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Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.
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Riñón/inmunología , Nefritis Lúpica/inmunología , Biomarcadores , Biopsia , Análisis por Conglomerados , Biología Computacional/métodos , Células Epiteliales/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunofenotipificación , Interferones/metabolismo , Riñón/metabolismo , Riñón/patología , Leucocitos/inmunología , Leucocitos/metabolismo , Nefritis Lúpica/genética , Nefritis Lúpica/metabolismo , Nefritis Lúpica/patología , Linfocitos/inmunología , Linfocitos/metabolismo , Anotación de Secuencia Molecular , Células Mieloides/inmunología , Células Mieloides/metabolismo , Análisis de la Célula Individual , TranscriptomaRESUMEN
All coronaviruses known to have recently emerged as human pathogens probably originated in bats1. Here we use a single experimental platform based on immunodeficient mice implanted with human lung tissue (hereafter, human lung-only mice (LoM)) to demonstrate the efficient in vivo replication of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as two endogenous SARS-like bat coronaviruses that show potential for emergence as human pathogens. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats contain endogenous coronaviruses that are capable of direct transmission to humans. Our detailed analysis of in vivo infection with SARS-CoV-2 in human lung tissue from LoM showed a predominant infection of human lung epithelial cells, including type-2 pneumocytes that are present in alveoli and ciliated airway cells. Acute infection with SARS-CoV-2 was highly cytopathic and induced a robust and sustained type-I interferon and inflammatory cytokine and chemokine response. Finally, we evaluated a therapeutic and pre-exposure prophylaxis strategy for SARS-CoV-2 infection. Our results show that therapeutic and prophylactic administration of EIDD-2801-an oral broad-spectrum antiviral agent that is currently in phase II/III clinical trials-markedly inhibited SARS-CoV-2 replication in vivo, and thus has considerable potential for the prevention and treatment of COVID-19.
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Tratamiento Farmacológico de COVID-19 , COVID-19/prevención & control , Citidina/análogos & derivados , Hidroxilaminas/administración & dosificación , Hidroxilaminas/uso terapéutico , Administración Oral , Células Epiteliales Alveolares/inmunología , Células Epiteliales Alveolares/patología , Células Epiteliales Alveolares/virología , Animales , COVID-19/inmunología , Quimioprevención , Quirópteros/virología , Ensayos Clínicos Fase II como Asunto , Ensayos Clínicos Fase III como Asunto , Citidina/administración & dosificación , Citidina/uso terapéutico , Citocinas/inmunología , Células Epiteliales/virología , Femenino , Xenoinjertos , Humanos , Inmunidad Innata , Interferón Tipo I/inmunología , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Trasplante de Pulmón , Masculino , Ratones , Profilaxis Posexposición , Profilaxis Pre-Exposición , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Replicación ViralRESUMEN
Understanding the mechanisms that drive HIV expression and latency is a key goal for achieving an HIV cure. Here we investigate the role of the SETD2 histone methyltransferase, which deposits H3K36 trimethylation (H3K36me3), in HIV infection. We show that prevention of H3K36me3 by a potent and selective inhibitor of SETD2 (EPZ-719) leads to reduced post-integration viral gene expression and accelerated emergence of latently infected cells. CRISPR/Cas9-mediated knockout of SETD2 in primary CD4 T cells confirmed the role of SETD2 in HIV expression. Transcriptomic profiling of EPZ-719-exposed HIV-infected cells identified numerous pathways impacted by EPZ-719. Notably, depletion of H3K36me3 prior to infection did not prevent HIV integration but resulted in a shift of integration sites from highly transcribed genes to quiescent chromatin regions and to polycomb repressed regions. We also observed that SETD2 inhibition did not apparently affect HIV RNA levels, indicating a post-transcriptional mechanism affecting HIV expression. Viral RNA splicing was modestly reduced in the presence of EPZ-719. Intriguingly, EPZ-719 exposure enhanced responsiveness of latent HIV to the HDAC inhibitor vorinostat, suggesting that H3K36me3 can contribute to a repressive chromatin state at the HIV locus. These results identify SETD2 and H3K36me3 as novel regulators of HIV integration, expression and latency.
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Infecciones por VIH , VIH-1 , N-Metiltransferasa de Histona-Lisina , Latencia del Virus , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Latencia del Virus/fisiología , Infecciones por VIH/virología , Infecciones por VIH/metabolismo , Infecciones por VIH/genética , VIH-1/fisiología , VIH-1/genética , Linfocitos T CD4-Positivos/virología , Linfocitos T CD4-Positivos/metabolismo , Regulación Viral de la Expresión GénicaRESUMEN
Long-lasting, latently infected resting CD4+ T cells are the greatest obstacle to obtaining a cure for HIV infection, as these cells can persist despite decades of treatment with antiretroviral therapy (ART). Estimates indicate that more than 70 years of continuous, fully suppressive ART are needed to eliminate the HIV reservoir1. Alternatively, induction of HIV from its latent state could accelerate the decrease in the reservoir, thus reducing the time to eradication. Previous attempts to reactivate latent HIV in preclinical animal models and in clinical trials have measured HIV induction in the peripheral blood with minimal focus on tissue reservoirs and have had limited effect2-9. Here we show that activation of the non-canonical NF-κB signalling pathway by AZD5582 results in the induction of HIV and SIV RNA expression in the blood and tissues of ART-suppressed bone-marrow-liver-thymus (BLT) humanized mice and rhesus macaques infected with HIV and SIV, respectively. Analysis of resting CD4+ T cells from tissues after AZD5582 treatment revealed increased SIV RNA expression in the lymph nodes of macaques and robust induction of HIV in almost all tissues analysed in humanized mice, including the lymph nodes, thymus, bone marrow, liver and lung. This promising approach to latency reversal-in combination with appropriate tools for systemic clearance of persistent HIV infection-greatly increases opportunities for HIV eradication.
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Infecciones por VIH/virología , VIH-1/fisiología , FN-kappa B/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Latencia del Virus , Alquinos/farmacología , Animales , Antirretrovirales/farmacología , Infecciones por VIH/metabolismo , VIH-1/efectos de los fármacos , Macaca mulatta , Ratones , Oligopéptidos/farmacología , Síndrome de Inmunodeficiencia Adquirida del Simio/metabolismo , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Latencia del Virus/efectos de los fármacosRESUMEN
Although antiretroviral therapy (ART) is effective at suppressing HIV replication, a viral reservoir persists that can reseed infection if ART is interrupted. Curing HIV will require elimination or containment of this reservoir, but the size of the HIV reservoir is highly variable between individuals. To evaluate the size of the HIV reservoir, several assays have been developed, including PCR-based assays for viral DNA, the intact proviral DNA assay, and the quantitative viral outgrowth assay (QVOA). QVOA is the gold standard assay for measuring inducible replication-competent proviruses, but this assay is technically challenging and time-consuming. To begin progress toward a more rapid and less laborious tool for quantifying cells infected with replication-competent HIV, we developed the Microwell Outgrowth Assay, in which infected CD4 T cells are co-cultured with an HIV-detecting reporter cell line in a polydimethylsiloxane (PDMS)/polystyrene array of nanoliter-sized wells. Transmission of HIV from infected cells to the reporter cell line induces fluorescent reporter protein expression that is detected by automated scanning across the array. Using this approach, we were able to detect HIV-infected cells from ART-naïve people with HIV (PWH) and from PWH on ART with large reservoirs. Furthermore, we demonstrate that infected cells can be recovered from individual rafts and used to analyze the diversity of viral sequences. Although additional development and optimization will be required for quantifying the reservoir in PWH with small latent reservoirs, this assay may be a useful prototype for microwell assays of infected cells.IMPORTANCEMeasuring the size of the HIV reservoir in people with HIV (PWH) will be important for determining the impact of HIV cure strategies. However, measuring this reservoir is challenging. We report a new method for quantifying HIV-infected cells that involves culturing cells from PWH in an array of microwells with a cell line that detects HIV infection. We show that this approach can detect rare HIV-infected cells and derive detailed virus sequence information for each infected cell.
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Infecciones por VIH , Virología , Humanos , Linfocitos T CD4-Positivos , Línea Celular , ADN Viral , Infecciones por VIH/virología , Provirus/genética , Carga Viral , Latencia del Virus , Virología/métodosRESUMEN
Transcriptional silencing of latent HIV-1 proviruses entails complex and overlapping mechanisms that pose a major barrier to in vivo elimination of HIV-1. We developed a new latency CRISPR screening strategy, called Latency HIV-CRISPR which uses the packaging of guideRNA-encoding lentiviral vector genomes into the supernatant of budding virions as a direct readout of factors involved in the maintenance of HIV-1 latency. We developed a custom guideRNA library targeting epigenetic regulatory genes and paired the screen with and without a latency reversal agent-AZD5582, an activator of the non-canonical NFκB pathway-to examine a combination of mechanisms controlling HIV-1 latency. A component of the Nucleosome Acetyltransferase of H4 histone acetylation (NuA4 HAT) complex, ING3, acts in concert with AZD5582 to activate proviruses in J-Lat cell lines and in a primary CD4+ T cell model of HIV-1 latency. We found that the knockout of ING3 reduces acetylation of the H4 histone tail and BRD4 occupancy on the HIV-1 LTR. However, the combination of ING3 knockout accompanied with the activation of the non-canonical NFκB pathway via AZD5582 resulted in a dramatic increase in initiation and elongation of RNA Polymerase II on the HIV-1 provirus in a manner that is nearly unique among all cellular promoters.
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Infecciones por VIH , Seropositividad para VIH , VIH-1 , Humanos , Histonas/metabolismo , Proteínas Nucleares/metabolismo , VIH-1/fisiología , Factores de Transcripción/metabolismo , Latencia del Virus/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Seropositividad para VIH/genética , Provirus/genética , Linfocitos T CD4-Positivos , Proteínas de Homeodominio/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMEN
BACKGROUND: Human immunodeficiency virus (HIV) infection remains incurable due to the persistence of a viral reservoir despite antiretroviral therapy (ART). Cannabis (CB) use is prevalent amongst people with HIV (PWH), but the impact of CB on the latent HIV reservoir has not been investigated. METHODS: Peripheral blood cells from a cohort of PWH who use CB and a matched cohort of PWH who do not use CB on ART were evaluated for expression of maturation/activation markers, HIV-specific T-cell responses, and intact proviral DNA. RESULTS: CB use was associated with increased abundance of naive T cells, reduced effector T cells, and reduced expression of activation markers. CB use was also associated with reduced levels of exhausted and senescent T cells compared to nonusing controls. HIV-specific T-cell responses were unaffected by CB use. CB use was not associated with intact or total HIV DNA frequency in CD4 T cells. CONCLUSIONS: This analysis is consistent with the hypothesis that CB use reduces activation, exhaustion, and senescence in the T cells of PWH, and does not impair HIV-specific CD8 T-cell responses. Longitudinal and interventional studies with evaluation of CB exposure are needed to fully evaluate the impact of CB use on the HIV reservoir.
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Cannabis , Infecciones por VIH , VIH-1 , Humanos , Cannabis/genética , VIH-1/genética , Latencia del Virus , Linfocitos T CD4-Positivos , ADN , Carga Viral , Antirretrovirales/uso terapéutico , ADN Viral/genéticaRESUMEN
Transcriptional silencing of HIV in CD4 T cells generates a reservoir of latently infected cells that can reseed infection after interruption of therapy. As such, these cells represent the principal barrier to curing HIV infection, but little is known about their characteristics. To further our understanding of the molecular mechanisms of latency, we characterized a primary cell model of HIV latency in which infected cells adopt heterogeneous transcriptional fates. In this model, we observed that latency is a stable, heritable state that is transmitted through cell division. Using Assay of Transposon-Accessible Chromatin sequencing (ATACseq) we found that latently infected cells exhibit greatly reduced proviral accessibility, indicating the presence of chromatin-based structural barriers to viral gene expression. By quantifying the activity of host cell transcription factors, we observe elevated activity of Forkhead and Kruppel-like factor transcription factors (TFs), and reduced activity of AP-1, RUNX and GATA TFs in latently infected cells. Interestingly, latency reversing agents with different mechanisms of action caused distinct patterns of chromatin reopening across the provirus. We observe that binding sites for the chromatin insulator CTCF are highly enriched in the differentially open chromatin of infected CD4 T cells. Furthermore, depletion of CTCF inhibited HIV latency, identifying this factor as playing a key role in the initiation or enforcement of latency. These data indicate that HIV latency develops preferentially in cells with a distinct pattern of TF activity that promotes a closed proviral structure and inhibits viral gene expression. Furthermore, these findings identify CTCF as a novel regulator of HIV latency.
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Linfocitos T CD4-Positivos/metabolismo , Cromatina/metabolismo , Epigenómica/métodos , VIH-1/fisiología , Interacciones Huésped-Patógeno , Factores de Transcripción/metabolismo , Latencia del Virus , Sitios de Unión , Linfocitos T CD4-Positivos/virología , Cromatina/genética , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Humanos , Células Jurkat , Factores de Transcripción/genética , Activación ViralRESUMEN
Targeting immune checkpoint pathways, such as programmed death ligand-1 (PD-L1, also known as CD274 or B7-H1) or its receptor programmed cell death-1 (PD-1) has shown improved survival for patients with numerous types of cancers, not limited to lung cancer, melanoma, renal cell carcinoma, and Hodgkin lymphoma. PD-L1 is a co-inhibitory molecule whose expression on the surface of tumor cells is associated with worse prognosis in many tumors. Here we describe a splice variant (secPD-L1) that does not splice into the transmembrane domain, but instead produces a secreted form of PD-L1 that has a unique 18 amino acid tail containing a cysteine that allows it to homodimerize and more effectively inhibit lymphocyte function than monomeric soluble PD-L1. We show that recombinant secPD-L1 can dimerize and inhibit T-cell proliferation and IFN-gamma production in vitro. The secPD-L1 variant is expressed by malignant cells in vitro that also express high levels of full-length PD-L1. Transcriptomic analysis of gene expression across The Cancer Genome Atlas found the strongest association of secPD-L1 with full-length PD-L1, but also with subsets of immunologic genes, such as in myeloid-derived suppressor cells. Moreover, the splice variant is also expressed in normal tissues and within normal peripheral blood cells it is preferentially expressed in activated myeloid cells. This is the first report of a form of secreted PD-L1 that homodimerizes and is functionally active. SecPD-L1 may function as a paracrine negative immune regulator within the tumor, since secPD-L1 does not require a cell-to-cell interaction to mediate its inhibitory effect.
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Antígeno B7-H1/genética , Inmunosupresores/farmacología , Multimerización de Proteína , Empalme del ARN , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/química , Antígeno B7-H1/farmacología , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Humanos , Células Supresoras de Origen Mieloide/fisiología , Placenta/metabolismo , Embarazo , Microambiente TumoralRESUMEN
Humans encode seven APOBEC3 proteins (A-H), with A3G, 3F and 3H as the major factors restricting HIV-1 replication. HIV-1, however, encodes Vif, which counteracts A3 proteins by chaperoning them to the proteasome where they are degraded. Vif polymorphisms found in HIV-1s isolated from infected patients have varying anti-A3G potency when assayed in vitro, but there are few studies demonstrating this in in vivo models. Here, we created Friend murine leukemia viruses encoding vif alleles that were previously shown to differentially neutralize A3G in cell culture or that were originally found in primary HIV-1 isolates. Infection of transgenic mice expressing different levels of human A3G showed that these naturally occurring Vif variants differed in their ability to counteract A3G during in vivo infection, although the effects on viral replication were not identical to those seen in cultured cells. We also found that the polymorphic Vifs that attenuated viral replication reverted to wild type only in A3G transgenic mice. Finally, we found that the level of A3G-mediated deamination was inversely correlated with the level of viral replication. This animal model should be useful for studying the functional significance of naturally occurring vif polymorphisms, as well as viral evolution in the presence of A3G.
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Desaminasa APOBEC-3G/metabolismo , Infecciones por VIH/virología , VIH-1/genética , Polimorfismo Genético , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Desaminasa APOBEC-3G/genética , Alelos , Animales , Modelos Animales de Enfermedad , Virus de la Leucemia Murina de Friend/genética , Virus de la Leucemia Murina de Friend/fisiología , Humanos , Ratones , Ratones Transgénicos , Mutación , Replicación ViralRESUMEN
UNLABELLED: Interleukin-1 beta (IL-1ß) is an inflammatory cytokine that is secreted in response to inflammasome activation by innate microbe-sensing pathways. Although some retroviruses can trigger IL-1ß secretion through the DNA-sensing molecule IFI16, the effect of IL-1ß on the course of infection is unknown. To test whether IL-1ß secretion affects retroviral replication in vivo, I constructed a novel murine leukemia virus strain (FMLV-IL-1ß) that encodes the mature form of IL-1ß. This virus replicated with kinetics similar to that of wild-type virus in tissue culture but caused a dramatically more aggressive infection of both C57BL/6 and BALB/c mice. By 7 days postinfection (PI), mice infected with FMLV-IL-1ß exhibited splenomegaly and viral loads 300-fold higher than those in mice infected with wild-type FMLV. Furthermore, the enlarged spleens of FMLV-IL-1ß-infected mice correlated with a large expansion of Gr-1(+) CD11b(+) myeloid-derived suppressor cells, as well as elevated levels of immune activation. Although FMLV-IL-1ß infection was controlled by C57BL/6 mice by 14 days p.i., FMLV-IL-1ß was able to establish a significant persistent infection and immune activation in BALB/c mice. These results demonstrate that IL-1ß secretion is a powerful positive regulator of retroviral infection and that FMLV-IL-1ß represents a new model of proinflammatory retroviral infection. IMPORTANCE: Interleukin-1 beta (IL-1ß) is an inflammatory cytokine released in response to activation of innate pathogen-sensing pathways during microbial infection. To examine the potential impact of IL-1ß on retroviral replication in vivo, I constructed a novel mouse retrovirus strain (FMLV-IL-1ß) that encodes IL-1ß and promotes abundant IL-1ß secretion from infected cells. This virus replicates with normal kinetics in cultured cells but displays a dramatically enhanced ability to replicate in mice and caused persistent infection and immune activation in the BALB/c strain of mice. These results establish IL-1ß as a positive regulator of retroviral replication and suggest that targeting this pathway may have therapeutic benefits in infections with proinflammatory retroviruses. This virus can also be used to further study the impact of inflammatory pathways on retroviral infection.
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Interleucina-1beta/metabolismo , Virus de la Leucemia Murina/inmunología , Virus de la Leucemia Murina/patogenicidad , Leucemia Experimental/patología , Infecciones por Retroviridae/patología , Infecciones Tumorales por Virus/patología , Animales , Interacciones Huésped-Patógeno , Interleucina-1beta/genética , Virus de la Leucemia Murina/genética , Virus de la Leucemia Murina/fisiología , Leucemia Experimental/virología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Infecciones por Retroviridae/virología , Esplenomegalia/patología , Infecciones Tumorales por Virus/virología , Carga Viral , Virulencia , Replicación ViralRESUMEN
The apolipoprotein B editing complex 3 (A3) cytidine deaminases are among the most highly evolutionarily selected retroviral restriction factors, both in terms of gene copy number and sequence diversity. Primate genomes encode seven A3 genes, and while A3F and 3G are widely recognized as important in the restriction of HIV, the role of the other genes, particularly A3A, is not as clear. Indeed, since human cells can express multiple A3 genes, and because of the lack of an experimentally tractable model, it is difficult to dissect the individual contribution of each gene to virus restriction in vivo. To overcome this problem, we generated human A3A and A3G transgenic mice on a mouse A3 knockout background. Using these mice, we demonstrate that both A3A and A3G restrict infection by murine retroviruses but by different mechanisms: A3G was packaged into virions and caused extensive deamination of the retrovirus genomes while A3A was not packaged and instead restricted infection when expressed in target cells. Additionally, we show that a murine leukemia virus engineered to express HIV Vif overcame the A3G-mediated restriction, thereby creating a novel model for studying the interaction between these proteins. We have thus developed an in vivo system for understanding how human A3 proteins use different modes of restriction, as well as a means for testing therapies that disrupt HIV Vif-A3G interactions.
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Citidina Desaminasa/fisiología , Proteínas/fisiología , Infecciones por Retroviridae/genética , Infecciones por Retroviridae/virología , Carga Viral/genética , Desaminasa APOBEC-3G , Animales , Células Cultivadas , VIH-1/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células 3T3 NIH , Retroviridae/fisiología , Ensamble de Virus/genética , Internalización del Virus , Replicación Viral/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Dynamical systems are frequently used to model biological systems. When these models are fit to data, it is necessary to ascertain the uncertainty in the model fit. Here, we present prediction deviation, a metric of uncertainty that determines the extent to which observed data have constrained the model's predictions. This is accomplished by solving an optimization problem that searches for a pair of models that each provides a good fit for the observed data, yet has maximally different predictions. We develop a method for estimating a priori the impact that additional experiments would have on the prediction deviation, allowing the experimenter to design a set of experiments that would most reduce uncertainty. We use prediction deviation to assess uncertainty in a model of interferon-alpha inhibition of viral infection, and to select a sequence of experiments that reduces this uncertainty. Finally, we prove a theoretical result which shows that prediction deviation provides bounds on the trajectories of the underlying true model. These results show that prediction deviation is a meaningful metric of uncertainty that can be used for optimal experimental design.
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Aprendizaje , Modelos Teóricos , Incertidumbre , Infecciones por VIH , HumanosRESUMEN
Early events during retroviral infection play a critical role in determining the course of infection and pathogenesis, but the mechanisms that regulate this phase of infection are poorly understood. Toll-like receptor 7 (TLR7) is required for promoting germinal center reactions and virus-specific neutralizing antibodies at later time points postinfection, but TLR7's role in early acute infection has not been determined. By infecting TLR7-deficient mice with a retroviral pathogen, Friend virus (FV), I determined that TLR7 potently inhibits retroviral replication during the first 5 days of infection and is required for rapid secretion of virus-specific IgM and interleukin-10 (IL-10) in response to infection. Although the IgM response was nonneutralizing, plasmas from wild-type mice but not TLR7-deficient mice inhibited FV replication when passively transferred to infected mice, suggesting an indirect mechanism of antibody function. Interestingly, IL-10 was secreted primarily by CD4 T cells, and IL-10-deficient mice also exhibited accelerated early virus spread, demonstrating that this response inhibits acute infection. Surprisingly, TLR7-deficient mice exhibited normal or elevated secretion of proinflammatory cytokines during acute infection, revealing the existence of a TLR7-independent retrovirus-sensing pathway that drives inflammatory cytokine secretion. Together, these results establish a previously unappreciated role for lymphocytes in mediating rapid TLR7-dependent inhibition of early retroviral infection through nonneutralizing IgM and IL-10.
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Linfocitos T CD4-Positivos/inmunología , Virus de la Leucemia Murina de Friend/inmunología , Glicoproteínas de Membrana/inmunología , Infecciones por Retroviridae/inmunología , Receptor Toll-Like 7/inmunología , Animales , Citocinas/inmunología , Citometría de Flujo , Inmunoglobulina M/inmunología , Interleucina-10/inmunología , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Receptor Toll-Like 7/genética , Replicación Viral/inmunologíaRESUMEN
Quantifying variable importance is essential for answering high-stakes questions in fields like genetics, public policy, and medicine. Current methods generally calculate variable importance for a given model trained on a given dataset. However, for a given dataset, there may be many models that explain the target outcome equally well; without accounting for all possible explanations, different researchers may arrive at many conflicting yet equally valid conclusions given the same data. Additionally, even when accounting for all possible explanations for a given dataset, these insights may not generalize because not all good explanations are stable across reasonable data perturbations. We propose a new variable importance framework that quantifies the importance of a variable across the set of all good models and is stable across the data distribution. Our framework is extremely flexible and can be integrated with most existing model classes and global variable importance metrics. We demonstrate through experiments that our framework recovers variable importance rankings for complex simulation setups where other methods fail. Further, we show that our framework accurately estimates the true importance of a variable for the underlying data distribution. We provide theoretical guarantees on the consistency and finite sample error rates for our estimator. Finally, we demonstrate its utility with a real-world case study exploring which genes are important for predicting HIV load in persons with HIV, highlighting an important gene that has not previously been studied in connection with HIV. Code is available at https://github.com/jdonnelly36/Rashomon_Importance_Distribution.
RESUMEN
Despite the success of antiretroviral therapy, human immunodeficiency virus (HIV) cannot be cured because of a reservoir of latently infected cells that evades therapy. To understand the mechanisms of HIV latency, we employed an integrated single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) approach to simultaneously profile the transcriptomic and epigenomic characteristics of â¼ 125,000 latently infected primary CD4+ T cells after reactivation using three different latency reversing agents. Differentially expressed genes and differentially accessible motifs were used to examine transcriptional pathways and transcription factor (TF) activities across the cell population. We identified cellular transcripts and TFs whose expression/activity was correlated with viral reactivation and demonstrated that a machine learning model trained on these data was 75%-79% accurate at predicting viral reactivation. Finally, we validated the role of two candidate HIV-regulating factors, FOXP1 and GATA3, in viral transcription. These data demonstrate the power of integrated multimodal single-cell analysis to uncover novel relationships between host cell factors and HIV latency.
Asunto(s)
Linfocitos T CD4-Positivos , Factor de Transcripción GATA3 , VIH-1 , Análisis de la Célula Individual , Activación Viral , Latencia del Virus , Latencia del Virus/genética , Humanos , Activación Viral/genética , Análisis de la Célula Individual/métodos , VIH-1/genética , VIH-1/fisiología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD4-Positivos/metabolismo , Factor de Transcripción GATA3/metabolismo , Factor de Transcripción GATA3/genética , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Infecciones por VIH/virología , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Transcriptoma/genética , Regulación Viral de la Expresión GénicaRESUMEN
The HIV reservoir is more dynamic than previously thought with around 70% of the latent reservoir originating from viruses circulating within 1 year of the initiation of antiretroviral therapy (ART). In an ex vivo model system of HIV latency, it was reported that early exposure to class I histone deacetylase (HDAC) inhibitors might prevent these more recently infected cells from entering a state of stable viral latency. This finding raises the possibility that co-administration of HDAC inhibitors at the time of ART initiation may prevent the establishment of much of the HIV reservoir. Here, we tested the effects of the HDAC inhibitors suberoylanilide hydroxamic acid (SAHA) and panobinostat co-administered at the time of ART initiation on the formation of the viral reservoir in HIV-infected humanized mice. As previously shown, SAHA and panobinostat were well tolerated in humanized mice. Unexpectedly, co-administration of SAHA resulted in an increase in the frequency of CD4+ cells carrying HIV DNA but did not alter the frequency of cell-associated HIV RNA in HIV-infected, ART-treated humanized mice. Co-administration of panobinostat did not alter levels of cell-associated HIV DNA or RNA. Our in vivo findings indicate that co-administration of HDAC inhibitors initiated at the same time of ART treatment does not prevent recently infected cells from entering latency.IMPORTANCECurrent antiretroviral therapy (ART) does not eradicate cells harboring replication-competent HIV reservoir. Withdrawal of ART inevitably results in a rapid viremia rebound. The HIV reservoir is more dynamic than previously thought. Early exposure to class I histone deacetylase (HDAC) inhibitors inhibit these more recently infected cells from entering a state of stable viral latency in an ex vivo model of latency, raising the possibility that co-administration of HDAC inhibitors at the time of ART initiation may reduce much of the HIV reservoir. Here, we tested the effects of the HDAC inhibitors suberoylanilide hydroxamic acid or panobinostat during ART initiation on the formation of the viral reservoir in HIV-infected humanized mice. Our in vivo study indicates that in contrast to in vitro observations, the co-administration of HDAC inhibitors at the same time of ART initiation does not prevent recently infected cells from entering latency.
RESUMEN
The development of vaccines that can enhance immunity to viral pathogens is an important goal. However, the innate molecular pathways that regulate the strength and quality of the immune response remain largely uncharacterized. To define the role of Toll-like receptor (TLR) signaling in control of a model retroviral pathogen, Friend virus (FV), I generated mice in which the TLR signaling adapter Myd88 was selectively deleted in dendritic cell (DC) or in B cell lineages. Deletion of Myd88 in DCs had little effect on immune control of FV, while B cell specific deletion of Myd88 caused a dramatic increase in viral infectious centers and a significantly reduced antibody response, indicating that B cell-intrinsic TLR signaling plays a crucial role, while TLR signaling in DCs is less important. I then identified the single-stranded RNA sensing protein TLR7 as being required for antibody-mediated control of FV by analyzing mice deficient in TLR7. Remarkably, B cells in infected TLR7-deficient mice upregulated CD69 and CD86 early in infection, but failed to develop into germinal center B cells. CD4 T cell responses were also attenuated in the absence of TLR7, but CD8 responses were TLR7 independent, suggesting the existence of additional pathways for detection of retroviral particles. Together these results demonstrate that the vertebrate immune system detects retroviruses in vivo via TLR7 and that this pathway regulates a key checkpoint controlling development of germinal center B cells.
Asunto(s)
Centro Germinal/inmunología , Centro Germinal/metabolismo , Glicoproteínas de Membrana/metabolismo , Infecciones por Retroviridae/inmunología , Receptor Toll-Like 7/metabolismo , Animales , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Virus de la Leucemia Murina de Friend/metabolismo , Virus de la Leucemia Murina de Friend/patogenicidad , Centro Germinal/virología , Interferón gamma/genética , Interferón gamma/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal , Receptor Toll-Like 7/genética , Regulación hacia ArribaRESUMEN
Antiretroviral therapy (ART) has dramatically improved the prognosis for people living with HIV-1, but a cure remains elusive. The largest barrier to a cure is the presence of a long-lived latent reservoir that persists within a heterogenous mix of cell types and anatomical compartments. Efforts to eradicate the latent reservoir have primarily focused on latency reversal strategies. However, new work has demonstrated that the majority of the long-lived latent reservoir is established near the time of ART initiation, suggesting that it may be possible to pair an intervention with ART initiation to prevent the formation of a sizable fraction of the latent reservoir. Subsequent treatment with latency reversal agents, in combination with immune clearance agents, may then be a more tractable strategy for fully clearing the latent reservoir in people newly initiating ART. Here, we summarize molecular mechanisms of latency establishment and maintenance, ongoing efforts to develop effective latency reversal agents, and newer efforts to design latency prevention agents. An improved understanding of the molecular mechanisms involved in both the establishment and maintenance of latency will aid in the development of new latency prevention and reversal approaches to ultimately eradicate the latent reservoir.