Human keratinocyte cultures (HaCaT) can be infected by DENV, triggering innate immune responses that include IFNλ and LL37
Introduction
Dengue viruses (DENV) are transmitted to humans by the Aedes mosquito, and all four serotypes (DENV 1–4) can cause disease (Gubler and Kuno, 1997). The skin is the first organ that DENV encounters after mosquitoe probing and blood feeding. Two of the most abundant cell types in the skin are keratinocytes and fibroblasts. We hypothesized that these cells could help restrict replication during DENV infection, depending on the inoculation route and intrinsic host variability (Garcia et al., 2017; Kupper and Fuhlbrigge, 2004; Nestle et al., 2009). Although dendritic cells and monocytes in the skin are considered important targets of DENV infection, the abundance of these cells is considerably low compared to keratinocytes and fibroblasts (Wu et al., 2000). In the dermis, fibroblasts have been found to be permissive to DENV infection and capable of establishing an antiviral microenvironment (Bustos-Arriaga et al., 2011; Kurane et al., 1992; Limon-Flores et al., 2005). Furthermore, data from a model of non-cadaveric human skin explants infected with DENV-2 suggested that keratinocytes were permissive to DENV infection in situ (Limon-Flores et al., 2005). Additionally, Surasombatpattana et. al. confirmed that primary cultures of keratinocytes are permissive to DENV infection (Surasombatpattana et al., 2011, 2012). The autocrine and paracrine activity of type I and type III interferons during viral infection is critical for restricting virus dissemination. In a previous study from our group, we observed that the release of IFN-β appears to be important part of the early immune response and effective control of DENV infection in primary fibroblasts (Bustos-Arriaga et al., 2011).
Three classes of IFN-λ have been identified (IFN-λ1, IFN-λ2, and IFN-λ3) and all of them play a role in the innate immune response to viral infections. Their receptors are largely restricted to cells of epithelial origin, such as keratinocytes (Ank et al., 2006; Donnelly and Kotenko, 2010; Kotenko et al., 2003). It has been shown that IFN-λ has antiviral activity against chikungunya virus, West Nile virus, and hepatitis C virus-the last two being members of the Flaviviridae family (Hsu et al., 2016; Kotenko et al., 2003; Liu et al., 2015; Ma et al., 2009). Recent reports indicate that human dendritic cells and human lung epithelial cells infected with DENV trigger the production of IFN-λ1 dependent toll-like receptor (TLR)-3, signaling pathway (Castaneda-Sanchez et al., 2016; Hsu et al., 2016; Palma-Ocampo et al., 2015) Type I, II, and III IFNs can induce transcription of thousands of IFN-stimulated genes (ISGs) (Helbig et al., 2013; MacMicking, 2012; Sadler and Williams, 2008). Some of these ISGs have broad-spectrum antiviral activity and can interfere with multiple steps of the virus life cycle (Chan et al., 2012; Jiang et al., 2010; Onoguchi et al., 2007).
Antimicrobial peptides (AMPs) constitute an early defence in epithelial and mucosal tissues, providing a rapid and broad-spectrum effect against pathogens, including viruses (Klotman and Chang, 2006; Nestle et al., 2009). AMPs can also modulate the immune response (Quinones-Mateu et al., 2003; Yang et al., 1999). Among the AMPs present in the skin, defensins and cathelicidin LL-37 are primarily expressed by keratinocytes. In addition to its microbicide activity, LL-37 also acts as a chemoattractant for neutrophils, monocytes, and T-cells (De et al., 2000). It has been reported that both THP-1 cells and human neutrophils produce HBD-1 and LL-37 following DENV infection (Castaneda-Sanchez et al., 2016). Furthermore, the treatment of DENV with LL37 prior infection of Vero cells significantly reduced levels of DENV genomic RNA and viral antigen (Alagarasu et al., 2017).
HaCaT cells are a widely used model in research involving keratinocytes and can produce type I and II IFNs, AMPs, HBDs, and LL-37. Therefore, to enrich the currently available information regarding the antiviral response in the skin and particularly the role of keratinocytes in the early stages of DENV infection, we evaluated the innate immune response to DENV infection of HaCaT cells.
Section snippets
Cells lines and virus
HaCaT cells, a non-tumorigenic, spontaneously immortalised human skin keratinocyte line were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Gibco) and l-glutamine (Gibco) in a humidified atmosphere of 5% CO2 at 37 °C.
C6/36 cells (Aedes albopictus mosquito cells) were maintained in supplemented minimum essential medium (MEM; Invitrogen, Grand Island, NY, USA) at 34 °C in the absence of CO2. The stock preparation and titration of
Infection of keratinocytes by DENV-2
To confirm productive infection of HaCaT cells with DENV-2, we performed immunofluorescence analysis at different time points. We observed the presence of the non-structural proteins the NS3 protease in the cytoplasm and NS5 RNA polymerase in the nucleus and cytoplasm. No signal was detected in mock-infected and uninfected controls (Fig. 1A). The percentage of infected cells was quantified by flow cytometry. At 24 h post infection, 10% of the cells expressed viral E protein and this increased
Discussion
Elucidating the contributions of each cell type present in the skin and their active interplay with DENV-infected and resident bystander cells will provide more insight into how DENV establishes infection in the skin (Bustos-Arriaga et al., 2015). Early stages of the in situ innate immunity to DENV infection might be crucial not just for the local response but may also be involved in defining the quality and intensity of the subsequent adaptive immunity.
Keratinocytes are crucial for innate
Competing interests
We declare that all authors, have no financial or personal relationships with other people or organizations that could create a potential conflict of interest or the appearance of a conflict of interest with regard to the work.
Acknowledgments
The authors would like to thanks to Victor Rosales-Garcia for the technical assistance Leticia Aleman Lazarini for the microscopy services and Sarah Alicia Gutierrez Cedillo for illustrate the Fig. 7 model. The National Council for Science and Technology (CONACyT Grant 0115401 CB). Additionally, MLG and DEMS received fellowships from CONACyT. JGC, LSA, LFR and LCB, are members of the National System of Researchers, SNI.
References (47)
- et al.
In-vitro effect of human cathelicidin antimicrobial peptide LL-37 on dengue virus type 2
Peptides
(2017) - et al.
The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus
Cell
(2009) - et al.
The role of viperin in the innate antiviral response
J. Mol. Biol.
(2014) Convergence of the NF-kappaB and IRF pathways in the regulation of the innate antiviral response
Cytokine Growth Factor Rev.
(2007)- et al.
Antiviral effect of interferon lambda against West Nile virus
Antivir. Res.
(2009) - et al.
Organotypic cocultures with genetically modified mouse fibroblasts as a tool to dissect molecular mechanisms regulating keratinocyte growth and differentiation
J. Invest. Dermatol.
(2001) - et al.
Viral infections activate types I and III interferon genes through a common mechanism
J. Biol. Chem.
(2007) - et al.
Dengue virus replication in infected human keratinocytes leads to activation of antiviral innate immune responses
Infect. Genet. Evol.
(2011) - et al.
Aedes aegypti saliva enhances dengue virus infection of human keratinocytes by suppressing innate immune responses
J. Invest. Dermatol.
(2012) - et al.
Lambda interferon (IFN-lambda), a type III IFN, is induced by viruses and IFNs and displays potent antiviral activity against select virus infections in vivo
J. Virol.
(2006)
Activation of the innate immune response against DENV in normal non-transformed human fibroblasts
PLoS Negl. Trop. Dis.
Soluble mediators produced by the crosstalk between microvascular endothelial cells and dengue-infected primary dermal fibroblasts inhibit dengue virus replication and increase leukocyte transmigration
Immunol. Res.
Expression of antimicrobial peptides in human monocytic cells and neutrophils in response to dengue virus type 2
Intervirology
IFITM proteins restrict antibody-dependent enhancement of dengue virus infection
PLoS One
LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells
J. Exp. Med.
Modulation of dengue virus infection in human cells by alpha, beta, and gamma interferons
J. Virol.
Interferon-lambda: a new addition to an old family
J. Interferon Cytokine Res.
Interplay between keratinocytes and myeloid cells drives dengue virus spread in human skin
J. Invest. Dermatol.
The impact of the interferon-lambda family on the innate and adaptive immune response to viral infections
Emerg. Microbes Infect.
Skin innate immune response to flaviviral infection
Eur. Cytokine Netw.
Production and characterization of a monoclonal antibody specific for NS3 protease and the ATPase region of dengue-2 virus
Hybridoma (Larchmt)
Generation and characterization of a rat monoclonal antibody against the RNA polymerase protein from dengue virus-2
Immunol. Invest.
Dengue and Dengue Hemorrhagic Fever
Cited by (17)
Immunomodulatory effect of vitamin D on immune response to dengue virus infection
2021, Vitamins and HormonesCitation Excerpt :LL-37 is a cathelicidin antimicrobial peptide in humans and vitamin D is a potent inducer of expression of CAMP gene which codes for LL-37 (Martineau et al., 2007). LL-37 also inhibits DENV by interacting with its envelope protein and LL-37 is also a potent immunomodulator of immune response against DENV (Alagarasu et al., 2017; Jadhav, Patil, & Alagarasu, 2020; López-González et al., 2018). Thus, vitamin D could contribute to the immune response against DENV by upregulating the expression of antimicrobial peptides.
Crosstalk Between Dermal Fibroblasts and Dendritic Cells During Dengue Virus Infection
2020, Frontiers in ImmunologyLL-37-Induced Autophagy Contributed to the Elimination of Live Porphyromonas gingivalis Internalized in Keratinocytes
2020, Frontiers in Cellular and Infection Microbiology