Image_1_The cutaneous beta human papillomavirus type 8 E6 protein induces CCL2 through the CEBPα/miR-203/p63 pathway to support an inflammatory microenvironment in epidermodysplasia verruciformis skin lesions.pdf

Human papillomavirus type 8 (HPV8), a cutaneous genus beta HPV type, has co-carcinogenic potential at sun-exposed sites in patients suffering from the inherited skin disease epidermodysplasia verruciformis (EV). We had previously shown that Langerhans cells responsible for epithelial immunosurveillance were strongly reduced at infected sites and that the HPV8 E7 protein interferes with the CCAAT/enhancer-binding protein (C/EBP)β to suppress the Langerhans cell chemokine CCL20. At the same time, however, we observed that EV lesions are heavily infiltrated with inflammatory immune cells, which is similar to the situation in HPV8 E6 transgenic mice. To identify critical inflammatory factors, we used a broad multiplex approach and found that the monocyte attracting chemokine CCL2 was significantly and strongly induced by HPV8 E6 but not E7-expressing HaCaT cells, which were used as a model for UV-damaged skin keratinocytes. Conditioned media from HPV8 E6-expressing keratinocytes enhanced CCL2-receptor (CCR2)-dependent monocyte recruitment in vitro, and macrophages predominated in the stroma but were also detected in the epidermal compartment of EV lesions in vivo. CCL2 induction by HPV8 E6 was even stronger than stimulation with the proinflammatory cytokine TNF-α, and both HPV8 E6 and TNF-α resulted in substantial suppression of the transcription factor C/EBPα. Using RNAi-mediated knockdown and overexpression approaches, we demonstrated a mechanistic role of the recently identified C/EBPα/miR-203/p63 pathway for HPV8 E6-mediated CCL2 induction at protein and transcriptional levels. Epithelial co-expression of p63 and CCL2 was confirmed in HPV8 E6-expressing organotypic air–liquid interface cultures and in lesional EV epidermis in vivo. In summary, our data demonstrate that HPV8 oncoproteins actively deregulate epidermal immune homeostasis through modulation of C/EBP factor-dependent pathways. While HPV8 E7 suppresses immunosurveillance required for viral persistence, the present study provides evidence that E6 involves the stemness-promoting factor p63 to support an inflammatory microenvironment that may fuel carcinogenesis in EV lesions.</p

Schematic presentation of the molecular mechanism how human papillomavirus type 8 interferes with a novel C/EBPβ-mediated mechanism of keratinocyte CCL20 chemokine expression and Langerhans cell migration.

<p>Schematic presentation of the molecular mechanism how human papillomavirus type 8 interferes with a novel C/EBPβ-mediated mechanism of keratinocyte CCL20 chemokine expression and Langerhans cell migration.</p

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

<div><p>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 <em>CCL20</em> 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 <em>CCL20</em> 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.</p> </div

Langerin-positive cells and CCL20 expression are reduced in lesional skin of EV patients.

<p>Sections of normal human skin (A, B, C) and lesional skin from EV patients (D–J) were stained using antibodies against langerin (A, D) or CCL20 (B, C, E, F) and hematoxylin. To verify the specificity of the immunohistochemical reaction, recombinant CCL20 protein was used in a blocking experiment (C). Lesion positive for HPV8 only (D, E, G–J). Lesion coinfected with HPV5 and HPV8 (F). Validation of active infection was visualized by hematoxylin eosin staining to reveal the characteristic E4 cytoplasmic inclusions typically found in EV lesions (G) along with immunofluorescence staining for the HPV8 E4 protein (green (H)) and FISH staining to identify the presence of amplified HPV genomes (red (I)). The merged image showing the onset of genome amplification in E4-positive cells is shown in J. Cell nuclei are visualized in J by counterstaining with DAPI. Selected cells double positive for E4 protein and HPV8 DNA are marked with arrows. Bars correspond to 100 µm in A–E and G–J, and to 200 µm in F.</p

Table_4_The cutaneous beta human papillomavirus type 8 E6 protein induces CCL2 through the CEBPα/miR-203/p63 pathway to support an inflammatory microenvironment in epidermodysplasia verruciformis skin lesions.docx

Human papillomavirus type 8 (HPV8), a cutaneous genus beta HPV type, has co-carcinogenic potential at sun-exposed sites in patients suffering from the inherited skin disease epidermodysplasia verruciformis (EV). We had previously shown that Langerhans cells responsible for epithelial immunosurveillance were strongly reduced at infected sites and that the HPV8 E7 protein interferes with the CCAAT/enhancer-binding protein (C/EBP)β to suppress the Langerhans cell chemokine CCL20. At the same time, however, we observed that EV lesions are heavily infiltrated with inflammatory immune cells, which is similar to the situation in HPV8 E6 transgenic mice. To identify critical inflammatory factors, we used a broad multiplex approach and found that the monocyte attracting chemokine CCL2 was significantly and strongly induced by HPV8 E6 but not E7-expressing HaCaT cells, which were used as a model for UV-damaged skin keratinocytes. Conditioned media from HPV8 E6-expressing keratinocytes enhanced CCL2-receptor (CCR2)-dependent monocyte recruitment in vitro, and macrophages predominated in the stroma but were also detected in the epidermal compartment of EV lesions in vivo. CCL2 induction by HPV8 E6 was even stronger than stimulation with the proinflammatory cytokine TNF-α, and both HPV8 E6 and TNF-α resulted in substantial suppression of the transcription factor C/EBPα. Using RNAi-mediated knockdown and overexpression approaches, we demonstrated a mechanistic role of the recently identified C/EBPα/miR-203/p63 pathway for HPV8 E6-mediated CCL2 induction at protein and transcriptional levels. Epithelial co-expression of p63 and CCL2 was confirmed in HPV8 E6-expressing organotypic air–liquid interface cultures and in lesional EV epidermis in vivo. In summary, our data demonstrate that HPV8 oncoproteins actively deregulate epidermal immune homeostasis through modulation of C/EBP factor-dependent pathways. While HPV8 E7 suppresses immunosurveillance required for viral persistence, the present study provides evidence that E6 involves the stemness-promoting factor p63 to support an inflammatory microenvironment that may fuel carcinogenesis in EV lesions.</p

Table_3_The cutaneous beta human papillomavirus type 8 E6 protein induces CCL2 through the CEBPα/miR-203/p63 pathway to support an inflammatory microenvironment in epidermodysplasia verruciformis skin lesions.docx

Human papillomavirus type 8 (HPV8), a cutaneous genus beta HPV type, has co-carcinogenic potential at sun-exposed sites in patients suffering from the inherited skin disease epidermodysplasia verruciformis (EV). We had previously shown that Langerhans cells responsible for epithelial immunosurveillance were strongly reduced at infected sites and that the HPV8 E7 protein interferes with the CCAAT/enhancer-binding protein (C/EBP)β to suppress the Langerhans cell chemokine CCL20. At the same time, however, we observed that EV lesions are heavily infiltrated with inflammatory immune cells, which is similar to the situation in HPV8 E6 transgenic mice. To identify critical inflammatory factors, we used a broad multiplex approach and found that the monocyte attracting chemokine CCL2 was significantly and strongly induced by HPV8 E6 but not E7-expressing HaCaT cells, which were used as a model for UV-damaged skin keratinocytes. Conditioned media from HPV8 E6-expressing keratinocytes enhanced CCL2-receptor (CCR2)-dependent monocyte recruitment in vitro, and macrophages predominated in the stroma but were also detected in the epidermal compartment of EV lesions in vivo. CCL2 induction by HPV8 E6 was even stronger than stimulation with the proinflammatory cytokine TNF-α, and both HPV8 E6 and TNF-α resulted in substantial suppression of the transcription factor C/EBPα. Using RNAi-mediated knockdown and overexpression approaches, we demonstrated a mechanistic role of the recently identified C/EBPα/miR-203/p63 pathway for HPV8 E6-mediated CCL2 induction at protein and transcriptional levels. Epithelial co-expression of p63 and CCL2 was confirmed in HPV8 E6-expressing organotypic air–liquid interface cultures and in lesional EV epidermis in vivo. In summary, our data demonstrate that HPV8 oncoproteins actively deregulate epidermal immune homeostasis through modulation of C/EBP factor-dependent pathways. While HPV8 E7 suppresses immunosurveillance required for viral persistence, the present study provides evidence that E6 involves the stemness-promoting factor p63 to support an inflammatory microenvironment that may fuel carcinogenesis in EV lesions.</p

Table_2_The cutaneous beta human papillomavirus type 8 E6 protein induces CCL2 through the CEBPα/miR-203/p63 pathway to support an inflammatory microenvironment in epidermodysplasia verruciformis skin lesions.docx

Human papillomavirus type 8 (HPV8), a cutaneous genus beta HPV type, has co-carcinogenic potential at sun-exposed sites in patients suffering from the inherited skin disease epidermodysplasia verruciformis (EV). We had previously shown that Langerhans cells responsible for epithelial immunosurveillance were strongly reduced at infected sites and that the HPV8 E7 protein interferes with the CCAAT/enhancer-binding protein (C/EBP)β to suppress the Langerhans cell chemokine CCL20. At the same time, however, we observed that EV lesions are heavily infiltrated with inflammatory immune cells, which is similar to the situation in HPV8 E6 transgenic mice. To identify critical inflammatory factors, we used a broad multiplex approach and found that the monocyte attracting chemokine CCL2 was significantly and strongly induced by HPV8 E6 but not E7-expressing HaCaT cells, which were used as a model for UV-damaged skin keratinocytes. Conditioned media from HPV8 E6-expressing keratinocytes enhanced CCL2-receptor (CCR2)-dependent monocyte recruitment in vitro, and macrophages predominated in the stroma but were also detected in the epidermal compartment of EV lesions in vivo. CCL2 induction by HPV8 E6 was even stronger than stimulation with the proinflammatory cytokine TNF-α, and both HPV8 E6 and TNF-α resulted in substantial suppression of the transcription factor C/EBPα. Using RNAi-mediated knockdown and overexpression approaches, we demonstrated a mechanistic role of the recently identified C/EBPα/miR-203/p63 pathway for HPV8 E6-mediated CCL2 induction at protein and transcriptional levels. Epithelial co-expression of p63 and CCL2 was confirmed in HPV8 E6-expressing organotypic air–liquid interface cultures and in lesional EV epidermis in vivo. In summary, our data demonstrate that HPV8 oncoproteins actively deregulate epidermal immune homeostasis through modulation of C/EBP factor-dependent pathways. While HPV8 E7 suppresses immunosurveillance required for viral persistence, the present study provides evidence that E6 involves the stemness-promoting factor p63 to support an inflammatory microenvironment that may fuel carcinogenesis in EV lesions.</p

Table_1_The cutaneous beta human papillomavirus type 8 E6 protein induces CCL2 through the CEBPα/miR-203/p63 pathway to support an inflammatory microenvironment in epidermodysplasia verruciformis skin lesions.docx

Human papillomavirus type 8 (HPV8), a cutaneous genus beta HPV type, has co-carcinogenic potential at sun-exposed sites in patients suffering from the inherited skin disease epidermodysplasia verruciformis (EV). We had previously shown that Langerhans cells responsible for epithelial immunosurveillance were strongly reduced at infected sites and that the HPV8 E7 protein interferes with the CCAAT/enhancer-binding protein (C/EBP)β to suppress the Langerhans cell chemokine CCL20. At the same time, however, we observed that EV lesions are heavily infiltrated with inflammatory immune cells, which is similar to the situation in HPV8 E6 transgenic mice. To identify critical inflammatory factors, we used a broad multiplex approach and found that the monocyte attracting chemokine CCL2 was significantly and strongly induced by HPV8 E6 but not E7-expressing HaCaT cells, which were used as a model for UV-damaged skin keratinocytes. Conditioned media from HPV8 E6-expressing keratinocytes enhanced CCL2-receptor (CCR2)-dependent monocyte recruitment in vitro, and macrophages predominated in the stroma but were also detected in the epidermal compartment of EV lesions in vivo. CCL2 induction by HPV8 E6 was even stronger than stimulation with the proinflammatory cytokine TNF-α, and both HPV8 E6 and TNF-α resulted in substantial suppression of the transcription factor C/EBPα. Using RNAi-mediated knockdown and overexpression approaches, we demonstrated a mechanistic role of the recently identified C/EBPα/miR-203/p63 pathway for HPV8 E6-mediated CCL2 induction at protein and transcriptional levels. Epithelial co-expression of p63 and CCL2 was confirmed in HPV8 E6-expressing organotypic air–liquid interface cultures and in lesional EV epidermis in vivo. In summary, our data demonstrate that HPV8 oncoproteins actively deregulate epidermal immune homeostasis through modulation of C/EBP factor-dependent pathways. While HPV8 E7 suppresses immunosurveillance required for viral persistence, the present study provides evidence that E6 involves the stemness-promoting factor p63 to support an inflammatory microenvironment that may fuel carcinogenesis in EV lesions.</p

Image_2_The cutaneous beta human papillomavirus type 8 E6 protein induces CCL2 through the CEBPα/miR-203/p63 pathway to support an inflammatory microenvironment in epidermodysplasia verruciformis skin lesions.pdf

Human papillomavirus type 8 (HPV8), a cutaneous genus beta HPV type, has co-carcinogenic potential at sun-exposed sites in patients suffering from the inherited skin disease epidermodysplasia verruciformis (EV). We had previously shown that Langerhans cells responsible for epithelial immunosurveillance were strongly reduced at infected sites and that the HPV8 E7 protein interferes with the CCAAT/enhancer-binding protein (C/EBP)β to suppress the Langerhans cell chemokine CCL20. At the same time, however, we observed that EV lesions are heavily infiltrated with inflammatory immune cells, which is similar to the situation in HPV8 E6 transgenic mice. To identify critical inflammatory factors, we used a broad multiplex approach and found that the monocyte attracting chemokine CCL2 was significantly and strongly induced by HPV8 E6 but not E7-expressing HaCaT cells, which were used as a model for UV-damaged skin keratinocytes. Conditioned media from HPV8 E6-expressing keratinocytes enhanced CCL2-receptor (CCR2)-dependent monocyte recruitment in vitro, and macrophages predominated in the stroma but were also detected in the epidermal compartment of EV lesions in vivo. CCL2 induction by HPV8 E6 was even stronger than stimulation with the proinflammatory cytokine TNF-α, and both HPV8 E6 and TNF-α resulted in substantial suppression of the transcription factor C/EBPα. Using RNAi-mediated knockdown and overexpression approaches, we demonstrated a mechanistic role of the recently identified C/EBPα/miR-203/p63 pathway for HPV8 E6-mediated CCL2 induction at protein and transcriptional levels. Epithelial co-expression of p63 and CCL2 was confirmed in HPV8 E6-expressing organotypic air–liquid interface cultures and in lesional EV epidermis in vivo. In summary, our data demonstrate that HPV8 oncoproteins actively deregulate epidermal immune homeostasis through modulation of C/EBP factor-dependent pathways. While HPV8 E7 suppresses immunosurveillance required for viral persistence, the present study provides evidence that E6 involves the stemness-promoting factor p63 to support an inflammatory microenvironment that may fuel carcinogenesis in EV lesions.</p

Image_3_The cutaneous beta human papillomavirus type 8 E6 protein induces CCL2 through the CEBPα/miR-203/p63 pathway to support an inflammatory microenvironment in epidermodysplasia verruciformis skin lesions.pdf

Human papillomavirus type 8 (HPV8), a cutaneous genus beta HPV type, has co-carcinogenic potential at sun-exposed sites in patients suffering from the inherited skin disease epidermodysplasia verruciformis (EV). We had previously shown that Langerhans cells responsible for epithelial immunosurveillance were strongly reduced at infected sites and that the HPV8 E7 protein interferes with the CCAAT/enhancer-binding protein (C/EBP)β to suppress the Langerhans cell chemokine CCL20. At the same time, however, we observed that EV lesions are heavily infiltrated with inflammatory immune cells, which is similar to the situation in HPV8 E6 transgenic mice. To identify critical inflammatory factors, we used a broad multiplex approach and found that the monocyte attracting chemokine CCL2 was significantly and strongly induced by HPV8 E6 but not E7-expressing HaCaT cells, which were used as a model for UV-damaged skin keratinocytes. Conditioned media from HPV8 E6-expressing keratinocytes enhanced CCL2-receptor (CCR2)-dependent monocyte recruitment in vitro, and macrophages predominated in the stroma but were also detected in the epidermal compartment of EV lesions in vivo. CCL2 induction by HPV8 E6 was even stronger than stimulation with the proinflammatory cytokine TNF-α, and both HPV8 E6 and TNF-α resulted in substantial suppression of the transcription factor C/EBPα. Using RNAi-mediated knockdown and overexpression approaches, we demonstrated a mechanistic role of the recently identified C/EBPα/miR-203/p63 pathway for HPV8 E6-mediated CCL2 induction at protein and transcriptional levels. Epithelial co-expression of p63 and CCL2 was confirmed in HPV8 E6-expressing organotypic air–liquid interface cultures and in lesional EV epidermis in vivo. In summary, our data demonstrate that HPV8 oncoproteins actively deregulate epidermal immune homeostasis through modulation of C/EBP factor-dependent pathways. While HPV8 E7 suppresses immunosurveillance required for viral persistence, the present study provides evidence that E6 involves the stemness-promoting factor p63 to support an inflammatory microenvironment that may fuel carcinogenesis in EV lesions.</p