Discussion
In most cases, the immune system succeeds in controlling hrHPV infections but this process takes time and requires the presence of strong IFNγ and TNFα-associated HPV-specific T cell responses (16). Here, we show that hrHPV-infected KCs resist the immune system by interfering with the regulation of intracellular growth and cell death programs of infected cells. Under normal circumstances, these would be activated in response to the effector molecules of the adaptive immune system and function as a host defense mechanism to control viral spread (54). Using a unique in vitro model, we showed that hrHPV infection renders KCs resistant to IFNγ/TNFα-induced necroptosis and arrest of cell growth. HrHPV infection is associated with the upregulation of nine methyltransferases, including EZH2, and downregulation of the expression of RIPK3, which results in an impaired induction of necroptosis by IFNγ/TNFα stimulation. Use of DZNep, a global inhibitor of methyltransferases and a pharmacological compound that depletes EZH2 (45, 46), restored the expression of RIPK3 and the sensitivity of hrHPV-infected KCs to IFNγ/TNFα-mediated necroptosis. Use of the catalytic EZH2 inhibitor GSK503 did not restore RIPK3 expression, suggesting that either EZH2 is indirectly responsible for suppressing RIPK3-mediated necroptosis or that one or more of the other overexpressed methyltransferases are involved in the downregulation of RIPK3. Furthermore, hrHPV effectively downregulated the basal expression of the negative regulator of cell growth IFITM1, resulting in an impaired IFNγ-mediated increase in the expression of the antiproliferative RARRES1 gene and decrease of the proliferative gene PCNA as well as impaired arrest of cells in the S-phase. Knockdown of IFITM1 with siRNA in normal KCs recapitulated the effects on RARRES1 and PCNA expression and cell proliferation observed in hrHPV + KCs.
Apoptosis and necroptosis play an important role in controlling viral infections (54). We did not observe any HPV-induced differences in the expression of caspase-8, FLIP, or FADD, nor did we observe differences in cleavage of caspase-8 upon stimulation with IFNγ and TNFα. Notably, stimulation with IFNγ and TNFα did not lead to activation of caspase-8, reflected by the absence of fully cleaved caspase-8 in undifferentiated normal or hrHPV-positive KCs. However, upon differentiation, KCs become sensitive to Fas- and caspase-8-mediated apoptosis following stimulation with IFNγ and TNFα (29, 30). In contrast, stimulation of undifferentiated KCs did result in necroptosis following an increase of RIPK3, most notably when both IFNγ and TNFα were used. The induction of necroptosis has shown to be important for the control of vaccinia virus (55) and herpes simplex virus type 1 (56). Consequently, viruses have developed strategies to resist this immune control mechanism. The murine cytomegalovirus expresses the M45-encoded inhibitor of RIP activation (vIRA) that targets RIPK3 and disrupts RIPK1–RIPK3 interactions characteristic for necroptosis (57). Rather than by interrupting necroptosis, hrHPV prevents the formation of the necrosome by reducing the levels of RIPK3 via the upregulation of histone methyltransferases.
The production of new HPV particles requires proliferation and differentiation of infected basal KCs. An arrest in cell proliferation, therefore, is an effective means to control viral infection. IFITM1 plays an essential role in the antiproliferative action of IFNγ (47), thus lowering its expression – as observed in hrHPV-infected KCs – may allow viral escape. Indeed, hepatitis C virus was found to decrease the expression of IFITM1 via the upregulation of mIR-130a in order to sustain its replication (58). We showed that the basal expression of IFITM1 is downregulated in hrHPV + KCs, but its downstream partner RARRES1 is not. This might be explained by the fact that the basal expression of RARRES1 in uninfected KCs is already low. The IFNγ-induced increase in expression of IFITM1 and RARRES1 requires signaling via the IFNGR1 and STAT1. Overexpression of EZH2 has been reported to suppress the expression of IFNGR1 in MYC- transformed cells but not phosphatidylinositol 3-kinase (PI3K)-transformed cells, despite the fact that in both types of transformed cells EZH2 was overexpressed (59). Notwithstanding the ectopic expression of EZH2 in hrHPV-positive KCs, there were no specific differences in the expression of IFNGR1 between non-infected and hrHPV-positive cells. This is in line with the observation that the PI3K pathway is a major target for the hrHPV proteins (59, 60) and suggests that the overexpression of EZH2 does not play a role in the escape of hrHPV-positive cells at this level. It was previously reported that the HPV early proteins E6 and E7 downregulate the expression of STAT1 (4–6). Our data confirm that infection with hrHPV decreases basal STAT1 protein levels in KCs but also show that hrHPV does not interfere with IFNγ-induced STAT1 activation per se, as reflected by STAT1 phosphorylation and increase in RARRES1 and IFITM1 expression. Still, as total STAT1 levels are lower in hrHPV + KCs, the reduced total amount of activated STAT1 may explain why in hrHPV + KCs the increase in RARRES1 and IFITM1 expression does not reach the levels observed in uninfected KCs. This is also demonstrated by the data showing that the effect of IFITM1 knockdown on proliferation of uninfected KCs is not similar to what hrHPV has on KCs. While the effect of IFITM1 in uninfected KCs is apparent and antiproliferative, indicated by the retained expression of PCNA and RARRES1 in KCs stimulated with a low dose of IFNγ when IFITM1 was knocked down, clearly the downregulation of STAT1 as well as the positive growth signals as delivered by hrHPV (52, 53) are missing in these cells. Hence, differences in IFNγ-stimulated arrest of proliferation are less noticeable. This shows that, whereas the decreased basal level of IFITM1 is already providing resistance to the IFNγ-stimulated arrest of proliferation, the downregulation of STAT1 is likely to exaggerate this effect. Mechanistically, IFITM1 inhibits the phosphorylation of ERK and thereby regulates mitogen-activated protein (MAP) kinase signaling. Furthermore, IFITM1 mediates the de-phosphorylation of p53 at Thr55, resulting in increased p53 stability and transcriptional activity and the upregulated expression of p21. Consequently, there is an arrest in cell cycle progression and hence a stop in proliferation (47). This was also observed in this study and reflected by the retained PCNA expression when IFITM1 was knocked down in low dose IFNγ-stimulated KCs.
In conclusion, hrHPV controls proliferation by regulating the expression of (anti-)proliferative genes via STAT1 and IFITM1 and resists the induction of necroptotic cell death by downregulation of RIPK3 expression. This allows infected KCs to partly resist immune pressure by IFNγ/TNFα and explains how hrHPV can partially evade the effector mechanisms of the immune system, which may ultimately lead to progression of hrHPV-induced lesions.