) mice relative to Sftpc(+/+) mice before and after RSV infection. Consistent with the increased TLR3 expression, BAL inflammatory cells were increased in the Sftpc(-/-) mice after exposure to a TLR3-specific ligand, poly(I:C). Preparations of purified SP-C and synthetic phospholipids blocked poly(I:C)-induced TLR3 signaling in vitro. SP-C deficiency increases the severity of RSV-induced pulmonary inflammation through regulation of TLR3 signaling.
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Proteína C Asociada a Surfactante Pulmonar/deficiencia , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/patología , Animales , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/virología , Recuento de Células , Línea Celular , Colectinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Regulación Viral de la Expresión Génica , Células Caliciformes/patología , Células Caliciformes/virología , Humanos , Hipertrofia , Ligandos , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Ratones , Neumonía/complicaciones , Neumonía/patología , Neumonía/virología , Proteína C Asociada a Surfactante Pulmonar/metabolismo , ARN Bicatenario/metabolismo , Infecciones por Virus Sincitial Respiratorio/complicaciones , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/genética , Factores de Tiempo , Receptor Toll-Like 3/metabolismoRESUMEN
OBJECTIVE: To determine the effect of feline herpesvirus type 1 (FHV-1) on tear film breakup time (TFBUT) and Schirmer tear test (STT) values in cats with primary experimental infection and to determine the relationship between TFBUT and STT values and conjunctival goblet cell density (GCD). SAMPLE POPULATION: 9 specific-pathogen-free cats of approximately 6 months of age. PROCEDURES: 6 cats were inoculated with FHV-1; 3 control cats were sham inoculated. Clinical and histologic evidence of conjunctivitis and TFBUT, GCD, and STT values were assessed at multiple times until postinoculation day (PID) 29. RESULTS: In infected cats, mean clinical and histologic conjunctivitis scores peaked at PID 7 and remained above baseline at PID 29. In control cats, these 2 variables did not change from baseline throughout the study. Mean TFBUT declined rapidly in infected cats up to PID 15 and at PID 29 remained less than baseline, less than for control cats, and below reference range values. Mean STT value for infected cats at PID 29 was increased from baseline but was within the reference range and not different from the value for control cats. Mean GCD in infected cats declined precipitously by PID 7 and remained below reference range values at PID 29. Mean GCD in control cats remained unchanged for the duration of the study period. CONCLUSIONS AND CLINICAL RELEVANCE: FHV-1 induced qualitative tear film abnormalities in experimentally infected cats, as measured by TFBUT and GCD. Assessment of TFBUT provided a reasonable clinical estimate of GCD.
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Enfermedades de los Gatos/virología , Conjuntiva/citología , Células Caliciformes/virología , Infecciones por Herpesviridae/veterinaria , Herpesviridae/clasificación , Lágrimas/química , Animales , Gatos , Conjuntivitis Viral/veterinaria , Técnicas de Diagnóstico Oftalmológico/veterinaria , Femenino , Infecciones por Herpesviridae/virología , Masculino , Organismos Libres de Patógenos EspecíficosRESUMEN
In this study we performed comparisons of pulmonary responses between two different respiratory syncytial virus (RSV) antigenic subgroup A strains, A2 and Line 19. Line 19 strain induced significant dose-responsive airway hyperreactivity (AHR) in BALB/c mice at days 6 and 9 after infection, whereas the A2 strain induced no AHR at any dose. Histological examination indicated that A2 induced no goblet cell hyper/metaplasia, whereas the Line 19 induced goblet cell expansion and significant increases in gob5 and MUC5AC mRNA and protein levels in vivo. When examining cytokine responses, A2 strain induced significant interleukin (IL)-10 expression, whereas Line 19 strain induced significant IL-13 expression. When IL-13-/- mice were infected with Line 19 RSV, the AHR responses were abrogated along with gob5 gene expression. There was little difference in viral titer throughout the infection between the line 19- and A2-infected mice. However, the A2 strain grew to significantly higher titers than the Line 19 strain in HEp-2 cells in vitro. Thus, RSV Line 19-induced airway dysfunction does not correlate with viral load in vivo. These data demonstrate that different RSV strains of the same antigenic subgroup can elicit differential immune responses that impact the phenotypic expression of RSV-induced illness.
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Hiperreactividad Bronquial/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/inmunología , Animales , Hiperreactividad Bronquial/patología , Hiperreactividad Bronquial/fisiopatología , Hiperreactividad Bronquial/virología , Línea Celular , Relación Dosis-Respuesta Inmunológica , Regulación de la Expresión Génica/inmunología , Células Caliciformes/inmunología , Células Caliciformes/patología , Células Caliciformes/virología , Hiperplasia/inmunología , Hiperplasia/patología , Hiperplasia/fisiopatología , Hiperplasia/virología , Interleucina-13/deficiencia , Interleucina-13/inmunología , Ratones , Ratones Noqueados , Mucina 5AC , Mucinas/inmunología , Infecciones por Virus Sincitial Respiratorio/patología , Infecciones por Virus Sincitial Respiratorio/fisiopatología , Especificidad de la EspecieRESUMEN
Hantavirus pulmonary syndrome (HPS) is an acute disease resulting from infection with any one of a number of New World hantaviruses. HPS has a mortality rate of 40% and, unlike many other severe respiratory diseases, often occurs in young, healthy adults. Infection is usually initiated after inhalation of rodent excreta containing virus particles, but human-to-human transmission has been documented. Postmortem tissue samples show high levels of viral antigen within the respiratory endothelium, but it is not clear how the virus can traverse the respiratory epithelium in order to initiate infection in the microvasculature. We have utilized Andes virus infection of primary, differentiated airway epithelial cells to investigate the ability of the virus to interact with and cross the respiratory epithelium. Andes virus infects the Clara and goblet cell populations but not the ciliated cells, and this infection pattern corresponds to the expression of beta(3) integrin, the viral receptor. The virus can infect via the apical or basolateral membrane, and progeny virus particles are secreted bidirectionally. There is no obvious cytopathology associated with infection, and beta(3) integrins do not appear to be critical for respiratory epithelial cell monolayer integrity. Our data suggest that hantavirus infection of the respiratory epithelium may play an important role in the early or prodrome phase of disease as well as serving as a source of virus involved in transmission.
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Síndrome Pulmonar por Hantavirus/etiología , Síndrome Pulmonar por Hantavirus/virología , Orthohantavirus/patogenicidad , Sistema Respiratorio/virología , Animales , Secuencia de Bases , Células Cultivadas , Cricetinae , ADN Viral/genética , Células Epiteliales/patología , Células Epiteliales/virología , Células Caliciformes/patología , Células Caliciformes/virología , Orthohantavirus/genética , Orthohantavirus/fisiología , Humanos , Integrina beta3/metabolismo , Ratones , Especificidad de Órganos , Sistema Respiratorio/patología , Tráquea/patología , Tráquea/virología , Replicación ViralRESUMEN
Rotaviruses are the leading cause of severe viral gastroenteritis in young children. To gain insight in goblet cell homeostasis and intestinal mucin expression during rotavirus infection, 6-day-old mice were inoculated with murine rotavirus. To determine epithelial cell migration, mice were injected with BrdU just before inoculation. Small intestines were isolated at different days postinfection (dpi) and evaluated for rotavirus and goblet cell-specific gene expression. Small intestinal mucins of control and infected animals at 1, 2, and 4 dpi were isolated and tested for their capability to neutralize rotavirus infection in vitro. After inoculation, two peaks of viral replication were observed at 1 and 4 dpi. During infection, the number of goblet cells in infected mice was decreased in duodenum and jejunum, but was unaffected in the ileum. Goblet cells in infected animals accumulated at the tips of the villi. Muc2 mRNA levels were increased during the peak of viral replication at 1 dpi, whereas at other time points Muc2 and Tff3 mRNA levels were maintained at control levels. Muc2 protein levels in the tissue were also maintained, however Tff3 protein levels were strongly decreased. The number of goblet cells containing sulfated mucins was reduced during the two peaks of infection. Mucins isolated at 1 and 2 dpi from control and infected mice efficiently neutralized rotavirus infection in vitro. Moreover, mucins isolated from infected mice at 4 dpi were more potent in inhibiting rotavirus infection than mucins from control mice at 4 dpi. In conclusion, these data show that during rotavirus infection, goblet cells, in contrast to enterocytes, are relatively spared from apoptosis especially in the ileum. Goblet cell-specific Muc2 expression is increased and mucin structure is modified in the course of infection. This suggests that goblet cells and mucins play a role in the active defense against rotavirus infection and that age-dependent differences in mucin quantities, composition, and/or structure alter the anti-viral capabilities of small intestinal mucins.
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Células Caliciformes/patología , Células Caliciformes/fisiología , Infecciones por Rotavirus/patología , Animales , Movimiento Celular , Modelos Animales de Enfermedad , Células Caliciformes/virología , Homeostasis , Íleon/patología , Íleon/virología , Mucosa Intestinal/patología , Mucosa Intestinal/virología , Yeyuno/patología , Yeyuno/virología , Ratones , Infecciones por Rotavirus/virologíaRESUMEN
Most infections with respiratory viruses induce Th1 responses characterized by the generation of Th1 and CD8(+) T cells secreting IFN-gamma, which in turn have been shown to inhibit the development of Th2 cells. Therefore, it could be expected that respiratory viral infections mediate protection against asthma. However, the opposite seems to be true, because viral infections are often associated with the exacerbation of asthma. For this reason, we investigated what effect an influenza A (flu) virus infection has on the development of asthma. We found that flu infection 1, 3, 6, or 9 wk before allergen airway challenge resulted in a strong suppression of allergen-induced airway eosinophilia. This effect was associated with strongly reduced numbers of Th2 cells in the airways and was not observed in IFN-gamma- or IL-12 p35-deficient mice. Mice infected with flu virus and immunized with OVA showed decreased IL-5 and increased IFN-gamma, eotaxin/CC chemokine ligand (CCL)11, RANTES/CCL5, and monocyte chemoattractant protein-1/CCL2 levels in the bronchoalveolar lavage fluid, and increased airway hyperreactivity compared with OVA-immunized mice. These results suggest that the flu virus infection reduced airway eosinophilia by inducing Th1 responses, which lead to the inefficient recruitment of Th2 cells into the airways. However, OVA-specific IgE and IgG1 serum levels, blood eosinophilia, and goblet cell metaplasia in the lung were not reduced by the flu infection. Flu virus infection also directly induced AHR and goblet cell metaplasia. Taken together, our results show that flu virus infections can induce, exacerbate, and suppress features of asthmatic disease in mice.