Altered α-defensin 5 expression in cervical squamocolumnar junction: implication in the formation of a viral/tumour-permissive microenvironment.

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix, which mainly develops at the squamocolumnar (SC) junction. The progression of cervical HPV infections into (pre)neoplastic lesions suggests that viral antigens are not adequately recognized by innate immunity or presented to the adaptive immune system. Members of the defensin family have recently been found to inhibit viral and bacterial pathogens, to stimulate the migration of immune cells and to play a role in anticancer responses. In the present study, we focused on the poorly characterized human α-defensin 5 (HD-5) and its possible role in these processes. We showed that HD-5 was able to prevent HPV virion entry into cervical keratinocytes and to influence adaptive immunity. Indeed, this peptide specifically induced the chemoattraction and proliferation of both activated T lymphocytes and immature dendritic cells in a CCR2/CCR6-dependent manner and stimulated the infiltration of these professional antigen-presenting cells in a (pre)neoplastic epithelium transplanted in vivo in immunodeficient mice. No chemotactic effect was observed with plasmacytoid dendritic cells, macrophages or natural killer cells. Proliferative and angiogenic effects of HD-5 were also assessed in vitro and in vivo. However there was a striking regional disparity in expression of HD-5, being prominent in ectocervical, vaginal and vulvar neoplasia, while absent, or nearly so, in the cervical SC junction. Taken together, these results suggest one possible explanation for why the SC junction is uniquely vulnerable to both high-risk HPV infection (via reduced HD-5 expression and viral entry) and progression of neoplasia (via altered cell-mediated immune responses and altered microenvironment).

The L1 major capsid protein of HPV16 differentially modulates APC trafficking according to the vaccination or natural infection context.

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix. The progression of cervical lesions suggests that viral antigens are not adequately presented to the immune system. The aim of this study was to determine whether HPV16 viral particles can influence the trafficking of human DC/Langerhans cells (LC), either by direct interactions with DC or following incubation with human normal keratinocytes that are in close contact with LC in the squamous epithelium. We first demonstrated that HPV16 L1 major capsid protein, when self-assembled into virus-like particles (VLP), is able to induce in DC an over-expression of CXC receptor 4 (CXCR4) via the activation of the NF-κB signaling pathway and to enhance DC motility in the presence of CXCL12, suggesting an ability to migrate towards lymph nodes. We also showed that conditioned media of HPV16 VLP-treated keratinocytes induce a lower LC migration than those from untreated keratinocytes and that prostaglandin E2 (PGE(2)), detected in HPV16 VLP-treated keratinocyte supernatants, may reduce LC recruitment into the squamous epithelium. Taken together, our data demonstrate that HPV16 VLP may differentially regulate the immune protective response according to their target cells.

High expression of PGE2 enzymatic pathways in cervical (pre)neoplastic lesions and functional consequences for antigen-presenting cells.

Although human papillomavirus (HPV) DNA is detected in the majority of squamous intraepithelial lesions (SIL) and carcinoma (SCC) of the uterine cervix, the persistence or progression of cervical lesions suggest that viral antigens are not adequately presented to the immune system. This hypothesis is reinforced by the observation that most SIL show quantitative and functional alterations of Langerhans cells (LC). The aim of this study was to determine whether prostaglandins (PG) may affect LC density in the cervical (pre)neoplastic epithelium. We first demonstrated that the epithelial expression of PGE(2) enzymatic pathways, including cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES-1), is higher in SIL and SCC compared to the normal exocervical epithelium and inversely correlated to the density of CD1a-positive LC. By using cell migration assays, we next showed that the motility of immature dendritic cells (DC) and DC partially differentiated in vitro in the presence of PGE(2) are differentially affected by PGE(2). Immature DC had a lower ability to migrate in the presence of PGE(2) compared to DC generated in vitro in the presence of PGE(2). Finally, we showed that PGE(2) induced a cytokine production profile and phenotypical features of tolerogenic DC, suggesting that the altered expression of PGE(2) enzymatic pathways may promote the cervical carcinogenesis by favouring (pre)cancer immunotolerance.

Increased migration of Langerhans cells in response to HPV16 E6 and E7 oncogene silencing: role of CCL20.

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix. The persistence or progression of cervical lesions suggests that viral antigens are not adequately presented to the immune system. This hypothesis is reinforced by the observation that most squamous intraepithelial lesions (SILs) show quantitative and functional alterations of Langerhans cells (LC). The infiltration of immature LC in the squamous epithelium is mainly controlled by Macrophage Inflammatory Protein 3alpha/CCL20. After having shown that CCL20 production is altered in HPV-transformed keratinocytes (KC), the possible role of HPV16 E6 and E7 viral oncoproteins in the reduced CCL20 levels observed in SILs was investigated by silencing HPV16 E6 and E7 oncogenes by RNA interference (siRNA). This treatment not only increased CCL20 secretion but also resulted in the modulation of NF-kappaB p50, p52 and p65 precursor localization. Moreover, silencing of E6 and E7 oncogenes in HPV16-transformed KC induced a significantly higher migratory capacity of LC in a Boyden chamber assay and in an in vitro formed (pre)neoplastic epithelium reminiscent of high-grade SILs. Anti-CCL20 neutralizing antibody experiments showed that the increased migration of LC is due to the re-expression of CCL20 in E6 and E7 siRNA transfected KC. These data suggest that HPV16 E6/E7-induced down-regulation of CCL20 observed during the cervical carcinogenesis may contribute to a diminished capacity of the immune system to control HPV infection.

Defensins induce the recruitment of dendritic cells in cervical human papillomavirus-associated (pre)neoplastic lesions formed in vitro and transplanted in vivo.

In addition to their direct antimicrobial activity, defensins might also influence adaptive immunity by attracting immature dendritic cells (DC). As these cells have been shown to be deficient in uterine cervix carcinogenesis, we evaluated the ability of alpha-defensin (HNP-2, human neutrophil defensin 2) and beta-defensin (HbetaD2, human beta defensin 2) to stimulate their migration in human papillomavirus (HPV)-associated (pre)cancers. We first observed, using RT-PCR and immunohistology, that HbetaD2 is absent in HPV-transformed keratinocytes and that it is weakly expressed in cervical (pre)neoplastic lesions in comparison with normal keratinocytes. We next demonstrated that defensins exert a chemotactic activity for DC in a Boyden Chamber assay and stimulate their infiltration in an in vitro-formed (pre)neoplastic epithelium (organotypic culture of HPV-transformed keratinocytes). To evaluate the ability of defensins also to recruit DC in vivo, we developed a model of immunodeficient mice grafted with organotypic cultures of HPV+ keratinocytes, which form an epithelium similar to a high-grade neoplastic lesion, with tumoral invasion and neovascularization. Intravenously injected human DC were able to infiltrate grafts of HPV+ keratinocytes after administration of HNP-2 in the transplantation chamber. Taken together, these results suggest that defensins could reverse a frequent immune alteration observed in cancer development.

Role of hormone cofactors in the human papillomavirus-induced carcinogenesis of the uterine cervix.

If human papillomavirus (HPV) is necessary for the development of (pre)neoplastic lesions of the uterine cervix, it is not sufficient. Among the cofactors involved in the malignant transformation of cells infected by HPV, sex hormones may facilitate the cervical carcinogenesis by different mechanisms, including the induction of squamous metaplasia in the transformation zone of the cervix, interactions between steroid hormones and HPV gene expression and alterations of the local immune microenvironment.

MIP3 alpha stimulates the migration of Langerhans cells in models of human papillomavirus (HPV)-associated (pre)neoplastic epithelium.

Although human papillomavirus (HPV) DNA is detected in the majority of cervical cancers and their precursors (squamous intraepithelial lesions; SIL), the persistence or progression of cervical lesions could be associated with quantitative and functional alterations of dendritic/Langerhans cells (DC/LC). As LC abnormalities have been associated with a decreased expression of macrophage inflammatory protein 3alpha (MIP3alpha) in cervical SIL, we tested the effect of exogenous MIP3alpha on the migration of LC in a (pre)neoplastic epithelium formed in vitro. By using a Boyden chamber assay, we first showed that the migratory capacity of LC generated in vitro is significantly increased in the presence of MIP3alpha compared to control medium. We next demonstrated that MIP3alpha is able to increase the 3D infiltration of LC in organotypic cultures of HPV-transformed keratinocytes. This property to stimulate LC migration was not altered after inclusion of MIP3alpha in a bioadhesive polycarbophil gel. Moreover, the function of DC to exert cytostatic effects and to present alloantigens was not altered in the presence of MIP3alpha.