Vaccination with an HIV T-Cell Immunogen (HTI) Using DNA Primes Followed by a ChAdOx1-MVA Boost Is Immunogenic in Gut Microbiota-Depleted Mice despite Low IL-22 Serum Levels.

Despite the important role of gut microbiota in the maturation of the immune system, little is known about its impact on the development of T-cell responses to vaccination. Here, we immunized C57BL/6 mice with a prime-boost regimen using DNA plasmid, the Chimpanzee Adenovirus, and the modified Vaccinia Ankara virus expressing a candidate HIV T-cell immunogen and compared the T-cell responses between individuals with an intact or antibiotic-depleted microbiota. Overall, the depletion of the gut microbiota did not result in significant differences in the magnitude or breadth of the immunogen-specific IFNγ T-cell response after vaccination. However, we observed marked changes in the serum levels of four cytokines after vaccinating microbiota-depleted animals, particularly a significant reduction in IL-22 levels. Interestingly, the level of IL-22 in serum correlated with the abundance of Roseburia in the large intestine of mice in the mock and vaccinated groups with intact microbiota. This short-chain fatty acid (SCFA)-producing bacterium was significantly reduced in the vaccinated, microbiota-depleted group. Therefore, our results indicate that, although microbiota depletion reduces serum levels of IL-22, the powerful vaccine regime used could have overcome the impact of microbiota depletion on IFNγ-producing T-cell responses.

Vaccination with an HIV T-cell immunogen induces alterations in the mouse gut microbiota.

The gut microbiota is emerging as a crucial factor modulating vaccine responses; however, few studies have investigated if vaccines, in turn, can alter the microbiota and to what extent such changes may improve vaccine efficacy. To understand the effect of T-cell vaccination on the gut microbiome, we administered an HIV-1 T-cell immunogen (HTI arm) or PBS (control, Mock arm) to C57Bl/6 mice following a heterologous prime-boost scheme. The longitudinal dynamics of the mice gut microbiota was characterized by 16 S ribosomal RNA sequencing in fecal samples collected from cages, as well as from three gut sections (cecum, small and large intestine). Serum and spleen cells were obtained at the last time point of the study to assess immune correlates using IFNγ ELISPOT and cytokine Luminex® assays. Compared with Mock, HTI-vaccinated mice were enriched in Clostridiales genera (Eubacterium xylanophilum group, Roseburia and Ruminococcus) known as primary contributors of anti-inflammatory metabolites, such as short-chain fatty acids. Such shift was observed after the first HTI dose and remained throughout the study follow-up (18 weeks). However, the enriched Clostridiales genera were different between feces and gut sections. The abundance of bacteria enriched in vaccinated animals positively correlated with HTI-specific T-cell responses and a set of pro-inflammatory cytokines, such as IL-6. This longitudinal analysis indicates that, in mice, T-cell vaccination may promote an increase in gut bacteria known to produce anti-inflammatory molecules, which in turn correlate with proinflammatory cytokines, suggesting an adaptation of the gut microbial milieu to T-cell-induced systemic inflammation.

Gut microbiome signatures linked to HIV-1 reservoir size and viremia control.

BACKGROUND: The potential role of the gut microbiome as a predictor of immune-mediated HIV-1 control in the absence of antiretroviral therapy (ART) is still unknown. In the BCN02 clinical trial, which combined the MVA.HIVconsv immunogen with the latency-reversing agent romidepsin in early-ART treated HIV-1 infected individuals, 23% (3/13) of participants showed sustained low-levels of plasma viremia during 32 weeks of a monitored ART pause (MAP). Here, we present a multi-omics analysis to identify compositional and functional gut microbiome patterns associated with HIV-1 control in the BCN02 trial. RESULTS: Viremic controllers during the MAP (controllers) exhibited higher Bacteroidales/Clostridiales ratio and lower microbial gene richness before vaccination and throughout the study intervention when compared to non-controllers. Longitudinal assessment indicated that the gut microbiome of controllers was enriched in pro-inflammatory bacteria and depleted in butyrate-producing bacteria and methanogenic archaea. Functional profiling also showed that metabolic pathways related to fatty acid and lipid biosynthesis were significantly increased in controllers. Fecal metaproteome analyses confirmed that baseline functional differences were mainly driven by Clostridiales. Participants with high baseline Bacteroidales/Clostridiales ratio had increased pre-existing immune activation-related transcripts. The Bacteroidales/Clostridiales ratio as well as host immune-activation signatures inversely correlated with HIV-1 reservoir size. CONCLUSIONS: The present proof-of-concept study suggests the Bacteroidales/Clostridiales ratio as a novel gut microbiome signature associated with HIV-1 reservoir size and immune-mediated viral control after ART interruption. Video abstract.

Performance of 16S Metagenomic Profiling in Formalin-Fixed Paraffin-Embedded versus Fresh-Frozen Colorectal Cancer Tissues.

Formalin-fixed, paraffin-embedded (FFPE) tissues represent the most widely available clinical material to study colorectal cancer (CRC). However, the accuracy and clinical validity of FFPE microbiome profiling in CRC is uncertain. Here, we compared the microbial composition of 10 paired fresh-frozen (FF) and FFPE CRC tissues using 16S rRNA sequencing and RNA-ISH. Both sample types showed different microbial diversity and composition. FF samples were enriched in archaea and representative CRC-associated bacteria, such as Firmicutes, Bacteroidetes and Fusobacteria. Conversely, FFPE samples were mainly enriched in typical contaminants, such as Sphingomonadales and Rhodobacterales. RNA-ISH in FFPE tissues confirmed the presence of CRC-associated bacteria, such as Fusobacterium and Bacteroides, as well as Propionibacterium allowing discrimination between tumor-associated and contaminant taxa. An internal quality index showed that the degree of similarity within sample pairs inversely correlated with the dominance of contaminant taxa. Given the importance of FFPE specimens for larger studies in human cancer genomics, our findings may provide useful indications on potential confounding factors to consider for accurate and reproducible metagenomics analyses.

Long-lasting blood pressure lowering effects of nitrite are NO-independent and mediated by hydrogen peroxide, persulfides, and oxidation of protein kinase G1α redox signalling.

AIMS: Under hypoxic conditions, nitrite (NO2-) can be reduced to nitric oxide (NO) eliciting vasorelaxation. However, nitrite also exerts vasorelaxant effects of potential therapeutic relevance under normal physiological conditions via undetermined mechanisms. We, therefore, sought to investigate the mechanism(s) by which nitrite regulates the vascular system in normoxia and, specifically, whether the biological effects are a result of NO generation (as in hypoxia) or mediated via alternative mechanisms involving classical downstream targets of NO [e.g. effects on protein kinase G1α (PKG1α)]. METHODS AND RESULTS: Ex vivo myography revealed that, unlike in thoracic aorta (conduit vessels), the vasorelaxant effects of nitrite in mesenteric resistance vessels from wild-type (WT) mice were NO-independent. Oxidants such as H2O2 promote disulfide formation of PKG1α, resulting in NO- cyclic guanosine monophosphate (cGMP) independent kinase activation. To explore whether the microvascular effects of nitrite were associated with PKG1α oxidation, we used a Cys42Ser PKG1α knock-in (C42S PKG1α KI; 'redox-dead') mouse that cannot transduce oxidant signals. Resistance vessels from these C42S PKG1α KI mice were markedly less responsive to nitrite-induced vasodilation. Intraperitoneal (i.p.) bolus application of nitrite in conscious WT mice induced a rapid yet transient increase in plasma nitrite and cGMP concentrations followed by prolonged hypotensive effects, as assessed using in vivo telemetry. In the C42S PKG1α KI mice, the blood pressure lowering effects of nitrite were lower compared to WT. Increased H2O2 concentrations were detected in WT resistance vessel tissue challenged with nitrite. Consistent with this, increased cysteine and glutathione persulfide levels were detected in these vessels by mass spectrometry, matching the temporal profile of nitrite's effects on H2O2 and blood pressure. CONCLUSION: Under physiological conditions, nitrite induces a delayed and long-lasting blood pressure lowering effect, which is NO-independent and occurs via a new redox mechanism involving H2O2, persulfides, and PKG1α oxidation/activation. Targeting this novel pathway may provide new prospects for anti-hypertensive therapy.

Distribution, Characteristics, and Regulatory Potential of Long Noncoding RNAs in Brown-Rot Fungi.

Long noncoding RNAs have been thoroughly studied in plants, animals, and yeasts, where they play important roles as regulators of transcription. Nevertheless, almost nothing is known about their presence and characteristics in filamentous fungi, especially in basidiomycetes. In the present study, we have carried out an exhaustive annotation and characterization of lncRNAs in two lignin degrader basidiomycetes, Coniophora puteana and Serpula lacrymans. We identified 2,712 putative lncRNAs in the former and 2,242 in the latter, mainly originating from intergenic locations of transposon-sparse genomic regions. The lncRNA length, GC content, expression levels, and stability of the secondary structure differ from coding transcripts but are similar in these two species and resemble that of other eukaryotes. Nevertheless, they lack sequence conservation. Also, we found that lncRNAs are transcriptionally regulated in the same proportion as genes when the fungus actively decomposes soil organic matter. Finally, up to 7% of the upstream gene regions of Coniophora puteana and Serpula lacrymans are transcribed and produce lncRNAs. The study of expression trends in these gene-lncRNA pairs uncovered groups with similar and opposite transcriptional profiles which may be the result of cis-transcriptional regulation.

Transposon-associated epigenetic silencing during Pleurotus ostreatus life cycle.

Transposable elements constitute an important fraction of eukaryotic genomes. Given their mutagenic potential, host-genomes have evolved epigenetic defense mechanisms to limit their expansion. In fungi, epigenetic modifications have been widely studied in ascomycetes, although we lack a global picture of the epigenetic landscape in basidiomycetes. In this study, we analysed the genome-wide epigenetic and transcriptional patterns of the white-rot basidiomycete Pleurotus ostreatus throughout its life cycle. Our results performed by using high-throughput sequencing analyses revealed that strain-specific DNA methylation profiles are primarily involved in the repression of transposon activity and suggest that 21 nt small RNAs play a key role in transposon silencing. Furthermore, we provide evidence that transposon-associated DNA methylation, but not sRNA production, is directly involved in the silencing of genes surrounded by transposons. Remarkably, we found that nucleus-specific methylation levels varied in dikaryotic strains sharing identical genetic complement but different subculture conditions. Finally, we identified key genes activated in the fruiting process through the comparative analysis of transcriptomes. This study provides an integrated picture of epigenetic defense mechanisms leading to the transcriptional silencing of transposons and surrounding genes in basidiomycetes. Moreover, our findings suggest that transcriptional but not methylation reprogramming triggers fruitbody development in P. ostreatus.

Nitrite circumvents platelet resistance to nitric oxide in patients with heart failure preserved ejection fraction and chronic atrial fibrillation.

Aims: Heart failure (HF) is a pro-thrombotic state. Both platelet and vascular responses to nitric oxide (NO) donors are impaired in HF patients with reduced ejection fraction (HFrEF) compared with healthy volunteers (HVs) due to scavenging of NO, and possibly also reduced activity of the principal NO sensor, soluble guanylate cyclase (sGC), limiting the therapeutic potential of NO donors as anti-aggregatory agents. Previous studies have shown that nitrite inhibits platelet activation presumptively after its reduction to NO, but the mechanism(s) involved remain poorly characterized. Our aim was to compare the effects of nitrite on platelet function in HV vs. HF patients with preserved ejection fraction (HFpEF) and chronic atrial fibrillation (HFpEF-AF), vs. patients with chronic AF without HF, and to assess whether these effects occur independent of the interaction with other formed elements of blood. Methods and results: Platelet responses to nitrite and the NO donor sodium nitroprusside (SNP) were compared in age-matched HV controls (n = 12), HFpEF-AF patients (n = 29), and chronic AF patients (n = 8). Anti-aggregatory effects of nitrite in the presence of NO scavengers/sGC inhibitor were determined and vasodilator-stimulated phosphoprotein (VASP) phosphorylation was assessed using western blotting. In HV and chronic AF, both nitrite and SNP inhibited platelet aggregation in a concentration-dependent manner. Inhibition of platelet aggregation by the NO donor SNP was impaired in HFpEF-AF patients compared with healthy and chronic AF individuals, but there was no impairment of the anti-aggregatory effects of nitrite. Nitrite circumvented platelet NO resistance independently of other blood cells by directly activating sGC and phosphorylating VASP. Conclusion: We here show for the first time that HFpEF-AF (but not chronic AF without HF) is associated with marked impairment of platelet NO responses due to sGC dysfunction and nitrite circumvents the 'platelet NO resistance' phenomenon in human HFpEF, at least partly, by acting as a direct sGC activator independent of NO.

Somatic transposition and meiotically driven elimination of an active helitron family in Pleurotus ostreatus.

Helitrons constitute a superfamily of DNA transposons that were discovered in silico and are widespread in most eukaryotic genomes. They are postulated to mobilize through a "rolling-circle" mechanism, but the experimental evidence of their transposition has been described only recently. Here, we present the inheritance patterns of HELPO1 and HELPO2 helitron families in meiotically derived progeny of the basidiomycete Pleurotus ostreatus. We found distorted segregation patterns of HELPO2 helitrons that led to a strong under-representation of these elements in the progeny. Further investigation of HELPO2 flanking sites showed that gene conversion may contribute to the elimination of such repetitive elements in meiosis, favouring the presence of HELPO2 vacant loci. In addition, the analysis of HELPO2 content in a reconstructed pedigree of subclones maintained under different culture conditions revealed an event of helitron somatic transposition. Additional analyses of genome and transcriptome data indicated that P. ostreatus carries active RNAi machinery that could be involved in the control of transposable element proliferation. Our results provide the first evidence of helitron mobilization in the fungal kingdom and highlight the interaction between genome defence mechanisms and invasive DNA.

Biology, dynamics, and applications of transposable elements in basidiomycete fungi.

The phylum Basidiomycota includes filamentous fungi and yeast species with different ecological and genomic characteristics. Transposable elements (TEs) are abundant components of most eukaryotic genomes, and their transition from being genomic parasites to key drivers of genomic architecture, functionality, and evolution is a subject receiving much attention. In light of the abundant genomic information released during the last decade, the aims of this mini-review are to discuss the dynamics and impact of TEs in basidiomycete fungi. To do this, we surveyed and explored data from 75 genomes, which encompass the phylogenetic diversity of the phylum Basidiomycota. We describe annotation approaches and analyze TE distribution in the context of species phylogeny and genome size. Further, we review the most relevant literature about the role of TEs in species lifestyle, their impact on genome architecture and functionality, and the defense mechanisms evolved to control their proliferation. Finally, we discuss potential applications of TEs that can drive future innovations in fungal research.

Transposable Elements versus the Fungal Genome: Impact on Whole-Genome Architecture and Transcriptional Profiles.

Transposable elements (TEs) are exceptional contributors to eukaryotic genome diversity. Their ubiquitous presence impacts the genomes of nearly all species and mediates genome evolution by causing mutations and chromosomal rearrangements and by modulating gene expression. We performed an exhaustive analysis of the TE content in 18 fungal genomes, including strains of the same species and species of the same genera. Our results depicted a scenario of exceptional variability, with species having 0.02 to 29.8% of their genome consisting of transposable elements. A detailed analysis performed on two strains of Pleurotus ostreatus uncovered a genome that is populated mainly by Class I elements, especially LTR-retrotransposons amplified in recent bursts from 0 to 2 million years (My) ago. The preferential accumulation of TEs in clusters led to the presence of genomic regions that lacked intra- and inter-specific conservation. In addition, we investigated the effect of TE insertions on the expression of their nearby upstream and downstream genes. Our results showed that an important number of genes under TE influence are significantly repressed, with stronger repression when genes are localized within transposon clusters. Our transcriptional analysis performed in four additional fungal models revealed that this TE-mediated silencing was present only in species with active cytosine methylation machinery. We hypothesize that this phenomenon is related to epigenetic defense mechanisms that are aimed to suppress TE expression and control their proliferation.

Role of aldehyde dehydrogenase in hypoxic vasodilator effects of nitrite in rats and humans.

BACKGROUND AND PURPOSE: Hypoxic conditions favour the reduction of nitrite to nitric oxide (NO) to elicit vasodilatation, but the mechanism(s) responsible for bioconversion remains ill defined. In the present study, we assess the role of aldehyde dehydrogenase 2 (ALDH2) in nitrite bioactivation under normoxia and hypoxia in the rat and human vasculature. EXPERIMENTAL APPROACH: The role of ALDH2 in vascular responses to nitrite was studied using rat thoracic aorta and gluteal subcutaneous fat resistance vessels from patients with heart failure (HF; 16 patients) in vitro and by measurement of changes in forearm blood flow (FBF) during intra-arterial nitrite infusion (21 patients) in vivo. Specifically, we investigated the effects of (i) ALDH2 inhibition by cyanamide or propionaldehyde and the (ii) tolerance-independent inactivation of ALDH2 by glyceryl trinitrate (GTN) on the vasodilator activity of nitrite. In each setting, nitrite effects were measured via evaluation of the concentration-response relationship under normoxic and hypoxic conditions in the absence or presence of ALDH2 inhibitors. KEY RESULTS: Both in rat aorta and human resistance vessels, dilatation to nitrite was diminished following ALDH2 inhibition, in particular under hypoxia. In humans there was a non-significant trend towards attenuation of nitrite-mediated increases in FBF. CONCLUSIONS AND IMPLICATIONS: In human and rat vascular tissue in vitro, hypoxic nitrite-mediated vasodilatation involves ALDH2. In patients with HF in vivo, the role of this enzyme in nitrite bioactivation is at the most, modest, suggesting the involvement of other more important mechanisms.

Aspirin influences megakaryocytic gene expression leading to up-regulation of multidrug resistance protein-4 in human platelets.

AIM: The aim of the study was to investigate whether human megakaryocytic cells have an adaptive response to aspirin treatment, leading to an enhancement of multidrug resistance protein-4 (MRP4) expression in circulating platelets responsible for a reduced aspirin action. We recently found that platelet MRP4 overexpression has a role in reducing aspirin action in patients after by-pass surgery. Aspirin enhances MRP4-mRNA levels in rat liver and drug administration transcriptionally regulates MRP4 gene expression through peroxisome proliferator-activated receptor-α (PPARα). METHODS: The effects induced by aspirin or PPARα agonist (WY14643) on MRP4 modulation were evaluated in vitro in a human megakaryoblastic DAMI cell line, in megakaryocytes (MKs) and in platelets obtained from human haematopoietic progenitor cell (HPC) cultures, and in vivo platelets obtained from aspirin treated healthy volunteers (HV). RESULTS: In DAMI cells, aspirin and WY14643 treatment induced a significant increase in MRP4 and PPARα expression. In human MKs grown in the presence of either aspirin or WY14643, MRP4 and PPARα-mRNA were higher than in control cultures and derived platelets showed an enhancement in MRP4 protein expression. The ability of aspirin to modulate MRP4 expression in MKs and to transfer it to platelets was also confirmed in vivo. In fact, we found the highest MRP4 mRNA and protein expression in platelets obtained from HV after 15 days' aspirin treatment. CONCLUSIONS: The present study provides evidence, for the first time, that aspirin treatment affects the platelet protein pattern through MK genomic modulation. This work represents an innovative and attractive approach, useful both to identify patients less sensitive to aspirin and to improve pharmacological treatment in cardiovascular high-risk patients.

Natriuretic peptides induce weak VASP phosphorylation at Serine 239 in platelets.

Cyclic guanosine-3',5'-monophoshate (cGMP) is the common second messenger for the cardiovascular effects of nitric oxide (NO) and natriuretic peptides (NP; e.g. atrial NP [ANP]), which activate soluble and particulate guanylyl cyclases, respectively. The role of NO in regulating cGMP and platelet function is well documented, whereas there is little evidence supporting a role for NPs in regulating platelet reactivity. By studying platelet aggregation and secretion in response to a PAR-1 peptide, collagen and ADP, and phosphorylation of the cGMP-dependent protein kinase (PKG) substrate vasodilator-stimulated phosphoprotein (VASP) at serine 239, we evaluated the effects of NPs in the absence or presence of the non-selective cGMP and cAMP phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Our results show that NPs, possibly through the clearance receptor (natriuretic peptide receptor-C) expressed on platelet membranes, increase VASP phosphorylation but only following PDE inhibition, indicating a small, localised cGMP synthesis. As platelet aggregation and secretion measured under the same conditions were not affected, we conclude that the magnitude of PKG activation achieved by NPs in platelets per se is not sufficient to exert functional inhibition of platelet involvement in haemostasis.

Tyrosine phosphorylation on spleen tyrosine kinase (Syk) is differentially regulated in human and murine platelets by protein kinase C isoforms.

Protein kinase C (PKC) isoforms differentially regulate platelet functional responses downstream of glycoprotein VI (GPVI) signaling, but the role of PKCs regulating upstream effectors such as Syk is not known. We investigated the role of PKC on Syk tyrosine phosphorylation using the pan-PKC inhibitor GF109203X (GFX). GPVI-mediated phosphorylation on Syk Tyr-323, Tyr-352, and Tyr-525/526 was rapidly dephosphorylated, but GFX treatment inhibited this dephosphorylation on Tyr-525/526 in human platelets but not in wild type murine platelets. GFX treatment did not affect tyrosine phosphorylation on FcRγ chain or Src family kinases. Phosphorylation of Lat Tyr-191 and PLCγ2 Tyr-759 was also increased upon treatment with GFX. We evaluated whether secreted ADP is required for such dephosphorylation. Exogenous addition of ADP to GFX-treated platelets did not affect tyrosine phosphorylation on Syk. FcγRIIA- or CLEC-2-mediated Syk tyrosine phosphorylation was also potentiated with GFX in human platelets. Because potentiation of Syk phosphorylation is not observed in murine platelets, PKC-deficient mice cannot be used to identify the PKC isoform regulating Syk phosphorylation. We therefore used selective inhibitors of PKC isoforms. Only PKCß inhibition resulted in Syk hyperphosphorylation similar to that in platelets treated with GFX. This result indicates that PKCß is the isoform responsible for Syk negative regulation in human platelets. In conclusion, we have elucidated a novel pathway of Syk regulation by PKCß in human platelets.

Reduction of cAMP and cGMP inhibitory effects in human platelets by MRP4-mediated transport.

Cyclic nucleotide-dependent inhibition of platelets represents the most important physiological way to limit thrombus formation. cAMP and cGMP increase in platelets as a consequence of prostacyclin and nitric oxide production by endothelial cells and act through PKA and PKG, respectively. The cytosolic concentration of cyclic nucleotides in platelets is regulated by AC- and GC-dependent synthesis and PDE-dependent degradation. In some cells cyclic nucleotides are eliminated also through MRP4/5/8-dependent efflux. As only MRP4 is expressed in platelets, at high levels in dense granules, we determined its role in the elimination of cyclic nucleotides from platelet cytosol. We studied the effects of MRP4 inhibition on cAMP/cGMP effects in platelets. Cyclic nucleotide inhibitory effects triggered by cAMP and cGMP-elevating agents on platelet aggregation are strongly enhanced by MRP4 inhibition and so is cyclic nucleotide-dependent phosphorylation of the common substrate VASP. MRP4 inhibition decreases cAMP concentration in platelet granules and both cAMP and cGMP compete with an established substrate of MRP4 (fluo-cAMP) for entrance in granules. Here we provide the first evidence of the transport of cyclic nucleotides mediated by MRP4 as part of their physiological mechanism of elimination in human platelets, which might represent a novel target to increase cyclic nucleotide-dependent inhibition.