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Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. We established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We then validated differences in key pathways through functional studies and determined whether these cultures recapitulate known features of the infant intestinal epithelium. RNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell, and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. HIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex vivo model to advance studies of infant-specific diseases and drug discovery for this population. IMPORTANCE: Tissue or biopsy stem cell-derived human intestinal enteroids are increasingly recognized as physiologically relevant models of the human gastrointestinal epithelium. While enteroids from adults and fetal tissues have been extensively used for studying many infectious and non-infectious diseases, there are few reports on enteroids from infants. We show that infant enteroids exhibit both transcriptomic and morphological differences compared to adult cultures. They also differ in functional responses to barrier disruption and innate immune responses to infection, suggesting that infant and adult enteroids are distinct model systems. Considering the dramatic changes in body composition and physiology that begin during infancy, tools that appropriately reflect intestinal development and diseases are critical. Infant enteroids exhibit key features of the infant gastrointestinal epithelium. This study is significant in establishing infant enteroids as age-appropriate models for infant intestinal physiology, infant-specific diseases, and responses to pathogens.
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Mucosa Intestinal , Humanos , Lactante , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Adulto , Diferenciación Celular , Yeyuno/citología , Yeyuno/inmunología , Transcriptoma , Organoides , Inmunidad Innata , Femenino , Masculino , Recién Nacido , EnterocitosRESUMEN
Respiratory syncytial virus (RSV) is a common cause of respiratory infections, causing significant morbidity and mortality, especially in young children. Why RSV infection in children is more severe as compared to healthy adults is not fully understood. In the present study, we infect both pediatric and adult human nose organoid-air liquid interface (HNO-ALIs) cell lines with two contemporary RSV isolates and demonstrate how they differ in virus replication, induction of the epithelial cytokine response, cell injury, and remodeling. Pediatric HNO-ALIs were more susceptible to early RSV replication, elicited a greater overall cytokine response, demonstrated enhanced mucous production, and manifested greater cellular damage compared to their adult counterparts. Adult HNO-ALIs displayed enhanced mucus production and robust cytokine response that was well controlled by superior regulatory cytokine response and possibly resulted in lower cellular damage than in pediatric lines. Taken together, our data suggest substantial differences in how pediatric and adult upper respiratory tract epithelium responds to RSV infection. These differences in epithelial cellular response can lead to poor mucociliary clearance and predispose infants to a worse respiratory outcome of RSV infection.
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Background & Aims: Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. Methods: We established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We validated differences in key pathways through functional studies and determined if these cultures recapitulate known features of the infant intestinal epithelium. Results: RNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. Conclusions: HIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex-vivo model to advance studies of infant-specific diseases and drug discovery for this population.
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Cronkhite-Canada Syndrome (CCS) is a rare, noninherited polyposis syndrome affecting 1 in every million individuals. Despite over 50 years of CCS cases, the etiopathogenesis and optimal treatment for CCS remains unknown due to the rarity of the disease and lack of model systems. To better understand the etiology of CCS, we generated human intestinal organoids (HIOs) from intestinal stem cells isolated from 2 patients. We discovered that CCS HIOs are highly proliferative and have increased numbers of enteroendocrine cells producing serotonin (also known as 5-hydroxytryptamine or 5HT). These features were also confirmed in patient tissue biopsies. Recombinant 5HT increased proliferation of non-CCS donor HIOs and inhibition of 5HT production in the CCS HIOs resulted in decreased proliferation, suggesting a link between local epithelial 5HT production and control of epithelial stem cell proliferation. This link was confirmed in genetically engineered HIOs with an increased number of enteroendocrine cells. This work provides a new mechanism to explain the pathogenesis of CCS and illustrates the important contribution of HIO cultures to understanding disease etiology and in the identification of novel therapies. Our work demonstrates the principle of using organoids for personalized medicine and sheds light on how intestinal hormones can play a role in intestinal epithelial proliferation.
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Neoplasias Colorrectales , Poliposis Intestinal , Humanos , Serotonina , Intestinos , Organoides/patología , Neoplasias Colorrectales/patología , Poliposis Intestinal/genética , Poliposis Intestinal/patologíaRESUMEN
The mammalian gut secretes a family of multifunctional peptides that affect appetite, intestinal secretions, and motility whereas others regulate the microbiota. We have found that peptide YY (PYY1-36), but not endocrine PYY3-36, acts as an antimicrobial peptide (AMP) expressed by gut epithelial paneth cells (PC). PC-PYY is packaged into secretory granules and is secreted into and retained by surface mucus, which optimizes PC-PYY activity. Although PC-PYY shows some antibacterial activity, it displays selective antifungal activity against virulent Candida albicans hyphae-but not the yeast form. PC-PYY is a cationic molecule that interacts with the anionic surfaces of fungal hyphae to cause membrane disruption and transcriptional reprogramming that selects for the yeast phenotype. Hence, PC-PYY is an antifungal AMP that contributes to the maintenance of gut fungal commensalism.
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Antifúngicos , Péptidos Antimicrobianos , Candida , Células de Paneth , Fragmentos de Péptidos , Péptido YY , Animales , Antifúngicos/metabolismo , Péptidos Antimicrobianos/metabolismo , Candida/efectos de los fármacos , Candida/fisiología , Células de Paneth/metabolismo , Fragmentos de Péptidos/metabolismo , Péptido YY/metabolismo , Simbiosis , Humanos , RatonesRESUMEN
We describe the epidemiology and clinical characteristics of 29 patients with cancer and diarrhea in whom Enteroaggregative Escherichia coli (EAEC) was initially identified by GI BioFire panel multiplex. E. coli strains were successfully isolated from fecal cultures in 14 of 29 patients. Six of the 14 strains were identified as EAEC and 8 belonged to other diverse E. coli groups of unknown pathogenesis. We investigated these strains by their adherence to human intestinal organoids, cytotoxic responses, antibiotic resistance profile, full sequencing of their genomes, and annotation of their functional virulome. Interestingly, we discovered novel and enhanced adherence and aggregative patterns for several diarrheagenic pathotypes that were not previously seen when co-cultured with immortalized cell lines. EAEC isolates displayed exceptional adherence and aggregation to human colonoids compared not only to diverse GI E. coli , but also compared to prototype strains of other diarrheagenic E. coli . Some of the diverse E. coli strains that could not be classified as a conventional pathotype also showed an enhanced aggregative and cytotoxic response. Notably, we found a high carriage rate of antibiotic resistance genes in both EAEC strains and diverse GI E. coli isolates and observed a positive correlation between adherence to colonoids and the number of metal acquisition genes carried in both EAEC and the diverse E. coli strains. This work indicates that E. coli from cancer patients constitute strains of remarkable pathotypic and genomic divergence, including strains of unknown disease etiology with unique virulomes. Future studies will allow for the opportunity to re-define E. coli pathotypes with greater diagnostic accuracy and into more clinically relevant groupings.
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Gut microbial diurnal oscillations are important diet-dependent drivers of host circadian rhythms and metabolism ensuring optimal energy balance. However, the interplay between diet, microbes, and host factors sustaining intestinal oscillations is complex and poorly understood. Here, using a mouse model, we report the host C-type lectin antimicrobial peptide Reg3γ works with key ileal microbes to orchestrate these interactions in a bidirectional manner and does not correlate with the intestinal core circadian clock. High-fat diet is the primary driver of microbial oscillators that impair host metabolic homeostasis, resulting in arrhythmic host Reg3γ expression that secondarily drives abundance and oscillation of key gut microbes. This illustrates transkingdom coordination of biological rhythms primarily influenced by diet and reciprocal sensor-effector signals between host and microbial components, ultimately driving metabolism. Restoring the gut microbiota's capacity to sense dietary signals mediated by specific host factors such as Reg3γ could be harnessed to improve metabolic dysfunction.
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Relojes Circadianos , Microbioma Gastrointestinal , Ritmo Circadiano , Dieta , Dieta Alta en Grasa/efectos adversos , Metabolismo de los LípidosRESUMEN
Elevated risk of developing Alzheimer's disease (AD) is associated with hypomorphic variants of TREM2, a surface receptor required for microglial responses to neurodegeneration, including proliferation, survival, clustering, and phagocytosis. How TREM2 promotes such diverse responses is unknown. Here, we find that microglia in AD patients carrying TREM2 risk variants and TREM2-deficient mice with AD-like pathology have abundant autophagic vesicles, as do TREM2-deficient macrophages under growth-factor limitation or endoplasmic reticulum (ER) stress. Combined metabolomics and RNA sequencing (RNA-seq) linked this anomalous autophagy to defective mammalian target of rapamycin (mTOR) signaling, which affects ATP levels and biosynthetic pathways. Metabolic derailment and autophagy were offset in vitro through Dectin-1, a receptor that elicits TREM2-like intracellular signals, and cyclocreatine, a creatine analog that can supply ATP. Dietary cyclocreatine tempered autophagy, restored microglial clustering around plaques, and decreased plaque-adjacent neuronal dystrophy in TREM2-deficient mice with amyloid-ß pathology. Thus, TREM2 enables microglial responses during AD by sustaining cellular energetic and biosynthetic metabolism.
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Enfermedad de Alzheimer/patología , Metabolismo Energético , Glicoproteínas de Membrana/metabolismo , Microglía/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Autofagia , Creatinina/análogos & derivados , Creatinina/metabolismo , Modelos Animales de Enfermedad , Humanos , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Microglía/patología , Neuritas/metabolismo , Placa Amiloide/metabolismo , Receptores Inmunológicos/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Inflammatory bowel disease (IBD) is associated with risk variants in the human genome and dysbiosis of the gut microbiome, though unifying principles for these findings remain largely undescribed. The human commensal Bacteroides fragilis delivers immunomodulatory molecules to immune cells via secretion of outer membrane vesicles (OMVs). We reveal that OMVs require IBD-associated genes, ATG16L1 and NOD2, to activate a noncanonical autophagy pathway during protection from colitis. ATG16L1-deficient dendritic cells do not induce regulatory T cells (T(regs)) to suppress mucosal inflammation. Immune cells from human subjects with a major risk variant in ATG16L1 are defective in T(reg) responses to OMVs. We propose that polymorphisms in susceptibility genes promote disease through defects in "sensing" protective signals from the microbiome, defining a potentially critical gene-environment etiology for IBD.
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Bacteroides fragilis/inmunología , Proteínas Portadoras/genética , Microbioma Gastrointestinal/inmunología , Interacción Gen-Ambiente , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/microbiología , Proteína Adaptadora de Señalización NOD2/genética , Adulto , Anciano , Animales , Autofagia/inmunología , Proteínas Relacionadas con la Autofagia , Células Dendríticas/inmunología , Vesículas Extracelulares/inmunología , Femenino , Predisposición Genética a la Enfermedad , Genoma Humano , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Polimorfismo Genético , Linfocitos T Reguladores/inmunologíaRESUMEN
Immune responses differ between laboratory mice and humans. Chronic infection with viruses and parasites are common in humans, but are absent in laboratory mice, and thus represent potential contributors to inter-species differences in immunity. To test this, we sequentially infected laboratory mice with herpesviruses, influenza, and an intestinal helminth and compared their blood immune signatures to mock-infected mice before and after vaccination against yellow fever virus (YFV-17D). Sequential infection altered pre- and post-vaccination gene expression, cytokines, and antibodies in blood. Sequential pathogen exposure induced gene signatures that recapitulated those seen in blood from pet store-raised versus laboratory mice, and adult versus cord blood in humans. Therefore, basal and vaccine-induced murine immune responses are altered by infection with agents common outside of barrier facilities. This raises the possibility that we can improve mouse models of vaccination and immunity by selective microbial exposure of laboratory animals to mimic that of humans.
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Helmintiasis/inmunología , Infecciones por Herpesviridae/inmunología , Herpesviridae/inmunología , Parasitosis Intestinales/inmunología , Vacuna contra la Fiebre Amarilla/inmunología , Fiebre Amarilla/inmunología , Fiebre Amarilla/prevención & control , Virus de la Fiebre Amarilla/inmunología , Animales , Anticuerpos/sangre , Anticuerpos Antivirales/inmunología , Coinfección/inmunología , Coinfección/parasitología , Coinfección/virología , Citocinas/sangre , Modelos Animales de Enfermedad , Sangre Fetal/inmunología , Expresión Génica , Helmintiasis/prevención & control , Helmintiasis/virología , Infecciones por Herpesviridae/prevención & control , Humanos , Inmunidad Innata , Inmunoglobulina G/sangre , Gripe Humana/inmunología , Gripe Humana/prevención & control , Parasitosis Intestinales/prevención & control , Parasitosis Intestinales/virología , Ratones , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/parasitología , Infecciones por Orthomyxoviridae/prevención & control , Fiebre Amarilla/parasitología , Fiebre Amarilla/virología , Vacuna contra la Fiebre Amarilla/farmacologíaRESUMEN
CLEC16A is in a locus genetically linked to autoimmune diseases including multiple sclerosis, but the function of this gene in the nervous system is unknown. Here we show that two mouse strains carrying independent Clec16a mutations developed neurodegenerative disease characterized by motor impairments and loss of Purkinje cells. Neurons from Clec16a-mutant mice exhibited increased expression of the autophagy substrate p62, accumulation of abnormal intra-axonal membranous structures bearing the autophagy protein LC3, and abnormal Golgi morphology. Multiple aspects of endocytosis, lysosome and Golgi function were normal in Clec16a-deficient murine embryonic fibroblasts and HeLa cells. However, these cells displayed abnormal bulk autophagy despite unimpaired autophagosome formation. Cultured Clec16a-deficient cells exhibited a striking accumulation of LC3 and LAMP-1 positive autolysosomes containing undigested cytoplasmic contents. Therefore Clec16a, an autophagy protein that is critical for autolysosome function and clearance, is required for Purkinje cell survival.
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Lectinas Tipo C/genética , Lisosomas/fisiología , Proteínas de Transporte de Monosacáridos/genética , Enfermedad de la Neurona Motora/patología , Mutación , Células de Purkinje/citología , Animales , Autofagia , Supervivencia Celular , Células Cultivadas , Aparato de Golgi/patología , Células HeLa , Humanos , Lectinas Tipo C/metabolismo , Ratones , Proteínas de Transporte de Monosacáridos/metabolismo , Enfermedad de la Neurona Motora/genéticaRESUMEN
Mutations in the autophagy gene EPG5 are linked to the multisystem human disease Vici syndrome, which is characterized in part by pulmonary abnormalities, including recurrent infections. We found that Epg5-deficient mice exhibited elevated baseline innate immune cellular and cytokine-based lung inflammation and were resistant to lethal influenza virus infection. Lung transcriptomics, bone marrow transplantation experiments, and analysis of cellular cytokine expression indicated that Epg5 plays a role in lung physiology through its function in macrophages. Deletion of other autophagy genes including Atg14, Fip200, Atg5, and Atg7 in myeloid cells also led to elevated basal lung inflammation and influenza resistance. This suggests that Epg5 and other Atg genes function in macrophages to limit innate immune inflammation in the lung. Disruption of this normal homeostatic dampening of lung inflammation results in increased resistance to influenza, suggesting that normal homeostatic mechanisms that limit basal tissue inflammation support some infectious diseases.
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Inmunidad Innata , Subtipo H1N1 del Virus de la Influenza A/fisiología , Gripe Humana/inmunología , Neumonía/inmunología , Proteínas/inmunología , Animales , Proteína 7 Relacionada con la Autofagia , Proteínas Relacionadas con la Autofagia , Homeostasis , Humanos , Gripe Humana/genética , Gripe Humana/virología , Macrófagos/inmunología , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/inmunología , Neumonía/genética , Neumonía/virología , Proteínas/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/inmunologíaRESUMEN
Mycobacterium tuberculosis, a major global health threat, replicates in macrophages in part by inhibiting phagosome-lysosome fusion, until interferon-γ (IFNγ) activates the macrophage to traffic M. tuberculosis to the lysosome. How IFNγ elicits this effect is unknown, but many studies suggest a role for macroautophagy (herein termed autophagy), a process by which cytoplasmic contents are targeted for lysosomal degradation. The involvement of autophagy has been defined based on studies in cultured cells where M. tuberculosis co-localizes with autophagy factors ATG5, ATG12, ATG16L1, p62, NDP52, BECN1 and LC3 (refs 2-6), stimulation of autophagy increases bacterial killing, and inhibition of autophagy increases bacterial survival. Notably, these studies reveal modest (~1.5-3-fold change) effects on M. tuberculosis replication. By contrast, mice lacking ATG5 in monocyte-derived cells and neutrophils (polymorponuclear cells, PMNs) succumb to M. tuberculosis within 30 days, an extremely severe phenotype similar to mice lacking IFNγ signalling. Importantly, ATG5 is the only autophagy factor that has been studied during M. tuberculosis infection in vivo and autophagy-independent functions of ATG5 have been described. For this reason, we used a genetic approach to elucidate the role for multiple autophagy-related genes and the requirement for autophagy in resistance to M. tuberculosis infection in vivo. Here we show that, contrary to expectation, autophagic capacity does not correlate with the outcome of M. tuberculosis infection. Instead, ATG5 plays a unique role in protection against M. tuberculosis by preventing PMN-mediated immunopathology. Furthermore, while Atg5 is dispensable in alveolar macrophages during M. tuberculosis infection, loss of Atg5 in PMNs can sensitize mice to M. tuberculosis. These findings shift our understanding of the role of ATG5 during M. tuberculosis infection, reveal new outcomes of ATG5 activity, and shed light on early events in innate immunity that are required to regulate disease pathology and bacterial replication.
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Proteínas Asociadas a Microtúbulos/metabolismo , Mycobacterium tuberculosis , Neutrófilos/inmunología , Tuberculosis/inmunología , Tuberculosis/patología , Animales , Autofagia/genética , Proteína 5 Relacionada con la Autofagia , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Inmunidad Innata/inmunología , Interferón gamma/deficiencia , Interferón gamma/inmunología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/deficiencia , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/fisiología , Neutrófilos/metabolismo , Tuberculosis/microbiologíaRESUMEN
Decreases in the diversity of enteric bacterial populations are observed in patients with Crohn's disease (CD) and ulcerative colitis (UC). Less is known about the virome in these diseases. We show that the enteric virome is abnormal in CD and UC patients. In-depth analysis of preparations enriched for free virions in the intestine revealed that CD and UC were associated with a significant expansion of Caudovirales bacteriophages. The viromes of CD and UC patients were disease and cohort specific. Importantly, it did not appear that expansion and diversification of the enteric virome was secondary to changes in bacterial populations. These data support a model in which changes in the virome may contribute to intestinal inflammation and bacterial dysbiosis. We conclude that the virome is a candidate for contributing to, or being a biomarker for, human inflammatory bowel disease and speculate that the enteric virome may play a role in other diseases.
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Caudovirales/aislamiento & purificación , Colitis Ulcerosa/virología , Enfermedad de Crohn/virología , Disbiosis/virología , Microviridae/aislamiento & purificación , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Estudios de Casos y Controles , Caudovirales/genética , Estudios de Cohortes , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/patología , Colitis Ulcerosa/terapia , Enfermedad de Crohn/microbiología , Enfermedad de Crohn/patología , Enfermedad de Crohn/terapia , Disbiosis/microbiología , Disbiosis/patología , Disbiosis/terapia , Heces/microbiología , Heces/virología , Humanos , Metagenoma , Microviridae/genéticaRESUMEN
Variation in the presentation of hereditary immunodeficiencies may be explained by genetic or environmental factors. Patients with mutations in HOIL1 (RBCK1) present with amylopectinosis-associated myopathy with or without hyper-inflammation and immunodeficiency. We report that barrier-raised HOIL-1-deficient mice exhibit amylopectin-like deposits in the myocardium but show minimal signs of hyper-inflammation. However, they show immunodeficiency upon acute infection with Listeria monocytogenes, Toxoplasma gondii or Citrobacter rodentium. Increased susceptibility to Listeria was due to HOIL-1 function in hematopoietic cells and macrophages in production of protective cytokines. In contrast, HOIL-1-deficient mice showed enhanced control of chronic Mycobacterium tuberculosis or murine γ-herpesvirus 68 (MHV68), and these infections conferred a hyper-inflammatory phenotype. Surprisingly, chronic infection with MHV68 complemented the immunodeficiency of HOIL-1, IL-6, Caspase-1 and Caspase-1;Caspase-11-deficient mice following Listeria infection. Thus chronic herpesvirus infection generates signs of auto-inflammation and complements genetic immunodeficiency in mutant mice, highlighting the importance of accounting for the virome in genotype-phenotype studies.
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Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/patología , Herpesviridae/fisiología , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/virología , Enfermedad Aguda , Animales , Células de la Médula Ósea/citología , Caspasa 1/metabolismo , Compartimento Celular , Enfermedad Crónica , Citrobacter/fisiología , Citocinas/biosíntesis , Prueba de Complementación Genética , Infecciones por Herpesviridae/virología , Humanos , Inmunidad Innata , Inflamación/patología , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Listeria monocytogenes/fisiología , Listeriosis/inmunología , Listeriosis/microbiología , Listeriosis/patología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Mycobacterium tuberculosis/fisiología , Fenotipo , Rhadinovirus/fisiología , Toxoplasma , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The capacity of human norovirus (NoV), which causes >90% of global epidemic nonbacterial gastroenteritis, to infect a subset of people persistently may contribute to its spread. How such enteric viruses establish persistent infections is not well understood. We found that antibiotics prevented persistent murine norovirus (MNoV) infection, an effect that was reversed by replenishment of the bacterial microbiota. Antibiotics did not prevent tissue infection or affect systemic viral replication but acted specifically in the intestine. The receptor for the antiviral cytokine interferon-λ, Ifnlr1, as well as the transcription factors Stat1 and Irf3, were required for antibiotics to prevent viral persistence. Thus, the bacterial microbiome fosters enteric viral persistence in a manner counteracted by specific components of the innate immune system.
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Infecciones por Caliciviridae/virología , Citocinas/fisiología , Gastroenteritis/virología , Intestinos/microbiología , Microbiota , Norovirus/fisiología , Simbiosis , Animales , Antibacterianos/farmacología , Infecciones por Caliciviridae/tratamiento farmacológico , Infecciones por Caliciviridae/inmunología , Infecciones por Caliciviridae/microbiología , Femenino , Gastroenteritis/tratamiento farmacológico , Gastroenteritis/inmunología , Gastroenteritis/microbiología , Intestinos/virología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Microbiota/efectos de los fármacos , Norovirus/inmunología , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Transducción de Señal , Carga Viral , Replicación Viral , Esparcimiento de VirusRESUMEN
Pervasive transcription is observed in a wide range of organisms, including humans, mice, and viruses, but the functional significance of the resulting transcripts remains uncertain. Current genetic approaches are often limited by their emphasis on protein-coding open reading frames (ORFs). We previously identified extensive pervasive transcription from the murine gammaherpesvirus 68 (MHV68) genome outside known ORFs and antisense to known genes (termed expressed genomic regions [EGRs]). Similar antisense transcripts have been identified in many other herpesviruses, including Kaposi's sarcoma-associated herpesvirus and human and murine cytomegalovirus. Despite their prevalence, whether these RNAs have any functional importance in the viral life cycle is unknown, and one interpretation is that these are merely "noise" generated by functionally unimportant transcriptional events. To determine whether pervasive transcription of a herpesvirus genome generates RNA molecules that are functionally important, we used a strand-specific functional approach to target transcripts from thirteen EGRs in MHV68. We found that targeting transcripts from six EGRs reduced viral protein expression, proving that pervasive transcription can generate functionally important RNAs. We characterized transcripts emanating from EGRs 26 and 27 in detail using several methods, including RNA sequencing, and identified several novel polyadenylated transcripts that were enriched in the nuclei of infected cells. These data provide the first evidence of the functional importance of regions of pervasive transcription emanating from MHV68 EGRs. Therefore, studies utilizing mutation of a herpesvirus genome must account for possible effects on RNAs generated by pervasive transcription. IMPORTANCE The fact that pervasive transcription produces functionally important RNAs has profound implications for design and interpretation of genetic studies in herpesviruses, since such studies often involve mutating both strands of the genome. This is a common potential problem; for example, a conservative estimate is that there are an additional 73,000 nucleotides transcribed antisense to annotated ORFs from the 119,450-bp MHV68 genome. Recognizing the importance of considering the function of each strand of the viral genome independently, we used strand-specific approaches to identify six regions of the genome encoding transcripts that promoted viral protein expression. For two of these regions, we mapped novel transcripts and determined that targeting transcripts from these regions reduced viral replication and the expression of other viral genes. This is the first description of a function for these RNAs and suggests that novel transcripts emanating from regions of pervasive transcription are critical for the viral life cycle.
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ARN Viral/biosíntesis , Rhadinovirus/fisiología , Transcripción Genética , Animales , Ratones , Rhadinovirus/genéticaRESUMEN
Human noroviruses (HuNoV) are the major cause of epidemic, nonbacterial gastroenteritis in the world. The short course of HuNoV-induced symptoms has implicated innate immunity in control of norovirus (NoV) infection. Studies using murine norovirus (MNV) confirm the importance of innate immune responses during NoV infection. Type I alpha and beta interferons (IFN-α/ß) limit HuNoV replicon function, restrict MNV replication in cultured cells, and control MNV replication in vivo. Therefore, the cell types and transcription factors involved in antiviral immune responses and IFN-α/ß-mediated control of NoV infection are important to define. We used mice with floxed alleles of the IFNAR1 chain of the IFN-α/ß receptor to identify cells expressing lysozyme M or CD11c as cells that respond to IFN-α/ß to restrict MNV replication in vivo. Furthermore, we show that the transcription factors IRF-3 and IRF-7 work in concert to initiate unique and overlapping antiviral responses to restrict MNV replication in vivo. IRF-3 and IRF-7 restrict MNV replication in both cultured macrophages and dendritic cells, are required for induction of IFN-α/ß in macrophages but not dendritic cells, and are dispensable for the antiviral effects of IFN-α/ß that block MNV replication. These studies suggest that expression of the IFN-α/ß receptor on macrophages/neutrophils and dendritic cells, as well as of IRF-3 and IRF-7, is critical for innate immune responses to NoV infection.
Asunto(s)
Factor 3 Regulador del Interferón/fisiología , Factor 7 Regulador del Interferón/fisiología , Interferón Tipo I/fisiología , Norovirus/fisiología , Replicación Viral/fisiología , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Células Dendríticas/inmunología , Inmunidad Innata , Macrófagos/inmunología , Ratones , Norovirus/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Pathogenic simian immunodeficiency virus (SIV) infection is associated with enteropathy, which likely contributes to AIDS progression. To identify candidate etiologies for AIDS enteropathy, we used next-generation sequencing to define the enteric virome during SIV infection in nonhuman primates. Pathogenic, but not nonpathogenic, SIV infection was associated with significant expansion of the enteric virome. We identified at least 32 previously undescribed enteric viruses during pathogenic SIV infection and confirmed their presence by using viral culture and PCR testing. We detected unsuspected mucosal adenovirus infection associated with enteritis as well as parvovirus viremia in animals with advanced AIDS, indicating the pathogenic potential of SIV-associated expansion of the enteric virome. No association between pathogenic SIV infection and the family-level taxonomy of enteric bacteria was detected. Thus, enteric viral infections may contribute to AIDS enteropathy and disease progression. These findings underline the importance of metagenomic analysis of the virome for understanding AIDS pathogenesis.
Asunto(s)
Caliciviridae/aislamiento & purificación , Intestinos/virología , Parvoviridae/aislamiento & purificación , Picornaviridae/aislamiento & purificación , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Animales , Caliciviridae/clasificación , Caliciviridae/genética , Chlorocebus aethiops , Heces/microbiología , Heces/virología , Intestinos/microbiología , Datos de Secuencia Molecular , Parvoviridae/clasificación , Parvoviridae/genética , Filogenia , Picornaviridae/clasificación , Picornaviridae/genética , Reacción en Cadena de la Polimerasa , Síndrome de Inmunodeficiencia Adquirida del Simio/microbiología , Virus de la Inmunodeficiencia de los Simios/patogenicidadRESUMEN
There is much interest in the use of mesenchymal stem cells/marrow stromal cells (MSC) to treat neurodegenerative disorders, in particular those that are fatal and difficult to treat, such as Huntington's disease. MSC present a promising tool for cell therapy and are currently being tested in FDA-approved phase I-III clinical trials for many disorders. In preclinical studies of neurodegenerative disorders, MSC have demonstrated efficacy, when used as delivery vehicles for neural growth factors. A number of investigators have examined the potential benefits of innate MSC-secreted trophic support and augmented growth factors to support injured neurons. These include overexpression of brain-derived neurotrophic factor and glial-derived neurotrophic factor, using genetically engineered MSC as a vehicle to deliver the cytokines directly into the microenvironment. Proposed regenerative approaches to neurological diseases using MSC include cell therapies in which cells are delivered via intracerebral or intrathecal injection. Upon transplantation, MSC in the brain promote endogenous neuronal growth, encourage synaptic connection from damaged neurons, decrease apoptosis, reduce levels of free radicals, and regulate inflammation. These abilities are primarily modulated through paracrine actions. Clinical trials for MSC injection into the central nervous system to treat amyotrophic lateral sclerosis, traumatic brain injury, and stroke are currently ongoing. The current data in support of applying MSC-based cellular therapies to the treatment of Huntington's disease is discussed.