RESUMEN
Airway epithelial cells and macrophages differ markedly in their responses to influenza A virus (IAV) infection. To investigate transcriptional responses underlying these differences, purified subsets of type II airway epithelial cells (ATII) and alveolar macrophages (AM) recovered from the lungs of mock- or IAV-infected mice at 9 h postinfection were subjected to RNA sequencing. This time point was chosen to allow for characterization of cell types first infected with the virus inoculum, prior to multicycle virus replication and the infiltration of inflammatory cells into the airways. In the absence of infection, AM predominantly expressed genes related to immunity, whereas ATII expressed genes consistent with their physiological roles in the lung. Following IAV infection, AM almost exclusively activated cell-intrinsic antiviral pathways that were dependent on interferon (IFN) regulatory factor 3/7 (IRF3/7) and/or type I IFN signaling. In contrast, IAV-infected ATII activated a broader range of physiological responses, including cell-intrinsic antiviral pathways, which were both independent of and dependent on IRF3/7 and/or type I IFN. These data suggest that transcriptional profiles hardwired during development are a major determinant underlying the different responses of ATII and AM to IAV infection.IMPORTANCE Airway epithelial cells (AEC) and airway macrophages (AM) represent major targets of influenza A virus (IAV) infection in the lung, yet the two cell types respond very differently to IAV infection. We have used RNA sequencing to define the host transcriptional responses in each cell type under steady-state conditions as well as following IAV infection. To do this, different cell subsets isolated from the lungs of mock- and IAV-infected mice were subjected to RNA sequencing. Under steady-state conditions, AM and AEC express distinct transcriptional activities, consistent with distinct physiological roles in the airways. Not surprisingly, these cells also exhibited major differences in transcriptional responses following IAV infection. These studies shed light on how the different transcriptional architectures of airway cells from two different lineages drive transcriptional responses to IAV infection.
Asunto(s)
Células Epiteliales/virología , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Pulmón/virología , Macrófagos/virología , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/virología , Animales , Línea Celular , Enfermedades Transmisibles/metabolismo , Enfermedades Transmisibles/virología , Perros , Células Epiteliales/metabolismo , Humanos , Gripe Humana/metabolismo , Gripe Humana/virología , Factor 3 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/metabolismo , Interferones/metabolismo , Pulmón/metabolismo , Macrófagos/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Replicación Viral/fisiologíaRESUMEN
Resolution of virus infections depends on the priming of virus-specific CD8+ T cells by dendritic cells (DC). While this process requires major histocompatibility complex (MHC) class I-restricted antigen presentation by DC, the relative contribution to CD8+ T cell priming by infected DC is less clear. We have addressed this question in the context of a peripheral infection with herpes simplex virus 1 (HSV). Assessing the endogenous, polyclonal HSV-specific CD8+ T cell response, we found that effective in vivo T cell priming depended on the presence of DC subsets specialized in cross-presentation, while Langerhans cells and plasmacytoid DC were dispensable. Utilizing a novel mouse model that allows for the in vivo elimination of infected DC, we also demonstrated in vivo that this requirement for cross-presenting DC was not related to their infection but instead reflected their capacity to cross-present HSV-derived antigen. Taking the results together, this study shows that infected DC are not required for effective CD8+ T cell priming during a peripheral virus infection.IMPORTANCE The ability of some DC to present viral antigen to CD8+ T cells without being infected is thought to enable the host to induce killer T cells even when viruses evade or kill infected DC. However, direct experimental in vivo proof for this notion has remained elusive. The work described in this study characterizes the role that different DC play in the induction of virus-specific killer T cell responses and, critically, introduces a novel mouse model that allows for the selective elimination of infected DC in vivo Our finding that HSV-specific CD8+ T cells can be fully primed in the absence of DC infection shows that cross-presentation by DC is indeed sufficient for effective CD8+ T cell priming during a peripheral virus infection.
Asunto(s)
Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Reactividad Cruzada , Células Dendríticas/inmunología , Herpes Simple/inmunología , Animales , Presentación de Antígeno , Linfocitos T CD8-positivos/citología , Células Dendríticas/citología , Citometría de Flujo , Herpesvirus Humano 1 , Ratones , Ratones Endogámicos C57BLRESUMEN
Lymph nodes (LNs) are constructed of intricate networks of endothelial and mesenchymal stromal cells. How these lymphoid stromal cells (LSCs) regulate lymphoid tissue remodeling and contribute to immune responses remains poorly understood. We performed a comprehensive functional and transcriptional analysis of LSC responses to skin viral infection and found that LSC subsets responded robustly, with different kinetics for distinct pathogens. Recruitment of cells to inflamed LNs induced LSC expansion, while B cells sustained stromal responses in an antigen-independent manner. Infection induced rapid transcriptional responses in LSCs. This transcriptional program was transient, returning to homeostasis within 1 month of infection, yet expanded fibroblastic reticular cell networks persisted for more than 3 months after infection, and this altered LN composition reduced the magnitude of LSC responses to subsequent heterologous infection. Our results reveal the complexity of LSC responses during infection and suggest that amplified networks of LN stromal cells support successive immune responses.
Asunto(s)
Ganglios Linfáticos/patología , Virosis/inmunología , Virosis/patología , Animales , Antígenos Virales/inmunología , Linfocitos B/inmunología , Proliferación Celular , Coinfección/inmunología , Regulación de la Expresión Génica , Cinética , Ratones Endogámicos C57BL , Células del Estroma/patología , Transcripción Genética , Virosis/genéticaRESUMEN
In recent years, various intervention strategies have reduced malaria morbidity and mortality, but further improvements probably depend upon development of a broadly protective vaccine. To better understand immune requirement for protection, we examined liver-stage immunity after vaccination with irradiated sporozoites, an effective though logistically difficult vaccine. We identified a population of memory CD8+ T cells that expressed the gene signature of tissue-resident memory T (Trm) cells and remained permanently within the liver, where they patrolled the sinusoids. Exploring the requirements for liver Trm cell induction, we showed that by combining dendritic cell-targeted priming with liver inflammation and antigen recognition on hepatocytes, high frequencies of Trm cells could be induced and these cells were essential for protection against malaria sporozoite challenge. Our study highlights the immune potential of liver Trm cells and provides approaches for their selective transfer, expansion, or depletion, which may be harnessed to control liver infections or autoimmunity.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Hígado/inmunología , Malaria/inmunología , Animales , Linfocitos T CD8-positivos/parasitología , Culicidae , Células Dendríticas/inmunología , Células Dendríticas/parasitología , Hepatocitos/inmunología , Hepatocitos/parasitología , Hígado/parasitología , Hepatopatías/inmunología , Hepatopatías/parasitología , Vacunas contra la Malaria/inmunología , Ratones , Plasmodium berghei/inmunología , Esporozoítos/inmunología , Esporozoítos/parasitología , Vacunación/métodosRESUMEN
Human skin contains various populations of memory T cells in permanent residence and in transit. Arguably, the best characterized of the skin subsets are the CD8(+) permanently resident memory T cells (TRM) expressing the integrin subunit, CD103. In order to investigate the remaining skin T cells, we isolated skin-tropic (CLA(+)) helper T cells, regulatory T cells, and CD8(+) CD103(-) T cells from skin and blood for RNA microarray analysis to compare the transcriptional profiles of these groups. We found that despite their common tropism, the T cells isolated from skin were transcriptionally distinct from blood-derived CLA(+) T cells. A shared pool of genes contributed to the skin/blood discrepancy, with substantial overlap in differentially expressed genes between each T cell subset. Gene set enrichment analysis further showed that the differential gene profiles of each human skin T cell subset were significantly enriched for previously identified TRM core signature genes. Our results support the hypothesis that human skin may contain additional TRM or TRM-like populations.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Piel/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Reguladores/metabolismo , Transcripción Genética , Adolescente , Adulto , Anciano , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos CD8/genética , Antígenos CD8/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunofenotipificación , Cadenas alfa de Integrinas/genética , Cadenas alfa de Integrinas/inmunología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Persona de Mediana Edad , Anotación de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de Órganos , Piel/citología , Piel/inmunología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunologíaRESUMEN
Herpes simplex viruses (HSV) are significant human pathogens that provide one of the best-described examples of viral latency and reactivation. HSV latency occurs in sensory neurons, being characterized by the absence of virus replication and only fragmentary evidence of protein production. In mouse models, HSV latency is especially stable but the detection of some lytic gene transcription and the ongoing presence of activated immune cells in latent ganglia have been used to suggest that this state is not entirely quiescent. Alternatively, these findings can be interpreted as signs of a low, but constant level of abortive reactivation punctuating otherwise silent latency. Using single cell analysis of transcription in mouse dorsal root ganglia, we reveal that HSV-1 latency is highly dynamic in the majority of neurons. Specifically, transcription from areas of the HSV genome associated with at least one viral lytic gene occurs in nearly two thirds of latently-infected neurons and more than half of these have RNA from more than one lytic gene locus. Further, bioinformatics analyses of host transcription showed that progressive appearance of these lytic transcripts correlated with alterations in expression of cellular genes. These data show for the first time that transcription consistent with lytic gene expression is a frequent event, taking place in the majority of HSV latently-infected neurons. Furthermore, this transcription is of biological significance in that it influences host gene expression. We suggest that the maintenance of HSV latency involves an active host response to frequent viral activity.
Asunto(s)
Herpes Simple/inmunología , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno/inmunología , Células Receptoras Sensoriales/inmunología , Latencia del Virus/inmunología , Liberación del Virus/inmunología , Animales , Modelos Animales de Enfermedad , Herpes Simple/patología , Humanos , Ratones , Ratones Transgénicos , Células Receptoras Sensoriales/patología , Células Receptoras Sensoriales/virologíaRESUMEN
Tissue-resident memory T cells (T(RM) cells) provide superior protection against infection in extralymphoid tissues. Here we found that CD103(+)CD8(+) T(RM) cells developed in the skin from epithelium-infiltrating precursor cells that lacked expression of the effector-cell marker KLRG1. A combination of entry into the epithelium plus local signaling by interleukin 15 (IL-15) and transforming growth factor-ß (TGF-ß) was required for the formation of these long-lived memory cells. Notably, differentiation into T(RM) cells resulted in the progressive acquisition of a unique transcriptional profile that differed from that of circulating memory cells and other types of T cells that permanently reside in skin epithelium. We provide a comprehensive molecular framework for the local differentiation of a distinct peripheral population of memory cells that forms a first-line immunological defense system in barrier tissues.
Asunto(s)
Antígenos CD/inmunología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Cadenas alfa de Integrinas/inmunología , Transducción de Señal/inmunología , Piel/inmunología , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos de Diferenciación de Linfocitos T/metabolismo , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Citometría de Flujo , Herpes Simple/inmunología , Herpes Simple/virología , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno/inmunología , Cadenas alfa de Integrinas/genética , Cadenas alfa de Integrinas/metabolismo , Interleucina-15/genética , Interleucina-15/inmunología , Interleucina-15/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Piel/metabolismo , Piel/virología , Transcriptoma/genética , Transcriptoma/inmunologíaRESUMEN
MicroRNA-224 (miR-224) is frequently over-expressed in liver and colorectal cancers. We and others have previously described the role of miR-224 over-expression in cell proliferation in vitro but we have yet to identify the relevant miR-224 direct target. In this study, we further demonstrated that miR-224 up-regulation promotes cell proliferation using both in vitro assays and in vivo tumor growth models. We systematically screened for high confidence miR-224 targets by overlapping in silico predicted targets from multiple algorithms and significantly down-regulated genes in miR-224-expressing cells from whole genome expression microarrays. A total of 72 high confidence miR-224 targets were identified and found to be enriched in various cancer-related processes. SMAD family member 4 (SMAD4) is experimentally validated as the direct cellular target through which miR-224 promotes cell proliferation. The clinical relevance of our experimental observations was supported by a statistically significant inverse correlation between miR-224 and SMAD4 transcript expression in tumor versus paired adjacent non-tumorous tissues from HCC patients (p<0.001, r=â-0.45, R(2)â=0.122). Furthermore, miR-224 up-regulation and SMAD4 down-regulation is significantly associated with poorer patient survival (p<0.05). In summary, miR-224/SMAD4 pathway is a clinically relevant pathway to provide new insights in understanding HCC. (191 words).
Asunto(s)
MicroARNs/metabolismo , Proteína Smad4/metabolismo , Animales , Secuencia de Bases , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proliferación Celular , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica , Ontología de Genes , Células HCT116 , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/genética , Datos de Secuencia Molecular , Análisis de SupervivenciaRESUMEN
Although circulating memory T cells provide enhanced protection against pathogen challenge, they often fail to do so if infection is localized to peripheral or extralymphoid compartments. In those cases, it is T cells already resident at the site of virus challenge that offer superior immune protection. These tissue-resident memory T (T(RM)) cells are identified by their expression of the α-chain from the integrin α(E)(CD103)ß(7), and can exist in disequilibrium with the blood, remaining in the local environment long after peripheral infections subside. In this study, we demonstrate that long-lived intraepithelial CD103(+)CD8(+) T(RM) cells can be generated in the absence of in situ antigen recognition. Local inflammation in skin and mucosa alone resulted in enhanced recruitment of effector populations and their conversion to the T(RM) phenotype. The CD8(+) T(RM) cells lodged in these barrier tissues provided long-lived protection against local challenge with herpes simplex virus in skin and vagina challenge models, and were clearly superior to the circulating memory T-cell cohort. The results demonstrate that peripheral T(RM) cells can be generated and survive in the absence of local antigen presentation and provide a powerful means of achieving immune protection against peripheral infection.
Asunto(s)
Subgrupos de Linfocitos T/inmunología , Traslado Adoptivo , Animales , Presentación de Antígeno , Linfocitos T CD8-positivos/inmunología , Epitelio/inmunología , Epitelio/virología , Femenino , Herpes Genital/inmunología , Herpes Genital/prevención & control , Herpes Simple/inmunología , Herpes Simple/prevención & control , Memoria Inmunológica , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Piel/inmunología , Piel/virología , Enfermedades Cutáneas Virales/inmunología , Enfermedades Cutáneas Virales/prevención & control , Factores de TiempoRESUMEN
Like other cancers, aberrant gene regulation features significantly in hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) were recently found to regulate gene expression at the post-transcriptional/translational levels. The expression profiles of 157 miRNAs were examined in 19 HCC patients, and 19 up-regulated and 3 down-regulated miRNAs were found to be associated with HCC. Putative gene targets of these 22 miRNAs were predicted in silico and were significantly enriched in 34 biological pathways, most of which are frequently dysregulated during carcinogenesis. Further characterization of microRNA-224 (miR-224), the most significantly up-regulated miRNA in HCC patients, revealed that miR-224 increases apoptotic cell death as well as proliferation and targets apoptosis inhibitor-5 (API-5) to inhibit API-5 transcript expression. Significantly, miR-224 expression was found to be inversely correlated with API-5 expression in HCC patients (p < 0.05). Hence, our findings define a true in vivo target of miR-224 and reaffirm the important role of miRNAs in the dysregulation of cellular processes that may ultimately lead to tumorigenesis.