Anatomic and functional mapping of human uterine innervation.

OBJECTIVE: To better understand the physiology of pain in pelvic pain pathological conditions, such as endometriosis, in which alterations of uterine innervation have been highlighted, we performed an anatomic and functional mapping of the macro- and microinnervation of the human uterus. Our aim was to provide a 3-dimensional reconstruction model of uterine innervation. DESIGN: This was an experimental study. We dissected the pelvises of 4 human female fetuses into serial sections, and treated them with hematoxylin and eosin staining before immunostaining. SETTING: Academic Research Unit. PATIENTS: None. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Detection of nerves (S100 +) and characterization of the types of nerves. The slices obtained were aligned to construct a 3-dimensional model. RESULTS: A 3-dimensional model of uterine innervation was constructed. The nerve fibers appeared to have a centripetal path from the uterine serosa to the endometrium. Within the myometrium, innervation was dense. Endometrial innervation was sparse but present in the functional layer of the endometrium. Overall innervation was richest in the supravaginal cervix and rarer in the body of the uterus. Innervation was rich particularly laterally to the cervix next to the parametrium and paracervix. Four types of nerve fibers were identified: autonomic sympathetic (TH+), parasympathetic (VIP+), and sensitive (NPY+, CGRP1+ and VIP+). They were found in the 3 portions and the 3 layers of the uterus. CONCLUSIONS: We constructed a 3-dimensional model of the human uterine innervation. This model could provide a solid base for studying uterine innervation in pathologic situations, in order to find new therapeutic approaches.

Usutu Virus escapes langerin-induced restriction to productively infect human Langerhans cells, unlike West Nile virus.

Usutu virus (USUV) and West Nile virus (WNV) are phylogenetically close emerging arboviruses and constitute a global public health threat. Since USUV and WNV are transmitted by mosquitoes, the first immune cells they encounter are skin-resident dendritic cells, the most peripheral outpost of immune defense. This unique network is composed of Langerhans cells (LCs) and dermal DCs, which reside in the epidermis and the dermis, respectively. Using human skin explants, we show that while both viruses can replicate in keratinocytes, they can also infect resident DCs with distinct tropism: WNV preferentially infects DCs in the dermis, whereas USUV has a greater propensity to infect LCs. Using both purified human epidermal LCs (eLCs) and monocyte derived LCs (MoLCs), we confirm that LCs sustain a faster and more efficient replication of USUV than WNV and that this correlates with a more intense innate immune response to USUV compared with WNV. Next, we show that ectopic expression of the LC-specific C-type lectin receptor (CLR), langerin, in HEK293T cells allows WNV and USUV to bind and enter, but supports the subsequent replication of USUV only. Conversely, blocking or silencing langerin in MoLCs or eLCs made them resistant to USUV infection, thus demonstrating that USUV uses langerin to enter and replicate in LCs. Altogether, our results demonstrate that LCs constitute privileged target cells for USUV in human skin, because langerin favours its entry and replication. Intriguingly, this suggests that USUV efficiently escapes the antiviral functions of langerin, which normally safeguards LCs from most viral infections.

Well-differentiated liver cancers reveal the potential link between ACE2 dysfunction and metabolic breakdown.

Angiotensin-converting enzyme 2 (ACE2) is the receptor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causing Coronavirus disease 2019 (COVID-19). Transmembrane serine protease 2 (TMPRSS2) is a coreceptor. Abnormal hepatic function in COVID-19 suggests specific or bystander liver disease. Because liver cancer cells express the ACE2 viral receptor, they are widely used as models of SARS-CoV-2 infection in vitro. Therefore, the purpose of this study was to analyze ACE2 and TMPRSS2 expression and localization in human liver cancers and in non-tumor livers. We studied ACE2 and TMPRSS2 in transcriptomic datasets totaling 1503 liver cancers, followed by high-resolution confocal multiplex immunohistochemistry and quantitative image analysis of a 41-HCC tissue microarray. In cancers, we detected ACE2 and TMPRSS2 at the biliary pole of tumor hepatocytes. In whole mount sections of five normal liver samples, we identified ACE2 in hepatocyte's bile canaliculi, biliary epithelium, sinusoidal and capillary endothelial cells. Tumors carrying mutated ß-catenin showed ACE2 DNA hypomethylation and higher mRNA and protein expression, consistently with predicted ß-catenin response sites in the ACE2 promoter. Finally, ACE2 and TMPRSS2 co-expression networks highlighted hepatocyte-specific functions, oxidative stress and inflammation, suggesting a link between inflammation, ACE2 dysfunction and metabolic breakdown.

HLA-G Is Widely Expressed by Mast Cells in Regions of Organ Fibrosis in the Liver, Lung and Kidney.

We previously demonstrated that mast cells expressing HLA-G are associated with regions of hepatitis C virus-induced liver fibrosis. Here, we aimed to determine whether HLA-G expression in mast cells is specific to viral etiology, the liver, or to the general process of fibrosis. We enumerated HLA-G+ cells and mast cells by the immunohistochemistry of (i) liver blocks from 41 cases of alcoholic cirrhosis, (ii) 10 of idiopathic pulmonary fibrosis (IPF), and (iii) 10 of renal fibrosis. The nature of the HLA-G+ cells was specified by multiplex immunofluorescence using software. More than half of all HLA-G+ cells were mast cells in fibrotic areas of alcoholic cirrhosis and IPF. In the kidneys, subjected to fibrosis, the HLA-G+ cells were indeed mast cells but could not be counted. Moreover, in certain cases of the liver and lung, we observed a number of cellular nodes, which were secondary or tertiary follicles, in which HLA-G was highly expressed by B lymphocytes. In conclusion, HLA-G+ mast cells could be observed in the fibrotic regions of all organs studied. Previous studies suggest a protective role for HLA-G+ mast cells against inflammation and fibrosis. The observed follicles with B lymphocytes that express HLA-G may also reinforce their antifibrotic role.

USF1 defect drives p53 degradation during Helicobacter pylori infection and accelerates gastric carcinogenesis.

OBJECTIVE: Helicobacter pylori (Hp) is a major risk factor for gastric cancer (GC). Hp promotes DNA damage and proteasomal degradation of p53, the guardian of genome stability. Hp reduces the expression of the transcription factor USF1 shown to stabilise p53 in response to genotoxic stress. We investigated whether Hp-mediated USF1 deregulation impacts p53-response and consequently genetic instability. We also explored in vivo the role of USF1 in gastric carcinogenesis. DESIGN: Human gastric epithelial cell lines were infected with Hp7.13, exposed or not to a DNA-damaging agent camptothecin (CPT), to mimic a genetic instability context. We quantified the expression of USF1, p53 and their target genes, we determined their subcellular localisation by immunofluorescence and examined USF1/p53 interaction. Usf1-/- and INS-GAS mice were used to strengthen the findings in vivo and patient data examined for clinical relevance. RESULTS: In vivo we revealed the dominant role of USF1 in protecting gastric cells against Hp-induced carcinogenesis and its impact on p53 levels. In vitro, Hp delocalises USF1 into foci close to cell membranes. Hp prevents USF1/p53 nuclear built up and relocates these complexes in the cytoplasm, thereby impairing their transcriptional function. Hp also inhibits CPT-induced USF1/p53 nuclear complexes, exacerbating CPT-dependent DNA damaging effects. CONCLUSION: Our data reveal that the depletion of USF1 and its de-localisation in the vicinity of cell membranes are essential events associated to the genotoxic activity of Hp infection, thus promoting gastric carcinogenesis. These findings are also of clinical relevance, supporting USF1 expression as a potential marker of GC susceptibility.

Sustained activation of the Aryl hydrocarbon Receptor transcription factor promotes resistance to BRAF-inhibitors in melanoma.

BRAF inhibitors target the BRAF-V600E/K mutated kinase, the driver mutation found in 50% of cutaneous melanoma. They give unprecedented anti-tumor responses but acquisition of resistance ultimately limits their clinical benefit. The master regulators driving the expression of resistance-genes remain poorly understood. Here, we demonstrate that the Aryl hydrocarbon Receptor (AhR) transcription factor is constitutively activated in a subset of melanoma cells, promoting the dedifferentiation of melanoma cells and the expression of BRAFi-resistance genes. Typically, under BRAFi pressure, death of BRAFi-sensitive cells leads to an enrichment of a small subpopulation of AhR-activated and BRAFi-persister cells, responsible for relapse. Also, differentiated and BRAFi-sensitive cells can be redirected towards an AhR-dependent resistant program using AhR agonists. We thus identify Resveratrol, a clinically compatible AhR-antagonist that abrogates deleterious AhR sustained-activation. Combined with BRAFi, Resveratrol reduces the number of BRAFi-resistant cells and delays tumor growth. We thus propose AhR-impairment as a strategy to overcome melanoma resistance.

A non-coding function of TYRP1 mRNA promotes melanoma growth.

Competition among RNAs to bind miRNA is proposed to influence biological systems. However, the role of this competition in disease onset is unclear. Here, we report that TYRP1 mRNA, in addition to encoding tyrosinase-related protein 1 (TYRP1), indirectly promotes cell proliferation by sequestering miR-16 on non-canonical miRNA response elements. Consequently, the sequestered miR-16 is no longer able to repress its mRNA targets, such as RAB17, which is involved in melanoma cell proliferation and tumour growth. Restoration of miR-16 tumour-suppressor function can be achieved in vitro by silencing TYRP1 or increasing miR-16 expression. Importantly, TYRP1-dependent miR-16 sequestration can also be overcome in vivo by using small oligonucleotides that mask miR-16-binding sites on TYRP1 mRNA. Together, our findings assign a pathogenic non-coding function to TYRP1 mRNA and highlight miRNA displacement as a promising targeted therapeutic approach for melanoma.

Integrating targeted gene expression and a skin model system to identify functional inhibitors of the UV activated p38 MAP kinase.

The stress-activated p38α MAP Kinase is an integral and critical component of the UV-induced inflammatory response. Despite the advances in recent years in the development of p38 kinase inhibitors, validation of these compounds in the diseased models remains limited. Based on the pharmacological profile of p38α inhibitor lead compound, SB203580, we synthesized a series of pyrrole-derivatives. Using UV-irradiated human skin punch-biopsies and cell cultures, we identified and validated the inhibitory activity of the derivatives by quantitatively measuring their effect on the expression of p38α target genes using real-time PCR. This approach not only identified pyrrole-2 as a unique derivative of this series that specifically inhibited the UV-activated p38α kinase, but also documented the skin permeation, bioavailability and reversible properties of this derivative in a 3D structure. The successful skin permeation of pyrrole-2 and its impact on AREG, COX-2 and MMP-9 gene expression demonstrates its potential use in modulating inflammatory processes in the skin. This study underscored the importance of using adapted biological models to identify accurate bioactive compounds.

Comparative transcriptional profiling of human Merkel cells and Merkel cell carcinoma.

Merkel cell carcinoma is believed to be derived from Merkel cells after infection by Merkel cell polyomavirus (MCPyV) and other poorly understood events. Transcriptional profiling using cDNA microarrays was performed on cells from MCPy-negative and MCPy-positive Merkel cell carcinomas and isolated normal Merkel cells. This microarray revealed numerous significantly upregulated genes and some downregulated genes. The extensive list of genes that were identified in these experiments provides a large body of potentially valuable information of Merkel cell carcinoma carcinogenesis and could represent a source of potential targets for cancer therapy.

p53 requires the stress sensor USF1 to direct appropriate cell fate decision.

Genomic instability is a major hallmark of cancer. To maintain genomic integrity, cells are equipped with dedicated sensors to monitor DNA repair or to force damaged cells into death programs. The tumor suppressor p53 is central in this process. Here, we report that the ubiquitous transcription factor Upstream Stimulatory factor 1 (USF1) coordinates p53 function in making proper cell fate decisions. USF1 stabilizes the p53 protein and promotes a transient cell cycle arrest, in the presence of DNA damage. Thus, cell proliferation is maintained inappropriately in Usf1 KO mice and in USF1-deficient melanoma cells challenged by genotoxic stress. We further demonstrate that the loss of USF1 compromises p53 stability by enhancing p53-MDM2 complex formation and MDM2-mediated degradation of p53. In USF1-deficient cells, the level of p53 can be restored by the re-expression of full-length USF1 protein similarly to what is observed using Nutlin-3, a specific inhibitor that prevents p53-MDM2 interaction. Consistent with a new function for USF1, a USF1 truncated protein lacking its DNA-binding and transactivation domains can also restore the induction and activity of p53. These findings establish that p53 function requires the ubiquitous stress sensor USF1 for appropriate cell fate decisions in response to DNA-damage. They underscore the new role of USF1 and give new clues of how p53 loss of function can occur in any cell type. Finally, these findings are of clinical relevance because they provide new therapeutic prospects in stabilizing and reactivating the p53 pathway.

Testing chemical carcinogenicity by using a transcriptomics HepaRG-based model?

The EU FP6 project carcinoGENOMICS explored the combination of toxicogenomics and in vitro cell culture models for identifying organotypical genotoxic- and non-genotoxic carcinogen-specific gene signatures. Here the performance of its gene classifier, derived from exposure of metabolically competent human HepaRG cells to prototypical non-carcinogens (10 compounds) and hepatocarcinogens (20 compounds), is reported. Analysis of the data at the gene and the pathway level by using independent biostatistical approaches showed a distinct separation of genotoxic from non-genotoxic hepatocarcinogens and non-carcinogens (up to 88 % correct prediction). The most characteristic pathway responding to genotoxic exposure was DNA damage. Interlaboratory reproducibility was assessed by blindly testing of three compounds, from the set of 30 compounds, by three independent laboratories. Subsequent classification of these compounds resulted in correct prediction of the genotoxicants. As expected, results on the non-genotoxic carcinogens and the non-carcinogens were less predictive. In conclusion, the combination of transcriptomics with the HepaRG in vitro cell model provides a potential weight of evidence approach for the evaluation of the genotoxic potential of chemical substances.

USF-1 is critical for maintaining genome integrity in response to UV-induced DNA photolesions.

An important function of all organisms is to ensure that their genetic material remains intact and unaltered through generations. This is an extremely challenging task since the cell's DNA is constantly under assault by endogenous and environmental agents. To protect against this, cells have evolved effective mechanisms to recognize DNA damage, signal its presence, and mediate its repair. While these responses are expected to be highly regulated because they are critical to avoid human diseases, very little is known about the regulation of the expression of genes involved in mediating their effects. The Nucleotide Excision Repair (NER) is the major DNA-repair process involved in the recognition and removal of UV-mediated DNA damage. Here we use a combination of in vitro and in vivo assays with an intermittent UV-irradiation protocol to investigate the regulation of key players in the DNA-damage recognition step of NER sub-pathways (TCR and GGR). We show an up-regulation in gene expression of CSA and HR23A, which are involved in TCR and GGR, respectively. Importantly, we show that this occurs through a p53 independent mechanism and that it is coordinated by the stress-responsive transcription factor USF-1. Furthermore, using a mouse model we show that the loss of USF-1 compromises DNA repair, which suggests that USF-1 plays an important role in maintaining genomic stability.

Merkel cell polyomavirus small T antigen mRNA level is increased following in vivo UV-radiation.

Merkel cell carcinoma (MCC) is a rare but aggressive skin cancer involving Merkel cells. Recently, a new human polyomavirus was implicated in MCC, being present in 80% of the samples analyzed. In virus-positive MCC, the Merkel cell polyomavirus (MCPyV) is clonally integrated into the patients DNA, and carries mutations in its large T antigen, leading to a truncated protein. In non-symptomatic tissue MCPyV can reside at very low levels. MCC is also associated with older age, immunosuppression and sun exposure. However, the link with solar exposure remains unknown, as the precise mechanism and steps involved between time of infection by MCPyV and the development of MCC. We thus investigated the potential impact of solar simulated radiation (SSR) on MCPyV transcriptional activity. We screened skin samples of 20 healthy patients enrolled in a photodermatological protocol based on in vivo-administered 2 and 4 J/cm(2) SSR. Two patients were infected with two new variants of MCPyV, present in their episomal form and RT-QPCR analyses on SSR-irradiated skin samples showed a specific and unique dose-dependent increase of MCPyV small t antigen transcript. A luciferase based in vitro assay confirmed that small t promoter is indeed UV-inducible. These findings demonstrate that solar radiation has an impact on MCPyV mRNA levels that may explain the association between MCC and solar exposure.

In vivo identification of solar radiation-responsive gene network: role of the p38 stress-dependent kinase.

Solar radiation is one of the most common threats to the skin, with exposure eliciting a specific protective cellular response. To decrypt the underlying mechanism, we used whole genome microarrays (Agilent 44K) to study epidermis gene expression in vivo in skin exposed to simulated solar radiation (SSR). We procured epidermis samples from healthy Caucasian patients, with phototypes II or III, and used two different SSR doses (2 and 4 J/cm(2)), the lower of which corresponded to the minimal erythemal dose. Analyses were carried out five hours after irradiation to identify early gene expression events in the photoprotective response. About 1.5% of genes from the human genome showed significant changes in gene expression. The annotations of these affected genes were assessed. They indicated a strengthening of the inflammation process and up-regulation of the JAK-STAT pathway and other pathways. Parallel to the p53 pathway, the p38 stress-responsive pathway was affected, supporting and mediating p53 function. We used an ex vivo assay with a specific inhibitor of p38 (SB203580) to investigate genes the expression of which was associated with active p38 kinase. We identified new direct p38 target genes and further characterized the role of p38. Our findings provide further insight into the physiological response to UV, including cell-cell interactions and cross-talk effects.

Alpha-melanocyte-stimulating hormone counteracts the suppressive effect of UVB on Nrf2 and Nrf-dependent gene expression in human skin.

Human skin is constantly exposed to UV light, the most ubiquitous environmental stressor. Here, we investigated the expression and regulation of Nrf1-3, transcription factors crucially involved in protection against oxidative stress in human skin cells in vitro, ex vivo, and in situ. In particular, we examined whether alpha-MSH, a UV-induced peptide, is capable of modulating Nrf2 and Nrf-dependent gene expression. Nrf1, -2, and -3 were found to be expressed in various cutaneous cell types in vitro. Surprisingly, UVB irradiation at physiological doses (10 mJ/cm(2)) reduced Nrf2 and Nrf-dependent gene expression in normal keratinocytes and melanocytes in vitro as well as ex vivo in skin organ cultures. alpha-MSH alone significantly increased Nrf2 as well as Nrf-dependent heme oxygenase-1, gamma-glutamylcysteine-synthetase, and glutathione-S-transferase Pi gene expression in both keratinocytes and melanocytes. This effect of alpha-MSH occurred at physiological doses and was due to transcriptional induction, mimicked by the artificial cAMP inducer forskolin, and blocked by protein kinase A pathway inhibition. In silico promoter analysis of Nrf2 further identified several putative binding sites for activator protein 1 and cAMP response element-binding protein, transcription factors typically activated by alpha-MSH. Importantly, alpha-MSH prevented or even overcompensated the UVB-induced suppression of Nrf2 and Nrf-dependent genes not only in normal keratinocytes and melanocytes in vitro but also in skin organ cultures. These findings, for the first time, show regulation of Nrf2 and Nrf-dependent genes by alpha-MSH. Our data also highlight a novel facet in the cytoprotective and antioxidative effector mechanisms of alpha-MSH and perhaps of related melanocortin peptides.