Human keratinocyte cultures (HaCaT) can be infected by DENV, triggering innate immune responses that include IFNλ and LL37

Abstract

The skin is the first anatomical region that dengue virus (DENV) encounters during the natural infection. Although the role of some skin resident cells like dendritic cells and fibroblasts has been demonstrated to be crucial to elucidate the role of resident cells and molecules participating during the early events of the innate immune response, the participation of keratinocytes during DENV infection has not been fully elucidated. In this paper we aimed to evaluate the use of the HaCaT cell line as a model to study the immune responses of skin keratinocytes to DENV infection. We demonstrated productive DENV-2 infection of HaCaT cells and their capability to establish an antiviral response through production of type I and type III interferons (IFN-β and IFN-λ). The production of these cytokines by HaCaT cells correlated with upregulation of IFN-inducible transmembrane protein-3 (IFITM3) and viperin in bystander, uninfected cells. We also observed an increase in secretion of IL-6 and IL-8. Skin keratinocytes are known to secrete antimicrobial peptides (AMPs) during viral infections. In our model, DENV-2 infected HaCaT cells upregulate the production of cytoplasmic LL-37. We evaluated the dual role of LL-37, HBD2, and HBD3 antiviral activity and immunoregulation during DENV-2 infection of HaCaT cells and found that LL-37 significantly reduced DENV-2 replication. This indicates that the HaCaT cell line can be used as a model for studying the innate response of keratinocytes to DENV infection. Our results also suggest that skin keratinocytes play an important role in the skin microenvironment after DENV infection by secreting molecules like type I and type III IFNs, pro-inflammatory molecules, and LL-37, which may contribute to the protection against arboviral infections.

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.