The profiles of TLRs expression and changes after HSV-2 infection in the human genital epithelial cells
Two kinds of typical human genital cell lines, HEC-1-A and VK2 were mainly employed and TLRs expression profiles were investigated via RT-PCR. As shown in Fig. 1a, TLR1, TLR2 and TLR4 mRNAs were highly expressing in both two cell lines, and TLR5 and TLR6 were moderate in mRNA transcription level. TLR3, TLR7 and TLR8 mRNA transcription were weak in both of cell lines. The mRNA level of TLR9 was distinct in these two cell lines, with moderate in HEC-1-A cells but weak in VK2/E6E7. After TLRs expression profiling in human genital epithelial cells, we considered whether these TLRs gene expression would be regulated by HSV-2 infection. In this study, we focused on TLR1, TLR2, TLR4, TLR5, TLR6 and TLR9 expression fold changes in two genital epithelial cells (HEC-1-A and VK2) after viral infection. The mRNA change threshold is set to 300%. The results illustrated that TLR2, TLR4 and TLR9 were up-regulated by over 300% in HEC-1-A cell 24 h post-infection (p.i.) (Fig. 1b), and only TLR4 transcription was enhanced by over 700% in VK2 cells (Fig. 1c). Although these two cell lines were with different genetic background, TLR4 expression was up-regulated after HSV-2 infection in both HEC-1-A and VK2/E6E7 cells. So we further focused on TLR4 expression and its function in genital epithelial cells in HSV-2 infection.
We also investigated the TLR4 expression in normal human genital epithelial cells comprehensively. VK2/E6E7, Ect1/E6E7 and End1/E6E7 represented normal vaginal mucosal, ectocervical and endocervical epithelial cell lines, respectively. As shown in Fig. 1d, all of the genital epithelial cell lines from different anatomical positions constitutively expressed TLR4 mRNA. We hypothesized that TLR4 might play a role in epithelial cell response to pathogens infection, and further studies would be focused on the relationship between TLR4 and HSV-2 infection in human genital epithelial cells.
HSV-2 infection up-regulated TLR4 expression in genital epithelial cells
Investigated above showed that HSV-2 infection augment TLR4 transcription level in HEC-1-A and VK2 cells. To validate these results, we studied the TLR4 expression fold change at different time-points during HSV-2 infection. As shown in Fig. 2 a-b, HSV-2-infection enhance TLR4 mRNA transcription in a time-dependent manner in both cell lines. In Fig. 2c, HSV-2 infection could cause significant cytopathic effect (CPE) with time-dependent manner. We also determined the effect of viral infection on TLR4 expression change via western blot, and illustrated that HSV-2 infection could up-regulate TLR4 expression in protein level (Fig. 2d). Then, we primarily considered which steps of viral life cycles were essential for TLR4 expression up-regulating in genital epithelial cells. Acyclovir was a typical inhibitor against herpes viral DNA polymerases, and it could suppress viral productive cycle, but no or less influence on initial viral immediate-early (IE) or certain early (E) genes expression. UV treatment could destroy viral DNA structure and the virions would be not persistently infectious, but UV-treated virions still retain their ability to adhere to host cells or enter into them. The results was shown in Fig. 2e–f, illustrating that UV treatment could impede TLR4 up-regulation completely. But by contrast, acyclovir treatment would enhance TLR4 mRNA transcription activity significantly, which compared with that of HSV-2-infected HEC-1-A cells. Similar results were obtained via detecting TLR4-promoter activity with the same treatments. It was concluded that certain IE and certain E genes products might accumulate after acyclovir treatment, and thus activate TLR4 promoter activity and up-regulate its expression.
HSV-2 triggered AP-1 activation via TLR4-MyD88/TRIF pathway in genital epithelial cells
In our previous study, we validated that HSV infection was able to induce MAPK activation, especially stimulate p38 and JNK pathway [18]. Firstly, we examined the effect of HSV-2 infection on cellular AP-1 activation in genital epithelial cells. AP-1 could be activated by HSV-2, and not UV-treated HSV-2, demonstrating that virus entry or post-entry steps of viral lifecycle might be essential for AP-1 activation. However, acyclovir treatment could not impede AP-1 activation, implicating that certain viral post-entry events mainly contributed to this effect (Fig. 3a). To rule out the possibility of LPS contamination in HSV-2 virus stock to activate AP-1, high concentration of LPS (20 μg/ml) was exposure to HEC-1-A cells transfected with AP-1-luc plasmid, and no response was observed (Data not shown, HEC-1-A is not sensitive for ~ng/ml LPS exposure, possible reason is that HEC-1-A did not express CD14 molecules), showing that HSV-2 infection might be the main stimuli for AP-1 activation. AP-1 was a heterodimer composed with c-Jun and c-Fos. So we also examined one of the monomer, c-Jun and its phosphorylation level after HSV-2 infection via western blot. As shown in Fig. 3b, HSV-2 infection could induce c-Jun phosphorylation, which was parallel with the results displayed in Fig. 3a.
After that, we considered whether TLR4 expression was necessary for virus-induced AP-1 activation in genital epithelial cells. TLR4-specific siRNA was employed to knockdown its expression in HEC-1-A cells and the results showed that the knockdown efficiency was over 90% and would be then employed in the next experiments (Fig. 3c). We evaluated the effect of TLR4-specific siRNA transfection on HSV-2-induced AP-1 activation and the phosphorylation level of c-Jun, and illustrated that knockdown of TLR4 would attenuate virus-mediated AP-1 activation and c-Jun phosphorylation significantly, concluding that TLR4 might play a role in viral-mediated AP-1 activation pathways (Fig. 3d). Whether silencing TLR4 would impede HSV-2 infection in HEC-1-A cells was examined via quantifying HSV-2 infectious virions. As shown in Fig. 3e, knockdown of TLR4 expression would moderately impede virions production. HSV-2 gD is a kind of viral late gene products which could represent viral replication efficiency [18, 20]. So its effect on HSV-2 gD expression was evaluated via In-cell western and the results was in parallel (Fig. 3f). It was concluded that TLR4 signaling might be associated with viral replication in human genital epithelial cells.
Myeloid differentiation factor 88 (MyD88) and Toll/IL-1R domain-containing adaptor inducing interferon-β factor (TRIF) are two important adaptors for TLR4 to activate downstream AP-1 pathways [21]. We would validate whether HSV-2-induced AP-1 activation was dependent on MyD88/TRIF. Also firstly, we validated the knockdown efficiency of the specific siRNA on MyD88 and TRIF in our culture systems, which were both more than 90% knockdown efficiency (Fig. 3g). Either knockdown of MyD88 or TRIF could suppress HSV-2-mediated AP-1 activation significantly (Fig. 3h). Similarly, knockdown of MyD88 and TRIF would also attenuate HSV-2 gD expression and viral replication (Fig. 3i). It was concluded that HSV-2-mediated AP-1 activation was driven via TLR4-MyD88/TRIF pathway, and also HSV-2 replication might be partly dependent on this axis.
MD2 is essential for TLR4-mediated AP-1 activation induced by HSV-2 infection
Typically, myeloid differentiation protein 2 (MD2) is reported as the accessory protein for TLR4 mediating endotoxin/lipopolysaccharide (LPS) signaling. Then whether MD2 was involved in TLR4-mediated virus-induced AP-1 signaling in human genital epithelial cells was estimated. As shown in Fig. 4a, it was validated that 4 different kinds of genital epithelial cells from different anatomical positions used in this study constitutively expressed MD2 mRNA. We also examined whether HSV-2 infection could modulate MD2 expression in genital epithelial cells. The result was that unlike TLR4, MD2 expression was not up-regulated during the viral infection (Fig. 4b). The similar results were also obtained in VK2 cells (data not shown). Moreover, the overexpression of TLR4, MD2 or both in HEC-1-A cells could enhance HSV-2-induced AP-1 activation (Fig. 4c), demonstrating that TLR4/MD2 complex was necessary for virus-mediated downstream pathway activation.
HSV-2 infection increases epithelial cell membrane-associated TLR4 anchoring
Functional TLR4 are located on the surface of cell membrane for sensing extracellular stimuli, such as bacteria LPS. We then extracted total proteins from the membrane or cytoplasm of U937 cells (human leukemic monocyte lymphoma cell line) and HEC-1-A cells, and examined the TLR4 expression localization. The result showed that almost all of TLR4 molecules are expressed in the cell membrane component, and a small amount of TLR4 molecules are localized in cytoplasm in U937 cells, which represented the typical immune cell (Fig. 5a). We also validated cytoplasm/membrane fractionation efficiency via detecting MEK1/2, which was marker of cytoplasm protein. As shown in Fig. 5a, the extraction approach was reliable to separate cytoplasm/membrane proteins. TLR4 molecules were both distributed in cytoplasm and membrane in HEC-1-A cells (Fig. 5b), implying that TLR4 distribution and innate immune status in monocytes and genital epithelial cells are totally different. Then we also investigated whether HSV-2 infection would change TLR4 localization on subcellular structure level in HEC-1-A cells. As shown in Fig. 5b, HSV-2 could augment membrane-associated TLR4 localization, but had less effect on cytoplasmic TLR4. We concluded that this effect would cause genital epithelial cells to transform from static state to immune activation status.
As observed above, HSV-2 infection could change the distribution of TLR4 molecules in HEC-1-A cells, which might activate immune response for genital epithelial cells. To verify this, we employed TLR4 ligand, LPS to activate AP-1 activation in HEC-1-A cells. As shown in Fig. 5c, LPS showed no effect on AP-1 activation in uninfected cells, but it can enhance AP-1-driven transcription activation in HSV-2 infected cells. It came to the conclusion that HSV-2 infection in HEC-1-A cells could increase TLR4 expression on the surface of cell membrane, which might cause enhancement of TLR4-mediated AP-1 activation.
HSV-2 ICP0 augments AP-1 transcriptional activity
The previous data exhibited that acyclovir treatment could enhance not only AP-1 transcriptional activity, but also TLR4 promoter activity, implicating that certain virus IE or E gene products might be as the trigger for this effect. After that, we found that overexpression of ICP0 in HEC-1-A cells could induce AP-1-driven transcriptional and TLR4-promoter activation significantly (Fig. 6a–b). Its effect on c-Jun phosphorylation was also investigated via western blot. And the result showed that overexpression of HSV-2 ICP0 would increase c-Jun phosphorylation level (Fig. 6a). TLR4 expression on mRNA and protein levels were also investigated. As shown in Fig. 6c, overexpression of ICP0 could enhance TLR4 protein and mRNA expression in HEC-1-A cells. These findings fitted our previous experiments. We hypothesized that HSV-2 firstly entry into host cells, and then initiated ICP0 expression which stimulated AP-1 activation and TLR4 promoter transcription.
AP-1 might be essential for TLR4 promoter transcriptional activity
To investigate the relationship between AP-1 and TLR4 expression regulation, a potent specific JNK inhibitor, SP600225 was chosen to evaluate its inhibitory effect on HSV-2-induced TLR4 promoter activation. As shown in Fig. 7a, SP600125 could inhibit virus-mediated TLR4 promoter activation significantly, implicating that JNK and its downstream AP-1 was important for the initiation of TLR4 expression in genital epithelial cells.
Further, we employed Alibaba 2.1 online software to predict transcriptional factors binding sites in TLR4 promoter, and AP-1 and NF-κB binding sites were labeled in TLR4 promoter schematic in Fig. 7b. We then constructed some truncated TLR4-promoter luciferase reporter plasmid to evaluate the contribution of these predicted transcriptional factor binding sites to TLR4 promoter activation. The results demonstrated that predicted AP-1 binding site (− 566~ − 556) might play an important role in AP-1-driven TLR4-promoter activation, thus up-regulated TLR4 expression (Fig. 7b). Other AP-1 and NF-κB binding sites did not exhibit any significant effect on it. In conclusion, AP-1 might be essential for TLR4-promoter activation and its expression modulation.