Caspase cleavage of gasdermin E causes neuronal pyroptosis in HIV-associated neurocognitive disorder.

Despite effective antiretroviral therapies, 20-30% of persons with treated HIV infection develop a neurodegenerative syndrome termed HIV-associated neurocognitive disorder (HAND). HAND is driven by HIV expression coupled with inflammation in the brain but the mechanisms underlying neuronal damage and death are uncertain. The inflammasome-pyroptosis axis coordinates an inflammatory type of regulated lytic cell death that is underpinned by the caspase-activated pore-forming gasdermin proteins. The mechanisms driving neuronal pyroptosis were investigated herein in models of HAND, using multi-platform molecular and morphological approaches that included brain tissues from persons with HAND and simian immunodeficiency virus (SIV)-infected non-human primates as well as cultured human neurons. Neurons in the frontal cortices from persons with HAND showed increased cleaved gasdermin E (GSDME), which was associated with ß-III tubulin degradation and increased HIV levels. Exposure of cultured human neurons to the HIV-encoded viral protein R (Vpr) elicited time-dependent cleavage of GSDME and Ninjurin-1 (NINJ1) induction with associated cell lysis that was inhibited by siRNA suppression of both proteins. Upstream of GSDME cleavage, Vpr exposure resulted in activation of caspases-1 and 3. Pretreatment of Vpr-exposed neurons with the caspase-1 inhibitor, VX-765, reduced cleavage of both caspase-3 and GSDME, resulting in diminished cell death. To validate these findings, we examined frontal cortical tissues from SIV-infected macaques, disclosing increased expression of GSDME and NINJ1 in cortical neurons, which was co-localized with caspase-3 detection in animals with neurological disease. Thus, HIV infection of the brain triggers the convergent activation of caspases-1 and -3, which results in GSDME-mediated neuronal pyroptosis in persons with HAND. These findings demonstrate a novel mechanism by which a viral infection causes pyroptotic death in neurons while also offering new diagnostic and therapeutic strategies for HAND and other neurodegenerative disorders.

A low dose of RBD and TLR7/8 agonist displayed on influenza virosome particles protects rhesus macaque against SARS-CoV-2 challenge.

Influenza virosomes serve as antigen delivery vehicles and pre-existing immunity toward influenza improves the immune responses toward antigens. Here, vaccine efficacy was evaluated in non-human primates with a COVID-19 virosome-based vaccine containing a low dose of RBD protein (15 µg) and the adjuvant 3M-052 (1 µg), displayed together on virosomes. Vaccinated animals (n = 6) received two intramuscular administrations at week 0 and 4 and challenged with SARS-CoV-2 at week 8, together with unvaccinated control animals (n = 4). The vaccine was safe and well tolerated and serum RBD IgG antibodies were induced in all animals and in the nasal washes and bronchoalveolar lavages in the three youngest animals. All control animals became strongly sgRNA positive in BAL, while all vaccinated animals were protected, although the oldest vaccinated animal (V1) was transiently weakly positive. The three youngest animals had also no detectable sgRNA in nasal wash and throat. Cross-strain serum neutralizing antibodies toward Wuhan-like, Alpha, Beta, and Delta viruses were observed in animals with the highest serum titers. Pro-inflammatory cytokines IL-8, CXCL-10 and IL-6 were increased in BALs of infected control animals but not in vaccinated animals. Virosomes-RBD/3M-052 prevented severe SARS-CoV-2, as shown by a lower total lung inflammatory pathology score than control animals.

The molecular evolution of spermatogenesis across mammals.

The testis produces gametes through spermatogenesis and evolves rapidly at both the morphological and molecular level in mammals1-6, probably owing to the evolutionary pressure on males to be reproductively successful7. However, the molecular evolution of individual spermatogenic cell types across mammals remains largely uncharacterized. Here we report evolutionary analyses of single-nucleus transcriptome data for testes from 11 species that cover the three main mammalian lineages (eutherians, marsupials and monotremes) and birds (the evolutionary outgroup), and include seven primates. We find that the rapid evolution of the testis was driven by accelerated fixation rates of gene expression changes, amino acid substitutions and new genes in late spermatogenic stages, probably facilitated by reduced pleiotropic constraints, haploid selection and transcriptionally permissive chromatin. We identify temporal expression changes of individual genes across species and conserved expression programs controlling ancestral spermatogenic processes. Genes predominantly expressed in spermatogonia (germ cells fuelling spermatogenesis) and Sertoli (somatic support) cells accumulated on X chromosomes during evolution, presumably owing to male-beneficial selective forces. Further work identified transcriptomal differences between X- and Y-bearing spermatids and uncovered that meiotic sex-chromosome inactivation (MSCI) also occurs in monotremes and hence is common to mammalian sex-chromosome systems. Thus, the mechanism of meiotic silencing of unsynapsed chromatin, which underlies MSCI, is an ancestral mammalian feature. Our study illuminates the molecular evolution of spermatogenesis and associated selective forces, and provides a resource for investigating the biology of the testis across mammals.

Microbial molecule ingress promotes neuroinflammation and brain CCR5 expression in persons with HIV-associated neurocognitive disorders.

BACKGROUND: Systemic inflammation accompanies HIV-1 infection, resulting in microbial translocation from different tissues. We investigated interactions between lentivirus infections, neuroinflammation and microbial molecule presence in the brain. METHODS: Brain tissues from adult humans with (n = 22) and without HIV-1 (n = 11) infection as well as adult nonhuman primates (NHPs) with (n = 11) and without (n = 4) SIVmac251 infection were investigated by RT-PCR/ddPCR, immunofluorescence and western blotting. Studies of viral infectivity, host immune gene expression and viability were performed in primary human neural cells. FINDINGS: Among NHPs, SIV DNA quantitation in brain showed increased levels among animals with SIV encephalitis (n = 5) that was associated with bacterial genomic copy number as well as CCR5 and CASP1 expression in brain. Microbial DnaK and peptidoglycan were immunodetected in brains from uninfected and SIV-infected animals, chiefly in glial cells. Human microglia infected by HIV-1 showed increased p24 production after exposure to peptidoglycan that was associated CCR5 induction. HIV-1 Vpr application to human neurons followed by peptidoglycan exposure resulted in reduced mitochondrial function and diminished beta-III tubulin expression. In human brains, bacterial genome copies (250-550 copies/gm of tissue), were correlated with increased bacterial rRNA and GroEL transcript levels in patients with HIV-associated neurocognitive disorders (HAND). Glial cells displayed microbial GroEL and peptidoglycan immunoreactivity accompanied by CCR5 induction in brains from patients with HAND. INTERPRETATION: Increased microbial genomes and proteins were evident in brain tissues from lentivirus-infected humans and animals and associated with neurological disease. Microbial molecule translocation into the brain might exacerbate neuroinflammatory disease severity and represent a driver of lentivirus-associated brain disease.

Viral biogeography of the mammalian gut and parenchymal organs.

The mammalian virome has been linked to health and disease but our understanding of how it is structured along the longitudinal axis of the mammalian gastrointestinal tract (GIT) and other organs is limited. Here, we report a metagenomic analysis of the prokaryotic and eukaryotic virome occupying luminal and mucosa-associated habitats along the GIT, as well as parenchymal organs (liver, lung and spleen), in two representative mammalian species, the domestic pig and rhesus macaque (six animals per species). Luminal samples from the large intestine of both mammals harboured the highest loads and diversity of bacteriophages (class Caudoviricetes, family Microviridae and others). Mucosal samples contained much lower viral loads but a higher proportion of eukaryotic viruses (families Astroviridae, Caliciviridae, Parvoviridae). Parenchymal organs contained bacteriophages of gut origin, in addition to some eukaryotic viruses. Overall, GIT virome composition was specific to anatomical region and host species. Upper GIT and mucosa-specific viruses were greatly under-represented in distal colon samples (a proxy for faeces). Nonetheless, certain viral and phage species were ubiquitous in all samples from the oral cavity to the distal colon. The dataset and its accompanying methodology may provide an important resource for future work investigating the biogeography of the mammalian gut virome.

Poxvirus MVA Expressing SARS-CoV-2 S Protein Induces Robust Immunity and Protects Rhesus Macaques From SARS-CoV-2.

Novel safe, immunogenic, and effective vaccines are needed to control the COVID-19 pandemic, caused by SARS-CoV-2. Here, we describe the safety, robust immunogenicity, and potent efficacy elicited in rhesus macaques by a modified vaccinia virus Ankara (MVA) vector expressing a full-length SARS-CoV-2 spike (S) protein (MVA-S). MVA-S vaccination was well tolerated and induced S and receptor-binding domain (RBD)-binding IgG antibodies and neutralizing antibodies against SARS-CoV-2 and several variants of concern. S-specific IFNγ, but not IL-4, -producing cells were also elicited. After SARS-CoV-2 challenge, vaccinated animals showed a significant strong reduction of virus loads in bronchoalveolar lavages (BAL) and decreased levels in throat and nasal mucosa. Remarkably, MVA-S also protected macaques from fever and infection-induced cytokine storm. Computed tomography and histological examination of the lungs showed reduced lung pathology in MVA-S-vaccinated animals. These findings favor the use of MVA-S as a potential vaccine for SARS-CoV-2 in clinical trials.

Light-Induced Smooth Endoplasmic Reticulum Rearrangement in a Unique Interlaced Compartmental Pattern in Macaca mulatta RPE.

Purpose: To investigate light-induced modifications of the smooth endoplasmic reticulum of the RPE in primates. Methods: Eyes of three terminally anesthetized Rhesus monkeys were exposed to 5000 lux for 10 minutes or kept in the dark. Transmission electron microscopy and electron tomography were conducted on small fragments of retina sampled from different regions of the retina. Results: RPE cells smooth endoplasmic reticulum shows a previously unknown arrangement characterized by an interlaced compartmental pattern (ICP). Electron tomograms and 3D-modelling demonstrated that the smooth endoplasmic reticulum with an ICP (ICPSER) consisted of four parallel, independent and interwoven networks of tubules arranged as interconnected coiled coils. Its architecture realized a compact labyrinthine structure of tightly packed tubules stabilized by intertubular filamentous tethers. On average, the ICPSER is present in about 14.6% of RPE cells. Although ICPSER was preferentially found in cells located in the peripheral and in the para/perifoveal retina, ICPSER cells significantly increased in number upon light exposure in the para/perifovea and in the fovea. Conclusions: An ICPSER is apparently a unique feature to primate RPE. Its rapid appearance in the area centralis of the retina upon light exposure suggests a function related to the foveate structure of primate retina or to the diurnal habits of animals that may require additional protection from photo-oxidation or enhanced requests of visual pigments regeneration.

Comparable Infection Level and Tropism of Measles Virus and Canine Distemper Virus in Organotypic Brain Slice Cultures Obtained from Natural Host Species.

Measles virus (MV) and canine distemper virus (CDV) are closely related members of the family Paramyxoviridae, genus Morbillivirus. MV infection of humans and non-human primates (NHPs) results in a self-limiting disease, which rarely involves central nervous system (CNS) complications. In contrast, infection of carnivores with CDV usually results in severe disease, in which CNS complications are common and the case-fatality rate is high. To compare the neurovirulence and neurotropism of MV and CDV, we established a short-term organotypic brain slice culture system of the olfactory bulb, hippocampus, or cortex obtained from NHPs, dogs, and ferrets. Slices were inoculated ex vivo with wild-type-based recombinant CDV or MV expressing a fluorescent reporter protein. The infection level of both morbilliviruses was determined at different times post-infection. We observed equivalent infection levels and identified microglia as main target cells in CDV-inoculated carnivore and MV-inoculated NHP brain tissue slices. Neurons were also susceptible to MV infection in NHP brain slice cultures. Our findings suggest that MV and CDV have comparable neurotropism and intrinsic capacity to infect CNS-resident cells of their natural host species.

The Post-Acute Phase of SARS-CoV-2 Infection in Two Macaque Species Is Associated with Signs of Ongoing Virus Replication and Pathology in Pulmonary and Extrapulmonary Tissues.

The post-acute phase of SARS-CoV-2 infection was investigated in rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis). During the acute phase of infection, SARS-CoV-2 was shed via the nose and throat, and viral RNA was occasionally detected in feces. This phase coincided with a transient change in systemic immune activation. Even after the alleged resolution of the infection, computed tomography (CT) and positron emission tomography (PET)-CT revealed pulmonary lesions and activated tracheobronchial lymph nodes in all animals. Post-mortem histological examination of the lung tissue revealed mostly marginal or resolving minimal lesions that were indicative of SARS-CoV-2 infection. Evidence for SARS-CoV-2-induced histopathology was also found in extrapulmonary tissue samples, such as conjunctiva, cervical, and mesenteric lymph nodes. However, 5-6 weeks after SARS-CoV-2 exposure, upon necropsy, viral RNA was still detectable in a wide range of tissue samples in 50% of the macaques and included amongst others the heart, the respiratory tract and surrounding lymph nodes, salivary gland, and conjunctiva. Subgenomic messenger RNA was detected in the lungs and tracheobronchial lymph nodes, indicative of ongoing virus replication during the post-acute phase. These results could be relevant for understanding the long-term consequences of COVID-19 in humans.

Immunogenicity and efficacy of one and two doses of Ad26.COV2.S COVID vaccine in adult and aged NHP.

Safe and effective coronavirus disease-19 (COVID-19) vaccines are urgently needed to control the ongoing pandemic. While single-dose vaccine regimens would provide multiple advantages, two doses may improve the magnitude and durability of immunity and protective efficacy. We assessed one- and two-dose regimens of the Ad26.COV2.S vaccine candidate in adult and aged nonhuman primates (NHPs). A two-dose Ad26.COV2.S regimen induced higher peak binding and neutralizing antibody responses compared with a single dose. In one-dose regimens, neutralizing antibody responses were stable for at least 14 wk, providing an early indication of durability. Ad26.COV2.S induced humoral immunity and T helper cell (Th cell) 1-skewed cellular responses in aged NHPs that were comparable to those in adult animals. Aged Ad26.COV2.S-vaccinated animals challenged 3 mo after dose 1 with a SARS-CoV-2 spike G614 variant showed near complete lower and substantial upper respiratory tract protection for both regimens. Neutralization of variants of concern by NHP sera was reduced for B.1.351 lineages while maintained for the B.1.1.7 lineage independent of Ad26.COV2.S vaccine regimen.

Aerosolized Exposure to H5N1 Influenza Virus Causes Less Severe Disease Than Infection via Combined Intrabronchial, Oral, and Nasal Inoculation in Cynomolgus Macaques.

Infection with highly pathogenic avian H5N1 influenza virus in humans often leads to severe respiratory disease with high mortality. Experimental infection in non-human primates can provide additional insight into disease pathogenesis. However, such a model should recapitulate the disease symptoms observed in humans, such as pneumonia and inflammatory cytokine response. While previous studies in macaques have demonstrated the occurrence of typical lesions in the lungs early after infection and a high level of immune activation, progression to severe disease and lethality were rarely observed. Here, we evaluated a routinely used combined route of infection via intra-bronchial, oral, and intra-nasal virus inoculation with aerosolized H5N1 exposure, with or without the regular collection of bronchoalveolar lavages early after infection. Both combined route and aerosol exposure resulted in similar levels of virus replication in nose and throat and similar levels of immune activation, cytokine, and chemokine release in the blood. However, while animals exposed to H5N1 by combined-route inoculation developed severe disease with high lethality, aerosolized exposure resulted in less lesions, as measured by consecutive computed tomography and less fever and lethal disease. In conclusion, not virus levels or immune activation, but route of infection determines fatal outcome for highly pathogenic avian H5N1 influenza infection.

Recently Evolved Enhancers Emerge with High Interindividual Variability and Less Frequently Associate with Disease.

Mutations in non-coding regulatory DNA such as enhancers underlie a wide variety of diseases including developmental disorders and cancer. As enhancers rapidly evolve, understanding their function and configuration in non-human disease models can have important clinical applications. Here, we analyze enhancer configurations in tissues isolated from the common marmoset, a widely used primate model for human disease. Integrating these data with human and mouse data, we find that enhancers containing trait-associated variants are preferentially conserved. In contrast, most human-specific enhancers are highly variable between individuals, with a subset failing to contact promoters. These are located further away from genes and more often reside in inactive B-compartments. Our data show that enhancers typically emerge as instable elements with minimal biological impact prior to their integration in a transcriptional program. Furthermore, our data provide insight into which trait variations in enhancers can be faithfully modeled using the common marmoset.

Differential DNA methylation of vocal and facial anatomy genes in modern humans.

Changes in potential regulatory elements are thought to be key drivers of phenotypic divergence. However, identifying changes to regulatory elements that underlie human-specific traits has proven very challenging. Here, we use 63 reconstructed and experimentally measured DNA methylation maps of ancient and present-day humans, as well as of six chimpanzees, to detect differentially methylated regions that likely emerged in modern humans after the split from Neanderthals and Denisovans. We show that genes associated with face and vocal tract anatomy went through particularly extensive methylation changes. Specifically, we identify widespread hypermethylation in a network of face- and voice-associated genes (SOX9, ACAN, COL2A1, NFIX and XYLT1). We propose that these repression patterns appeared after the split from Neanderthals and Denisovans, and that they might have played a key role in shaping the modern human face and vocal tract.

Immunohistochemical distribution of 10 GABAA receptor subunits in the forebrain of the rhesus monkey Macaca mulatta.

GABAA receptors are composed of five subunits arranged around a central chloride channel. Their subunits originate from different genes or gene families. The majority of GABAA receptors in the mammalian brain consist of two α-, two ß- and one γ- or δ-subunit. This subunit organization crucially determines the physiological and pharmacological properties of the GABAA receptors. Using immunohistochemistry, we investigated the distribution of 10 GABAA receptor subunits (α1, α2, α3, α4, α5, ß1, ß2, ß3, γ2, and δ) in the fore brain of three female rhesus monkeys (Macaca mulatta). Within the cerebral cortex, subunits α1, α5, ß2, ß3, and γ2 were found in all layers, α2, α3, and ß1 were more concentrated in the inner and outer layers. The caudate/putamen was rich in α1, α2, α5, all three ß-subunits, γ2, and δ. Subunits α3 and α5 were more concentrated in the caudate than in the putamen. In contrast, α1, α2, ß1, ß2, γ2, and δ were highest in the pallidum. Most dorsal thalamic nuclei contained subunits α1, α2, α4, ß2, ß3, and γ2, whereas α1, α3, ß1, and γ2 were most abundant in the reticular nucleus. Within the amygdala, subunits α1, α2, α5, ß1, ß3, γ2, and δ were concentrated in the cortical nucleus, whereas in the lateral and basolateral amygdala α1, α2, α5, ß1, ß3, and δ, and in the central amygdala α1, α2, ß3, and γ2 were most abundant. Interestingly, subunit α3-IR outlined the intercalated nuclei of the amygdala. In the hippocampus, subunits α1, α2, α5, ß2, ß3, γ2, and δ were highly expressed in the dentate molecular layer, whereas α1, α2, α3, α5, ß1, ß2, ß3, and γ2 were concentrated in sector CA1 and the subiculum. The distribution of GABAA receptor subunits in the rhesus monkey was highly heterogeneous indicating a high number of differently assembled receptors. In most areas investigated, notably in the striatum/pallidum, amygdaloid nuclei and in the hippocampus it was more diverse than in the rat and mouse indicating a more heterogeneous and less defined receptor assembly in the monkey than in rodent brain.

Hominin-specific regulatory elements selectively emerged in oligodendrocytes and are disrupted in autism patients.

Speciation is associated with substantial rewiring of the regulatory circuitry underlying the expression of genes. Determining which changes are relevant and underlie the emergence of the human brain or its unique susceptibility to neural disease has been challenging. Here we annotate changes to gene regulatory elements (GREs) at cell type resolution in the brains of multiple primate species spanning most of primate evolution. We identify a unique set of regulatory elements that emerged in hominins prior to the separation of humans and chimpanzees. We demonstrate that these hominin gains perferentially affect oligodendrocyte function postnatally and are preferentially affected in the brains of autism patients. This preference is also observed for human-specific GREs suggesting this system is under continued selective pressure. Our data provide a roadmap of regulatory rewiring across primate evolution providing insight into the genomic changes that underlie the emergence of the brain and its susceptibility to neural disease.

Prevention of tuberculosis infection and disease by local BCG in repeatedly exposed rhesus macaques.

Tuberculosis (TB) remains the deadliest infectious disease1, and the widely used Bacillus Calmette-Guérin (BCG) vaccine fails to curb the epidemic. An improved vaccination strategy could provide a cost-effective intervention to break the transmission cycle and prevent antimicrobial resistance2,3. Limited knowledge of the host responses critically involved in protective immunity hampers the development of improved TB vaccination regimens. Therefore, assessment of new strategies in preclinical models to select the best candidate vaccines before clinical vaccine testing remains indispensable. We have previously established in rhesus macaques (Macaca mulatta) that pulmonary mucosal BCG delivery reduces TB disease where standard intradermal injection fails4,5. Here, we show that pulmonary BCG prevents infection by using a repeated limiting-dose Mycobacterium tuberculosis challenge model and identify polyfunctional T-helper type 17 (TH17) cells, interleukin-10 and immunoglobulin A as correlates of local protective immunity. These findings warrant further research into mucosal immunization strategies and their translation to clinical application to more effectively prevent the spread of TB.

Gene expression variability across cells and species shapes innate immunity.

As the first line of defence against pathogens, cells mount an innate immune response, which varies widely from cell to cell. The response must be potent but carefully controlled to avoid self-damage. How these constraints have shaped the evolution of innate immunity remains poorly understood. Here we characterize the innate immune response's transcriptional divergence between species and variability in expression among cells. Using bulk and single-cell transcriptomics in fibroblasts and mononuclear phagocytes from different species, challenged with immune stimuli, we map the architecture of the innate immune response. Transcriptionally diverging genes, including those that encode cytokines and chemokines, vary across cells and have distinct promoter structures. Conversely, genes that are involved in the regulation of this response, such as those that encode transcription factors and kinases, are conserved between species and display low cell-to-cell variability in expression. We suggest that this expression pattern, which is observed across species and conditions, has evolved as a mechanism for fine-tuned regulation to achieve an effective but balanced response.

Cell Type and Species-specific Patterns in Neuronal and Non-neuronal Methylomes of Human and Chimpanzee Cortices.

Epigenetic changes have likely contributed to the large size and enhanced cognitive abilities of the human brain which evolved within the last 2 million years after the human-chimpanzee split. Using reduced representation bisulfite sequencing, we have compared the methylomes of neuronal and non-neuronal cells from 3 human and 3 chimpanzee cortices. Differentially methylated regions (DMRs) with genome-wide significance were enriched in specific genomic regions. Intraspecific methylation differences between neuronal and non-neuronal cells were approximately 3 times more abundant than interspecific methylation differences between human and chimpanzee cell types. The vast majority (>90%) of human intraspecific DMRs (including DMRs in retrotransposons) were hypomethylated in neurons, compared with glia. Intraspecific DMRs were enriched in genes associated with different neuropsychiatric disorders. Interspecific DMRs were enriched in genes showing human-specific brain histone modifications. Human-chimpanzee methylation differences were much more frequent in non-neuronal cells (n. DMRs = 666) than in neurons (n. DMRs = 96). More than 95% of interspecific DMRs in glia were hypermethylated in humans. Although without an outgroup we cannot assign whether a change in methylation occurred in the human or chimpanzee lineage, our results are consistent with a wave of methylation affecting several hundred non-neuronal genes during human brain evolution.

The C-Type Lectin Receptor DC-SIGN Has an Anti-Inflammatory Role in Human M(IL-4) Macrophages in Response to Mycobacterium tuberculosis.

DC-SIGN (CD209/CLEC4L) is a C-type lectin receptor (CLR) that serves as a reliable cell-surface marker of interleukin 4 (IL-4)-activated human macrophages [M(IL-4)], which historically represent the most studied subset within the M2 spectrum of macrophage activation. Although DC-SIGN plays important roles in Mycobacterium tuberculosis (Mtb) interactions with dendritic cells, its contribution to the Mtb-macrophage interaction remains poorly understood. Since high levels of IL-4 are correlated with tuberculosis (TB) susceptibility and progression, we investigated the role of DC-SIGN in M(IL-4) macrophages in the TB context. First, we demonstrate that DC-SIGN expression is present both in CD68+ macrophages found in tuberculous pulmonary lesions of non-human primates, and in the CD14+ cell population isolated from pleural effusions obtained from TB patients (TB-PE). Likewise, we show that DC-SIGN expression is accentuated in M(IL-4) macrophages derived from peripheral blood CD14+ monocytes isolated from TB patients, or in macrophages stimulated with acellular TB-PE, arguing for the pertinence of DC-SIGN-expressing macrophages in TB. Second, using a siRNA-mediated gene silencing approach, we performed a transcriptomic analysis of DC-SIGN-depleted M(IL-4) macrophages and revealed the upregulation of pro-inflammatory signals in response to challenge with Mtb, as compared to control cells. This pro-inflammatory gene signature was confirmed by RT-qPCR, cytokine/chemokine-based protein array, and ELISA analyses. We also found that inactivation of DC-SIGN renders M(IL-4) macrophages less permissive to Mtb intracellular growth compared to control cells, despite the equal level of bacteria uptake. Last, at the molecular level, we show that DC-SIGN interferes negatively with the pro-inflammatory response and control of Mtb intracellular growth mediated by another CLR, Dectin-1 (CLEC7A). Collectively, this study highlights a dual role for DC-SIGN as, on the one hand, being a host factor granting advantage for Mtb to parasitize macrophages and, on the other hand, representing a molecular switch to turn off the pro-inflammatory response in these cells to prevent potential immunopathology associated to TB.

RNA editing independently occurs at three mir-376a-1 sites and may compromise the stability of the microRNA hairpin.

RNA editing is being recognized as an important post-transcriptional mechanism that may have crucial roles in introducing genetic variation and phenotypic diversity. Despite microRNA editing recurrence, defining its biological relevance is still under extended debate. To better understand microRNA editing function and regulation we performed an exhaustive characterization of the A-to-I site-specific patterns in mir-376a-1, a mammalian microRNA which RNA editing is involved in the regulation of development and in disease. Thorough an integrative approach based on high-throughput small RNA sequencing, Sanger sequencing and computer simulations we explored mir-376a-1 editing in samples from various individuals and primate species including human placenta and macaque, gorilla, chimpanzee and human brain cortex. We observed that mir-376a-1 editing is a common phenomenon in the mature and primary microRNA molecules and it is more frequently detected in brain than in placenta. Primary mir-376a-1 is edited at three positions, -1, +4 and +44. Editing frequency estimations and in silico simulations indicated that editing was not equally recurrent along the three mir-376a-1 sites, nevertheless no epistatic interactions among them were observed. Particularly, the +4 site, located in the seed region of the mature miR-376a-5p, reached the highest editing frequency in all samples. Secondary structure predictions revealed that the +4 position was the one that conferred the highest stability to the mir-376a-1 hairpin. We suggest that molecular stability might partially explain the editing recurrence observed in certain microRNAs and that editing events conferring new functional regulatory roles in particular tissues and species could have been conserved along evolution, as it might be the case of mir-376a-1 in primate brain cortex.