Dottorato in MEDICINA SPERIMENTALE

Titolo della tesi: Determination of Interferon related genes controlling papillomavirus replication in vitro and mediating ex vivo anti-papillomavirus response

Introduction More than 5% of all cancers are attributable to HPV infections. Despite the well-established antiviral and antiproliferative activity of the innate immune system, especially type I and III IFNs, as well as NK cells, the evasion mechanism employed by HPV may support the progression of anogenital cancer, mostly in HIV-1 infected MSM, due to sexual behavior and HIV-1 related immunosuppression, which reduces the ability to control the oncogenic processes of HPV. To date, our understanding of the early stages of HPV infection are limited by difficulties in identifying incident HPV infections. In this context, the MmuPV1 mouse model has provided new insights into how the early host response can control PV replication. We hypothesized that HPV, through evasion strategies adopted to overcome the host immune defense, might modulate levels of IFN-pathway related genes, as well as NK cells. In the light of these consideration, my PhD project planned i) to evaluate the gene expression level of specific TLRs and ISGs in anal cells of HIV-1 positive MSM patients, ii) to analyze the frequencies of NK cell subsets in anal biopsies obtained from a subgroup of HIV-1 infected MSM with dysplasia, iii) to measure in vitro the expression levels of selected ISGs in MPEK-BL6 cells infected with MmuPV1. Materials and Methods HIV-1 infected MSM (n=159) underwent anal canal brushing at baseline and follow up (n=45) during routine proctology visit, to allow HPV-DNA detection by PCR and genotyping by sequencing, and analysis of TLRs (TLR2, TLR3, TLR4, TLR8 and TLR9) and ISGs (ISG15, ISG56, MXA, PKR) gene expression by RT/Real time PCR assay. Anal biopsies from abnormal areas and normal mucosa were obtained from a subgroup of HPV-HIV-1 coinfected MSM (n=18), in order to evaluate any differences in the frequencies of CD56+, CD56dim, CD56bright and NKT cells by flow cytometric analysis. MmuPV1 infected-MPEK-BL6 cells were cultured over 144 hours, and early and late viral gene expression, as well as transcription of cellular genes coding for ISGs (STAT1, DDX58, IFIH1, ISG15, HERC6, IFI27, IFI35, IRF7, OAS1, OAS3, OASL1, PLSCR1, SP100, BST2, and SP100), were quantified at serial timepoints by RT/Real time PCR assay. Results Prevalence of patients tested positive for HPV DNA was 71.07%, with HPV6 and HPV16 as the most common genotypes. Patients who resolved and mantained the infection at the follow-up were 13.33% and 86.67%, respectively. Results showed a decreased expression of TLR9 in HPV positive samples compared to negative ones (p=0.021), especially in Lr infections (p=0.004), while no differential expression of the ISGs was recorded. TLR4 and TLR9 transcripts were higher in persistent Hr HPV infections (p=0.041 and p=0.033, respectively), whereas MXA was more activated during low-risks (p=0.0079). A difference in the expression of TLR3 (p=0.046) and TLR8 (p=0.028) was detected among Hr HPV-HIV-1+ positive, compared HIV-1 negative individuals, as well as for ISG15 (p=0.0180) and PKR (p=0.0443). Patients with dysplastic and normal mucosa showed similar levels of the CD56+, CD56dim, and NKT cells; moreover, a significant downregulation of CD56bright in LSIL was caused by Hr HPV. Finally, persistence and cleared infections did not revealed differential frequencies. Compared to undetectable levels of MmuPV1 transcripts in the control cells, in infected MPEK-BL6 we detected ~50.000 copies/µg of early transcripts between 24-72 hours post infection (p.i.), which was then suppressed to ~2.000 copies/µg at later time points. Compared to IFNα induced upregulation of ISGs, the expression of IFIH1, ISG15, OAS1, OASL1 and SP100 unchanged or were downregulated in MmuPV1 infected MPEK-BL6 cells. Conversely, RIG-I (DDX58), HERC6, IFI27, IRF7, OAS3, PLSCR1, STAT1 and IFI35 were significantly (>3-fold) upregulated at late time points p.i., 96, 120 and 144 hours, coincident with the suppression of early MmuPV1 mRNA. Conclusion Results obtained during my PhD project underlined a key role played by PV on the regulation of the early innate immune response. The low levels of TLRs activation may be related with the long time necessary for anal HPV clearance; however, TLR over-expression may be associated with pathological roles and may enhance the risk of HPV-related cancers. Moreover, we demonstrated that anal Hr HPV infection could inactivate IFN-mediated innate response due to lack of activation of ISGs. Interestingly, Hr HPV infection might inhibit the innate immune response by reducing NK cells activation, favouring the development of high-grade HPV-related lesions. Finally, our in vitro findings suggested that ISGs complex are triggered by MmuPV1 infection and act to downregulate MmuPV1 early mRNA in the tissue culture model.