Human Papillomavirus Type 8 Interferes with a Novel C/EBPβ-Mediated Mechanism of Keratinocyte CCL20 Chemokine Expression and Langerhans Cell Migration

Abstract

Infection with genus beta human papillomaviruses (HPV) is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and 8 in patients with epidermodysplasia verruciformis (EV), a genetic skin disease. So far, it has been unknown how these viruses overcome cutaneous immune control allowing their persistence in lesional epidermis of these patients. Here we demonstrate that Langerhans cells, essential for skin immunosurveillance, are strongly reduced in HPV8-positive lesional epidermis from EV patients. Interestingly, the same lesions were largely devoid of the important Langerhans cells chemoattractant protein CCL20. Applying bioinformatic tools, chromatin immunoprecipitation assays and functional studies we identified the differentiation-associated transcription factor CCAAT/enhancer binding protein β (C/EBPβ) as a critical regulator of CCL20 gene expression in normal human keratinocytes. The physiological relevance of this finding is supported by our in vivo studies showing that the expression patterns of CCL20 and nuclear C/EBPβ converge spatially in the most differentiated layers of human epidermis. Our analyses further identified C/EBPβ as a novel target of the HPV8 E7 oncoprotein, which co-localizes with C/EBPβ in the nucleus, co-precipitates with it and interferes with its binding to the CCL20 promoter in vivo. As a consequence, the HPV8 E7 but not E6 oncoprotein suppressed C/EBPβ-inducible and constitutive CCL20 gene expression as well as Langerhans cell migration. In conclusion, our study unraveled a novel molecular mechanism central to cutaneous host defense. Interference of the HPV8 E7 oncoprotein with this regulatory pathway allows the virus to disrupt the immune barrier, a major prerequisite for its epithelial persistence and procarcinogenic activity.

Author Summary

Human papillomaviruses (HPVs) infect squamous epithelial cells of skin or mucosa, giving rise to hyperproliferative lesions. A subgroup of “high-risk” genus alpha HPVs is associated with human anogenital malignancies, e.g. cervical cancer. The skin carcinogenic potential of genus beta HPV types, such as HPV8, is fully accepted in epidermodysplasia verruciformis (EV) patients and their contribution to the development of non-melanoma skin cancer in the general population is under investigation. This genetic disorder serves as a model disease for studying the immunobiology, viral persistence and molecular mechanisms of HPV-induced skin carcinogenesis. Here, we demonstrate that antigen-presenting Langerhans cells and the Langerhans cell attracting chemokine CCL20 are strongly reduced in lesional skin of EV patients. We show that the HPV8 encoded E7 oncoprotein substantially contributes to this disturbance of cutaneous innate immune control. Our data define a novel mechanism of C/EBPβ-dependent CCL20 gene regulation. HPV8 E7 directly interacts with this transcription factor, interferes with its binding to the CCL20 promoter and suppresses keratinocyte CCL20 expression as well as Langerhans cell migration. Our study unravels a molecular mechanism of virus-host interaction critical for evading host immune defense and providing a microenvironment that is conducive to persistent HPV infection and skin carcinogenesis.