Biology subjects

Boilard, E.

Publications and source records attributed to Boilard, E..

11 recordsLinked to original sources

bioRxiv2026

Megakaryocyte emperipolesis arms neutrophils via intracellular provisioning

Neutrophils are phenotypically heterogenous cells that mediate host defense and tissue homeostasis. Here, we identify emperipolesis - the evolutionarily conserved process by which neutrophils pass through megakaryocytes - as a phenotypically transformative route of egress from bone marrow. By intravital microscopy and 3-D histology, we show that the rapid form emperipolesis is markedly enhanced under inflammatory conditions. Neutrophils exit from megakaryocytes directly to the blood, acquiring exosomes enriched in proteins related to metabolism, migration, and immune function. This transfer induces a distinct neutrophil phenotype characterized by enhanced glycolysis, oxidative phosphorylation, cytokine release, and longevity. Correspondingly, emperipolesis-educated neutrophils display accelerated migration in vitro and in vivo. Disrupting emperipolesis does not alter circulating neutrophil abundance but impairs neutrophil infiltration into inflamed tissues, including Pseudomonas aeruginosa-infected lung. These findings establish emperipolesis as a mechanism by which megakaryocytes amplify neutrophil-mediated immunity.

immunology↗

bioRxiv2026

Immunoglobulins are rapidly internalized by neurons after CNS injury and cleared through lysosomal degradation

Spinal cord injury (SCI) causes hemorrhage and blood-spinal cord barrier disruption, allowing blood-derived molecules to infiltrate the parenchyma. While immunoglobulins (Ig) are abundant plasma proteins, their distribution and cellular targets within the injured spinal cord remain poorly defined. Here, we show that circulating non-autoimmune immunoglobulins rapidly infiltrate the spinal cord after injury in mice and disseminate beyond the lesion core. IgG, IgM, and IgA accumulate within the parenchyma early post-injury, with IgG displaying the widest spatial distribution, reaching distant spinal segments within hours. Neurons are the predominant cell type internalizing immunoglobulins in the gray matter, whereas astrocytes exhibit moderate uptake in white matter. Intra-cisterna magna administration of fluorescent serum-derived IgG reveals that neurons and astrocytes internalize IgG under physiological conditions, independently of Fc receptor engagement. Although the neonatal Fc receptor (FcRn) has minimal impact on CNS IgG recycling, its genetic deletion significantly improves locomotor recovery after SCI. Both in vitro and in vivo, neurons clear IgG through lysosomal degradation. Following SCI, inhibition of lysosomal proteases with the clinically approved drug E64d increases CNS IgG retention without compromising locomotor recovery. These findings establish neurons as key targets of circulating immunoglobulins after CNS injury and reveal IgG uptake and clearance pathways that may be leveraged to improve therapeutic performance of monoclonal antibody treatments.

neuroscience↗

bioRxiv2026

Blood-brain barrier model architecture shapes peripheral immune cell trafficking in Parkinson's disease

Parkinsons disease (PD) is a neurodegenerative disorder traditionally characterized by dopaminergic neuron loss in the substantial nigra pars compacta, but peripheral immune dysregulation and blood-brain barrier (BBB) dysfunction have been increasingly implicated in disease etiology. However, how circulating immune cells interact with the human BBB and how these interactions are captured across experimental models remains poorly understood. In particular, human BBB models offer multiple platforms to interrogate these biological questions, with organ-on-chip approaches attracting significant interest. In this context, it is essential to determine whether model architecture influences the assessment of immune-endothelial interactions in PD, and if it may lead to fundamentally different interpretations of immune cell trafficking at the human BBB. Here, we compared peripheral blood mononuclear cells (PBMCs) from control donors or individuals with PD in human induced pluripotent stem cell (iPSC)-derived BBB models to determine how static and dynamic BBB systems influence immune cell behavior. To do so, we leveraged our brain chip platform to establish a model based on expression of the PD-associated SNCA triplication mutation. Using a two-dimensional transwell system and a three-dimensional (3D) microfluidic BBB chip, we evaluated PBMC attachment and transmigration under conditions of PBMC disease status, endothelial genotype associated with SNCA triplication, and exposure to -synuclein (-Syn) monomers or preformed fibrils (PFFs). PBMCs from PD donors showed increased baseline reactivity and altered endothelial interactions compared with controls. In transwell models, SNCA triplication increased PBMC attachment and selectively enhanced PD PBMC transmigration, while PFF increased attachment without affecting transmigration. In contrast, in the microfluidic BBB chip, attachment was largely unchanged by endothelial genotype or -Syn exposure, whereas transmigration increased following -Syn monomer pre-treatment. Together, PBMC-BBB interactions in PD appear to be shaped by immune cell status, endothelial genotype, and -Syn exposure, but are strongly influenced by BBB model dimensionality and flow. This study underscores the importance of physiologically relevant multicellular and flow-based BBB systems and provides a human-focused framework for studying peripheral immune cell trafficking across the diseased BBB. These findings also emphasize that biological insights into BBB function are inherently shaped by the experimental model used, underscoring the need for complementary human BBB platforms.

neuroscience↗

bioRxiv2026

Mitochondrial Transplantation in the Eye: A Review and Evaluation of Surgical Approaches

PurposeMitochondrial dysfunction contributes to major blinding diseases, including age-related macular degeneration and glaucoma. Although mitochondrial transplantation has shown therapeutic potential in multiple organ systems, translation to the eye remains limited, partly due to uncertainty regarding optimal delivery. We summarize the biologic rationale and preclinical evidence supporting ocular mitochondrial transplantation and present feasibility data evaluating clinically relevant delivery routes. MethodsWe conducted a focused narrative review of ocular mitochondrial transplantation. For feasibility experiments, mitochondria with an endogenous fluorescent dye were isolated from liver donor mice. Postnatal day 7 pups received subretinal injections, and adult CD1 mice received intravitreal injections, including optic nerve head directed delivery. Eyes were analyzed using fluorescence microscopy and immunohistochemistry. Mitochondrial uptake was assessed in cultured retinal pigmental epithelial (RPE) cells using co-incubation assays. Suprachoroidal delivery feasibility was evaluated in cadaveric human near-real surgical specimens using a novel dedicated suprachoroidal injector. ResultsThe literature on ocular mitochondrial transplantation remains limited and consists primarily of small preclinical studies using intravitreal delivery and imaging-based detection. In our experiments, intravitreal delivery produced donor signals predominantly within inner retinal layers, with enrichment along retinal nerve fiber bundles when directed toward the optic nerve head. Cultured RPE cells demonstrated dose-dependent uptake of exogenous mitochondria. Subretinal delivery localized donors signal to the RPE and adjacent outer retina. Suprachoroidal injections demonstrated procedural feasibility with reliable access to the suprachoroidal space and visible injectate distribution. ConclusionsOcular mitochondrial transplantation is in an early stage of investigation. Our feasibility data indicate that established posterior-segment delivery routes expose distinct retinal compartments and that route selection strongly influences anatomic distribution. Further studies are needed to verify intracellular uptake, define dosing and durability, and evaluate safety in disease-relevant models.

cell biology↗

bioRxiv2025

Parkinson's disease risk factors are expressed at brain barriers.

Parkinsons disease (PD) is characterized by the selective loss of dopaminergic neurons in the substantia nigra pars compacta, but whether its etiology is cell autonomous remains unclear. Increasing evidence implicates the blood-central nervous system (CNS) barriers in disease development, highlighting the importance of identifying genetic risk factors linked to cells forming the cerebrovasculature to advance this emerging area of research. The objective of this study is to identify PD genetic risk factors associated with blood-brain (BBB) and blood-cerebrospinal fluid (BCSFB) barriers, and to validate protein localization in human tissue and experimental models. To do so, we integrated genome-wide association studies and single nuclei RNA-sequencing datasets from the human postmortem substantia nigra (SN), midbrain, or cortical samples from control and PD donors. An in-depth bioinformatics analysis identified genes enriched in cell types that form the multicellular architecture of brain barriers, including CAVIN2, ANXA1, ANO2, and LRP1B. We further validated whether corresponding proteins were present in cell types associated with the blood-CNS barriers in human and mouse post-mortem tissues, as well as in iPSC-differentiated cells and choroid plexus organoids. Results showed that quantifying the proportion of endothelial cells expressing PD-related genes was under-evaluated at the transcript level compared to immunofluorescence analyses. In addition, we observed that CAVIN2 and ANXA1 proteins were more abundant at the vasculature of the substantia nigra vs. cortex, and CAVIN2 protein levels were reduced in PD vs. control human postmortem tissues. In contrast, the investigation of mouse postmortem samples demonstrated that the CAVIN2 protein is only present in a subset of mouse blood vessels, compared to nearly all vessels in human tissue. Similarly, mouse ANXA1 protein localizes to dopaminergic neurons of the substantia nigra and not at the vasculature, as seen in human tissue. The primary outcome of this study is the identification of PD-relevant risk genes specifically expressed at brain barriers and enriched in PD-relevant brain regions. The secondary outcome is the demonstration of poor transcript-protein correlation in - at least - a subset of PD risk factors, and a low interspecies conservation of protein localization for the selected candidates. In conclusion, the BBB and BCSFB may represent understudied contributors to PD, endothelial-specific proteins appear differentially regulated compared to transcripts, and experimental models require comprehensive validation to ensure relevance to the human condition.

neuroscience↗

bioRxiv2024

IgG hexamers initiate acute lung injury

Antibodies can initiate lung injury in a variety of disease states such as autoimmunity, transfusion reactions, or after organ transplantation, but the key factors determining in vivo pathogenicity of injury-inducing antibodies are unclear. A previously overlooked step in complement activation by IgG antibodies has been elucidated involving interactions between IgG Fc domains that enable assembly of IgG hexamers, which can optimally activate the complement cascade. Here, we tested the in vivo relevance of IgG hexamers in a complement-dependent alloantibody model of acute lung injury. We used three approaches to block alloantibody hexamerization (antibody carbamylation, the K439E Fc mutation, or treatment with domain B from Staphylococcal protein A), all of which reduced acute lung injury. Conversely, Fc mutations promoting spontaneous hexamerization made a harmful alloantibody into a more potent inducer of acute lung injury and rendered an innocuous alloantibody pathogenic. Treatment with a recombinant Fc hexamer decoy therapeutic protected mice from lung injury, including in a model with transgenic human FCGR2A expression that exacerbated pathology. These results indicate a direct in vivo role of IgG hexamerization in initiating acute lung injury and the potential for therapeutics that inhibit or mimic hexamerization to treat antibody-mediated diseases. Brief summaryIgG antibodies can form hexamers. This study shows that hexamer assembly is an important event determining the ability of IgG to trigger acute lung injury. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/577129v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@15b0346org.highwire.dtl.DTLVardef@33f39eorg.highwire.dtl.DTLVardef@bf0f4dorg.highwire.dtl.DTLVardef@185b5bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Efficient megakaryopoiesis and platelet production require phospholipid remodeling and PUFA uptake through CD36

Lipids contribute to hematopoiesis and membrane properties and dynamics, however, little is known about the role of lipids in megakaryopoiesis. Here, a lipidomic analysis of megakaryocyte progenitors, megakaryocytes, and platelets revealed a unique lipidome progressively enriched in polyunsaturated fatty acid (PUFA)-containing phospholipids. In vitro, inhibition of both exogenous fatty acid functionalization and uptake and de novo lipogenesis impaired megakaryocyte differentiation and proplatelet production. In vivo, mice on a high saturated fatty acid diet had significantly lower platelet counts, which was prevented by eating a PUFA-enriched diet. Fatty acid uptake was largely dependent on CD36, and its deletion in mice resulted in thrombocytopenia. Moreover, patients with a CD36 loss-of-function mutation exhibited thrombocytopenia and increased bleeding. Our results suggest that fatty acid uptake and regulation is essential for megakaryocyte maturation and platelet production, and that changes in dietary fatty acids may be a novel and viable target to modulate platelet counts.

cell biology↗

bioRxiv2022

SARS-CoV-2 type I Interferon modulation by non structural proteins 1 and 2

Since the beginning of the COVID-19 pandemic, enormous efforts were devoted to understanding how SARS-CoV-2 escapes the antiviral response. Yet, modulation of type I interferons (IFNs) by this virus is not completely understood. Using in vitro and in vivo approaches, we have characterized the type I IFN response during SARS-CoV-2 infection as well as immune evasion mechanisms. The transcriptional and translational expression of IFNs, cytokines and chemokines were measured in lung homogenates of Wuhan-like, Beta, and Delta SARS-CoV-2 K18-ACE2 transgenic mice. Using in vitro experiments, we measured SARS-CoV-2 and its non-structural proteins 1 and 2 (Nsp1-2) to modulate expression of IFN{beta} and interferon-stimulated genes (ISG). Our data show that infection of mice with Wuhan-like virus induces robust expression of Ifna and Ifnb1 mRNA and limited type I production. In contrast, Beta and Delta variant infected mice failed to activate and produce IFN. Using in vitro systems, Ifn{beta} gene translation inhibition was observed using an Nsp1 expression vector. Conversely, SARS-CoV-2 and its variants induce robust expression of NF-{kappa}B-driven genes such as those encoding CCL2 ans CXCL10 chemokines. We also identified Nsp2 as an activator of NF-{kappa}B that partially counteracts the inhibitory actions of Nsp1. In summary, our work indicates that SARS-CoV-2 skews the antiviral response in favor of an NF-{kappa}B-driven inflammatory response, a hallmark of acute COVID-19, and that Nsp2 is partly responsible for this effect. ImportanceSeveral studies suggest that SARS-CoV-2 possess multiple mechanisms aimed shunting the type I interferon response. However, few studies have studied type I IFN modulation in the context of infection. Our work indicates that mice and human cells infected with SARS-CoV-2 produce sufficient type I IFN to activate an antiviral response, despite Nsp1 translational blockade of IFNB1 mRNA. In contrast to Wuhan-like virus, Beta and Delta variants failed to induce Ifna gene expression. Our work also showcases the importance of studying protein functions in the context of infection, as demonstrated by the partial antagonizing properties of the Nsp2 protein on the activities of Nsp1. Our studies also highlight that the innate immune response triggered by SARS-CoV-2 is chiefly driven by NF-{kappa}B responsive genes for which Nsp2 is partially responsible.

microbiology↗

bioRxiv2020

IgG-aggregates rapidly up-regulate FcgRI expression at the surface of human neutrophils in a FcgRII-dependent fashion: A crucial role for FcgRI in cellular responsiveness.

Autoimmune complexes are an important feature of several autoimmune diseases such as lupus, as they contribute to tissue damage through the activation of immune cells. Neutrophils, key players in lupus, interact with immune complexes through Fc gamma receptors (FcgR). Incubation of neutrophils with aggregated-IgGs caused degranulation and increased the surface expression of FcgRI within minutes in a concentration-dependent fashion. After 30 min, IgG aggregates (1 mg/ml) up-regulated FcgRI by 4.95 {+/-} 0.45-fold. FcgRI-positive neutrophils reached 67.24% {+/-} 6.88% on HA-IgGs stimulated neutrophils, from 3.12% {+/-} 1.62% in non-stimulated cells, ranking IgG-aggregates among the most potent known agonists. FcgRIIa, and possibly FcgRIIIa, appeared to mediate this up-regulation. Also, FcgRI-dependent signaling proved necessary for reactive oxygen species (ROS) production in response to IgG-aggregates. Finally, combinations of bacterial materials with aggregates dramatically boosted ROS production. This work suggests FcgRI as an essential component in the response of human neutrophils to immune complexes leading to the production of ROS, which may help explain how neutrophils contribute to tissue damage associated with immune complex-associated diseases, such as lupus.

immunology↗

bioRxiv2018

Megakaryocyte emperipolesis mediates membrane transfer from intracytoplasmic neutrophils to platelets

Bone marrow megakaryocytes engulf neutrophils in a phenomenon termed emperipolesis. We show here that emperipolesis is a dynamic process mediated actively by both lineages, in part through the {beta}2-integrin/ICAM-1/ezrin pathway. Tethered neutrophils enter in membrane-bound vesicles before penetrating into the megakaryocyte cytoplasm. Intracytoplasmic neutrophils develop membrane contiguity with the demarcation membrane system, thereby transferring membrane to the megakaryocyte and to daughter platelets. This phenomenon occurs in otherwise unmanipulated marrow in vivo, resulting in circulating platelets that bear membrane from non-megakaryocytic hematopoietic donors. Transit through megakaryocytes can be completed as rapidly as minutes, after which neutrophils egress intact. Emperipolesis is amplified in models of inflammation associated with platelet overproduction, contributing to platelet production in vitro and in vivo. These findings identify emperipolesis as a new cell-in-cell interaction that enables neutrophils and potentially other cells passing through the megakaryocyte cytoplasm to modulate the production and membrane content of platelets.

cell biology↗

bioRxiv2018

Differential attenuation of β2 integrin-dependent and -independent neutrophil migration by Ly6G ligation

Abstract\n\nAntibody ligation of the murine neutrophil surface protein Ly6G disrupts neutrophil migration in some contexts but not others. We tested whether this variability reflected divergent dependence of neutrophil migration on {beta}2 integrins, adhesion molecules that interact with Ly6G at the neutrophil surface. In integrin-dependent murine arthritis, Ly6G ligation attenuated joint inflammation, even though mice lacking Ly6G altogether developed arthritis normally. By contrast, Ly6G ligation had no impact on integrin- independent neutrophil migration into inflamed lung. In peritoneum, the role of {beta}2 integrins varied with stimulus, proving dispensable for neutrophil entry in E. coli peritonitis but contributory in IL-1-mediated sterile peritonitis. Correspondingly, Ly6G ligation attenuated only IL-1 peritonitis, disrupting the molecular association between integrins and Ly6G and inducing cell-intrinsic blockade restricted to integrin-dependent migration. Consistent with this observation, Ly6G ligation impaired integrin-mediated postadhesion strengthening for neutrophils arresting on activated cremaster endothelium in vivo. Together, these findings identify selective inhibition of integrin- mediated neutrophil emigration through Ly6G ligation, highlighting the marked site and stimulus specificity of {beta}2 integrin dependence in neutrophil migration.\n\nKEY POINTSO_LIThe contribution of {beta}2 integrins to neutrophil migration into inflamed tissues varies with site and stimulus.\nC_LIO_LILigation of Ly6G, a GPI-linked neutrophil surface protein, selectively attenuates {beta}2 integrin-dependent neutrophil migration in vivo.\nC_LIO_LIBlockade correlates with disrupted interaction between Ly6G and {beta}2 integrins and impaired integrin-mediated postadhesion strengthening.\nC_LI

immunology↗