Innate Immune Response of Primary Human Keratinocytes to West Nile Virus Infection and Its Modulation by Mosquito Saliva

Abstract

West Nile Virus (WNV) is a flavivirus involved in many human infections worldwide. This arthropod-borne virus is directly co-inoculated with mosquito saliva through the epidermis and the dermis during blood meal. WNV starts replicating in the skin before migrating to the draining lymph node, leading to widespread viremia and in some cases to neurological symptoms. Skin is a complex organ composed of different cell types that together perform essential functions such as pathogen sensing, barrier maintenance and immunity. Keratinocytes, which represent 90% of the cells of the epidermis, are the organism's first line of defense, initiating innate immune response by recognizing pathogens through their pattern recognition receptors. Although WNV was previously known to replicate in human primary keratinocytes, the induced inflammatory response remains unknown. The aim of this study was first to characterize the inflammatory response of human primary keratinocytes to WNV infection and then, to assess the potential role of co-inoculated mosquito saliva on the keratinocyte immune response and viral replication. A type I and III interferon inflammatory response associated with an increase of IRF7 but not IRF3 mRNA expression, and dependent on infectious dose, was observed during keratinocyte infection with WNV. Expression of several interferon-stimulated gene mRNA was also increased at 24 h post-infection (p.i.); they included CXCL10 and interferon-induced proteins with tetratricopeptide repeats (IFIT)-2 sustained up until 48 h p.i. Moreover, WNV infection of keratinocyte resulted in a significant increase of pro-inflammatory cytokines (TNFα, IL-6) and various chemokines (CXCL1, CXCL2, CXCL8 and CCL20) expression. The addition of Aedes aegypti or Culex quinquefasciatus mosquito saliva, two vectors of WNV infection, to infected keratinocytes led to a decrease of inflammatory response at 24 h p.i. However, only Ae. Aegypti saliva adjunction induced modulation of viral replication. In conclusion, this work describes for the first time the inflammatory response of human primary keratinocytes to WNV infection and its modulation in presence of vector mosquito saliva. The effects of mosquito saliva assessed in this work could be involved in the early steps of WNV replication in skin promoting viral spread through the body.

Aedes Mosquito Salivary Components and Their Effect on the Immune Response to Arboviruses

“…Upon stimulation, they promote pro-inflammatory responses, along with the production of interferons (IFNs), chemokines, and cytokines (Miller, 2008 ; Briant et al, 2014 ). In a recent study, Garcia et al ( 2018 ) observed that keratinocytes produce a type I and III interferon inflammatory response when infected with WNV that is associated with viral load. In addition, the expression of pro-inflammatory cytokines, such as TNF-α and IL-6, and various chemokines by keratinocytes increased significantly after viral infection.…”

Opossum Cathelicidins Exhibit Antimicrobial Activity Against a Broad Spectrum of Pathogens Including West Nile Virus

“…Viral quantification in cell supernatants and keratinocytes was performed using a previously described one-step real time RT-PCR assay (23). Total RNA (5 µL) was added to the reaction mixture containing 12.5 µL of Master Mix (Invitrogen), 0.5 µL (0.2 µM) of forward (5 -GTGCGGTCTACGATCAGTTT-3 ) and reverse primers (5 -CACTAAGGTCCACACCATTCTC-3 ), 0.25 µL (0.1 µM) of 5 FAM and 3 Dark Quencher probe (5 -AATGTGGGAAGCAGTGAAGGACGA-3 ), 0.5 µL of SuperScript III reverse transcriptase (Invitrogen) and DNA polymerase platinum Taq (Invitrogen), 0.5 µL of RNase out (Invitrogen), and 5.25 µL of water.…”

“…At the initial site of viral inoculation, the skin serves as a firstline host defense against flaviviruses and leads to the initiation of the early innate immune response (22). Keratinocytes, the most abundant epidermal cells, are permissive to WNV and express inflammatory and antiviral proteins upon infection (23). Thus far, no antiviral agent to combat WNV infections or vaccines are available.…”

Skin Viral Infections: Host Antiviral Innate Immunity and Viral Immune Evasion

“…In CHIKV, mosquito saliva suppresses Th1 cytokine (IFN- γ and IL-2), TLR3, and chemokine expression while simultaneously pushing toward a Th2 polarity—which, as we have discussed, is a less advantageous antiviral profile from the host perspective ( 147 , 148 ). Decreases in expression of PRRs and antiviral proteins with specific targeting of flaviviruses (OAS1, MX1, and ISG20) were also observed in WNV-infected keratinocytes ( 149 , 150 ).…”