Up until now, mainly TLR3 and TLR7 have been implicated in sensing respectively transient dsRNA and ssRNA generated during the flavivirus replicative cycle.
It has been previously described that DENV infection of HUH-7 (hepatocarcinoma cell line) cells resulted in the upregulation of TLR3 expression and that TLR3 in HEK293 (human embryonic kidney 293) cells recognized DENV2 RNA after endosomal acidification, which resulted in strong IL8 and IFNα/β responses [56, 75]. Nasirudeen et al. also demonstrated that downregulation of TLR3 expression by siRNA silencing in HUH7 cells resulted in higher DENV1 replication. Moreover, overexpression of TLR3 in these cells inhibited DENV1 infection through induction of high levels of IFNβ production . There are, however, few data concerning flaviviral sensing in human skin cells. HDFs and human primary keratinocytes infected with DENV2 and HDFs infected by ZIKV showed an increased level of TLR3 expression [16, 17, 46]. Moreover, it has been shown that inhibition of TLR3 mRNA expression in the human skin fibroblast cell line HFF1 led to a strong increase in the ZIKV RNA load at 48 h p.i. without modulation of type I IFN mRNA expression . Hence, TLR3 seems to play a key role in ZIKV and DENV sensing.
The role of TLR3 in WNV sensing nonetheless remains controversial. TLR3 deficiency has been described as related to protection against neuro-invasive form of WNV infection in mice highlighted by a reduced viral load, inflammatory response and neuropathology in the brain, while impaired cytokine production and enhanced viral load have been observed in blood; this observation was in comparison with wild-type mice . On the contrary, Daffis et al. described enhanced WNV replication in the central nervous system of TLR3–/– mice after subcutaneous inoculation with only modest differences observed in peripheral viral load level and IFN production . It has also been demonstrated that TLR3 does not modulate WNV replication and IFN induction in vitro in myeloid DCs, macrophages and murine embryonic fibroblasts [77, 78]. Activation of the IRF3 transcription factor has been described as both dependent and independent of TLR3 [79, 80]. Fredericksen et al. suggested that highly virulent strains of WNV might have evolved to more efficiently stimulate the TLR3-mediated inflammatory response involved in increased blood–brain barrier permeability rather than to disrupt TLR3 signalling . Up until now, no data is available concerning WNV sensing by human skin cells. Unpublished data by our group show that TLR3 is upregulated following primary keratinocyte and dermal fibroblast infection by WNV.
TLR7 is an endosomal receptor involved in the recognition of ssRNA. It has been shown that TLR7 is involved in DENV recognition in plasmacytoid DCs, resulting in type I IFN production inversely proportional to the DENV titre, thereby suggesting an escape mechanism of the virus to immune response [81, 82]. On the other hand, skin fibroblasts have been reported as not expressing TLR7, even following flaviviral infection [17, 46, 83]. Mc Cracken et al. have demonstrated, using an original strategy of mice intradermal inoculation of DENV2 at sites where Aedes aegypti had or had not probed immediately or prior, downregulation of several genes including TLR7 in the presence of mosquito saliva . Consequently, even though TLR7 may be involved in DENV sensing, its role in skin cells remains to be demonstrated.
Concerning WNV, it has been reported that infection of keratinocytes from TLR7–/– mice resulted in higher viral replication and weaker expression of IFNα, IL1β, IL6 and IL12 than in keratinocytes from wild-type mice and that TLR7 response following cutaneous infection promotes LCs migration from the skin to the draining lymph nodes . Nonetheless, in vivo, no difference was observed concerning susceptibility to WNV encephalitis or in the peripheral and brain RNA viral loads between wild-type and TLR7–/– mice .
MDA5 and RIG-I are induced during HUH-7 cell and MEF infection by DENV1 and 2 respectively, and are involved in IFNβ production [56, 86]. Knockdown of these RLRs enhanced cellular permissiveness to DENV1 replication, increased virus propagation and led to higher ISG expression such as of the 2′,5′oligoadenylate synthetase (OAS)2, ISG15 and ISG56, and to strong activation of IRF3, suggesting that both RIG-I and MDA5 synergistically inhibit DENV replication in vitro [56,86]. In HDFs infected by DENV2, type I IFN induction has resulted in an IRF3 nuclear translocation, whereas IRF7 stayed mainly in the cytoplasm. An increased expression of RIG-I, but not MDA5, has been reported . Moreover, the incubation with RIG-I agonist 5′ triphosphorylated RNA prior or after challenge with DENV has been shown to protect against DENV infection by inducing a strong antiviral response via the RIG-I/IPS-1/TBK1/IRF3 signalling pathway . In contrast, during DENV infection of human primary keratinocytes, increased expression of IRF7 transcripts at 48 h p.i. has been reported, whereas no significant change in IRF3 mRNA expression levels was noted . In addition, several ISGs are induced after keratinocyte infection by DENV such as protein kinase R (PKR), OAS2, Ribonuclease L (RNase L) and RNase L inhibitor (RLI) from 6 h p.i., highlighting the broad antiviral response of the keratinocytes to flavivirus infection . Indeed, PKR expression is increased after cell priming with type I IFN and binds the dsRNA produced during the viral RNA replication, leading to activation of kinase and inhibition of viral translation and discontinuation of virus spread . OAS2 is involved in viral dsRNA sensing, leading to its cleavage by RNase L, generating smaller dsRNA, which could be detected by MDA5 and RIG-I in order to amplify innate immune response .
Cooperation between RIG-I and MDA5 sensing has been described during WNV infection, with viral recognition by these RLRs resulting in establishment of an antiviral response [78, 80]. It has been reported that RIG-I and MDA5 double-knockout mice were more susceptible to WNV infection and that disruption of MDA5 and RIG-I pathways abrogated activation of the antiviral response of MEFs to WNV [78, 86, 90]. In addition, RIG-I–/– MEFs showed enhanced viral titres but were still able to mount an innate antiviral response with delayed expression of several genes including IRF3, IRF7, type I IFN, ISG15 and STAT1 [78, 80]. Moreover, IPS-1, an essential RIG-I and MDA5 adaptor molecule, was shown to play a major role in induction of the innate antiviral response of MEFs infected by WNV both in vitro and in vivo, since mice deficient for IPS-1 were more susceptible to WNV infection [91, 92]. IPS-1–/– mice exhibited higher mortality, higher viral loads in the serum and in the peripheral tissues, earlier brain infection and presented impairment in T cell expansion, intense inflammatory response and increased IgM and IgG production despite weak functionality, suggesting that IPS-1 plays a central role in immunity against WNV, linking innate and adaptive responses [78, 80, 93]. Interestingly, IRF3 is induced only when WNV is replicative in cells and is essential along with IRF7 to control its replication [80, 91, 92]. Other ISGs have been described as displaying significant antiviral activity against WNV in vitro such as C6orf150, DDX24, HPSE, IFI44L, IFI6, IFITM2, IFITM3, IFRD1, IL13RA1, ISG20, MAFK, NAMPT, PAK3, PHF15, SAMD9L, SC4MOL and viperin . Hence, RIG-I and MDA-5 exert a complementary role, whereas RIG-I initiates WNV detection; MDA5 then sustains and enlarges innate immune response. Taken together, the results suggest that WNV and DENV-induced innate immune responses are both RIG-I- and MDA5-dependent .
Few data are available concerning skin innate immune sensing of ZIKV. Nonetheless, Hamel et al. have described that infection of primary HDFs with ZIKV resulted in delayed upregulation of RIG-I and MDA5 mRNA as compared with TLR3 expression. Inhibition of TLR3 expression in primary HDFs, unlike that of other PRRs, led to a strong increase of ZIKV RNA copy numbers at 48 h p.i. but had no consequence on type I IFN mRNA expression in the infected cells. These data suggest that, in primary HDFs, sensing of ZIKV is mainly related to TLR3, which is quickly expressed during infection and relayed by RIG-I and MDA5 with a major role in stemming viral replication . Similar findings have been observed in primary skin fibroblasts infected with DENV in which TLR3 may play a role in the early antiviral response while RIG-I might regulate the amplified response at later time points . Otherwise, human peripheral DCs infected by ZIKV have led to an induction of RIG-I, MDA5 and STAT1 and -2, and ISGs such as viperin and IFIT-1, -2 and -3 expression . Interestingly, ZIKV replication is restricted by RIG-I agonists but not by type I IFN .
Other proteins involved in flavivirus sensing such as LGP2 can contribute to cytosolic RNA sensing, but its role in innate immunity remains unclear . It has been reported that LGP2 was not essential for induction of innate immunity but was indeed required for controlling antigen-specific CD8+ T cell activation, survival and fitness during peripheral T cell-number expansion in response to WNV infection, independently of MDA5 [94, 96]. In addition, LGP2 mRNA expression is induced after ZIKV infection of human DCs .
Recently a cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS, also known as MB21D1) signalling through downstream signalling molecule STING to induce type I IFN production , was described as being involved in inhibition of replication of several RNA viruses such as WNV in a RIG-I-dependent manner and suggested as being important for basal expression of ISGs . Schoggins et al. demonstrated that ectopic expression of cGAS inhibited WNV replication and that cGAS–/– mice exhibited increased lethality after WNV infection . The mechanism by which cGAS becomes activated following WNV infection remains unknown.
Few data have been published concerning the role of inflammasome during infections by flaviviruses. Inflammasome is a multiprotein complex belonging to the innate immunity system. Inflammasome activation is a consequence of host cell's cytosolic NLRs such as NLRP (nucleotidic domain and leucine-rich repeat containing (NLR) protein)-1 and -3, NLRC4 (nlr family, caspase recruitment domain-containing 4) and AIM2 (absent in melanoma) having sensed “danger signals”, and it leads to IL1β, IL18 and IL33 pro-inflammatory cytokine secretion . NLRP3 inflammasome is activated following WNV and DENV infection; these infections in humans are associated with elevated levels of systemic IL1β [101, 102]. ZIKV has been shown to be responsible for activation of the inflammasome pathway in fibroblasts, since AIM2, which is a DNA sensor involved in caspase 1 activation, and IL1β mRNA were strongly induced after infection [46, 103].