RESUMEN
BACKGROUND: Virus-associated febrile lower respiratory tract infections (fLRIs) during infancy have been identified as risk factors for persistent wheeze development. We hypothesized that variations in innate immune defense capacity during this period, as exemplified by production of type 1 and 3 interferons (T1/3IFNs), might be an underlying determinant of risk. OBJECTIVE: We sought to investigate relationships between postnatal development of innate interferon response capacity and susceptibility to early infections and persistent wheeze. METHODS: We studied a subset of subjects from a birth cohort at high risk for asthma/allergy and determined the capacity of cord blood cells (n = 151) to produce any of a panel of 17 T1/3IFNs in response to the viral mimic polyinosinic-polycytidylic acid using a sensitive PCR assay. We investigated relationships between neonatal interferon responses and lower respiratory tract infection history during infancy, wheezing history to 5 age years, and ensuing maturation of innate immune capacity by age 4 years (n = 160) and 10 years (n = 125). RESULTS: Although cohort subjects produced an average of 2.6 ± 0.3 of the 17 innate interferons tested at birth, 24% showed no T1/3IFN production. This nonproducer subgroup showed increased risk for infant fLRIs (odds ratio, 2.62; 95% CI, 1.14-6.06; P = .024) and persistent wheeze (odds ratio, 4.24; 95% CI, 1.60-11.24; P = .004) at age 5 years relative to those producing 1 or more T1/3IFNs, whereas risk for infant wheezy lower respiratory tract infections or "transient early wheeze" was unaffected. Moreover, infants who experienced fLRIs subsequently demonstrated accelerated development of T1/3IFN response capacity between 1 and 4 years of age. CONCLUSIONS: T1/3IFN response capacity appears strongly developmentally constrained at birth. Infants in whom this negative regulation is strongest manifest increased risk for severe respiratory tract infections during infancy and subsequent persistent wheeze.
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Asma/inmunología , Interferones/inmunología , Ruidos Respiratorios/inmunología , Infecciones del Sistema Respiratorio/inmunología , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Leucocitos Mononucleares/inmunología , Masculino , Factores de RiesgoRESUMEN
UNLABELLED: Ebola virus (EBOV) causes a severe hemorrhagic fever with a deficient immune response, lymphopenia, and lymphocyte apoptosis. Dendritic cells (DC), which trigger the adaptive response, do not mature despite EBOV infection. We recently demonstrated that DC maturation is unblocked by disabling the innate response antagonizing domains (IRADs) in EBOV VP35 and VP24 by the mutations R312A and K142A, respectively. Here we analyzed the effects of VP35 and VP24 with the IRADs disabled on global gene expression in human DC. Human monocyte-derived DC were infected by wild-type (wt) EBOV or EBOVs carrying the mutation in VP35 (EBOV/VP35m), VP24 (EBOV/VP24m), or both (EBOV/VP35m/VP24m). Global gene expression at 8 and 24 h was analyzed by deep sequencing, and the expression of interferon (IFN) subtypes up to 5 days postinfection was analyzed by quantitative reverse transcription-PCR (qRT-PCR). wt EBOV induced a weak global gene expression response, including markers of DC maturation, cytokines, chemokines, chemokine receptors, and multiple IFNs. The VP35 mutation unblocked the expression, resulting in a dramatic increase in expression of these transcripts at 8 and 24 h. Surprisingly, DC infected with EBOV/VP24m expressed lower levels of many of these transcripts at 8 h after infection, compared to wt EBOV. In contrast, at 24 h, expression of the transcripts increased in DC infected with any of the three mutants, compared to wt EBOV. Moreover, sets of genes affected by the two mutations only partially overlapped. Pathway analysis demonstrated that the VP35 mutation unblocked pathways involved in antigen processing and presentation and IFN signaling. These data suggest that EBOV IRADs have profound effects on the host adaptive immune response through massive transcriptional downregulation of DC. IMPORTANCE: This study shows that infection of DC with EBOV, but not its mutant forms with the VP35 IRAD and/or VP24 IRAD disabled, causes a global block in expression of host genes. The temporal effects of mutations disrupting the two IRADs differ, and the lists of affected genes only partially overlap such that VP35 and VP24 IRADs each have profound effects on antigen presentation by exposed DC. The global modulation of DC gene expression and the resulting lack of their maturation represent a major mechanism by which EBOV disables the T cell response and suggests that these suppressive pathways are a therapeutic target that may unleash the T cell responses during EBOV infection.
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Células Dendríticas/metabolismo , Ebolavirus/metabolismo , Expresión Génica , Fiebre Hemorrágica Ebola/genética , Interferones/genética , Nucleoproteínas/metabolismo , Proteínas del Núcleo Viral/metabolismo , Proteínas Virales/metabolismo , Células Cultivadas , Células Dendríticas/virología , Ebolavirus/química , Ebolavirus/genética , Fiebre Hemorrágica Ebola/metabolismo , Fiebre Hemorrágica Ebola/virología , Humanos , Interferones/metabolismo , Proteínas de la Nucleocápside , Nucleoproteínas/química , Nucleoproteínas/genética , Estructura Terciaria de Proteína , Transducción de Señal , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genética , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Innate interferons (IFN) are comprised of multiple Type I and III subtypes. The in vivo kinetics of subtype responses during human immunodeficiency virus (HIV) infection is not well defined. Using the acute simian immunodeficiency virus (SIV) infection model, we show that plasma IFNα levels peak at day 10 post-infection (pi) after which they rapidly declined. The mRNA expression of Type I and III IFN subtypes were significantly elevated in the lymph nodes (LN) at day 10 pi. Though the expression levels of all subtypes declined by day 14-31 pi, numerous subtypes remained elevated suggesting that ongoing viral replication in LN continues to drive induction of these subtypes. Interestingly, treatment with reverse transcriptase (RT) inhibitors at day 7 pi significantly suppressed plasma IFNα responses by day 10 pi that significantly correlated with cell-associated SIV DNA loads suggesting that RT byproducts such as viral DNA likely plays a role in driving IFN responses during acute SIV infection. Quantification of Type I and III subtype transcripts in sorted subsets of LN CD4+ and CD8+ T cells, CD14+/CD14- monocytes/macrophages, and total CD11c/CD123+ dendritic cells (DC) at day 10 pi showed that DC expressed â¼3-4 log more subtype transcripts as compared to the other subsets. Taken together, our results provide new insights into the kinetics of innate interferon responses during early stages of infection, and provide evidence that DC's are a major in vivo source of innate IFN during acute SIV infection.
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Síndrome de Inmunodeficiencia Adquirida/terapia , Células Dendríticas/efectos de los fármacos , Interferón-alfa/biosíntesis , Ganglios Linfáticos/inmunología , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Virus de la Inmunodeficiencia de los Simios/inmunología , Síndrome de Inmunodeficiencia Adquirida/inmunología , Enfermedad Aguda , Animales , Células Cultivadas , ADN Viral/inmunología , Células Dendríticas/inmunología , Células Dendríticas/virología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunidad Innata/efectos de los fármacos , Terapia de Inmunosupresión , Interferón-alfa/sangre , Ganglios Linfáticos/virología , Macaca mulatta , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunologíaRESUMEN
Translocations resulting in ectopic expression of the TLX1 homeobox gene (previously known as HOX11) are recurrent events in human T-cell acute lymphoblastic leukemia (T-ALL). Transduction of primary murine hematopoietic stem/progenitor cells with retroviral vectors expressing TLX1 readily yields immortalized hematopoietic progenitor cell lines. Understanding the processes involved in TLX1-mediated cellular immortalization should yield insights into the growth and differentiation pathways altered by TLX1 during the development of T-ALL. In recent clinical gene therapy trials, hematopoietic clonal dominance or T-ALL-like diseases have occurred as a direct consequence of insertional activation of the EVI1, PRDM16 or LMO2 proto-oncogenes by the retroviral vectors used to deliver the therapeutic genes. Additionally, the generation of murine hematopoietic progenitor cell lines due to retroviral integrations into Evi1 or Prdm16 has also been recently reported. Here, we determined by linker-mediated nested polymerase chain reaction the integration sites in eight TLX1-immortalized hematopoietic cell lines. Notably, no common integration site was observed among the cell lines. Moreover, no insertions into the Evi1 or Prdm16 genes were identified although insertion near Lmo2 was observed in one instance. However, neither Lmo2 nor any of the other genes examined surrounding the integration sites showed differential vector-influenced expression compared to the cell lines lacking such insertions. While we cannot exclude the possibility that insertional side effects transiently provided a selective growth/survival advantage to the hematopoietic progenitor populations, our results unequivocally rule out insertions into Evi1 and Prdm16 as being integral to the TLX1-initiated immortalization process.
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Hematopoyesis/genética , Proteínas de Homeodominio/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogénicas/genética , Animales , Southern Blotting , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Diferenciación Celular , División Celular , Línea Celular Tumoral , Cartilla de ADN , Femenino , Genes Homeobox , Estudio de Asociación del Genoma Completo , Humanos , Cariotipificación , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante de Células Madre/métodosRESUMEN
Gender differences in Human immunodeficiency virus (HIV) disease progression and comorbidities have been extensively reported. Using the simian immunodeficiency virus (SIV) infected rhesus macaque model, we show that these differences are apparent very early during the course of infection. Though there were no major changes in the proportions of CD4 T cells or its subsets, central memory CD4 T cells from female macaques were found to differentially regulate a significantly larger number of genes at day 4 post-infection (PI) as compared to males. Pathway analysis revealed divergence of both canonical and biological pathways that persisted at day 10 PI. Changes in gene expression profiles were accompanied by a significant increase in plasma levels of pro-inflammatory mediators such as MCP-1/CCL2, I-TAC/CXCL11, and MIF. Though plasma levels of IFNα did not differ between male and female macaques, the expression levels of IFNα subtype-14, 16, IFNß, and IFNω were significantly upregulated in the lymph nodes of female macaques at day 10 PI as compared to male macaques. Our results suggest that the pathogenic sequelae seen during chronic infection may be shaped by gender differences in immune responses induced very early during the course of HIV infection.
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Linfocitos T CD4-Positivos/inmunología , Perfilación de la Expresión Génica , Inmunidad Innata , Caracteres Sexuales , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/fisiología , Enfermedad Aguda , Animales , Linfocitos T CD4-Positivos/metabolismo , Quimiocina CXCL11/sangre , Femenino , Interferones/sangre , Macaca mulatta , Masculino , ARN Mensajero/genética , Receptores CCR2/sangre , Síndrome de Inmunodeficiencia Adquirida del Simio/sangre , Síndrome de Inmunodeficiencia Adquirida del Simio/fisiopatologíaRESUMEN
Intestinal epithelial cells (IECs) are constantly exposed to commensal flora and pathogen challenges. How IECs regulate their innate immune response to maintain gut homeostasis remains unclear. Interferons (IFNs) are cytokines produced during infections. While type I IFN receptors are ubiquitously expressed, type III IFN receptors are expressed only on epithelial cells. This epithelium specificity strongly suggests exclusive functions at epithelial surfaces, but the relative roles of type I and III IFNs in the establishment of an antiviral innate immune response in human IECs are not clearly defined. Here, we used mini-gut organoids to define the functions of types I and III IFNs to protect the human gut against viral infection. We show that primary non-transformed human IECs, upon viral challenge, upregulate the expression of both type I and type III IFNs at the transcriptional level but only secrete type III IFN in the supernatant. However, human IECs respond to both type I and type III IFNs by producing IFN-stimulated genes that in turn induce an antiviral state. Using genetic ablation of either type I or type III IFN receptors, we show that either IFN can independently restrict virus infection in human IECs. Importantly, we report, for the first time, differences in the mechanisms by which each IFN establishes the antiviral state. Contrary to type I IFN, the antiviral activity induced by type III IFN is strongly dependent on the mitogen-activated protein kinases signaling pathway, suggesting a pathway used by type III IFNs that non-redundantly contributes to the antiviral state. In conclusion, we demonstrate that human intestinal epithelial cells specifically regulate their innate immune response favoring type III IFN-mediated signaling, which allows for efficient protection against pathogens without producing excessive inflammation. Our results strongly suggest that type III IFN constitutes the frontline of antiviral response in the human gut. We propose that mucosal surfaces, particularly the gastrointestinal tract, have evolved to favor type III IFN-mediated response to pathogen infections as it allows for spatial segregation of signaling and moderate production of inflammatory signals which we propose are key to maintain gut homeostasis.
RESUMEN
After viral infection, type I and III interferons (IFNs) are coexpressed by respiratory epithelial cells (RECs) and activate the ISGF3 transcription factor (TF) complex to induce expression of a cell-specific set of interferon-stimulated genes (ISGs). Type I and III IFNs share a canonical signaling pathway, suggesting that they are redundant. Animal and in vitro models, however, have shown that they are not redundant. Because TFs dictate cellular phenotype and function, we hypothesized that focusing on TF-ISG will reveal critical combinatorial and nonredundant functions of type I or III IFN. We treated BEAS-2B human RECs with increasing doses of IFNß or IFNλ1 and measured expression of TF-ISG. ISGs were expressed in a dose-dependent manner with a nonlinear jump at intermediate doses. At subsaturating combinations of IFNß and IFNλ1, many ISGs were expressed in a pattern that we modeled with a cubic equation that mathematically defines this threshold effect. Uniquely, IFNß alone induced early and transient IRF1 transcript and protein expression, while IFNλ1 alone induced IRF1 protein expression at low levels that were sustained through 24 h. In combination, saturating doses of these 2 IFNs together enhanced and sustained IRF1 expression. We conclude that the cubic model quantitates combinatorial effects of IFNß and IFNλ1 and that IRF1 may mediate nonredundancy of type I or III IFN in RECs.
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Interferón Tipo I/metabolismo , Interferón beta/metabolismo , Factores de Transcripción/genética , Células Cultivadas , Humanos , Factor 1 Regulador del Interferón/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-α subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-λ2 and -λ3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.
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Interferones/biosíntesis , Interferones/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Interferón Tipo I/biosíntesis , Interferón Tipo I/genética , TranscriptomaRESUMEN
INTRODUCTION: Inappropriate activation of the TLX1 (T-cell leukemia homeobox 1) gene by chromosomal translocation is a recurrent event in human T-cell Acute Lymphoblastic Leukemia (T-ALL). Ectopic expression of TLX1 in murine bone marrow progenitor cells using a conventional retroviral vector efficiently yields immortalized cell lines and induces T-ALL-like tumors in mice after long latency. METHODS: To eliminate a potential contribution of retroviral insertional mutagenesis to TLX1 immortalizing and transforming function, we incorporated the TLX1 gene into an insulated self-inactivating retroviral vector. RESULTS: Retrovirally transduced TLX1-expressing murine bone marrow progenitor cells had a growth/survival advantage and readily gave rise to immortalized cell lines. Extensive characterization of 15 newly established cell lines failed to reveal a common retroviral integration site. This comprehensive analysis greatly extends our previous study involving a limited number of cell lines, providing additional support for the view that constitutive TLX1 expression is sufficient to initiate the series of events culminating in hematopoietic progenitor cell immortalization. When TLX1-immortalized cells were co-cultured on OP9-DL1 monolayers under conditions permissive for T-cell differentiation, a latent T-lineage potential was revealed. However, the cells were unable to transit the DN2 myeloid-T (DN2mt)-DN2 T-lineage determined (DN2t) commitment step. The differentiation block coincided with failure to upregulate the zinc finger transcription factor gene Bcl11b, the human ortholog of which was shown to be a direct transcriptional target of TLX1 downregulated in the TLX1+ T-ALL cell line ALL-SIL. Other studies have described the ability of TLX1 to promote bypass of mitotic checkpoint arrest, leading to aneuploidy. We likewise found that diploid TLX1-expressing DN2mt cells treated with the mitotic inhibitor paclitaxel bypassed the mitotic checkpoint and displayed chromosomal instability. This was associated with elevated expression of TLX1 transcriptional targets involved in DNA replication and mitosis, including Ccna2 (cyclin A2), Ccnb1 (cyclin B1), Ccnb2 (cyclin B2) and Top2a (topoisomerase IIα). Notably, enforced expression of BCL11B in ALL-SIL T-ALL cells conferred resistance to the topoisomerase IIα poison etoposide. CONCLUSION: Taken together with previous findings, the data reinforce a mechanism of TLX1 oncogenic activity linked to chromosomal instability resulting from dysregulated expression of target genes involved in mitotic processes. We speculate that repression of BCL11B expression may provide part of the explanation for the observation that aneuploid DNA content in TLX1+ leukemic T cells does not necessarily portend an unfavorable prognosis. This TLX1 hematopoietic progenitor cell immortalization/T-cell differentiation assay should help further our understanding of the mechanisms of TLX1-mediated evolution to malignancy and has the potential to be a useful predictor of disease response to novel therapeutic agents in TLX1+ T-ALL.
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The main impediments to clinical application of haematopoietic stem cell (HSC) gene therapy for treatment of haemophilia A are the bone marrow transplant-related risks and the potential for insertional mutagenesis caused by retroviral vectors. To circumvent these limitations, we have adapted a non-myeloablative conditioning regimen and directed factor VIII (FVIII) protein synthesis to B lineage cells using an insulated lentiviral vector containing an immunoglobulin heavy chain enhancer-promoter. Transplantation of lentiviral vector-modified HSCs resulted in therapeutic levels of FVIII in the circulation of all transplanted mice for the duration of the study (six months). Immunostaining of spleen cells showed that the majority of FVIII was synthesised by B220+ B cells and CD138+ plasma cells. Subsequent challenge with recombinant FVIII elicited at most a minor anti-FVIII antibody response, demonstrating induction of immune hyporesponsiveness. All transplant recipients exhibited clot formation and survived tail clipping, indicating correction of their haemophilic phenotype. Therapeutic levels of FVIII could be transferred to secondary recipients by bone marrow transplantation, confirming gene transfer into long-term repopulating HSCs. Moreover, short-term therapeutic FVIII levels could also be achieved in secondary recipients by adoptive transfer of HSC-derived splenic B cells. Our findings support pursuit of B cell-directed protein delivery as a potential clinical approach to treat haemophilia A and other disorders correctable by systemically distributed proteins.
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Linfocitos B/metabolismo , Factor VIII/metabolismo , Hemofilia A/sangre , Hemofilia A/terapia , Lentivirus/genética , Traslado Adoptivo , Animales , Linfocitos B/citología , Linfocitos B/trasplante , Diferenciación Celular , Linaje de la Célula , Modelos Animales de Enfermedad , Factor VIII/genética , Terapia Genética , Vectores Genéticos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Hemofilia A/genética , Hemorragia/genética , Hemorragia/prevención & control , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Ratones , Ratones Mutantes , Regiones Promotoras Genéticas , Transgenes/genéticaRESUMEN
Despite considerable progress in the treatment of T cell acute lymphoblastic leukemia (T-ALL), it is still the highest risk malignancy among ALL. The outcome of relapsed patients remains dismal. The pro-survival role of NOTCH1 and NFκB in T-ALL is well documented; also, both factors were reported to be predictive of relapse. The NOTCH1 signaling pathway, commonly activated in T-ALL, was shown to enhance the transcriptional function of NFκB via several mechanisms. Thus, pharmacological inhibition of NOTCH1-NFκB signaling was suggested to be incorporated into existing T-ALL treatment protocols. However, conventional chemotherapy is based on activation of various types of stress, such as DNA damage, mitotic perturbations or endoplasmic reticulum overload. NFκB is frequently activated in response to stress and, depending on yet unknown mechanisms, it either protects cells from the drug action or mediates apoptosis. Here, we report that T-ALL cells respond to NFκB inhibition in opposite ways depending on whether they were treated with a stress-inducing chemotherapeutic agent or not. Moreover, we found that NOTCH1 enhances NFκB apoptotic function in the stressed cells. The data argue for further studies of NFκB status in T-ALL patients on different treatment protocols and the impact of activating NOTCH1 mutations on treatment response.