The immune response of skin keratinocytes to tick-borne encephalitis virus infection

Tick-borne encephalitis virus (TBEV) is a small enveloped virus with an unsegmented genome formed by an RNA molecule [(+) ssRNA] of polarity, belonging to the Flaviviridae family. In nature, this arbovirus is transmitted by ticks of the genus Ixodes and Dermacentor. TBEV is a neurotropic virus, its target organ is the central nervous system of the host. After infection, it induces febrile conditions, which often progress to encephalitis and meningitis [1]. However, the gateway is the skin. In the mouse system, the virus was detected in the skin, mainly in the Langerhans dendritic cells of the skin, in neutrophils, in macrophages, in monocytes, and also in skin fibroblasts [xxx]. In our experiment, we monitored the effect of TBEV on the expression of selected early immune response genes in the most numerous skin cells, namely in infected human skin keratinocytes. The experiment during the first three days after infection monitored the concentration dependence of the expression of the monitored genes on the infectious dose of TBEV. We infected the cell culture of skin keratinocytes - HaCaT (spontaneously transformed aneuploid cell line of keratinocytes, CLS) in vitro with the TBEV virus (strain-Hypr, collection of VÚ BMC, SAV), with different multiplicities of infection (MOI) - 0.1, 1 and 5. For the next three days after infection, cells were collected at 24-hour intervals. We isolated total RNA from the cells (Gen Jet RNA purification kit, Thermoscientific), which we subsequently transcribed using the RT-PCR method into the first strand of cDNA (First Strand cDNA Synthesis Kit, Themoscientific). On the cDNA template, we used semi-quantitative real-time PCR to monitor the expression of our selected genes (Maxima SYBR Green/ROX, Thermoscientific), using gene-specific primers designed by us. We compared the resulting Ct values against β-actin actin and against control-uninfected cells. These values indicate the fold of induction of individual genes in infected cells. In the experiment, we monitored the effect of TBEV on the expression of genes for TLR3, TLR4, RIG, MDA-5, IRF3, IFN β1, IFN λ1, IFNα-R1, IFNλ-R1, OAS1, MxA, ISG56, Myd88, and PKR. All these genes are associated with the antiviral immune response in human keratinocytes. Compared to uninfected cells, 24h after infection we noted an increase in the expression of genes for TLR3, IRF3, IFNα-R1 and IFNλ-R1, MxA, where the concentration dependence of the monitored gene expression on the virus dose was also manifested. We observed a decrease in expression depending on increasing MOI for the genes for TLR4, IFN β1, IFN λ1, OAS1, Myd88, and PKR. While a lower MOI rather induced the expression of the target gene, increasing the dose of the virus, on the contrary, dampened the expression to the level of control cells, or even suppressed it. In the case of ISG56, MDA5, and RIG-I, all used MOIs inhibited the expression of the genes under investigation. With regard to the previous time interval, 48h after infection of keratinocytes, and especially for MOI5, we noted an increase in the expression of the first subunit of the receptor for type III IFN - IFNλ-R1, the induction of the expression of IFNα-R1, TLR3, IRF3 and Myd88 persisted in infected cells. In the case of the remaining monitored genes, especially the genes for TLR4, MDA-5, RIG-I, IFN β1, IFN λ1, OAS1, ISG56 and PKR, we detected expression inhibition. With the exception of TLR4 and PKR, regardless of the infectious dose of TBEV, almost 100% inhibition of the expression of the monitored genes occurred, compared to uninfected cells. In the last time interval, we continued to observe an increase in the expression of IFNα-R1 and IFNλ-R1 genes, especially at MOI 5, even compared to both previous intervals. Activation of expression also occurred in the genes for TLR3, Myd88, and IRF3. For the rest of the monitored genes, we monitored the inhibition of expression, although in the case of RIG-I, OAS1, ISG56 and PKR, a concentration effect was manifested, and with the increase in MOI, the inhibitory effect of the virus on the expression of the monitored gene decreased. In conclusion, it can be stated that the induction of the interferons tested by us occurred immediately after infection, but the expression of genes for IFN-β and IFN-λ, MDA-5, and RIG-I gradually decreased. The genes for the receptors of these interferons reached higher expression values ​​even at later time intervals and at higher infectious doses (MOI 5). Arboviruses are not inducers of type I interferons, but in our experiment, we demonstrated the expression of IFN, but also type III. IFN-λ thus participates in the defense against invasion, e.g. arboviruses [2], providing a first-line defense that causes less collateral damage than a stronger type I IFN response. Sensory PRR molecules such as RIG-I and MDA-5 showed the highest levels of expression at the beginning of infection in the time interval 24h after infection. Most genes encoding antiviral proteins we tested did not show significant expression changes except Myd88. The role of Myd88 during TBEV infection was demonstrated in the infection of mouse astrocytes [3]. Due to CNS targeting by TBEV, studies on human skin interaction with TBEV are still rare. Keratinocytes, the main cells of the epidermis, are the first line of defence and yet the first site of replication of many viruses, such as arboviruses, enteroviruses, herpes viruses, human papillomaviruses or vaccinia virus amplifying the viral load and facilitating viral spread to the liver, the fetus or the CNS [4]. In our experiments, we demonstrated the ability of human keratinocytes to be infected with the TBE virus and to respond to the infection by increased expression of genes involved in the innate antiviral response. We recorded the highest expression ​​for most activated genes already 24h after infection. Some genes achieved higher expression values ​​due to higher virus concentration, but we observed a decrease in expression induction with increasing MOI for some genes. Our preliminary results suggest the importance of type I and III IFNs during TBEV infection of skin keratinocytes. A stepwise comprehensive unraveling of the interactions of a panel of compounds of the host's innate immune response to TBEV infection could reveal the common defense mechanisms of skin cells against TBEV infection.