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Early Dengue Virus Infection in Human Skin: A Cycle of Inflammation and Infectivity

  • Matthew O. Ivory
    Affiliations
    Departments of Dermatology and Academic Wound Healing, Institute of Infection and Immunity, Cardiff University and University Hospital of Wales, Heath Park, Cardiff, UK

    School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UK
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  • James C. Birchall
    Affiliations
    School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UK
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  • Vincent Piguet
    Correspondence
    Departments of Dermatology and Academic Wound Healing, Institute of Infection and Immunity, Cardiff University and University Hospital of Wales, Heath Park, Cardiff CF14 4XN, UK
    Affiliations
    Departments of Dermatology and Academic Wound Healing, Institute of Infection and Immunity, Cardiff University and University Hospital of Wales, Heath Park, Cardiff, UK
    Search for articles by this author
      Early events during dengue virus (DENV) infection remain poorly understood. In this issue, Schaeffer and colleagues employ ex vivo human skin cells to investigate viral infection. They show that skin-resident immune cells are infected by DENV and that their infectability is increased in the inflammatory skin conditions (especially those in which IL-4 is released) that accompany the mosquito bites transmitting the virus.
      Dengue virus (DENV) is a single-strand positive RNA virus, and it is the most common arthropod-transmitted virus (arbovirus) that is transmitted to humans exclusively via Aedes mosquitos. Dengue viral infection presents most commonly with fever, muscle/joint pains, and headache that are often accompanied by a characteristic rash. Serious infection can progress to hemorrhagic dengue fever or dengue shock syndrome, which may lead to death via hemorrhage and hypovolemic shock, respectively. Neither vaccines nor specific antiviral treatments are available, and the World Health Organization estimates that almost half of the world’s population are currently at a risk for the disease. The potential severity of symptoms, significant at-risk population, and lack of effective treatments have led to dengue disease being named as one of the Bill and Melinda Gates Foundation’s “neglected” tropical diseases that require an increase in research to improve global health. In the search for new treatment/prevention strategies, improved understanding of the viral pathogenesis is essential. The study by
      • Schaeffer E.
      • Flacher V.
      • Papageorgiou V.
      • et al.
      Dermal CD14+ dendritic cell and macrophage infection by dengue virus is stimulated by interleukin-4.
      in this issue employs an ex vivo human skin cell model to further our understanding of DENV infection.
      As DENV is transmitted via mosquito bites, the skin is the primary site of infection. Human skin contains a variety of immunological cells, including Langerhans cells (LCs) in the epidermis, and subsets of dermal dendritic cells (DCs), along with macrophages and T cells, in the dermis. These cells will be among the first to encounter the virus following transmission. Both LCs and DCs have been shown previously to be infected by DENV (
      • Cerny D.
      • Haniffa M.
      • Shin A.
      • et al.
      Selective susceptibility of human skin antigen presenting cells to productive dengue virus infection.
      ). In their study,
      • Schaeffer E.
      • Flacher V.
      • Papageorgiou V.
      • et al.
      Dermal CD14+ dendritic cell and macrophage infection by dengue virus is stimulated by interleukin-4.
      investigated the interactions between DENV and dermal DCs and macrophages under normal conditions and in a model of inflammation. Dermal DCs, unlike LCs, are heterogeneous. Three subsets are commonly cited in the literature: CD14+, CD1c+, and CD141+ DCs (
      • Chu C.C.
      • Ali N.
      • Karagiannis P.
      • et al.
      Resident CD141 (BDCA3)+ dendritic cells in human skin produce IL-10 and induce regulatory T cells that suppress skin inflammation.
      and
      • Haniffa M.
      • Shin A.
      • Bigley V.
      • et al.
      Human tissues contain CD141hi cross-presenting dendritic cells with functional homology to mouse CD103+ nonlymphoid dendritic cells.
      ). Each subset possesses individual properties in terms of viral infectability, antigen processing, and immune response stimulation.
      • Schaeffer E.
      • Flacher V.
      • Papageorgiou V.
      • et al.
      Dermal CD14+ dendritic cell and macrophage infection by dengue virus is stimulated by interleukin-4.
      focus on CD14+ and CD1c+ DCs alongside dermal macrophages to determine their susceptibility to infection following viral entry into the skin.
      C-type lectins such as Langerin (CD207), dendritic cell-Specific Intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN; CD209), and mannose receptor (MR/CD206) enable immunological cells to take up antigen from the extracellular environment (
      • Engering A.
      • Geijtenbeek T.B.
      • van Vliet S.J.
      • et al.
      The dendritic cell-specific adhesion receptor DC-SIGN internalizes antigen for presentation to T cells.
      and
      • de Witte L.
      • Nabatov A.
      • Pion M.
      • et al.
      Langerin is a natural barrier to HIV-1 transmission by Langerhans cells.
      ). This method of uptake is exploited by a number of viruses, such as HIV-1 and DENV, to gain entry into the cell that they can subsequently infect (
      • Arrighi J.F.
      • Pion M.
      • Garcia E.
      • et al.
      DC-SIGN-mediated infectious synapse formation enhances X4 HIV-1 transmission from dendritic cells to T cells.
      ). The role of DC-SIGN in DENV pathogenesis has been confirmed through the study of patients with certain DC-SIGN polymorphisms and who possess a higher risk of developing dengue hemorrhagic fever following viral infection (
      • Sakuntabhai A.
      • Turbpaiboon C.
      • Casademont I.
      • et al.
      A variant in the CD209 promoter is associated with severity of dengue disease.
      ).
      Given the proposed role of DC-SIGN in DENV infection,
      • Schaeffer E.
      • Flacher V.
      • Papageorgiou V.
      • et al.
      Dermal CD14+ dendritic cell and macrophage infection by dengue virus is stimulated by interleukin-4.
      initially determined the effect of skin inflammation on the number of DC-SIGN+ cells, using bullous pemphigoid, hypereosinophilic syndrome, mastocytosis, and scabies infection-affected skin as models for mosquito-bitten skin, on the basis of shared properties of basophil recruitment and eosinophilia. It was found that all of the inflammatory conditions studied possessed higher numbers of DC-SIGN+ cells in the skin, when compared with non-inflammed skin, leading the authors to suggest that the inflammation arising from a mosquito bite might enhance the recruitment of cells available for infection at the site.
      • Schaeffer E.
      • Flacher V.
      • Papageorgiou V.
      • et al.
      Dermal CD14+ dendritic cell and macrophage infection by dengue virus is stimulated by interleukin-4.
      obtained skin DCs (along with other migratory cells) using a well-established migratory cell protocol (
      • Chu C.C.
      • Ali N.
      • Karagiannis P.
      • et al.
      Resident CD141 (BDCA3)+ dendritic cells in human skin produce IL-10 and induce regulatory T cells that suppress skin inflammation.
      ). Cells were infected in an unsorted state, then the infection levels were determined via flow cytometry. They found that all of the skin DC subsets could be infected by DENV, with infectability being highest in CD14+ dermal DCs and lowest in LCs, with higher DC-SIGN expression in CD14+ cells suggested as a reason for this difference. The low infectability of LCs found by
      • Schaeffer E.
      • Flacher V.
      • Papageorgiou V.
      • et al.
      Dermal CD14+ dendritic cell and macrophage infection by dengue virus is stimulated by interleukin-4.
      contradicts a previous study that found LCs to be the most susceptible skin immune cell to DENV infection (
      • Cerny D.
      • Haniffa M.
      • Shin A.
      • et al.
      Selective susceptibility of human skin antigen presenting cells to productive dengue virus infection.
      ).
      Transmission of arboviruses is accompanied by trauma at the local site, as well as the delivery of insect salivary substances, which can elicit an inflammatory response in isolation. It is speculated that the Th2-type immune response that occurs following insect bites leads to increased rates of infection and clinical symptoms from DENV (
      • Cox J.
      • Mota J.
      • Sukupolvi-Petty S.
      • et al.
      Mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice.
      ).
      • Schaeffer E.
      • Flacher V.
      • Papageorgiou V.
      • et al.
      Dermal CD14+ dendritic cell and macrophage infection by dengue virus is stimulated by interleukin-4.
      focused on IL-4, which is released from basophils, mast cells, and T cells during the immune response to foreign antigens, to determine whether inflammation from the mosquito bite promotes viral infection of cutaneous immune cells. Exposing DCs to IL-4 led to a marked increase in CD14+ DC infection rates with the virus, but with little effect on other DCs/LCs, suggesting that CD14+ cell infection may become prominent following mosquito bite inflammation. Incubation of dermal DCs with IL-4 also enhanced their T-cell stimulatory ability as evidenced by proliferation of carboxyfluorescein succinimidyl ester-labeled naive T cells following co-culture. Their presence in the skin and expression of the C-type lectin MR make macrophages a cell population potentially susceptible to DENV infection. Dermal macrophage infection was studied following enzymatic digestion and cell sorting of skin specimens. This allowed the investigators to obtain a population of CD14+ macrophages; the investigators speculate that CD14+ DCs may have developed a macrophage-like phenotype in culture. It was found that IL-4, with or without GM-CSF, also increased their susceptibility to DENV infection. Infection led to the release of tumor necrosis factor (TNF-α), most markedly in the macrophages with higher levels of viral infection. This led the authors to speculate that DENV infection of DCs could lead to potent T-cell antiviral responses, whereas macrophage infection would most likely lead to the symptoms of inflammation associated with DENV infection.
      • Schaeffer E.
      • Flacher V.
      • Papageorgiou V.
      • et al.
      Dermal CD14+ dendritic cell and macrophage infection by dengue virus is stimulated by interleukin-4.
      reason that because CD14+ DCs are most readily infected, especially under inflammatory conditions, and because they tend to generate humoral immune responses, these cells are likely to be responsible for the increased severity of DENV disease upon subsequent exposure to a different serotype of the virus. This occurs because of cross-reactive antibodies that can bind to other DENV serotypes without fully neutralizing them, instead facilitating their entry into immune cells and leading to more severe infection (
      • Endy T.P.
      • Nisalak A.
      • Chunsuttitwat S.
      • et al.
      Relationship of preexisting dengue virus (DV) neutralizing antibody levels to viremia and severity of disease in a prospective cohort study of DV infection in Thailand.
      ). This could represent an important subject of further study, as moderating this response might lower the risk of serious DENV disease. The release of TNF-α by macrophages in the dermis, especially when exposed to IL-4, is also likely to be important because of the cytokine’s proposed central role in the development of dengue fever. It is also likely that the local inflammation caused by TNF-α release would lead to the release of IL-4, creating a cycle of increasing rates of infection by the local immune cells.
      The studies reported by
      • Schaeffer E.
      • Flacher V.
      • Papageorgiou V.
      • et al.
      Dermal CD14+ dendritic cell and macrophage infection by dengue virus is stimulated by interleukin-4.
      build upon earlier animal studies that have shown an increase in arbovirus infection rates because of simultaneous immune responses to mosquito salivary substances, but it widens the focus by employing primary human skin cells, a model that resembles arboviral infection more closely. Understanding how DENV can take advantage of inflammatory conditions for its propagation may reveal novel opportunities to block this virus when invading hosts via skin immune cells.

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