Phd in IINNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Thesis title: Mucosal microenvironment and host immunity in HPV-driven carcinogenesis

Background: Innate immunity pathways, especially those related to type I and III interferon (IFN) are involved in Human Papillomavirus (HPV) recognition and clearance. Among HIV-1 positive men who have sex with men (MSM), the extremely high incidence of HPV infection is strongly associated with an increased risk of squamous cell carcinoma of the anal canal. To date, few data are available on the complex interplay between persistent mucosal HPV infection, IFN signature, and their involvement in cancer development. At the same time, NK cells mediated cytotoxicity plays a pivotal role in mucosal response against HPV infection and dysplasia, but their activity is counteracted by several HPVs. We hypothesized that HPV, through evasion strategies adopted to overcome the host immune defense, might modulate levels of different type I and III IFN genes, as well as of NK cells in the anal mucosa of HIV-1 MSM patients. Moreover, a goal of this project is to evaluate the effects of oral bacteriotherapy supplementation on anal IFN transcript levels, the frequencies of intra-epithelial NK cell subsets and on immune activation status of HPV-HIV-1 infected MSM. Methods: Anal brushings were collected from 110 Caucasian HIV-1 infected MSM, with a median age of 47.4 (41–52.75) years, on long-term antiretroviral therapy (ART), attending Policlinico Umberto I Hospital in Rome. Detection of HPV DNA and genotyping were performed by PCR and sequencing. The mRNA levels of IFN-α, IFN-β, IFN-ε, an emerging component of innate immune defence at mucosal sites, IFNAR1 and IFNAR2 receptors, IFN-λ 1-3 subtypes and IL28R receptor, were measured in anal brushings by TaqMan RT–PCR. A subgroup of HPV-HIV-1 co-infected MSM underwent high resolution anoscopy (HRA), to obtain biopsies from areas consistent with SIL and healthy mucosa. Intra-Epithelial Lymphocytes (IELs) were isolated from anal biopsies and stained to identify CD56+, CD16+, CD56dim, CD56bright NK cells and NKT cells by multiparametric flow cytometry. In addition, type I and III IFN levels, NK cell subsets and the frequencies of CD4+ and CD8+ T lymphocytes expressing CD38 and HLADR were evaluated after six months of oral bacteriotherapy supplementation. Results: Anal HPV DNA was detected in 87 MSM patients (79%), with 49.4% of the cases having a high-risk (HR) HPV genotype, mainly HPV16. Out of 110 patients, 58.4% showed HSIL/LSIL. A decreased mucosal expression of IFN-β, IFN-ε, IFNAR1 and IFNAR2 as well as IFN-λ 1-3 was recorded in HR compared to low-risk (LR) HPV positive and HPV negative patients (Mann-Whitney U test p <0.05 for all genes). No differences were found on levels of type I and III components according to the presence or absence of SIL. By contrast, the expression of IFN-β, IFN-ε, IFNAR1, IFNAR2 and IFN-λ 1-3 was reduced in patients with a persistent HPV infection compared to those who spontaneously cleared the infection (Mann-Whitney U test p <0.01 for all genes). A reduction in the frequencies of CD16+ NK cells was found in the dysplastic mucosa (LSIL) as compared to the normal anal mucosa, while the levels of CD56+, CD56dim, CD56bright NK cells and NKT cells were similar between LSIL and normal anal mucosa of HPV positive patients. Oral bacteriotherapy supplementation resulted in changes in IFN-I and III levels and in frequency of peripheral CD38/HLA-DR T cell subsets evaluated by flow cytometry (p<0.05 for all variables). Moreover, an increase of CD16+ NK and NKT cell frequencies and a trend toward an upregulation of CD56+ NK cells (p=0.089) has been observed after probiotic intake. Interestingly, oral bacteriotherapy induce also the clearance of HPV anal lesions in one HR-HPV infected MSM. Conclusions: HPV persistent infection is able to regulate type I and III IFN and NK cells responses response and contribute to the establishment of an immunosuppressive microenvironment in mucosal epithelia, which is essential for precancerous anal lesions progression. Moreover, results suggest a potential role for oral probiotic supplementation in modulating mucosal immunity and promoting the regression of anal HPV related lesions in HIV-1 infected MSM.