Basal keratinocytes (KCs) are responsible for renewal of the epithelium and are the target cells for high risk human papilloma viruses (hrHPVs) which may cause KCs to become transformed. The immune system has developed means to counteract infections through several mechanisms, including the suppression of viral spread through the proliferation of infected cells via the production of the effector cytokines IFNγ and TNFα. hrHPV also have the ablility to resist cell death and cell senescence induced by IFNγ and TNFα.
Using an unique system for freshly established or persistent hrHPV infection, we first screened the gene expression related with cell growth and cell death of hrHPV16 positive keratinocytes(HPV+ KC) and normal keratinycytes treated with IFNγ by gene array. Then we checked the target in the TNFR1 and IFNγR pathway by Western blot and qPCR. SYTOX green assay was applied to check the necroptosis. DNA staining is applied to check the proliferation, as well as qPCR of certain proliferation markers.
We found that hrHPV escapes from IFNγ and TNFα induced cell death by impairing the expression of RIP3 at the gene and protein level. Subsequently, the phosphorylation of MLKL is also reduced. RIP3 is part of the necrosome and as a consequence stimulation of KC and HPV+ KC by BV6 and zVADfmk plus IFNγ and TNFα only marginally induced cell death in HPV+ KC while almost all non-infected KC die. Furthermore, we observed that hrHPV impairs the IFNγ growth inhibitory pathways by targeting the expression of the anti-proliferative gene IFITM1. The expression of IFITM1 was downregulated already at 48 hours after hrHPV infection. In non-infected KCs stimulation with IFNγ and TNFα induced the expression of the antiproliferative gene RARRES and suppressed the expression of the with proliferation associated gene PCNA. However, this was not the case in HPV+ KCs. In order to recapitulate the effect observed in HPV+KCs, we knocked down IFITM1 in non-infected KCs and showed that this provided resistance to the anti-proliferatory effects of IFNγ and TNFα.
Our study revealed that HPV downregulated the expression of RIP3 and IFITM1 to resist IFNγ and TNFα induced cell death and senescence.