Whole-Exome Sequencing in 10 Unrelated Patients with Syndromic Hidradenitis Suppurativa: A Preliminary Step for a Genotype-Phenotype Correlation.

BACKGROUND: The genetics of syndromic hidradenitis suppurativa (HS), an immune-mediated condition associated with systemic comorbidities such as inflammatory bowel diseases and arthritis, has not been completely elucidated. OBJECTIVE: To describe clinical features and genetic signature of patients with the main syndromic HS forms, i.e., PASH, PAPASH, and PASH/SAPHO overlapping. METHODS: Whole-exome sequencing (WES) approach was performed in ten patients with syndromic HS. RESULTS: Three clinical settings have been identified based on presence/absence of gut and joint inflammation. Four PASH patients who had also gut inflammation showed three different variants in NOD2 gene, two variants in OTULIN, and a variant in GJB2, respectively. Three PAPASH and three PASH/SAPHO overlapping patients who had also joint inflammation showed two different variants in NCSTN, one in WDR1 and PSTPIP1, and two variants in NLRC4, one of whom was present in a patient with a mixed phenotype characterized by gut and joint inflammation. LIMITATIONS: Limited number of patients that can be counterbalanced by the rarity of syndromic HS. CONCLUSION: Syndromic HS can be considered as a polygenic autoinflammatory condition; currently WES is a diagnostic tool allowing more accurate genotype-phenotype correlation.

Unraveling the Role of Sex Hormones on Keratinocyte Functions in Human Inflammatory Skin Diseases.

The skin exerts several fundamental functions that are the first physical, chemical and immune barriers to the human body. Keratinocytes, the main cell type of the epidermis, provide mechanical defense, support skin integrity and actively endorse cutaneous immune responses. Not surprisingly, considering these crucial activities, alterations in keratinocyte functions are associated with different inflammatory skin diseases. Recent findings indicate that the skin should not only be regarded as a target for hormones but that it should also be considered as an endocrine peripheral organ that is directly involved in the synthesis and metabolism of these chemical messengers. Sex hormones have multiple effects on the skin, attributed to the binding with intracellular receptors expressed by different skin cell populations, including keratinocytes, that activate downstream signaling routes that modulate specific cellular functions and activities. This review is aimed at reorganizing the current knowledge on the role exerted by sex hormones on keratinocyte function in five different inflammatory skin diseases: Hidradenitis suppurativa; Acne vulgaris; Atopic dermatitis; progesterone hypersensitivity; psoriasis. The results of our work aim to provide a deeper insight into common cellular mechanisms and molecular effectors that might constitute putative targets to address for the development of specific therapeutic interventions.

Multiomics Integration in Skin Diseases with Alterations in Notch Signaling Pathway: PlatOMICs Phase 1 Deployment.

The high volume of information produced in the age of omics was and still is an important step to understanding several pathological processes, providing the enlightenment of complex molecular networks and the identification of molecular targets associated with many diseases. Despite these remarkable scientific advances, the majority of the results are disconnected and divergent, making their use limited. Skin diseases with alterations in the Notch signaling pathway were extensively studied during the omics era. In the GWAS Catalog, considering only studies on genomics association (GWAS), several works were deposited, some of which with divergent results. In addition, there are thousands of scientific articles available about these skin diseases. In our study, we focused our attention on skin diseases characterized by the impairment of Notch signaling, this pathway being of pivotal importance in the context of epithelial disorders. We considered the pathologies of five human skin diseases, Hidradenitis Suppurativa, Dowling Degos Disease, Adams-Oliver Syndrome, Psoriasis, and Atopic Dermatitis, in which the molecular alterations in the Notch signaling pathway have been reported. To this end, we started developing a new multiomics platform, PlatOMICs, to integrate and re-analyze omics information, searching for the molecular interactions involved in the pathogenesis of skin diseases with alterations in the Notch signaling pathway.

Notch Signaling Regulation in Autoinflammatory Diseases.

Notch pathway is a highly conserved intracellular signaling route that modulates a vast variety of cellular processes including proliferation, differentiation, migration, cell fate and death. Recently, the presence of a strict crosstalk between Notch signaling and inflammation has been described, although the precise molecular mechanisms underlying this interplay have not yet been fully unravelled. Disruptions in Notch cascade, due both to direct mutations and/or to an altered regulation in the core components of Notch signaling, might lead to hypo- or hyperactivation of Notch target genes and signaling molecules, ultimately contributing to the onset of autoinflammatory diseases. To date, alterations in Notch signaling have been reported as associated with three autoinflammatory disorders, therefore, suggesting a possible role of Notch in the pathogenesis of the following diseases: hidradenitis suppurativa (HS), Behçet disease (BD), and giant cell arteritis (GCA). In this review, we aim at better characterizing the interplay between Notch and autoinflammatory diseases, trying to identify the role of this signaling route in the context of these disorders.

Pleiotropic Role of Notch Signaling in Human Skin Diseases.

Notch signaling orchestrates the regulation of cell proliferation, differentiation, migration and apoptosis of epidermal cells by strictly interacting with other cellular pathways. Any disruption of Notch signaling, either due to direct mutations or to an aberrant regulation of genes involved in the signaling route, might lead to both hyper- or hypo-activation of Notch signaling molecules and of target genes, ultimately inducing the onset of skin diseases. The mechanisms through which Notch contributes to the pathogenesis of skin diseases are multiple and still not fully understood. So far, Notch signaling alterations have been reported for five human skin diseases, suggesting the involvement of Notch in their pathogenesis: Hidradenitis Suppurativa, Dowling Degos Disease, Adams-Oliver Syndrome, Psoriasis and Atopic Dermatitis. In this review, we aim at describing the role of Notch signaling in the skin, particularly focusing on the principal consequences associated with its alterations in these five human skin diseases, in order to reorganize the current knowledge and to identify potential cellular mechanisms in common between these pathologies.

Autophagy in Zika Virus Infection: A Possible Therapeutic Target to Counteract Viral Replication.

Zika virus (ZIKV) still constitutes a public health concern, however, no vaccines or therapies are currently approved for treatment. A fundamental process involved in ZIKV infection is autophagy, a cellular catabolic pathway delivering cytoplasmic cargo to the lysosome for degradation-considered as a primordial form of innate immunity against invading microorganisms. ZIKV is thought to inhibit the Akt-mTOR signaling pathway, which causes aberrant activation of autophagy promoting viral replication and propagation. It is therefore appealing to study the role of autophagic molecular effectors during viral infection to identify potential targets for anti-ZIKV therapeutic intervention.

25-hydroxycholesterol reduces inflammation, viral load and cell death in ZIKV-infected U-87 MG glial cell line.

Zika virus (ZIKV) infection is a global health issue due to its worldwide diffusion and to the related effects on neural progenitor cells with severe consequences on developing brain as well as on the central nervous system of adults. Previous studies showed that ZIKV infection induces an increment of IL1B expression in the central nervous system and also in the blood. IL-1ß is a pro-inflammatory cytokine essential for cellular defence, tissue repair and neuroinflammation, a mechanism seen to be associated with several neuroinflammatory diseases. 25-hydroxycholesterol (25-HC) is a natural oxysterol, derived from hydroxylation of cholesterol, possessing important antiviral activity possibly correlated to its ability to alter host membrane structures. Furthermore, 25-HC is involved in the modulation of IL1B gene expression, being able to suppress IL-1ß driven inflammation probably by blocking the activation of the SREB proteins. In our study, we analysed the antiviral action of 25-HC in ZIKV-infected U-87 MG cells, also evaluating its impact on inflammation and cell death. We demonstrated that 25-HC is able to reduce inflammation and cell death caused by ZIKV infection and also to diminish intracellular ZIKV load in U-87 MG glial cell line. Considering its antiviral activity and its ability to penetrate blood-brain barrier, 25-HC could be proposed, based on our results and literature findings, as a potential anti-ZIKV agent.

Perforin gene PRF1 c.900C> T polymorphism and HIV-1 vertical transmission.

Perforin-1, a component of the immune system, is able to control Human Immunodeficiency Virus-1 (HIV-1) replication and could be involved in HIV-1 mother-to-child transmission (MTCT). This study aims at evaluating the role of the c.900C > T PRF1 gene (encoding for perforin-1) polymorphism (rs885822) in HIV-1 MTCT. The PRF1 c.900C > T polymorphism was genotyped in 331 children from Zambia using a Taqman probe on a Real Time PCR platform. The PRF1 c.900C > T C/T genotype was more frequent among HIV-1 exposed but non-infected children than in HIV-1 positive cases, and the results were confirmed among children infected during breastfeeding. PRF1 c.900C > T correlated with protection against HIV-1 MTCT, suggesting its role in HIV-1 vertical transmission.

Lack of Prenylated Proteins, Autophagy Impairment and Apoptosis in SH-SY5Y Neuronal Cell Model of Mevalonate Kinase Deficiency.

BACKGROUND/AIMS: Mevalonate Kinase Deficiency (MKD), is a hereditary disease due to mutations in mevalonate kinase gene (MVK). MKD has heterogeneous clinical phenotypes: the correlation between MVK mutations and MKD clinical phenotype is still to be fully elucidated. Deficiency of prenylated proteins has been hypothesized as possible MKD pathogenic mechanism. Based on this hypothesis and considering that neurologic impairment characterizes Mevalonic Aciduria (MA), the most severe form of MKD, we studied the effects of I268T and N301T MVK mutations on protein prenylation, autophagy and programmed cell death in SH-SY5Y neuroblastoma cell lines. METHODS: SH-SY5Y cells were transiently transfected, with the pCMV-6 plasmid containing MVK wild type and the two mutated sequences. Protein prenylation levels were evaluated using GFP-RhoA-F to assess farnesylation, and GFP-RhoA to evaluate geranylgeranylation; autophagy was measured by evaluating LC3 and p62 protein levels, while Annexin V-FITC and Propidium Iodide staining allowed apoptosis detection. RESULTS: MVK mutants' over-expression causes decreased levels of farnesylation and geranylgeranylation, and also increased LC3 lipidation in SH-SY5Y, with concomitant p62 accumulation. Treatment with bafilomycin A1 (an inhibitor of vacuolar H+-ATPase, a late autophagy inhibitor) further increase LC3-II and p62 levels, suggesting that degradation of autophagolysosome could be impaired. SH-SY5Y, with both MVK mutants, showed apoptosis increase; the presence of N301T associated with augmented cell death. CONCLUSIONS: We hypothesize that mevalonate pathway impairment causes alteration of farnesylation and geranylgeranylation proteins and alteration of the autophagic flux; these changes can induce apoptosis, possibly more relevant in the presence of N301T mutation.

Direct inactivation of SARS-CoV-2 by low level blue photobiomodulation LED at 470, 454 and 450 nm.

Blue light has been already reported as able to counteract different types of microorganisms including Gram-positive and Gram-negative bacteria, fungi and viruses, especially the enveloped ones. It has been reported that both blue and visible light can efficiently impact SARS-CoV-2 by affecting its ability to replicate in in vitro cellular models of infection. In this study, blue light at 450, 454 and 470 nm was tested on SARS-CoV-2 to evaluate the residual viral infectious potential on Vero E6, Caco-2 and Calu-3 cells, after the irradiation of viral particles. Following 12' of irradiation at 40 mW/cm2 , a drastic block of viral amplification was observed. Indeed, at 7 days post-irradiation/infection the viral load was the same as the one measured 1 day post-irradiation/infection, and cellular viability was maintained showing similar levels to the noninfected control cells. Taken together our results indicate that blue LED lamps can be considered as a cheap and convenient tool for SARS-CoV-2 disinfection.

A rare loss-of-function genetic mutation suggest a role of dermcidin deficiency in hidradenitis suppurativa pathogenesis.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a multifactorial aetiology that involves a strict interplay between genetic factors, immune dysregulation and lifestyle. Familial forms represent around 40% of total HS cases and show an autosomal dominant mode of inheritance of the disease. In this study, we conducted a whole-exome sequence analysis on an Italian family of 4 members encompassing a vertical transmission of HS. Focusing on rare damaging variants, we identified a rare insertion of one nucleotide (c.225dupA:p.A76Sfs*21) in the DCD gene encoding for the antimicrobial peptide dermcidin (DCD) that was shared by the proband, his affected father and his 11-years old daughter. Since several transcriptome studies have shown a significantly decreased expression of DCD in HS skin, we hypothesised that the identified frameshift insertion was a loss-of-function mutation that might be associated with HS susceptibility in this family. We thus confirmed by mass spectrometry that DCD levels were diminished in the affected members and showed that the antimicrobial activity of a synthetic DCD peptide resulting from the frameshift mutation was impaired. In order to define the consequences related to a decrease in DCD activity, skin microbiome analyses of different body sites were performed by comparing DCD mutant and wild type samples, and results highlighted significant differences between the groins of mutated and wild type groups. Starting from genetic analysis conducted on an HS family, our findings showed, confirming previous transcriptome results, the potential role of the antimicrobial DCD peptide as an actor playing a crucial part in the etio-pathogenesis of HS and in the maintenance of the skin's physiological microbiome composition; so, we can hypothesise that DCD could be used as a novel target for personalised therapeutic approach.

Blue photobiomodulation LED therapy impacts SARS-CoV-2 by limiting its replication in Vero cells.

The study of any intervention able to counteract SARS-CoV-2 pandemic is considerably envisaged. It was previously shown, in in vitro models of infections, that the LED blue light is able to decrease the viral load of HSV-1 and ZIKV. In our study, LED photobiomodulation therapy (PBMT) at blue wavelengths (450, 454 and 470 nm) was tested in an in vitro model of SARS-CoV-2 infection, employing three experimental settings: SARS-CoV-2 was irradiated and then transferred to cells; already infected cells were irradiated; cells were irradiated prior to infection. A decrement of the viral load was observed when previously infected cells were irradiated with all three tested wavelengths and relevant effects were registered especially at 48 hours post-infection, possibly suggesting that the blue light could interfere with the intracellular viral replication machinery. Our in vitro findings could represent the starting point for translational applications of PBMT as a supportive approach to fight SARS-CoV-2.

SARS-CoV-2 Short-Time Infection Produces Relevant Cytopathic Effects in Vero E6 Cell Line.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is mainly transmitted through respiratory droplets from positive subjects to susceptible hosts or by direct contact with an infected individual. Our study focuses on the in vitro minimal time of viral absorption as well as the minimal quantity of virus able to establish a persistent infection in Vero E6 cells. We observed that 1 min of in vitro virus exposure is sufficient to generate a cytopathic effect in cells after 7 days of infection, even at a multiplicity of infection (MOI) value of 0.01. Being aware that our findings have been obtained using an in vitro cellular model, we demonstrated that short-time exposures and low viral concentrations are able to cause infection, thus opening questions about the risk of SARS-CoV-2 transmissibility even following short contact times.

HIV-1 Infection Transcriptomics: Meta-Analysis of CD4+ T Cells Gene Expression Profiles.

HIV-1 infection elicits a complex dynamic of the expression various host genes. High throughput sequencing added an expressive amount of information regarding HIV-1 infections and pathogenesis. RNA sequencing (RNA-Seq) is currently the tool of choice to investigate gene expression in a several range of experimental setting. This study aims at performing a meta-analysis of RNA-Seq expression profiles in samples of HIV-1 infected CD4+ T cells compared to uninfected cells to assess consistently differentially expressed genes in the context of HIV-1 infection. We selected two studies (22 samples: 15 experimentally infected and 7 mock-infected). We found 208 differentially expressed genes in infected cells when compared to uninfected/mock-infected cells. This result had moderate overlap when compared to previous studies of HIV-1 infection transcriptomics, but we identified 64 genes already known to interact with HIV-1 according to the HIV-1 Human Interaction Database. A gene ontology (GO) analysis revealed enrichment of several pathways involved in immune response, cell adhesion, cell migration, inflammation, apoptosis, Wnt, Notch and ERK/MAPK signaling.

In Vitro Zika Virus Infection of Human Neural Progenitor Cells: Meta-Analysis of RNA-Seq Assays.

The Zika virus (ZIKV) is an emergent arthropod-borne virus (arbovirus) responsible for congenital Zika syndrome (CZS) and a range of other congenital malformations. Evidence shows that ZIKV infects human neural progenitor cells (hNPCs) in the fetal brain, prompting inflammation and tissue damage/loss. Despite recent advances, little is known about the pathways involved in CZS pathogenesis. We performed a meta-analysis, gene ontology (GO), and pathway analysis of whole transcriptome studies with the aim of clarifying the genes and pathways potentially altered during hNPCs infection with ZIKV. We selected three studies (17 samples of infected hPNCs compared to hPNCs uninfected controls) through a systematic search of the Gene Expression Omnibus (GEO) database. The raw reads were trimmed, counted, and normalized. Next, we performed a rank product meta-analysis to detect consistently differentially expressed genes (DEGs) in these independent experiments. We detected 13 statistically significant DEGs. GO ontology and reactome analysis showed an enrichment of interferon, pro-inflammatory, and chemokines signaling and apoptosis pathways in ZIKV-infected cells. Moreover, we detected three possible new candidate genes involved in hNPCs infection: APOL6, XAF1, and TNFRSF1. Our results confirm that interferon (IFN) signaling dominates the ZIKV response, and that a crucial contribution is given by apoptotic pathways, which might elicit the CZS phenotype.

Altered keratinization and vitamin D metabolism may be key pathogenetic pathways in syndromic hidradenitis suppurativa: a novel whole exome sequencing approach.

BACKGROUND: Diagnosis of pyoderma gangrenosum, acne and hidradenitis suppurativa (PASH) and pyogenic arthritis, pyoderma gangrenosum, acne, and hidradenitis suppurativa (PAPASH) patients, in spite of recently identified genetic variations, is just clinical, since most patients do not share the same mutations, and the mutations themselves are not informative of the biological pathways commonly disrupted in these patients. OBJECTIVE: To reveal genetic changes more closely related to PASH and PAPASH etiopathogenesis, identifying novel common pathways involved in these diseases. METHODS: Cohort study on PASH (n = 4) and PAPASH (n = 1) patients conducted using whole exome sequencing (WES) approach and a novel bioinformatic pipeline aimed at discovering potentially candidate genes selected from density mutations and involved in pathways relevant to the disease. RESULTS: WES results showed that patients presented 90 genes carrying mutations with deleterious and/or damage impact: 12 genes were in common among the 5 patients and bared 237 ns ExonVar (54 and 183 in homozygosis and heterozygosis, respectively). In the pathway enrichment analysis, only 10 genes were included, allowing us to retrieve 4 pathways shared by all patients: (1) Vitamin D metabolism, (2) keratinization, (3) formation of the cornified envelope and (4) steroid metabolism. Interestingly, all patients had vitamin D levels lower than normal, with a mean value of 10 ng/mL. CONCLUSION: Our findings, through a novel strategy for analysing the genetic background of syndromic HS patients, suggested that vitamin D metabolism dysfunctions seem to be crucial in PASH and PAPASH pathogenesis. Based on low vitamin D serum levels, its supplementation is envisaged.

Prolonged treatment with mevalonolactone induces oxidative stress response with reactive oxygen species production, mitochondrial depolarization and inflammation in human glioblastoma U-87 MG cells.

Mevalonate pathway impairment has been observed in diverse diseases, including Mevalonate Kinase Deficiency (MKD). MKD is a hereditary auto-inflammatory disorder, due to mutations at mevalonate kinase gene (MVK), encoding mevalonate kinase (MK) enzyme. To date, the most accredited MKD pathogenic hypothesis suggests that the typical MKD phenotypes might be due to a decreased isoprenoid production rather than to the excess and accumulation of mevalonic acid, as initially supported. Nevertheless, recent studies provide clear evidences that accumulating metabolites might be involved in MKD pathophysiology by exerting a toxic effect. Our work aims at describing the effects of accumulating mevalonolactone, mostly produced by a dehydration reaction due to mevalonic acid accumulation, using an in vitro cellular model mimicking the glial component of the central nervous system (human glioblastoma U-87 MG cells). In order to mimic its progressive increase, occurring during the disease, U-87 MG cells have been treated repeatedly with growing doses of mevalonolactone, followed by the assessment of oxidative stress response (evaluated by measuring SOD2 and HemeOX expression levels), ROS production, mitochondrial damage and inflammatory response (evaluated by measuring IL1B expression levels). Our results suggest that protracted treatments with mevalonolactone induce oxidative stress with augmented ROS production and mitochondrial damage accompanied by membrane depolarization. Furthermore, an increment in IL1B expression has been observed, thus correlating the accumulation of the metabolite with the development of a neuroinflammatory response. Our experimental work suggests to reconsider the presence of a possible synergy between the two major MKD pathogenic hypotheses in attempt of unravelling the different pathogenic pathways responsible for the disease.

Lopinavir/Ritonavir Treatment Induces Oxidative Stress and Caspaseindependent Apoptosis in Human Glioblastoma U-87 MG Cell Line.

BACKGROUND: Lopinavir and Ritonavir (LPV/r) treatment is widely used to prevent HIV mother-to-child transmission. Nevertheless, studies related to the impact of these compounds on patients, in particular in the foetus and newborns, are strictly required due to the controversial findings reported in the literature concerning possible neurologic side effects following the administration of these drugs. OBJECTIVES: In our study, we evaluated the impact of LPV/r treatment on the human glioblastoma U- 87 MG cell line. METHODS: In order to evaluate the influence of Lopinavir and Ritonavir in terms of oxidative stress (ROS production), mitochondrial morphology and apoptotic cell death, the latter either in the presence or in the absence of caspase-3 and -9 inhibitors, we treated U-87 MG with increasing doses (0.1-1-10-25-50 µM) of Lopinavir and Ritonavir for 24h, either in single formulation or in combination. ROS production was measured by flow cytometry using H2DCFDA dye, mitochondrial morphology was evaluated using MitoRed dye and apoptotic cell death was monitored by flow cytometry using Annexin V-FITC and Propidium Iodide. RESULTS: We observed that co-treatment with Lopinavir and Ritonavir (25 and 50 µM) promoted a significant increase in ROS production, caused mitochondrial network damage and induced apoptosis in a caspase-independent manner. CONCLUSION: Based on our findings, concordant with others reported in the literature, we hypothesize that LPV/r treatment could not be entirely free from side effects, being aware of the need of validation in in vivo models, necessary to confirm our results.