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Published on: July 8, 2011
Activation of the innate immune response against DENV in normal non-transformed human fibroblasts
José Bustos-Arriaga1, Jazmín García-Machorro, Moisés León-Juárez
1Departamento de Biomedicina Molecular Centro de Investigación y de Estudios Avanzados, México Distrito Federal, Mexico.
Background:
When mosquitoes infected with DENV are feeding, the proboscis must traverse the epidermis several times ("probing") before reaching a blood vessel in the dermis. During this process, the salivary glands release the virus, which is likely to interact first with cells of the various epidermal and dermal layers, cells which could be physiologically relevant to DENV infection and replication in humans. However, important questions are whether more abundant non-hematopoietic cells such as fibroblasts become infected, and whether they play any role in antiviral innate immunity in the very early stages of infection, or even if they might be used by DENV as primary replication cells.
Methodology/Principal Findings:
Fibroblasts freshly released from healthy skin and infected 12 hours after their isolation show a positive signal for DENV. In addition, when primary skin fibroblast cultures were established and subsequently infected, we showed DENV-2 antigen-positive intracellular signal at 24 hours and 48 hours post-infection. Moreover, the fibroblasts showed productive infection in a conventional plaque assay. The skin fibroblasts infected with DENV-2 underwent potent signaling through both TLR3 and RIG- 1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5) and β defensin 2 (HβD2). In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3), but not interferon regulatory factor 7 (IRF7), when compared with mock-infected fibroblasts.
Conclusions/Significance:
In this work, we demonstrated the high susceptibility to DENV infection by primary fibroblasts from normal human skin, both in situ and in vitro. Our results suggest that these cells may contribute to the pro-inflammatory and anti-viral microenvironment in the early stages of interaction with DENV-2. Furthermore, the data suggest that fibroblast may also be used as a primary site of DENV replication and provide viral particles that may contribute to subsequent viral dissemination.
Insights
Human skin fibroblasts are highly susceptible to Dengue virus (DENV) infection. These cells play a role in antiviral immunity and may serve as a primary site for DENV replication and dissemination.
Area of Science:
- Virology
- Immunology
- Dermatology
Background:
- Mosquitoes transmit Dengue virus (DENV) through skin probing.
- Early DENV interactions involve epidermal and dermal cells, but the role of abundant non-hematopoietic cells like fibroblasts is unclear.
- Fibroblasts' susceptibility and role in early antiviral immunity or viral replication remain to be elucidated.
Purpose of the Study:
- To investigate the susceptibility of human skin fibroblasts to DENV infection.
- To determine if fibroblasts play a role in the early innate immune response to DENV.
- To assess if fibroblasts can serve as primary replication sites for DENV.
Main Methods:
- Infection of freshly isolated and cultured primary human skin fibroblasts with DENV-2.
- Detection of DENV antigen using immunofluorescence assays.
- Assessment of productive infection via plaque assays.
- Analysis of innate immune signaling pathways (TLR3, RIG-I, Mda5) and downstream cytokine/chemokine production (IFNβ, TNFα, HβD2, HB5).
- Evaluation of IRF3 and IRF7 nuclear translocation.
Main Results:
- Primary human skin fibroblasts demonstrated susceptibility to DENV-2 infection both in situ and in vitro.
- Productive DENV-2 infection was confirmed by antigen detection and plaque assays.
- Infected fibroblasts exhibited robust signaling via TLR3 and RIG-I, leading to the upregulation of IFNβ, TNFα, HβD2, and HB5.
- Increased nuclear translocation of IRF3 was observed in DENV-2 infected fibroblasts.
Conclusions:
- Primary human skin fibroblasts are highly susceptible to DENV-2 infection.
- Fibroblasts contribute to the early pro-inflammatory and antiviral microenvironment.
- Fibroblasts may act as a primary replication site for DENV, facilitating viral dissemination.
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