Highlights d Wound microbiota was profiled longitudinally in patients with diabetic foot ulcers d Staphylococcus aureus strains were associated with poor outcomes d S. aureus and other wound isolates promoted differential wound healing responses d Debridement depleted anaerobic bacteria in wounds with favorable outcomes

Cutaneous beta-papillomaviruses are associated with non-melanoma skin cancers that arise in patients who suffer from a rare genetic disorder, Epidermodysplasia verruciformis (EV) or after immunosuppression following organ transplantation. Recent studies have shown that the E6 proteins of the cancer associated beta human papillomavirus (HPV) 5 and HPV8 inhibit NOTCH and TGF-β signaling. However, it is unclear whether disruption of these pathways may contribute to cutaneous HPV pathogenesis and carcinogenesis. A recently identified papillomavirus, MmuPV1, infects laboratory mouse strains and causes cutaneous skin warts that can progress to squamous cell carcinoma. To determine whether MmuPV1 may be an appropriate model to mechanistically dissect the molecular contributions of cutaneous HPV infections to skin carcinogenesis, we investigated whether MmuPV1 E6 shares biological and biochemical activities with HPV8 E6. We report that the HPV8 and MmuPV1 E6 proteins share the ability to bind to the MAML1 and SMAD2/SMAD3 transcriptional cofactors of NOTCH and TGF-beta signaling, respectively. Moreover, we demonstrate that these cutaneous papillomavirus E6 proteins inhibit these two tumor suppressor pathways and that this ability is linked to delayed differentiation and sustained proliferation of differentiating keratinocytes. Furthermore, we demonstrate that the ability of MmuPV1 E6 to bind MAML1 is necessary for papilloma formation in experimentally infected mice. Our results, therefore, suggest that experimental MmuPV1 infection in mice will be a robust and useful experimental system to model key aspects of cutaneous HPV infection, pathogenesis and carcinogenesis.

Human papillomaviruses are causally associated with 5% of human cancers. The recent discovery of a papillomavirus (MmuPV1) that infects laboratory mice provides unique opportunities to study the life cycle and pathogenesis of papillomaviruses in the context of a genetically manipulatable host organism. To date, MmuPV1-induced disease has been found largely to be restricted to severely immunodeficient strains of mice. In this study, we report that ultraviolet radiation (UVR), specifically UVB spectra, causes wild-type strains of mice to become highly susceptible to MmuPV1-induced disease. MmuPV1-infected mice treated with UVB develop warts that progress to squamous cell carcinoma. Our studies further indicate that UVB induces systemic immunosuppression in mice that correlates with susceptibility to MmuPV1-associated disease. These findings provide new insight into how MmuPV1 can be used to study the life cycle of papillomaviruses and their role in carcinogenesis, the role of host immunity in controlling papillomavirus-associated pathogenesis, and a basis for understanding in part the role of UVR in promoting HPV infection in humans.

Papillomaviruses exhibit species-specific tropism, thereby limiting understanding and research of several aspects of HPV infection and carcinogenesis. The discovery of a murine papillomavirus (MmuPV1) provides the opportunity to study papillomavirus infections in a tractable, in vivo laboratory model. MmuPV1 infects and causes disease in the cutaneous epithelium, as well as the mucosal epithelia of the oral cavity and anogenital tract. In this report, we describe a murine model of MmuPV1 infection and neoplastic disease in the female reproductive tracts of wild-type immunocompetent FVB mice. Low-grade dysplastic lesions developed in reproductive tracts of FVB mice infected with MmuPV1 for 4 months, and mice infected for 6 months developed significantly worse disease, including squamous cell carcinoma (SCC). We also tested the contribution of estrogen and/or UV radiation (UVR), two cofactors we previously identified as being involved in papillomavirus-mediated disease, to cervicovaginal disease. Similar to HPV16 transgenic mice, exogenous estrogen treatment induced high-grade precancerous lesions in the reproductive tracts of MmuPV1-infected mice by 4 months and together with MmuPV1 efficiently induced SCC by 6 months. UV radiation and exogenous estrogen cooperated to promote carcinogenesis in MmuPV1-infected mice. This murine infection model represents the first instance of de novo papillomavirus-mediated carcinogenesis in the female reproductive tract of wild-type mice resulting from active virus infection and is also the first report of the female hormone estrogen contributing to this process. This model will provide an additional platform for fundamental studies on papillomavirus infection, cervicovaginal disease, and the role of cellular cofactors during papillomavirus-induced carcinogenesis. IMPORTANCE Tractable and efficient models of papillomavirus-induced pathogenesis are limited due to the strict species-specific and tissue-specific tropism of these viruses. Here, we report a novel preclinical murine model of papillomavirus-induced cervicovaginal disease in wild-type, immunocompetent mice using the recently discovered murine papillomavirus, MmuPV1. In this model, MmuPV1 establishes persistent viral infections in the mucosal epithelia of the female reproductive tract, a necessary component needed to accurately mimic HPV-mediated neoplastic disease in humans. Persistent MmuPV1 infections were able to induce progressive neoplastic disease and carcinogenesis, either alone or in combination with previously identified cofactors of papillomavirus-induced disease. This new model will provide a much-needed platform for basic and translational studies on both papillomavirus infection and associated disease in immunocompetent mice.

Highlights d Germ-free mice have abnormal skin barrier structure and function d Keratinocyte aryl hydrocarbon receptor (AHR) mediates barrier function and repair d Human skin commensal microbes activate keratinocyte AHR d Targeting the microbiota-AHR axis promotes skin barrier repair in disease models

Mouse papillomavirus type 1 (MmuPV1) provides, for the first time, the opportunity to study infection and pathogenesis of papillomaviruses in the context of laboratory mice. In this report, we define the transcriptome of MmuPV1 genome present in papillomas arising in experimentally infected mice using a combination of RNA-seq, PacBio Iso-seq, 5’ RACE, 3’ RACE, primer-walking RT-PCR, RNase protection, Northern blot and in situ hybridization analyses. We demonstrate that the MmuPV1 genome is transcribed unidirectionally from five major promoters (P) or transcription start sites (TSS) and polyadenylates its transcripts at two major polyadenylation (pA) sites. We designate the P7503, P360 and P859 as “early” promoters because they give rise to transcripts mostly utilizing the polyadenylation signal at nt 3844 and therefore can only encode early genes, and P7107 and P533 as “late” promoters because they give rise to transcripts utilizing polyadenylation signals at either nt 3844 or nt 7047, the latter being able to encode late, capsid proteins. MmuPV1 genome contains five splice donor sites and three acceptor sites that produce thirty-six RNA isoforms deduced to express seven predicted early gene products (E6, E7, E1, E1^M1, E1^M2, E2 and E8^E2) and three predicted late gene products (E1^E4, L2 and L1). The majority of the viral early transcripts are spliced once from nt 757 to 3139, while viral late transcripts, which are predicted to encode L1, are spliced twice, first from nt 7243 to either nt 3139 (P7107) or nt 757 to 3139 (P533) and second from nt 3431 to nt 5372. Thirteen of these viral transcripts were detectable by Northern blot analysis, with the P533-derived late E1^E4 transcripts being the most abundant. The late transcripts could be detected in highly differentiated keratinocytes of MmuPV1-infected tissues as early as ten days after MmuPV1 inoculation and correlated with detection of L1 protein and viral DNA amplification. In mature warts, detection of L1 was also found in more poorly differentiated cells, as previously reported. Subclinical infections were also observed. The comprehensive transcription map of MmuPV1 generated in this study provides further evidence that MmuPV1 is similar to high-risk cutaneous beta human papillomaviruses. The knowledge revealed will facilitate the use of MmuPV1 as an animal virus model for understanding of human papillomavirus gene expression, pathogenesis and immunology.

Infections with cutaneous papillomaviruses have been linked to cutaneous squamous cell carcinomas that arise in patients who suffer from a rare genetic disorder, epidermodysplasia verruciformis, or those who have experienced long-term, systemic immunosuppression following organ transplantation. The E6 proteins of the prototypical cutaneous human papillomavirus (HPV) 5 and HPV8 inhibit TGF-β and NOTCH signaling. The Mus musculus papillomavirus 1, MmuPV1, infects laboratory mouse strains and causes cutaneous skin warts that can progress to squamous cell carcinomas. MmuPV1 E6 shares biological and biochemical activities with HPV8 E6 including the ability to inhibit TGF-β and NOTCH signaling by binding the SMAD2/SMAD3 and MAML1 transcription factors, respectively. Inhibition of TGF-β and NOTCH signaling is linked to delayed differentiation and sustained proliferation of differentiating keratinocytes. Furthermore, the ability of MmuPV1 E6 to bind MAML1 is necessary for wart and cancer formation in experimentally infected mice. Hence, experimental MmuPV1 infection in mice will be a robust and valuable experimental system to dissect key aspects of cutaneous HPV infection, pathogenesis, and carcinogenesis.

Preclinical infection model systems are extremely valuable tools to aid in our understanding of Human Papillomavirus (HPV) biology, disease progression, prevention, and treatments. In this context, rodent papillomaviruses and their respective infection models are useful tools but remain underutilized resources in the field of papillomavirus biology. Two rodent papillomaviruses, MnPV1, which infects the Mastomys species of multimammate rats, and MmuPV1, which infects laboratory mice, are currently the most studied rodent PVs. Both of these viruses cause malignancy in the skin and can provide attractive infection models to study the lesser understood cutaneous papillomaviruses that have been frequently associated with HPV-related skin cancers. Of these, MmuPV1 is the first reported rodent papillomavirus that can naturally infect the laboratory strain of mice. MmuPV1 is an attractive model virus to study papillomavirus pathogenesis because of the ubiquitous availability of lab mice and the fact that this mouse species is genetically modifiable. In this review, we have summarized the knowledge we have gained about PV biology from the study of rodent papillomaviruses and point out the remaining gaps that can provide new research opportunities.