Dengue virus inoculation to human skin explants: an effective approach to assess in situ the early infection and the effects on cutaneous dendritic cells

Dengue virus (DV) infections are serious causes of morbidity and mortality worldwide, and adaptive (secondary) immune response appears to influence the severity of this arboviral disease (Morens 1994; Halstead & O'Rourke 1977; Rigau-Perez et al. 1998). There are an estimated 50–100 million cases of dengue fever and 250,000-500,000 cases of dengue haemorrhagic fever (DHF) annually in the world (WHO 1997). The dengue shock syndrome (DSS), the most severe and potentially fatal form of the disease, especially in developing countries, is less frequent. In the model proposed here, we have chosen to examine DV2, because it is the prevalent serotype (Gomez-Dantes et al. 2004). The high seroprevalence together with the co-circulation of multiple serotypes indicates that Mexico could be in risk of an important DHF outbreak (Gomez-Dantes et al. 2004). The exact pathogenic mechanisms following DV infection are not well understood, especially in the severe forms of the infection (DHF and DSS). The immune response to DV seems at least partially involved in the process (Halstead 1988; Avirutnan et al. 1998; Diamond et al. 2000). Several theories have been advanced including facilitation of DV infection by non-neutralizing antibodies (Halstead & O'Rourke 1977; Halstead et al. 1977; Morens 1994), the potential differential virulence of each infecting serotype (Leitmeyer et al. 1999; Kawaguchi et al. 2003), CD4-CD8 T-cell cross-reactivity and host factors such as a particular, but yet undefined susceptibility (Klenerman & Zinkernagel 1998; Mongkolsapaya et al. 2003). While there is an excellent research on dengue about the molecular structure and epidemiology of this virus, the basic mechanisms of the immunopathology still remain obscure (Haywood 1994; Kuhn et al. 2002). For instance, until very recently (Johnston et al. 2000; Wu et al. 2000), it was unknown whether DV was able to replicate in cutaneous cells. Likewise, there are two other features largely unexplored regarding DV infection: (i) the very early stages of the infection, because, by necessity, patients are examined when disease is almost over, when the patients are under recovery or when they have entered into DSS and (ii) which type of responses, if any, occur cutaneously, in situ, following DV inoculation when mosquitoes feed. We wanted to develop an in situ approach that would enable us to study the early immune pathology of the cellular events regarding the infectious process and the first reactions of the peripheral immune system, the dendritic cells (DCs). DCs constitute a system of highly dynamic sentinel cells, whose most studied element in peripheral non-lymphoid tissues is the epidermal Langerhans cells (LCs). It is now known that LCs perform multiple tasks including antigen capturing and processing, antigen ferrying to regional lymphoid tissues, and ultimately, cognate antigen presentation to naive lymphocytes, once in secondary lymphoid organs (Flores-Romo 2001). Under the influence of a variety of stimuli such as cytokines, antigens or microbial products, LCs become activated, start to migrate into the dermis and concomitantly to up-regulate antigen-presenting molecules and to express co-stimulatory and maturation markers (Cumberbatch & Kimber 1992; Cumberbatch et al. 1997). Nevertheless, despite that LCs as sensors of the external antigenic world are the most exposed cells of the immune system during mosquito feeding, and the initial virus inoculation through skin, the interactions of these cells with DV and the outcome of such potential interplay regarding both the virus and the LC have received little attention in situ (Wu et al. 2000). We attempted to address some of these issues by establishing a model with fresh, healthy, non-cadaveric human skin explants exposed to DV, to analyse whether local infection and viral replication are feasible experimentally, to assess the most early phases of the initiation of the immune responses in situ and the immediate local repercussions of viral inoculation upon local antigen-presenting cells (APCs), particularly on the DCs, the sentinel posts of the immune system in the skin.