RESUMO
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.
Assuntos
Quimiocinas , Prepúcio do Pênis , Herpes Simples , Herpesvirus Humano 1 , Queratinócitos , Nectinas , Humanos , Masculino , Queratinócitos/virologia , Queratinócitos/metabolismo , Prepúcio do Pênis/virologia , Prepúcio do Pênis/citologia , Nectinas/metabolismo , Herpes Simples/virologia , Herpes Simples/metabolismo , Quimiocinas/metabolismo , Herpesvirus Humano 1/fisiologia , Moléculas de Adesão Celular/metabolismo , Internalização do VírusRESUMO
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.
Assuntos
Células Dendríticas , Infecções por HIV , Humanos , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Receptor Tirosina Quinase Axl , Masculino , HIV-1/imunologia , Feminino , Células Mieloides/metabolismo , Células Mieloides/imunologia , Pessoa de Meia-Idade , AdultoRESUMO
MOTIVATION: High parameter histological techniques have allowed for the identification of a variety of distinct cell types within an image, providing a comprehensive overview of the tissue environment. This allows the complex cellular architecture and environment of diseased tissue to be explored. While spatial analysis techniques have revealed how cell-cell interactions are important within the disease pathology, there remains a gap in exploring changes in these interactions within the disease process. Specifically, there are currently few established methods for performing inference on cell-type co-localization changes across images, hindering an understanding of how cellular environments change with a disease pathology. RESULTS: We have developed the spicyR R package to perform inference on changes in the spatial co-localization of types across groups of images. Application to simulated data demonstrates a high sensitivity and specificity. We the utility of spicyR by applying it to a type 1 diabetes imaging mass cytometry dataset, revealing changes in cellular associations that were relevant to the disease progression. Ultimately, spicyR allows changes in cellular environments to be explored under different pathologies or disease states. AVAILABILITY AND IMPLEMENTATION: R package is freely available at http://bioconductor.org/packages/release/bioc/html/spicyR.html and shiny app implementation at http://shiny.maths.usyd.edu.au/spicyR/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Software , Análise EspacialRESUMO
Skin mononuclear phagocytes (MNPs) provide the first interactions of invading viruses with the immune system. In addition to Langerhans cells (LCs), we recently described a second epidermal MNP population, Epi-cDC2s, in human anogenital epidermis that is closely related to dermal conventional dendritic cells type 2 (cDC2) and can be preferentially infected by HIV. Here we show that in epidermal explants topically infected with herpes simplex virus (HSV-1), both LCs and Epi-cDC2s interact with HSV-1 particles and infected keratinocytes. Isolated Epi-cDC2s support higher levels of infection than LCs in vitro, inhibited by acyclovir, but both MNP subtypes express similar levels of the HSV entry receptors nectin-1 and HVEM, and show similar levels of initial uptake. Using inhibitors of endosomal acidification, actin and cholesterol, we found that HSV-1 utilises different entry pathways in each cell type. HSV-1 predominantly infects LCs, and monocyte-derived MNPs, via a pH-dependent pathway. In contrast, Epi-cDC2s are mainly infected via a pH-independent pathway which may contribute to the enhanced infection of Epi-cDC2s. Both cells underwent apoptosis suggesting that Epi-cDC2s may follow the same dermal migration and uptake by dermal MNPs that we have previously shown for LCs. Thus, we hypothesize that the uptake of HSV and infection of Epi-cDC2s will stimulate immune responses via a different pathway to LCs, which in future may help guide HSV vaccine development and adjuvant targeting.
Assuntos
Herpesvirus Humano 1/fisiologia , Células de Langerhans/virologia , Internalização do Vírus , Adolescente , Animais , Células Cultivadas , Criança , Pré-Escolar , Chlorocebus aethiops , Epiderme/patologia , Epiderme/virologia , Células HaCaT , Células HeLa , Herpes Simples/patologia , Herpes Simples/virologia , Humanos , Lactente , Transdução de Sinais/fisiologia , Células VeroRESUMO
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.
Assuntos
Células Dendríticas/virologia , Infecções por HIV/virologia , HIV/imunologia , Interferon-alfa/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/imunologia , Citometria de Fluxo , HIV/genética , HIV/fisiologia , Proteína do Núcleo p24 do HIV/genética , Proteína do Núcleo p24 do HIV/metabolismo , Infecções por HIV/imunologia , Humanos , Células Mieloides/imunologia , Células Mieloides/virologia , FenótipoRESUMO
Highly multiplexed in situ imaging cytometry assays have made it possible to study the spatial organization of numerous cell types simultaneously. We have addressed the challenge of quantifying complex multi-cellular relationships by proposing a statistical method which clusters local indicators of spatial association. Our approach successfully identifies distinct tissue architectures in datasets generated from three state-of-the-art high-parameter assays demonstrating its value in summarizing the information-rich data generated from these technologies.
Assuntos
Citometria por Imagem , Análise EspacialRESUMO
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.
Assuntos
Linfócitos T CD8-Positivos , Intestinos , Feminino , Humanos , Citometria de Fluxo , Linfócitos T CD4-Positivos , Mucosa , Memória ImunológicaRESUMO
MOTIVATION: Autofluorescence is a long-standing problem that has hindered the analysis of images of tissues acquired by fluorescence microscopy. Current approaches to mitigate autofluorescence in tissue are lab-based and involve either chemical treatment of sections or specialized instrumentation and software to 'unmix' autofluorescent signals. Importantly, these approaches are pre-emptive and there are currently no methods to deal with autofluorescence in acquired fluorescence microscopy images. RESULTS: To address this, we developed Autofluorescence Identifier (AFid). AFid identifies autofluorescent pixels as discrete objects in multi-channel images post-acquisition. These objects can then be tagged for exclusion from downstream analysis. We validated AFid using images of FFPE human colorectal tissue stained for common immune markers. Further, we demonstrate its utility for image analysis where its implementation allows the accurate measurement of HIV-Dendritic cell interactions in a colorectal explant model of HIV transmission. Therefore, AFid represents a major leap forward in the extraction of useful data from images plagued by autofluorescence by offering an approach that is easily incorporated into existing workflows and that can be used with various samples, staining panels and image acquisition methods. We have implemented AFid in ImageJ, Matlab and R to accommodate the diverse image analysis community. AVAILABILITY AND IMPLEMENTATION: AFid software is available at https://ellispatrick.github.io/AFid. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Processamento de Imagem Assistida por Computador , Software , Técnicas Histológicas , Humanos , Microscopia de Fluorescência , Fluxo de TrabalhoRESUMO
HIV latency is the major barrier to a cure for people living with HIV (PLWH) on antiretroviral therapy (ART) because the virus persists in long-lived non-proliferating and proliferating latently infected CD4+ T cells. Latently infected CD4+ T cells do not express viral proteins and are therefore not visible to immune mediated clearance. Therefore, identifying interventions that can reverse latency and also enhance immune mediated clearance is of high interest. Interferons (IFNs) have multiple immune enhancing effects and can inhibit HIV replication in activated CD4+ T cells. However, the effects of IFNs on the establishment and reversal of HIV latency is not understood. Using an in vitro model of latency, we demonstrated that plasmacytoid dendritic cells (pDC) inhibit the establishment of HIV latency through secretion of type I IFNα, IFNß and IFNω but not IFNε or type III IFNλ1 and IFNλ3. However, once latency was established, IFNα but no other IFNs were able to efficiently reverse latency in both an in vitro model of latency and CD4+ T cells collected from PLWH on suppressive ART. Binding of IFNα to its receptor expressed on primary CD4+ T cells did not induce activation of the canonical or non-canonical NFκB pathway but did induce phosphorylation of STAT1, 3 and 5 proteins. STAT5 has been previously demonstrated to bind to the HIV long terminal repeat and activate HIV transcription. We demonstrate diverse effects of interferons on HIV latency with type I IFNα; inhibiting the establishment of latency but also reversing HIV latency once latency is established.
Assuntos
Linfócitos T CD4-Positivos , Repetição Terminal Longa de HIV/imunologia , HIV-1/fisiologia , Interferon-alfa/imunologia , Transcrição Gênica/imunologia , Latência Viral/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Células Dendríticas/virologia , Células HEK293 , Humanos , NF-kappa B/imunologia , Fatores de Transcrição STAT/imunologiaRESUMO
In HIV-hepatitis B virus (HBV) co-infection, adverse liver outcomes including liver fibrosis occur at higher frequency than in HBV-mono-infection, even following antiretroviral therapy (ART) that suppresses both HIV and HBV replication. To determine whether liver disease was associated with intrahepatic or circulating markers of inflammation or burden of HIV or HBV, liver biopsies and blood were collected from HIV-HBV co-infected individuals (n = 39) living in Bangkok, Thailand and naïve to ART. Transient elastography (TE) was performed. Intrahepatic and circulating markers of inflammation and microbial translocation were quantified by ELISA and bead arrays and HIV and HBV infection quantified by PCR. Liver fibrosis (measured by both transient elastography and liver biopsy) was statistically significantly associated with intrahepatic mRNA for CXCL10 and CXCR3 using linear and logistic regression analyses adjusted for CD4 T-cell count. There was no evidence of a relationship between liver fibrosis and circulating HBV DNA, qHBsAg, plasma HIV RNA or circulating cell-associated HIV RNA or DNA. Using immunohistochemistry of liver biopsies from this cohort, intrahepatic CXCL10 was detected in hepatocytes associated with inflammatory liver infiltrates in the portal tracts. In an in vitro model, we infected an HBV-infected hepatocyte cell line with HIV, followed by interferon-γ stimulation. HBV-infected cells lines produced significantly more CXCL10 than uninfected cells lines and this significantly increased in the presence of an increasing multiplicity of HIV infection. Conclusion: Enhanced production of CXCL10 following co-infection of hepatocytes with both HIV and HBV may contribute to accelerated liver disease in the setting of HIV-HBV co-infection.
Assuntos
Quimiocina CXCL10/metabolismo , Coinfecção/complicações , Infecções por HIV/complicações , HIV/isolamento & purificação , Vírus da Hepatite B/isolamento & purificação , Hepatite B/complicações , Cirrose Hepática/epidemiologia , Adulto , Austrália/epidemiologia , Estudos de Coortes , Coinfecção/virologia , Feminino , Infecções por HIV/virologia , Hepatite B/virologia , Humanos , Incidência , Cirrose Hepática/metabolismo , Cirrose Hepática/virologia , Masculino , Países Baixos/epidemiologia , Prognóstico , Tailândia/epidemiologiaRESUMO
We developed a 25-color flow cytometry panel to comprehensively interrogate innate lymphoid cells (ILC), mucosal-associated invariant T (MAIT) cells, natural killer (NK) cells and γδ T cells in human tissues. The ability to isolate and interrogate these cells from fresh human tissue is crucial in understanding the role these cells play at immune-privileged mucosal surfaces like the intestine in health and disease settings. However, liberating these cells from tissue is extremely challenging as many key surface identification markers are susceptible to enzymatic cleavage. Choosing the correct enzyme-antibody clone combination within a high-parameter panel is, therefore, a critical consideration. Here, we present a comprehensive, in-depth analysis of the effect different common digestive enzyme blends have on key surface markers used to identify these cell types. In addition, we compared multiple antibody clones for surface markers that are highly susceptible to enzymatic cleavage, such as CD127 and NKp44, to achieve the most consistent and superior staining patterns among donors.
Assuntos
Células T Invariantes Associadas à Mucosa , Biomarcadores , Citometria de Fluxo , Humanos , Imunidade Inata , Intestinos , Células Matadoras NaturaisRESUMO
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.
Assuntos
Regulação da Expressão Gênica , HIV-1/fisiologia , Interferons/metabolismo , Macrófagos/virologia , RNA Viral/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Dendríticas/virologia , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/metabolismo , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , RNA Interferente Pequeno , Proteínas de Ligação a RNA , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Infecções por HIV/transmissão , Herpes Genital/transmissão , Interações Hospedeiro-Patógeno , Células de Langerhans/imunologia , Células de Langerhans/virologia , HumanosRESUMO
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.
Assuntos
Células Dendríticas/virologia , Herpesvirus Humano 1/fisiologia , Células de Langerhans/virologia , Simplexvirus/patogenicidade , Pele/virologia , Movimento Celular , Citometria de Fluxo , Humanos , Microscopia de FluorescênciaRESUMO
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.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , HIV-1/fisiologia , Herpesvirus Humano 2/fisiologia , Receptores CCR5/genética , Fator de Necrose Tumoral alfa/metabolismo , Replicação Viral , Coinfecção , Meios de Cultivo Condicionados/metabolismo , Citocinas/biossíntese , Células Dendríticas/virologia , Regulação da Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Herpes Genital/genética , Herpes Genital/imunologia , Herpes Genital/metabolismo , Herpesvirus Humano 2/efeitos da radiação , Humanos , Modelos Biológicos , Receptores CCR5/metabolismo , Regulação para CimaRESUMO
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.
Assuntos
Células Dendríticas/imunologia , HIV-1/imunologia , Interações Hospedeiro-Patógeno , Macrófagos/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Células Cultivadas , Células Dendríticas/virologia , Humanos , Evasão da Resposta Imune , Interferons/antagonistas & inibidores , Macrófagos/virologia , Fosforilação , Processamento de Proteína Pós-Traducional , Transdução de Sinais , UbiquitinaçãoRESUMO
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.
Assuntos
Antígenos CD/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Células de Langerhans/imunologia , Lectinas Tipo C/imunologia , Lectinas de Ligação a Manose/imunologia , Linfócitos T/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Transporte Biológico/imunologia , Infecções por HIV/patologia , Humanos , Células de Langerhans/patologia , Células de Langerhans/virologia , Lectinas Tipo C/antagonistas & inibidores , Lectinas de Ligação a Manose/antagonistas & inibidores , Linfócitos T/patologia , Linfócitos T/virologia , Replicação ViralRESUMO
HIV-1 is taken up by immature monocyte derived dendritic cells (iMDDCs) into tetraspanin rich caves from which the virus can either be transferred to T lymphocytes or enter into endosomes resulting in degradation. HIV-1 binding and fusion with the DC membrane results in low level de novo infection that can also be transferred to T lymphocytes at a later stage. We have previously reported that HIV-1 can induce partial maturation of iMDDCs at both stages of trafficking. Here we show that CD45⺠microvesicles (MV) which contaminate purified HIV-1 inocula due to similar size and density, affect DC maturation, de novo HIV-1 infection and transfer to T lymphocytes. Comparing iMDDCs infected with CD45-depleted HIV-1BaL or matched non-depleted preparations, the presence of CD45⺠MVs was shown to enhance DC maturation and ICAM-1 (CD54) expression, which is involved in DCâ¶T lymphocyte interactions, while restricting HIV-1 infection of MDDCs. Furthermore, in the DC culture HIV-1 infected (p24âº) MDDCs were more mature than bystander cells. Depletion of MVs from the HIV-1 inoculum markedly inhibited DCâ¶T lymphocyte clustering and the induction of alloproliferation as well as limiting HIV-1 transfer from DCs to T lymphocytes. The effects of MV depletion on these functions were reversed by the re-addition of purified MVs from activated but not non-activated SUPT1.CCR5-CL.30 or primary T cells. Analysis of the protein complement of these MVs and of these HIV-1 inocula before and after MV depletion showed that Heat Shock Proteins (HSPs) and nef were the likely DC maturation candidates. Recombinant HSP90α and ß and nef all induced DC maturation and ICAM-1 expression, greater when combined. These results suggest that MVs contaminating HIV-1 released from infected T lymphocytes may be biologically important, especially in enhancing T cell activation, during uptake by DCs in vitro and in vivo, particularly as MVs have been detected in the circulation of HIV-1 infected subjects.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/virologia , Infecções por HIV/imunologia , HIV-1/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Adesão Celular/imunologia , Células Cultivadas , Células Dendríticas/patologia , Infecções por HIV/patologia , Humanos , Monócitos/imunologia , Monócitos/patologia , Linfócitos T/patologiaRESUMO
The lineage relationships and fate of human dendritic cells (DCs) have significance for a number of diseases including HIV where both blood and tissue DCs may be infected. We used gene expression profiling of human monocyte and DC subpopulations sorted directly from blood and skin to define the lineage relationships. We also compared these with monocyte-derived DCs (MDDCs) and MUTZ3 Langerhans cells (LCs) to investigate their relevance as model skin DCs. Hierarchical clustering analysis showed that myeloid DCs clustered according to anatomical origin rather than putative lineage. Plasmacytoid DCs formed the most discrete cluster, but ex vivo myeloid cells formed separate clusters of cells both in blood and in skin. Separate and specific DC populations could be determined within skin, and the proportion of CD14(+) dermal DCs (DDCs) was reduced and CD1a(+) DDCs increased during culture, suggesting conversion to CD1a(+)-expressing cells in situ. This is consistent with origin of the CD1a(+) DDCs from a local precursor rather than directly from circulating blood DCs or monocyte precursors. Consistent with their use as model skin DCs, the in vitro-derived MDDC and MUTZ3 LC populations grouped within the skin DC cluster. MDDCs clustered most closely to CD14(+) DDCs; furthermore, common unique patterns of C-type lectin receptor expression were identified between these two cell types. MUTZ3 LCs, however, did not cluster closely with ex vivo-derived LCs. We identified differential expression of novel genes in monocyte and DC subsets including genes related to DC surface receptors (including C-type lectin receptors, TLRs, and galectins).