Biology subjects

Commere, P.-H.

Publications and source records attributed to Commere, P.-H..

15 recordsLinked to original sources

bioRxiv2025

Purification protocol of hypertrophied hepatic stellate cells for their transcriptomic characterization from CDAHFD mice liver

Hepatic stellate cells (HSC) are known for their major role in hepatic fibrosis. Recently, it has been shown that different subpopulations of HSC co-exist during fibrogenesis and play different roles in the establishment of fibrosis. We previously highlighted, in murine model and human biopsies, a specific subpopulation of hypertrophied HSC (hypHSC) which exhibit exacerbated retinoid droplets and were closely associated to collagen fibers. The present study describes the purification protocol of hypHSC developed from a murine model of metabolic liver fibrosis. Liver dissociation followed by density gradient and fluorescence assisted cell sorting allowed us to obtain higlhly pure hypHSC preparations. Then, a transcriptomic analysis (bulk RNAseq) of hypHSCs versus quiescent HSCs purified from healthy mouse liver, was performed. This showed that hypHSCs molecular signature differs from HSC subtypes already described in the literature, with a "hybrid" profile of both inflammation and matrix remodeling. Our study highlights that a phenotype-to-molecular approach can provide complementary elements to single-cell molecular approaches.

cell biology↗

bioRxiv2025

Extracellular vesicle production and membrane uptake promote repair and antibiotic tolerance in E. coli

Bacterial extracellular vesicles (EVs) are nanosized lipid structures released in response to environmental stressors, such as phages and antibiotics. Despite their critical role in bacterial adaptability, the mechanisms by which EVs interact with membranes under stress remain poorly understood, due to challenges in visualizing these dynamic processes in live bacteria. Here, we use high-resolution fluorescence microscopy, flow cytometry, and cryo-electron microscopy to investigate EV production and uptake in Escherichia coli exposed to sub-minimum inhibitory concentration doses of polymyxin B (Pmb), a membrane-active antimicrobial peptide. Using fluorescently labeled Pmb and EVs, we track Pmb insertion and removal from membranes, EV production and uptake, and their effects on cell growth. Our findings demonstrate that EV production rapidly sequesters Pmb in the medium and facilitates its removal from bacterial membranes. For the first time, we demonstrated that EVs act as membrane plugs by adhering to or fusing with Pmb-damaged membranes. These dynamic processes work together to reduce the antibiotic load from the membranes, turn off the RcsA-mediated membrane stress response, and enable cells to resume growth. Although EVs do not provide resistance to Pmb, they enhance the survival and tolerance of bacterial populations. This study uncovers the dual role of EVs in Pmb sequestration and membrane repair, providing new insights into antibiotic tolerance mechanisms and paving the way for innovative approaches to combat antimicrobial resistance.

microbiology↗

bioRxiv2025

Altered RNA-processing provides a mechanistic framework delineating human sex reversal associated with pathogenic variants in the RNA-helicase DHX37

Recurrent heterozygous missense variants in the highly conserved RNA-helicase DHX37, which is required for ribosome biogenesis, are a frequent cause of 46,XY sex-reversal or testis regression syndrome. How these missense variants specifically disrupt testis formation is unknown. Here, we demonstrate that mutant DHX37 proteins retain their ATPase activity and are not associated with stabilization of cellular {beta}-catenin. Transfection of DHX37 p.R674Q mutant protein in an in-vitro cellular model recapitulating human Sertoli cell formation, showed a reduced activation of pro-testis genes compared to the WT protein. The expression of a DHX37 mutant protein in in-vitro derived human Sertoli-like cells (iSLCs) was also associated with global changes in gene expression, predicted to impact basic cellular functions. To define RNA transcripts interacting with either the WT or a mutant (p.R674Q) protein, we combined HyperTRIBE and single-cell full-length RNA-sequencing approaches using iSLCs. Gene ontology analysis indicated that transcripts targeted by WT DHX37 were primarily associated with cytoskeleton organization, including cell motility and cell adhesion. However, in contrast transcripts targeted by the mutated DHX37 protein, were not only associated with cytoskeleton organization but also with protein degradation and cell death. These data provide mechanistic framework that may explain how variants in the DHX37 protein can result in 46,XY sex-reversal through altered RNA networks that are required for the formation and maintenance of the supporting cell lineages of the human testis.

cell biology↗

bioRxiv2024

Biogenesis of DNA-carrying extracellular vesicles by the dominant human gut methanogenic archaeon

Extracellular vesicles (EVs) are membrane-bound particles secreted by cells from all domains of life and implicated in a variety of important processes, from intercellular communication to pathogenesis. Here, we characterize EVs produced by the dominant human gut methanogen, Methanobrevibacter smithii, which, unlike most archaea, contains a peptidoglycan cell wall. Using quantitative proteomics, we show that M. smithii EVs are enriched in various proteins responsible for chromatin structure, including histones, and DNA repair. Consistently, the M. smithii EVs carry DNA, with fragments covering the entire cellular chromosome. Notably, the EVs are strongly enriched in extrachromosomal circular DNA (eccDNA) molecules which originate from excision of a 2.9-kb chromosomal fragment and a proviral genome. The eccDNA encodes two of the key methanogenesis enzymes and could boost their expression inside the cells through the gene dosage effect. Furthermore, four of the top ten most abundant EV proteins are implicated in methanogenesis. Cryo-electron tomography (Cryo-ET) suggests that EVs are formed by budding from the cell membrane and are trapped under the cell wall prior to liberation through local disruptions in the cell wall. Collectively, our results reveal parallels with EV biogenesis in bacteria and suggest that M. smithii EVs facilitate the export of both cellular and viral DNA as well as key metabolic proteins in the gut environment, with potential impact on methane production.

microbiology↗

bioRxiv2023

Beyond 40 fluorescent probes for deep phenotyping of blood mononuclear cells, using spectral technology.

The analytical capability of flow cytometry is crucial for differentiating the growing number of cell subsets found in human blood. This is important for accurate immunophenotyping of patients with few cells and a large number of parameters to monitor. Here, we present a 43-parameter panel to analyze peripheral blood mononuclear cells from healthy individuals using 41 fluorescence-labelled monoclonal antibodies, an autofluorescent channel, and a viability dye. We demonstrate minimal population distortions that lead to optimized population identification and reproducible results. We have applied an advanced approach in panel design, in selection of sample acquisition parameters and in data analysis. Appropriate autofluorescence identification and integration in the unmixing matrix, allowed for resolution of unspecific signals and increased dimensionality. Addition of one laser without assigned fluorochrome resulted in decreased fluorescence spill over and improved discrimination of cell subsets. It also increased staining index when autofluorescence was integrated in the matrix. We conclude that spectral flow cytometry is highly valuable tool for high-end immunophenotyping, and that fine-tuning of major experimental steps is key for taking advantage of its full capacity.

cell biology↗

bioRxiv2023

High-dimensional spectral cytometry panels for whole blood immune phenotyping

The need to understand the mechanisms and pathways of immune responses in pathogenic conditions such as cancer and autoimmunity requires awareness of natural immune variability in healthy subjects. To this end, various systems immunology studies have been established. Among them, the Milieu Interieur (MI) study was established to define the boundaries of a healthy immune response and identify determinants of immune response variation. MI used immunophenotyping of a 1000 healthy donor cohort by flow cytometry as a principal outcome for immune variance at steady state. For the 10-year longitudinal MI study, we have developed two high-dimensional spectral flow cytometry panels that allow deep characterization of innate and adaptive whole blood immune cells (35 and 34 fluorescent markers, respectively) and standardized the protocol for sample handling, staining, acquisition, and data analysis. This permits the reproducible quantification of over 182 immune cell phenotypes through robust immunophenotyping at a single site. This highly standardized protocol was applied to samples from patients with autoimmune/inflammatory diseases. It is currently used for characterization of the impact of age and environmental factors on peripheral blood immune phenotypes of >400 donors from the initial MI cohort.

immunology↗

bioRxiv2023

Extracellular vesicles production regulates fluconazole resistance in Cryptococcus neoformans

Resistance to fluconazole (FLC), the most widely used antifungal drug, is typically achieved by altering the azole drug target and/or drug efflux pumps. Recent reports have suggested a link between vesicular trafficking and antifungal resistance. Here, we identified novel Cryptococcus neoformans regulators of extracellular vesicle (EV) biogenesis that impact FLC resistance. In particular, the transcription factor Hap2 does not affect the expression of the drug target or efflux pumps, yet it impacts the cellular sterol profile. Subinhibitory FLC concentrations also downregulate EV production. Moreover, in vitro spontaneous FLC-resistant colonies showed altered EV production, and the acquisition of FLC resistance was associated with decreased EV production in clinical isolates. Finally, the reversion of FLC resistance was associated with increased EV production. These data suggest a model in which fungal cells can regulate EV production in place of regulating the drug target gene expression as a first line of defense against antifungal assault in this fungal pathogen. IMPORTANCEExtracellular vesicles (EVs) are membrane-enveloped particles that are released by cells into the extracellular space. Fungal EVs can mediate community interactions and biofilm formation but thier functions remain poorly understood. Here, we report the identification of the first regulators of EV production in the major fungal pathogen Cryptococcus neoformans. Surprisingly, we uncover a novel role of EVs in modulating antifungal drug resistance. Disruption of EV production was associated with altered lipid composition and changes in fluconazole susceptibility. Spontaneous azole-resistant mutants were deficient in EV production, while loss of resistance restored initial EV production levels. These findings were recapitulated in C. neoformans clinical isolates, indicating that azole resistance and EV production are coregulated in diverse strains. Our study reveals a new mechanism of drug resistance in which cells adapt to azole stress by modulating EV production.

microbiology↗

bioRxiv2022

IRF8 regulates efficacy of therapeutic anti-CD20 monoclonal antibodies

Anti-CD20 monoclonal antibodies such as Rituximab, Ofatumumab and Obinutuzumab are widely used to treat lymphomas and autoimmune diseases. They act by depleting B cells, mainly through Fc-dependent effectors functions. Some patients develop resistance to treatment but the underlying mechanisms are poorly understood. Here, we performed a genome-wide CRISPR/Cas9 screen to identify genes regulating the efficacy of anti-CD20 antibodies. We used as a model the killing of RAJI B cells by Rituximab through complement-dependent-cytotoxicity (CDC). As expected, the screen identified MS4A1, encoding CD20, the target of Rituximab. Among other identified genes, the role of Interferon Regulatory Factor 8 (IRF8) was validated in two B-cell lines. IRF8 knockout also decreased the efficacy of antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP) induced by anti-CD20 antibodies. We further show that IRF8 is necessary for efficient CD20 transcription. Levels of IRF8 and CD20 RNA or proteins correlated in normal B cells and in hundreds of malignant B cells. Therefore, IRF8 regulates CD20 expression and controls the depleting-capacity of anti-CD20 antibodies. Our results bring novel insights into the pathways underlying resistance to CD20-targeting immunotherapies.

immunology↗

bioRxiv2022

Humanized mice for sustained Plasmodium vivax blood-stage infection and transmission

Plasmodium vivax is the most widespread human malaria parasite 1. The presence of extravascular reservoirs 2, the early circulation of infective stages (gametocytes), and relapsing infections arising from dormant liver stages 3 render this parasite particularly difficult to control and eliminate 4. Experimental research is limited by the lack of a continuous culture in vitro system that fulfills the parasites needs,5 namely its tropism for immature CD71+ red blood cells (RBCs). 5 Here, we report a humanized mice model, which upon engraftment of human hematopoietic progenitor and stem cells (HPSCs), exhibits efficient human erythropoiesis. Humanized HIS-HEry mice inoculated with cryopreserved P. vivax samples sustain long-lasting asexual parasite multiplication within CD71+ human RBCs and differentiation into mature gametocytes that can be efficiently transmitted to Anopheles mosquitoes, leading to formation salivary-gland sporozoites. Blood stages can be sequentially transferred to uninfected humanized mice by injection of fresh or frozen infected bone marrow cells, providing a unique murine model for the long-term maintenance of P. vivax isolates. This work offers a novel experimental platform to investigate the biology of RBC invasion and intraerythrocytic P. vivax development in vivo and evaluate new interventions against this elusive human parasite.

microbiology↗

bioRxiv2021

Plasmodium-encoded murine IL-6 impairs liver stage infection and elicits long-lasting sterilizing immunity

Plasmodium sporozoites inoculated by Anopheles mosquitoes into the skin of the mammalian host migrate to the liver before infecting hepatocytes. Previous work demonstrated that early production of IL-6 in the liver is detrimental for the parasite growth, contributing to the acquisition of a long-lasting immune protection after immunization with live attenuated parasites. Considering that IL-6 ais a critical pro-inflammatory signal, we explored a novel approach whereby the parasite itself encodes for the murine IL-6 gene. We generated transgenic P. berghei parasites that express murine IL-6 during liver stage development. Though IL-6 transgenic sporozoites develop into exo-erythrocytic forms in cultured hepatocytes in vitro and in vivo, these parasites were not capable of inducing a blood stage infection in mice. Furthermore, immunization of mice with transgenic IL-6-expressing P. berghei sporozoites elicited a long-lasting CD8+ T cell-mediated protective immunity against a subsequent infectious sporozoite challenge. Collectively, this study demonstrates that parasite-encoded IL-6 attenuates parasite virulence with abortive liver stage of Plasmodium infection, forming the basis of a novel suicide vaccine strategy to elicit protective antimalarial immunity. SummaryIL-6 was shown to control Plasmodium parasite development in the liver. Here, Belhimeur et al. generated a murine IL-6 transgenic Plasmodium berghei. These parasites show an arrest in hepatocyte development and protect mice against homologous and heterologous parasite challenge in a CD8-dependent manner.

immunology↗

bioRxiv2021

In-vitro cellular reprogramming to model gonad development and its disorders

During embryonic development, mutually antagonistic signaling cascades determine the fate of the bipotential gonad towards a testicular or ovarian identity. Errors in this process result in human Disorders of Sex Development (DSDs), where there is discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in-vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells towards gonadal progenitors. Transcriptomic analysis reveals that the in-vitro-derived murine gonadal cells are equivalent to E11.5 in-vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete AMH, migrate and form tubular structures. The cells derived from a 46,XY DSD female hiPSCs, carrying a NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR/Cas9-mediated correction of the variant rescued the phenotype. This is a robust tool to understand mechanisms of sex-determination and model DSDs.

developmental biology↗

bioRxiv2021

Archaeal extracellular vesicles are produced in an ESCRT-dependent manner and promote gene transfer and nutrient cycling in extreme environments

Membrane-bound extracellular vesicles (EVs), secreted by cells from all three domains of life, transport various molecules and act as agents of intercellular communication in diverse environments. Here we demonstrate that EVs produced by a hyperthermophilic and acidophilic archaeon Sulfolobus islandicus carry not only diverse proteins but also chromosomal and plasmid DNA, and can transfer this DNA to recipient cells. Furthermore, we show that EVs can support the heterotrophic growth of Sulfolobus in minimal medium, implicating EVs in carbon and nitrogen fluxes in extreme environments. Finally, our results suggest that, similar to eukaryotes, production of EVs in S. islandicus depends on the archaeal ESCRT machinery. Using synchronized S. islandicus cultures, we show that EV production is linked to cell division and appears to be triggered by increased expression of ESCRT proteins during this cell cycle phase. Using a CRISPR-based knockdown system, we show that archaeal ESCRT-III and AAA+ ATPase Vps4 are required for EV production, whereas archaea-specific component CdvA appears to be dispensable. Collectively, our results suggest that ESCRT-mediated EV biogenesis has deep evolutionary roots, likely predating the divergence of eukaryotes and archaea, and that EVs play an important role in horizontal gene transfer and nutrient cycling in extreme environments.

microbiology↗

bioRxiv2020

New insights into Cryptococcus extracellular vesicles suggest a new structural model and an antifungal vaccine strategy

Whereas extracellular vesicle (EV) research has become commonplace in different biomedical fields, this field of research is still in its infancy in mycology. Here we provide a robust set of data regarding the structural and compositional aspects of EVs isolated from the fungal pathogenic species Cryptococcus neoformans, C. deneoformans and C. deuterogattii. Using cutting-edge methodological approaches including cryogenic electron microscopy and cryogenic electron tomography, proteomics, and flow cytometry, we revisited cryptococcal EV features and suggest a new EV structural model, in which the vesicular lipid bilayer is covered by mannoprotein-based fibrillar decoration, bearing the capsule polysaccharide as its outer layer. About 10% of the EV population is devoid of fibrillar decoration, adding another aspect to EV diversity. By analyzing EV protein cargo from the three species, we characterized the typical Cryptococcus EV proteome. It contains several membrane-bound protein families, including some Tsh proteins bearing a SUR7/PalI motif. The presence of known protective antigens on the surface of Cryptococcus EVs, resembling the morphology of encapsulated virus structures, suggested their potential as a vaccine. Indeed, mice immunized with EVs obtained from an acapsular C. neoformans mutant strain rendered a strong antibody response in mice and significantly prolonged their survival upon C. neoformans infection.

microbiology↗

bioRxiv2020

Transcriptome and epigenome diversity and plasticity of muscle stem cells following transplantation

Adult skeletal muscles are maintained during homeostasis and regenerated upon injury by muscle stem cells (MuSCs). A heterogeneity in self-renewal, differentiation and regeneration properties has been reported for MuSCs based on their anatomical location. Although MuSCs derived from extraocular muscles (EOM) have a higher regenerative capacity than those derived from limb muscles, the molecular determinants that govern these differences remain undefined. Here we show that EOM and limb MuSCs have distinct DNA methylation signatures associated with enhancers of location-specific genes, and that the EOM transcriptome is reprogrammed following transplantation into a limb muscle environment. Notably, EOM MuSCs expressed host-site specific positional Hox codes after engraftment and self-renewal within the host muscle. However, about 10% of EOM-specific genes showed engraftment-resistant expression, pointing to cell-intrinsic molecular determinants of the higher engraftment potential of EOM MuSCs. Our results underscore the molecular diversity of distinct MuSC populations and molecularly define their plasticity in response to microenvironmental cues. These findings provide insights into strategies designed to improve the functional capacity of MuSCs in the context of regenerative medicine.

molecular biology↗

bioRxiv2018

Ageing affects DNA methylation drift and transcriptional cell-to-cell variability in muscle stem cells

Age-related tissue alterations have been associated with a decline in stem cell number and function1. Although increased cell-to-cell variability in transcription or epigenetic marks has been proposed to be a major hallmark of ageing2-5, little is known about the molecular diversity of stem cells during ageing. Here, by combined single-cell transcriptome and DNA methylome profiling in mouse muscle stem cells, we show a striking global increase of uncoordinated transcriptional heterogeneity together with context-dependent alterations of DNA methylation with age. Importantly, promoters with increased methylation heterogeneity are associated with increased transcriptional heterogeneity of the genes they drive. Notably, old cells that change the most with age reveal alterations in the transcription of genes regulating cell-niche interactions. These results indicate that epigenetic drift, by accumulation of stochastic DNA methylation changes in promoters, is a substantial driver of the degradation of coherent transcriptional networks with consequent stem cell functional decline during ageing.

genomics↗