HPV8-induced STAT3 activation led keratinocyte stem cell expansion in human actinic keratoses.

Despite epidermal turnover, the skin is host to a complex array of microbes including viruses, such as the human papillomavirus (HPV), which must infect and manipulate skin keratinocyte stem cells (KSC) to survive. This crosstalk between the virome and KSC populations remains largely unknown. Here, we investigated the effect of HPV8 on KSCs using various mouse models. We observed that the HPV8 early region gene E6 specifically caused Lrig1+ hair follicle junctional zone KSC proliferation and expansion, which would facilitate viral transmission. Within Lrig1+ KSCs specifically, HPV8 E6 bound intracellular p300 to phosphorylate the STAT3 transcriptional regulatory node. This induces ΔNp63 expression, resulting in KSC expansion into the overlying epidermis. HPV8 was associated with 70% of human actinic keratoses (AK). Together these results define the "hit and run" mechanism for HPV8 in human actinic keratosis as an expansion of KSCs, which lacks melanosome protection and is thus susceptible to sun-light-induced malignant transformation.

Enhanced Spontaneous Skin Tumorigenesis and Aberrant Inflammatory Response to UVB Exposure in Immunosuppressed Human Papillomavirus Type 8‒Transgenic Mice.

Human papillomaviruses (HPVs) from the beta genus are commensal viruses of the skin usually associated with asymptomatic infection in the general population. However, in individuals with specific genetic backgrounds, such as patients with epidermodysplasia verruciformis, or those with immune defects, such as organ transplant recipients, they are functionally involved in sunlight-induced skin cancer development, mainly keratinocyte carcinoma. Despite their well-established protumorigenic role, the cooperation between ß-HPV infection, impaired host immunosurveillance, and UVB exposure has never been formally shown in animal models. In this study, by crossing skin-specific HPV8-transgenic mice with Rag2-deficient mice, we have generated a preclinical mouse model, named Rag2‒/‒:K14-HPV8. These mice display an unhealthy skin phenotype and spontaneously develop papilloma-like lesions spreading to the entire skin much more rapidly compared with Rag2+/+:K14-HPV8 mice. Exposure to low doses of UVB radiation is sufficient to trigger severe skin inflammation in Rag2‒/‒:K14-HPV8 but not in Rag2+/+:K14-HPV8 mice. Their inflamed skin very much resembled that observed in cutaneous field cancerization in organ transplant recipients, showing high levels of UVB-damaged cells, enhanced production of proinflammatory cytokines, and mast cell recruitment to the dermis. Overall, this immunocompromised HPV8-transgenic mouse model shows that the coexistence of immune defects, ß-HPV, and UVB exposure promotes skin cancer development.

Cancer-Associated Fibroblasts Exert Proangiogenic Activity in Merkel Cell Carcinoma.

The tumor microenvironment is a complex niche enveloping a tumor formed by extracellular matrix, blood vessels, immune cells, and fibroblasts constantly interacting with cancer cells. Although tumor microenvironment is increasingly recognized as a major player in cancer initiation and progression in many tumor types, its involvement in Merkel cell carcinoma (MCC) pathogenesis is currently unknown. In this study, we provide a molecular and functional characterization of cancer-associated fibroblasts (CAFs), the major tumor microenvironment component, in patient-derived xenografts of patients with MCC. We show that subcutaneous coinjection of patient-derived CAFs and human MCC MKL-1 cells into severe combined immunodeficient mice significantly promotes tumor growth and metastasis. These fast-growing xenografts are characterized by areas densely populated with human CAFs, mainly localized around blood vessels. We provide evidence that the growth-promoting activity of MCC-derived CAFs is mediated by the aminopeptidase A/angiotensin II and III/angiotensin II type 1 receptor axis, with the expression of aminopeptidase A in CAFs being a triggering event. Together, our findings point to aminopeptidase A as a potential marker for MCC prognostic stratification and as a candidate for therapeutic intervention.

Patterns of neutralizing humoral response to SARS-CoV-2 infection among hematologic malignancy patients reveal a robust immune response in anti-cancer therapy-naive patients.

Understanding antibody-based SARS-CoV-2 immunity in hematologic malignancy (HM) patients following infection is crucial to inform vaccination strategies for this highly vulnerable population. This cross-sectional study documents the anti-SARS-CoV-2 humoral response and serum neutralizing activity in 189 HM patients recovering from a PCR-confirmed infection. The overall seroconversion rate was 85.7%, with the lowest values in patients with lymphoid malignancies or undergoing chemotherapy. Therapy-naive patients in the "watch and wait" status were more likely to seroconvert and display increased anti-s IgG titers. Enhanced serum neutralizing activity was observed in the following SARS-CoV-2-infected HM patient groups: (i) males; (ii) severe COVID-19; and (iii) "watch and wait" or "complete/partial response". The geometric mean (GeoMean) ID50 neutralization titers in patients analyzed before or after 6 months post-infection were 299.1 and 306.3, respectively, indicating that >50% of the patients in either group had a neutralization titer sufficient to provide 50% protection from symptomatic COVID-19. Altogether, our findings suggest that therapy-naive HM patients mount a far more robust immune response to SARS-CoV-2 infection vs. patients receiving anti-cancer treatment, raising the important question as to whether HM patients should be vaccinated before therapy and/or receive vaccine formats capable of better recapitulating the natural infection.

Evidence of BK Polyomavirus Infection in Urothelial but not Renal Tumors from a Single Center Cohort of Kidney Transplant Recipients.

Emerging evidence indicates that reactivation of BK polyomavirus (BKPyV) in the kidney and urothelial tract of kidney transplant recipients (KTRs) may be associated with cancer in these sites. In this retrospective study of a single center cohort of KTRs (n = 1307), 10 clear cell renal cell carcinomas and 5 urinary bladder carcinomas were analyzed from 15 KTRs for the presence of BKPyV infection through immunohistochemistry and fluorescent in situ hybridization (FISH). Three of these patients had already exhibited biopsy-proven polyomavirus-associated nephropathies (PyVAN). Although the presence of BKPyV large-T antigen was evident in the urothelium from a kidney removed soon after PyVAN diagnosis, it was undetectable in all the formalin-fixed and paraffin-embedded (FFPE) blocks obtained from the 10 kidney tumors. By contrast, large-T antigen (LT) labeling of tumor cells was detected in two out of five bladder carcinomas. Lastly, the proportion of BKPyV DNA-FISH-positive bladder carcinoma nuclei was much lower than that of LT-positive cells. Taken together, our findings further strengthen the association between BKPyV reactivation and cancer development in KTRs, especially bladder carcinoma.

Human Papillomavirus E7 Oncoprotein Subverts Host Innate Immunity via SUV39H1-Mediated Epigenetic Silencing of Immune Sensor Genes.

Subversion of innate immunity by oncoviruses, such as human papillomavirus (HPV), favors carcinogenesis because the mechanism(s) of viral immune evasion can also hamper cancer immunosurveillance. Previously, we demonstrated that high-risk (hr) HPVs trigger simultaneous epigenetic silencing of multiple effectors of innate immunity to promote viral persistence. Here, we expand on those observations and show that the HPV E7 oncoprotein upregulates the H3K9-specific methyltransferase, whose action shuts down the host innate immune response. Specifically, we demonstrate that SUV39H1 contributes to chromatin repression at the promoter regions of the viral nucleic acid sensors RIG-I and cGAS and the adaptor molecule STING in HPV-transformed cells. Inhibition of SUV39H1 leads to transcriptional activation of these genes, especially RIG-I, followed by increased beta interferon (IFN-ß) and IFN-λ1 production after poly(dA·dT) or RIG-I agonist M8 transfection. Collectively, our findings provide new evidence that the E7 oncoprotein plays a central role in dampening host innate immunity and raise the possibility that targeting the downstream effector SUV39H1 or the RIG-I pathway is a viable strategy to treat viral and neoplastic disease.IMPORTANCE High-risk HPVs are major viral human carcinogens responsible for approximately 5% of all human cancers. The growth of HPV-transformed cells depends on the ability of viral oncoproteins to manipulate a variety of cellular circuits, including those involved in innate immunity. Here, we show that one of these strategies relies on E7-mediated transcriptional activation of the chromatin repressor SUV39H1, which then promotes epigenetic silencing of RIG-I, cGAS, and STING genes, thereby shutting down interferon secretion in HPV-transformed cells. Pharmacological or genetic inhibition of SUV39H1 restored the innate response in HPV-transformed cells, mostly through activation of RIG-I signaling. We also show that IFN production upon transfection of poly(dA·dT) or the RIG-I agonist M8 predominantly occurs through RIG-I signaling. Altogether, the reversible nature of the modifications associated with E7-mediated SUV39H1 upregulation provides a rationale for the design of novel anticancer and antiviral therapies targeting these molecules.

Interleukin-1 primes human mesenchymal stem cells towards an anti-inflammatory and pro-trophic phenotype in vitro.

BACKGROUND: Inflammation is a key contributor to central nervous system (CNS) injury such as stroke, and is a major target for therapeutic intervention. Effective treatments for CNS injuries are limited and applicable to only a minority of patients. Stem cell-based therapies are increasingly considered for the treatment of CNS disease, because they can be used as in-situ regulators of inflammation, and improve tissue repair and recovery. One promising option is the use of bone marrow-derived mesenchymal stem cells (MSCs), which can secrete anti-inflammatory and trophic factors, can migrate towards inflamed and injured sites or can be implanted locally. Here we tested the hypothesis that pre-treatment with inflammatory cytokines can prime MSCs towards an anti-inflammatory and pro-trophic phenotype in vitro. METHODS: Human MSCs from three different donors were cultured in vitro and treated with inflammatory mediators as follows: interleukin (IL)-1α, IL-1ß, tumour necrosis factor alpha (TNF-α) or interferon-γ. After 24 h of treatment, cell supernatants were analysed by ELISA for expression of granulocyte-colony stimulating factor (G-CSF), IL-10, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), IL-1 receptor antagonist (IL-1Ra) and vascular endothelial growth factor (VEGF). To confirm the anti-inflammatory potential of MSCs, immortalised mouse microglial BV2 cells were treated with bacterial lipopolysaccharide (LPS) and exposed to conditioned media (CM) of naïve or IL-1-primed MSCs, and levels of secreted microglial-derived inflammatory mediators including TNF-α, IL-10, G-CSF and IL-6 were measured by ELISA. RESULTS: Unstimulated MSCs constitutively expressed anti-inflammatory cytokines and trophic factors (IL-10, VEGF, BDNF, G-CSF, NGF and IL-1Ra). MSCs primed with IL-1α or IL-1ß showed increased secretion of G-CSF, which was blocked by IL-1Ra. Furthermore, LPS-treated BV2 cells secreted less inflammatory and apoptotic markers, and showed increased secretion of the anti-inflammatory IL-10 in response to treatment with CM of IL-1-primed MSCs compared with CM of unprimed MSCs. CONCLUSIONS: Our results demonstrate that priming MSCs with IL-1 increases expression of trophic factor G-CSF through an IL-1 receptor type 1 (IL-1R1) mechanism, and induces a reduction in the secretion of inflammatory mediators in LPS-activated microglial cells. The results therefore support the potential use of preconditioning treatments of stem cells in future therapies.