RESUMEN
HSV infects keratinocytes in the epidermis of skin via nectin-1. We established a human foreskin explant infection model to investigate HSV entry and spread. HSV1 entry could only be achieved by the topical application of virus via high density microarray projections (HD-MAPs) to the epidermis, which penetrated beyond one third of its thickness, simulating in vivo microtrauma. Rapid lateral spread of HSV1 to a mean of 13 keratinocytes wide occurred after 24 hours and free virus particles were observed between keratinocytes, consistent with an intercellular route of spread. Nectin-1 staining was markedly decreased in foci of infection in the epidermis and in the human keratinocyte HaCaT cell line. Nectin-1 was redistributed, at the protein level, in adjacent uninfected cells surrounding infection, inducible by CCL3, IL-8 (or CXCL8), and possibly CXCL10 and IL-6, thus facilitating spread. These findings provide the first insights into HSV1 entry and spread in human inner foreskin in situ.
Asunto(s)
Quimiocinas , Prepucio , Herpes Simple , Herpesvirus Humano 1 , Queratinocitos , Nectinas , Humanos , Masculino , Queratinocitos/virología , Queratinocitos/metabolismo , Prepucio/virología , Prepucio/citología , Nectinas/metabolismo , Herpes Simple/virología , Herpes Simple/metabolismo , Quimiocinas/metabolismo , Herpesvirus Humano 1/fisiología , Moléculas de Adhesión Celular/metabolismo , Internalización del VirusRESUMEN
AXL+ Siglec-6+ dendritic cells (ASDC) are novel myeloid DCs which can be subdivided into CD11c+ and CD123+ expressing subsets. We showed for the first time that these two ASDC subsets are present in inflamed human anogenital tissues where HIV transmission occurs. Their presence in inflamed tissues was supported by single cell RNA analysis of public databases of such tissues including psoriasis diseased skin and colorectal cancer. Almost all previous studies have examined ASDCs as a combined population. Our data revealed that the two ASDC subsets differ markedly in their functions when compared with each other and to pDCs. Relative to their cell functions, both subsets of blood ASDCs but not pDCs expressed co-stimulatory and maturation markers which were more prevalent on CD11c+ ASDCs, thus inducing more T cell proliferation and activation than their CD123+ counterparts. There was also a significant polarisation of naïve T cells by both ASDC subsets toward Th2, Th9, Th22, Th17 and Treg but less toward a Th1 phenotype. Furthermore, we investigated the expression of chemokine receptors that facilitate ASDCs and pDCs migration from blood to inflamed tissues, their HIV binding receptors, and their interactions with HIV and CD4 T cells. For HIV infection, within 2 hours of HIV exposure, CD11c+ ASDCs showed a trend in more viral transfer to T cells than CD123+ ASDCs and pDCs for first phase transfer. However, for second phase transfer, CD123+ ASDCs showed a trend in transferring more HIV than CD11c+ ASDCs and there was no viral transfer from pDCs. As anogenital inflammation is a prerequisite for HIV transmission, strategies to inhibit ASDC recruitment into inflamed tissues and their ability to transmit HIV to CD4 T cells should be considered.
Asunto(s)
Células Dendríticas , Infecciones por VIH , Humanos , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Tirosina Quinasa del Receptor Axl , Masculino , VIH-1/inmunología , Femenino , Células Mieloides/metabolismo , Células Mieloides/inmunología , Persona de Mediana Edad , AdultoRESUMEN
Next-generation vaccines may be delivered via the skin and mucosa. The stratified squamous epithelium (SSE) represents the outermost layer of the skin (epidermis) and type II mucosa (epithelium). Langerhans cells (LCs) have been considered the sole antigen-presenting cells (APCs) to inhabit the SSE; however, it is now clear that dendritic cells (DCs) are also present. Importantly, there are functional differences in how LCs and DCs take up and process pathogens as well as their ability to activate and polarize T cells, though whether DCs participate in neuroimmune interactions like LCs is yet to be elucidated. A correct definition and functional characterization of APCs in the skin and anogenital tissues are of utmost importance for the design of better vaccines and blocking pathogen transmission. Here, we provide a historical perspective on the evolution of our understanding of the APCs that inhabit the SSE, including a detailed review of the most recent literature.
Asunto(s)
Células Dendríticas , Células de Langerhans , Vacunas , Células de Langerhans/inmunología , Humanos , Células Dendríticas/inmunología , Animales , Vacunas/inmunología , Membrana Mucosa/inmunología , Membrana Mucosa/citología , Células Epiteliales/inmunología , Piel/inmunologíaRESUMEN
There is a great need to understand human immune cells within tissue, where disease manifests and infection occurs. Tissue-resident memory T cells (TRMs) were discovered over a decade ago, there is a great need to understand their role in human disease. We developed a 24-color flow cytometry panel to comprehensively interrogate CD4+ and CD8+ TRMs isolated from human tissues. When interrogating cells within human tissue, enzymatic methods used to liberate cells from within the tissue can cause cleavage of cell surface markers needed to phenotype these cells. Here we carefully select antibody clones and evaluate the effect of enzymatic digestion on the expression of markers relevant to the identification of T cell residency, as well as markers relevant to the activation and immunoregulation status of these cells. We have designed this panel to be applicable across a range of human tissues including skin, intestine, and type II mucosae such as the vagina.
Asunto(s)
Linfocitos T CD8-positivos , Intestinos , Femenino , Humanos , Citometría de Flujo , Linfocitos T CD4-Positivos , Membrana Mucosa , Memoria InmunológicaRESUMEN
The initial immune response to HIV determines transmission. However, due to technical limitations we still do not have a comparative map of early mucosal transmission events. By combining RNAscope, cyclic immunofluorescence, and image analysis tools, we quantify HIV transmission signatures in intact human colorectal explants within 2 h of topical exposure. We map HIV enrichment to mucosal dendritic cells (DCs) and submucosal macrophages, but not CD4+ T cells, the primary targets of downstream infection. HIV+ DCs accumulate near and within lymphoid aggregates, which act as early sanctuaries of high viral titers while facilitating HIV passage to the submucosa. Finally, HIV entry induces recruitment and clustering of target cells, facilitating DC- and macrophage-mediated HIV transfer and enhanced infection of CD4+ T cells. These data demonstrate a rapid response to HIV structured to maximize the likelihood of mucosal infection and provide a framework for in situ studies of host-pathogen interactions and immune-mediated pathologies.
Asunto(s)
Neoplasias Colorrectales , Infecciones por VIH , VIH-1 , Linfocitos T CD4-Positivos , Neoplasias Colorrectales/patología , Células Dendríticas , Interacciones Huésped-Patógeno , HumanosRESUMEN
Although the advent of ART has significantly reduced the morbidity and mortality associated with HIV infection, the stable pool of HIV in latently infected cells requires lifelong treatment adherence, with the cessation of ART resulting in rapid reactivation of the virus and productive HIV infection. Therefore, these few cells containing replication-competent HIV, known as the latent HIV reservoir, act as the main barrier to immune clearance and HIV cure. While several strategies involving HIV silencing or its reactivation in latently infected cells for elimination by immune responses have been explored, exciting cell based immune therapies involving genetically engineered T cells expressing synthetic chimeric receptors (CAR T cells) are highly appealing and promising. CAR T cells, in contrast to endogenous cytotoxic T cells, can function independently of MHC to target HIV-infected cells, are efficacious and have demonstrated acceptable safety profiles and long-term persistence in peripheral blood. In this review, we present a comprehensive picture of the current efforts to target the HIV latent reservoir, with a focus on CAR T cell therapies. We highlight the current challenges and advances in this field, while discussing the importance of novel CAR designs in the efforts to find a HIV cure.
Asunto(s)
Infecciones por VIH , VIH-1 , Linfocitos T CD4-Positivos , Humanos , Inmunoterapia Adoptiva , Latencia del VirusRESUMEN
Despite long-term antiretroviral therapy (ART), HIV-1 persists within a reservoir of CD4+ T cells that contribute to viral rebound if treatment is interrupted. Identifying the cellular populations that contribute to the HIV-1 reservoir and understanding the mechanisms of viral persistence are necessary to achieve an effective cure. In this regard, through Full-Length Individual Proviral Sequencing, we observed that the HIV-1 proviral landscape was different and changed with time on ART across naive and memory CD4+ T cell subsets isolated from 24 participants. We found that the proportion of genetically intact HIV-1 proviruses was higher and persisted over time in effector memory CD4+ T cells when compared with naive, central, and transitional memory CD4+ T cells. Interestingly, we found that escape mutations remained stable over time within effector memory T cells during therapy. Finally, we provided evidence that Nef plays a role in the persistence of genetically intact HIV-1. These findings posit effector memory T cells as a key component of the HIV-1 reservoir and suggest Nef as an attractive therapeutic target.
Asunto(s)
Infecciones por VIH , VIH-1 , Linfocitos T CD4-Positivos , ADN Viral/genética , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , VIH-1/genética , Humanos , Provirus/genética , Carga Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/uso terapéuticoRESUMEN
Although HIV infection inhibits interferon responses in its target cells in vitro, interferon signatures can be detected in vivo soon after sexual transmission, mainly attributed to plasmacytoid dendritic cells (pDCs). In this study, we examined the physiological contributions of pDCs to early HIV acquisition using coculture models of pDCs with myeloid DCs, macrophages and the resting central, transitional and effector memory CD4 T cell subsets. pDCs impacted infection in a cell-specific manner. In myeloid cells, HIV infection was decreased via antiviral effects, cell maturation and downregulation of CCR5 expression. In contrast, in resting memory CD4 T cells, pDCs induced a subset-specific increase in intracellular HIV p24 protein expression without any activation or increase in CCR5 expression, as measured by flow cytometry. This increase was due to reactivation rather than enhanced viral spread, as blocking HIV entry via CCR5 did not alter the increased intracellular p24 expression. Furthermore, the load and proportion of cells expressing HIV DNA were restricted in the presence of pDCs while reverse transcriptase and p24 ELISA assays showed no increase in particle associated reverse transcriptase or extracellular p24 production. In addition, pDCs also markedly induced the expression of CD69 on infected CD4 T cells and other markers of CD4 T cell tissue retention. These phenotypic changes showed marked parallels with resident memory CD4 T cells isolated from anogenital tissue using enzymatic digestion. Production of IFNα by pDCs was the main driving factor for all these results. Thus, pDCs may reduce HIV spread during initial mucosal acquisition by inhibiting replication in myeloid cells while reactivating latent virus in resting memory CD4 T cells and retaining them for immune clearance.
Asunto(s)
Células Dendríticas/virología , Infecciones por VIH/virología , VIH/inmunología , Interferón-alfa/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Células Dendríticas/inmunología , Citometría de Flujo , VIH/genética , VIH/fisiología , Proteína p24 del Núcleo del VIH/genética , Proteína p24 del Núcleo del VIH/metabolismo , Infecciones por VIH/inmunología , Humanos , Células Mieloides/inmunología , Células Mieloides/virología , FenotipoRESUMEN
Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. As such they deliver HIV to its primary target cells; CD4 T cells. Most MNP HIV transmission studies have focused on epithelial MNPs. However, as mucosal trauma and inflammation are now known to be strongly associated with HIV transmission, here we examine the role of sub-epithelial MNPs which are present in a diverse array of subsets. We show that HIV can penetrate the epithelial surface to interact with sub-epithelial resident MNPs in anogenital explants and define the full array of subsets that are present in the human anogenital and colorectal tissues that HIV may encounter during sexual transmission. In doing so we identify two subsets that preferentially take up HIV, become infected and transmit the virus to CD4 T cells; CD14+CD1c+ monocyte-derived dendritic cells and langerin-expressing conventional dendritic cells 2 (cDC2).
Asunto(s)
Canal Anal/citología , Antígenos CD/metabolismo , Células Dendríticas/metabolismo , Genitales/citología , VIH-1/fisiología , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa/metabolismo , Monocitos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Forma de la Célula , Colagenasas/metabolismo , Dermis/metabolismo , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Humanos , Receptores de Lipopolisacáridos/metabolismo , Membrana Mucosa/metabolismo , Fagocitos/metabolismo , Fenotipo , Receptores CCR5/metabolismo , Lectina 1 Similar a Ig de Unión al Ácido Siálico/metabolismo , Transcripción GenéticaRESUMEN
Tissue-resident memory T cells (TRM) were first described in 2009. While initially the major focus was on CD8+ TRM, there has recently been increased interest in defining the phenotype and the role of CD4+ TRM in diseases. Circulating CD4+ T cells seed CD4+ TRM, but there also appears to be an equilibrium between CD4+ TRM and blood CD4+ T cells. CD4+ TRM are more mobile than CD8+ TRM, usually localized deeper within the dermis/lamina propria and yet may exhibit synergy with CD8+ TRM in disease control. This has been demonstrated in herpes simplex infections in mice. In human recurrent herpes infections, both CD4+ and CD8+ TRM persisting between lesions may control asymptomatic shedding through interferon-gamma secretion, although this has been more clearly shown for CD8+ T cells. The exact role of the CD4+/CD8+ TRM axis in the trigeminal ganglia and/or cornea in controlling recurrent herpetic keratitis is unknown. In HIV, CD4+ TRM have now been shown to be a major target for productive and latent infection in the cervix. In HSV and HIV co-infections, CD4+ TRM persisting in the dermis support HIV replication. Further understanding of the role of CD4+ TRM and their induction by vaccines may help control sexual transmission by both viruses.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/inmunología , Herpes Simple/inmunología , Memoria Inmunológica/inmunología , Animales , Coinfección/inmunología , Coinfección/virología , Infecciones por VIH/virología , Herpes Simple/virología , HumanosRESUMEN
Mononuclear phagocytes are antigen presenting cells that play a key role in linking the innate and adaptive immune systems. In tissue, these consist of Langerhans cells, dendritic cells and macrophages, all of which express the key HIV entry receptors CD4 and CCR5 making them directly infectible with HIV. Mononuclear phagocytes are the first cells of the immune system to interact with invading pathogens such as HIV. Each cell type expresses a specific repertoire of pathogen binding receptors which triggers pathogen uptake and the release of innate immune cytokines. Langerhans cells and dendritic cells migrate to lymph nodes and present antigens to CD4 T cells, whereas macrophages remain tissue resident. Here we review how HIV-1 manipulates these cells by blocking their ability to produce innate immune cytokines and taking advantage of their antigen presenting cell function in order to gain transport to its primary target cells, CD4 T cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Fagocitos/inmunología , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/virología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/virología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Células de Langerhans/inmunología , Células de Langerhans/metabolismo , Células de Langerhans/virología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Fagocitos/metabolismo , Fagocitos/virología , Receptores CCR5/inmunología , Receptores CCR5/metabolismoRESUMEN
Langerhans cells (LC) are thought to be the only mononuclear phagocyte population in the epidermis where they detect pathogens. Here, we show that CD11c+ dendritic cells (DCs) are also present. These cells are transcriptionally similar to dermal cDC2 but are more efficient antigen-presenting cells. Compared to LCs, epidermal CD11c+ DCs are enriched in anogenital tissues where they preferentially interact with HIV, express the higher levels of HIV entry receptor CCR5, support the higher levels of HIV uptake and replication and are more efficient at transmitting the virus to CD4 T cells. Importantly, these findings are observed using both a lab-adapted and transmitted/founder strain of HIV. We also describe a CD33low cell population, which is transcriptionally similar to LCs but does not appear to function as antigen-presenting cells or acts as HIV target cells. Our findings reveal that epidermal DCs in anogenital tissues potentially play a key role in sexual transmission of HIV.
Asunto(s)
Células Dendríticas/virología , Células Epidérmicas/virología , Infecciones por VIH/transmisión , VIH-1/inmunología , Presentación de Antígeno/inmunología , Antígeno CD11c/metabolismo , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Epidérmicas/inmunología , Células Epidérmicas/metabolismo , Epidermis/inmunología , Femenino , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/patogenicidad , Voluntarios Sanos , Humanos , Masculino , Cultivo Primario de Células , Receptores CCR5/metabolismo , Lectina 3 Similar a Ig de Unión al Ácido Siálico/metabolismo , Linfocitos T/inmunología , Internalización del VirusRESUMEN
Viruses manipulate the complex interferon and interferon-stimulated gene (ISG) system in different ways. We have previously shown that HIV inhibits type I and III interferons in its key target cells but directly stimulates a subset of >20 ISGs in macrophages and dendritic cells, many of which are antiviral. Here, we examine the mechanism of induction of ISGs and show this occurs in two phases. The first phase was transient (0 to 24 h postinfection [hpi]), induced mainly by extracellular vesicles and one of its component proteins, HSP90α, contained within the HIV inoculum. The second, dominant, and persistent phase (>48 hpi) was induced via newly transcribed HIV RNA and sensed via RIGI, as shown by the reduction in ISG expression after the knockdown of the RIGI adaptor, MAVS, by small interfering RNA (siRNA) and the inhibition of both the initiation and elongation of HIV transcription by short hairpin RNA (shRNA) transcriptional silencing. We further define the induction pathway, showing sequential HIV RNA stimulation via Tat, RIGI, MAVS, IRF1, and IRF7, also identified by siRNA knockdown. IRF1 also plays a key role in the first phase. We also show that the ISGs IFIT1 to -3 inhibit HIV production, measured as extracellular infectious virus. All induced antiviral ISGs probably lead to restriction of HIV replication in macrophages, contributing to a persistent, noncytopathic infection, while the inhibition of interferon facilitates spread to adjacent cells. Both may influence the size of macrophage HIV reservoirs in vivo Elucidating the mechanisms of ISG induction may help in devising immunotherapeutic strategies to limit the size of these reservoirs.IMPORTANCE HIV, like other viruses, manipulates the antiviral interferon and interferon-stimulated gene (ISG) system to facilitate its initial infection and establishment of viral reservoirs. HIV specifically inhibits all type I and III interferons in its target cells, including macrophages, dendritic cells, and T cells. It also induces a subset of over 20 ISGs of differing compositions in each cell target. This occurs in two temporal phases in macrophages. Extracellular vesicles contained within the inoculum induce the first, transient phase of ISGs. Newly transcribed HIV RNA induce the second, dominant ISG phase, and here, the full induction pathway is defined. Therefore, HIV nucleic acids, which are potent inducers of interferon and ISGs, are initially concealed, and antiviral ISGs are not fully induced until replication is well established. These antiviral ISGs may contribute to persistent infection in macrophages and to the establishment of viral reservoirs in vivo.
Asunto(s)
Regulación de la Expresión Génica , VIH-1/fisiología , Interferones/metabolismo , Macrófagos/virología , ARN Viral/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Dendríticas/virología , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , ARN Interferente Pequeño , Proteínas de Unión al ARN , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Transducción de SeñalRESUMEN
Langerhans cells (LCs) situated in stratified squamous epithelium of the skin and mucosal tissue are amongst the first cells that sexually transmitted pathogens encounter during transmission. They are potent antigen presenting cells and play a key role in the host mounting an appropriate immune response. As such, viruses have evolved complex strategies to manipulate these cells to facilitate successful transmission. One of best studied examples is HIV, which manipulates the natural function of these cells to interact with CD4 T cells, which are the main target cell for HIV in which rapid replication occurs. However, there is controversy in the literature as to the role that LCs play in this process. Langerhans cells also play a key role in the way the body mounts an immune response to HSV, and there is also a complex interplay between the transmission of HSV and HIV that involves LCs. In this article, we review both past and present literatures with a particular focus on a few very recent studies that shed new light on the role that LCs play in the transmission and immune response to these 2 pathogens.
Asunto(s)
Infecciones por VIH/transmisión , Herpes Genital/transmisión , Interacciones Huésped-Patógeno , Células de Langerhans/inmunología , Células de Langerhans/virología , HumanosRESUMEN
UNLABELLED: It is well established that influenza A virus (IAV) attachment to and infection of epithelial cells is dependent on sialic acid (SIA) at the cell surface, although the specific receptors that mediate IAV entry have not been defined and multiple receptors may exist. Lec2 Chinese hamster ovary (CHO) cells are SIA deficient and resistant to IAV infection. Here we demonstrate that the expression of the C-type lectin receptor langerin in Lec2 cells (Lec2-Lg) rendered them permissive to IAV infection, as measured by replication of the viral genome, transcription of viral mRNA, and synthesis of viral proteins. Unlike SIA-dependent infection of parental CHO cells, IAV attachment and infection of Lec2-Lg cells was mediated via lectin-mediated recognition of mannose-rich glycans expressed by the viral hemagglutinin glycoprotein. Lec2 cells expressing endocytosis-defective langerin bound IAV efficiently but remained resistant to IAV infection, confirming that internalization via langerin was essential for infectious entry. Langerin-mediated infection of Lec2-Lg cells was pH and dynamin dependent, occurred via clathrin- and caveolin-mediated endocytic pathways, and utilized early (Rab5(+)) but not late (Rab7(+)) endosomes. This study is the first to demonstrate that langerin represents an authentic receptor that binds and internalizes IAV to facilitate infection. Moreover, it describes a unique experimental system to probe specific pathways and compartments involved in infectious entry following recognition of IAV by a single cell surface receptor. IMPORTANCE: On the surface of host cells, sialic acid (SIA) functions as the major attachment factor for influenza A viruses (IAV). However, few studies have identified specific transmembrane receptors that bind and internalize IAV to facilitate infection. Here we identify human langerin as a transmembrane glycoprotein that can act as an attachment factor and a bone fide endocytic receptor for IAV infection. Expression of langerin by an SIA-deficient cell line resistant to IAV rendered cells permissive to infection. As langerin represented the sole receptor for IAV infection in this system, we have defined the pathways and compartments involved in infectious entry of IAV into cells following recognition by langerin.
Asunto(s)
Antígenos CD/metabolismo , Virus de la Influenza A/fisiología , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa/metabolismo , Receptores Virales/metabolismo , Acoplamiento Viral , Internalización del Virus , Animales , Células CHO , Cricetulus , Dinaminas/metabolismo , Endocitosis , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Polisacáridos/metabolismo , Unión ProteicaRESUMEN
The mechanism by which immunity to Herpes Simplex Virus (HSV) is initiated is not completely defined. HSV initially infects mucosal epidermis prior to entering nerve endings. In mice, epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to encounter HSV, but it is CD103(+) dermal DCs that carry viral antigen to lymph nodes for antigen presentation, suggesting DC cross-talk in skin. In this study, we compared topically HSV-1 infected human foreskin explants with biopsies of initial human genital herpes lesions to show LCs are initially infected then emigrate into the dermis. Here, LCs bearing markers of maturation and apoptosis formed large cell clusters with BDCA3(+) dermal DCs (thought to be equivalent to murine CD103(+) dermal DCs) and DC-SIGN(+) DCs/macrophages. HSV-expressing LC fragments were observed inside the dermal DCs/macrophages and the BDCA3(+) dermal DCs had up-regulated a damaged cell uptake receptor CLEC9A. No other infected epidermal cells interacted with dermal DCs. Correspondingly, LCs isolated from human skin and infected with HSV-1 in vitro also underwent apoptosis and were taken up by similarly isolated BDCA3(+) dermal DCs and DC-SIGN(+) cells. Thus, we conclude a viral antigen relay takes place where HSV infected LCs undergo apoptosis and are taken up by dermal DCs for subsequent antigen presentation. This provides a rationale for targeting these cells with mucosal or perhaps intradermal HSV immunization.
Asunto(s)
Células Dendríticas/virología , Herpesvirus Humano 1/fisiología , Células de Langerhans/virología , Simplexvirus/patogenicidad , Piel/virología , Movimiento Celular , Citometría de Flujo , Humanos , Microscopía FluorescenteRESUMEN
UNLABELLED: Dendritic cells (DCs) and macrophages are present in the tissues of the anogenital tract, where HIV-1 transmission occurs in almost all cases. These cells are both target cells for HIV-1 and represent the first opportunity for the virus to interfere with innate recognition. Previously we have shown that both cell types fail to produce type I interferons (IFNs) in response to HIV-1 but that, unlike T cells, the virus does not block IFN induction by targeting IFN regulatory factor 3 (IRF3) for cellular degradation. Thus, either HIV-1 inhibits IFN induction by an alternate mechanism or, less likely, these cells fail to sense HIV-1. Here we show that HIV-1 (but not herpes simplex virus 2 [HSV-2] or Sendai virus)-exposed DCs and macrophages fail to induce the expression of all known type I and III IFN genes. These cells do sense the virus, and pattern recognition receptor (PRR)-induced signaling pathways are triggered. The precise stage in the IFN-inducing signaling pathway that HIV-1 targets to block IFN induction was identified; phosphorylation but not K63 polyubiquitination of TANK-binding kinase 1 (TBK1) was completely inhibited. Two HIV-1 accessory proteins, Vpr and Vif, were shown to bind to TBK1, and their individual deletion partly restored IFN-ß expression. Thus, the inhibition of TBK1 autophosphorylation by binding of these proteins appears to be the principal mechanism by which HIV-1 blocks type I and III IFN induction in myeloid cells. IMPORTANCE: Dendritic cells (DCs) and macrophages are key HIV target cells. Therefore, definition of how HIV impairs innate immune responses to initially establish infection is essential to design preventative interventions, especially by restoring initial interferon production. Here we demonstrate how HIV-1 blocks interferon induction by inhibiting the function of a key kinase in the interferon signaling pathway, TBK1, via two different viral accessory proteins. Other viral proteins have been shown to target the general effects of TBK1, but this precise targeting between ubiquitination and phosphorylation of TBK1 is novel.
Asunto(s)
Células Dendríticas/inmunología , VIH-1/inmunología , Interacciones Huésped-Patógeno , Macrófagos/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Células Cultivadas , Células Dendríticas/virología , Humanos , Evasión Inmune , Interferones/antagonistas & inhibidores , Macrófagos/virología , Fosforilación , Procesamiento Proteico-Postraduccional , Transducción de Señal , UbiquitinaciónRESUMEN
Prior HSV-2 infection enhances the acquisition of HIV-1 >3-fold. In genital herpes lesions, the superficial layers of stratified squamous epithelium are disrupted, allowing easier access of HIV-1 to Langerhans cells (LC) in the epidermis and perhaps even dendritic cells (DCs) in the outer dermis, as well as to lesion infiltrating activated T lymphocytes and macrophages. Therefore, we examined the effects of coinfection with HIV-1 and HSV-2 on monocyte-derived DCs (MDDC). With simultaneous coinfection, HSV-2 significantly stimulated HIV-1 DNA production 5-fold compared with HIV-1 infection alone. Because <1% of cells were dually infected, this was a field effect. Virus-stripped supernatants from HSV-2-infected MDDCs were shown to enhance HIV-1 infection, as measured by HIV-1-DNA and p24 Ag in MDDCs. Furthermore these supernatants markedly stimulated CCR5 expression on both MDDCs and LCs. TNF-α was by far the most prominent cytokine in the supernatant and also within HSV-2-infected MDDCs. HSV-2 infection of isolated immature epidermal LCs, but not keratinocytes, also produced TNF-α (and low levels of IFN-ß). Neutralizing Ab to TNF-α and its receptor, TNF-R1, on MDDCs markedly inhibited the CCR5-stimulating effect of the supernatant. Therefore, these results suggest that HSV-2 infection of DCs in the skin during primary or recurrent genital herpes may enhance HIV-1 infection of adjacent DCs, thus contributing to acquisition of HIV-1 through herpetic lesions.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , VIH-1/fisiología , Herpesvirus Humano 2/fisiología , Receptores CCR5/genética , Factor de Necrosis Tumoral alfa/metabolismo , Replicación Viral , Coinfección , Medios de Cultivo Condicionados/metabolismo , Citocinas/biosíntesis , Células Dendríticas/virología , Regulación de la Expresión Génica , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Herpes Genital/genética , Herpes Genital/inmunología , Herpes Genital/metabolismo , Herpesvirus Humano 2/efectos de la radiación , Humanos , Modelos Biológicos , Receptores CCR5/metabolismo , Regulación hacia ArribaRESUMEN
Epidermal Langerhans cells (eLCs) uniquely express the C-type lectin receptor langerin in addition to the HIV entry receptors CD4 and CCR5. They are among the first target cells to encounter HIV in the anogenital stratified squamous mucosa during sexual transmission. Previous reports on the mechanism of HIV transfer to T cells and the role of langerin have been contradictory. In this study, we examined HIV replication and langerin-mediated viral transfer by authentic immature eLCs and model Mutz-3 LCs. eLCs were productively infected with HIV, whereas Mutz-3 LCs were not susceptible because of a lack of CCR5 expression. Two successive phases of HIV viral transfer to T cells via cave/vesicular trafficking and de novo replication were observed with eLCs as previously described in monocyte-derived or blood dendritic cells, but only first phase transfer was observed with Mutz-3 LCs. Langerin was expressed as trimers after cross-linking on the cell surface of Mutz-3 LCs and in this form preferentially bound HIV envelope protein gp140 and whole HIV particles via the carbohydrate recognition domain (CRD). Both phases of HIV transfer from eLCs to T cells were inhibited when eLCs were pretreated with a mAb to langerin CRD or when HIV was pretreated with a soluble langerin trimeric extracellular domain or by a CRD homolog. However, the langerin homolog did not inhibit direct HIV infection of T cells. These two novel soluble langerin inhibitors could be developed to prevent HIV uptake, infection, and subsequent transfer to T cells during early stages of infection.
Asunto(s)
Antígenos CD/inmunología , Infecciones por VIH/inmunología , VIH-1/fisiología , Células de Langerhans/inmunología , Lectinas Tipo C/inmunología , Lectinas de Unión a Manosa/inmunología , Linfocitos T/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Transporte Biológico/inmunología , Infecciones por VIH/patología , Humanos , Células de Langerhans/patología , Células de Langerhans/virología , Lectinas Tipo C/antagonistas & inhibidores , Lectinas de Unión a Manosa/antagonistas & inhibidores , Linfocitos T/patología , Linfocitos T/virología , Replicación ViralRESUMEN
Dendritic cells (DC) present in the genital tract are one of the first cells to encounter HIV during sexual mucosal transmission. In addition they are able to efficiently transfer the virus to its main target cells, CD4(+) T-lymphocytes. As such an understanding of how HIV interacts with and manipulates DCs is of key importance for the design of mucosal vaccines and microbicides. However working with these cells is difficult for several reasons. Firstly, immature DCs are difficult to infect due to their high endocytic capacity and mature DCs are usually resistant to infection. Secondly, tissue DCs are inherently difficult to isolate, which results in small yields and the cells are prone to maturation as a result of extraction. Here we describe how to isolate CD1a expressing Langerhans cells from the epidermis and CD1a(+), CD14(+) and perhaps BDCA3(+) DCs from the dermis. We also describe how to produce the model monocyte-derived DC (MDDC) by cytokine stimulation of CD14(+) monocytes, which results in the production of large numbers of immature cells. We also describe methods by which high titer HIV stocks can be generated to infect a significant proportion of DCs and also methods for determining the titer of such stocks.