Droplet-based forward genetic screening of astrocyte-microglia cross-talk.

Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms.

MCAM+ brain endothelial cells contribute to neuroinflammation by recruiting pathogenic CD4+ T lymphocytes.

The trafficking of autoreactive leucocytes across the blood-brain barrier endothelium is a hallmark of multiple sclerosis pathogenesis. Although the blood-brain barrier endothelium represents one of the main CNS borders to interact with the infiltrating leucocytes, its exact contribution to neuroinflammation remains understudied. Here, we show that Mcam identifies inflammatory brain endothelial cells with pro-migratory transcriptomic signature during experimental autoimmune encephalomyelitis. In addition, MCAM was preferentially upregulated on blood-brain barrier endothelial cells in multiple sclerosis lesions in situ and at experimental autoimmune encephalomyelitis disease onset by molecular MRI. In vitro and in vivo, we demonstrate that MCAM on blood-brain barrier endothelial cells contributes to experimental autoimmune encephalomyelitis development by promoting the cellular trafficking of TH1 and TH17 lymphocytes across the blood-brain barrier. Last, we showcase ST14 as an immune ligand to brain endothelial MCAM, enriched on CD4+ T lymphocytes that cross the blood-brain barrier in vitro, in vivo and in multiple sclerosis lesions as detected by flow cytometry on rapid autopsy derived brain tissue from multiple sclerosis patients. Collectively, our findings reveal that MCAM is at the centre of a pathological pathway used by brain endothelial cells to recruit pathogenic CD4+ T lymphocyte from circulation early during neuroinflammation. The therapeutic targeting of this mechanism is a promising avenue to treat multiple sclerosis.

CLMP Promotes Leukocyte Migration Across Brain Barriers in Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: In multiple sclerosis (MS), peripheral immune cells use various cell trafficking molecules to infiltrate the CNS where they cause damage.The objective of this study was to investigate the involvement of coxsackie and adenovirus receptor-like membrane protein (CLMP) in the migration of immune cells into the CNS of patients with MS. METHODS: Expression of CLMP was measured in primary cultures of human brain endothelial cells (HBECs) and human meningeal endothelial cells (HMECs), postmortem brain samples, and peripheral blood mononuclear cells (PBMCs) from patients with MS and controls by RNA sequencing, quantitative PCR, immunohistochemistry, and flow cytometry. In vitro migration assays using HBECs and HMECs were performed to evaluate the function of CLMP. RESULTS: Using bulk RNA sequencing of primary cultures of human brain and meningeal endothelial cells (ECs), we have identified CLMP as a new potential cell trafficking molecule upregulated in inflammatory conditions. We first confirmed the upregulation of CLMP at the protein level on TNFα-activated and IFNγ-activated primary cultures of human brain and meningeal ECs. In autopsy brain specimens from patients with MS, we demonstrated an overexpression of endothelial CLMP in active MS lesions when compared with normal control brain tissue. Flow cytometry of human PBMCs demonstrated an increased frequency of CLMP+ B lymphocytes and monocytes in patients with MS, when compared with that in healthy controls. The use of a blocking antibody against CLMP reduced the migration of immune cells across the human brain and meningeal ECs in vitro. Finally, we found CLMP+ immune cell infiltrates in the perivascular area of parenchymal lesions and in the meninges of patients with MS. DISCUSSION: Collectively, our data demonstrate that CLMP is an adhesion molecule used by immune cells to access the CNS during neuroinflammatory disorders such as MS. CLMP could represent a target for a new treatment of neuroinflammatory conditions.

Identification of SARS-CoV-2-specific immune alterations in acutely ill patients.

Dysregulated immune profiles have been described in symptomatic patients infected with SARS-CoV-2. Whether the reported immune alterations are specific to SARS-CoV-2 infection or also triggered by other acute illnesses remains unclear. We performed flow cytometry analysis on fresh peripheral blood from a consecutive cohort of (a) patients hospitalized with acute SARS-CoV-2 infection, (b) patients of comparable age and sex hospitalized for another acute disease (SARS-CoV-2 negative), and (c) healthy controls. Using both data-driven and hypothesis-driven analyses, we found several dysregulations in immune cell subsets (e.g., decreased proportion of T cells) that were similarly associated with acute SARS-CoV-2 infection and non-COVID-19-related acute illnesses. In contrast, we identified specific differences in myeloid and lymphocyte subsets that were associated with SARS-CoV-2 status (e.g., elevated proportion of ICAM-1+ mature/activated neutrophils, ALCAM+ monocytes, and CD38+CD8+ T cells). A subset of SARS-CoV-2-specific immune alterations correlated with disease severity, disease outcome at 30 days, and mortality. Our data provide an understanding of the immune dysregulation specifically associated with SARS-CoV-2 infection among acute care hospitalized patients. Our study lays the foundation for the development of specific biomarkers to stratify SARS-CoV-2-positive patients at risk of unfavorable outcomes and to uncover candidate molecules to investigate from a therapeutic perspective.

Overcoming the Brain Barriers: From Immune Cells to Nanoparticles.

Nanoparticulate carriers, often referred to as nanoparticles (NPs), represent an important pharmacological advance for drug protection and tissue-specific drug delivery. Accessing the central nervous system (CNS), however, is a complex process regulated by mainly three brain barriers. While some leukocyte (i.e., immune cell) subsets are equipped with the adequate molecular machinery to infiltrate the CNS in physiological and/or pathological contexts, the successful delivery of NPs into the CNS remains hindered by the tightness of the brain barriers. Here, we present an overview of the three major brain barriers and the mechanisms allowing leukocytes to migrate across each of them. We subsequently review different immune-inspired and -mediated strategies to deliver NPs into the CNS. Finally, we discuss the prospect of exploiting leukocyte trafficking mechanisms for further progress.

Isolation of endothelial cells, pericytes and astrocytes from mouse brain.

Primary cell isolation from the central nervous system (CNS) has allowed fundamental understanding of blood-brain barrier (BBB) properties. However, poorly described isolation techniques or suboptimal cellular purity has been a weak point of some published scientific articles. Here, we describe in detail how to isolate and enrich, using a common approach, endothelial cells (ECs) from adult mouse brains, as well as pericytes (PCs) and astrocytes (ACs) from newborn mouse brains. Our approach allowed the isolation of these three brain cell types with purities of around 90%. Furthermore, using our protocols, around 3 times more PCs and 2 times more ACs could be grown in culture, as compared to previously published protocols. The cells were identified and characterized using flow cytometry and confocal microscopy. The ability of ECs to form a tight monolayer was assessed for passages 0 to 3. The expression of claudin-5, occludin, zonula occludens-1, P-glycoprotein-1 and breast cancer resistance protein by ECs, as well as the ability of the cells to respond to cytokine stimuli (TNF-α, IFN-γ) was also investigated by q-PCR. The transcellular permeability of ECs was evaluated in the presence of pericytes or astrocytes in a Transwell® model by measuring the transendothelial electrical resistance (TEER), dextran-FITC and sodium fluorescein permeability. Overall, ECs at passages 0 and 1 featured the best properties valued in a BBB model. Furthermore, pericytes did not increase tightness of EC monolayers, whereas astrocytes did regardless of their seeding location. Finally, ECs resuspended in fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) could be cryopreserved in liquid nitrogen without affecting their phenotype nor their capacity to form a tight monolayer, thus allowing these primary cells to be used for various longitudinal in vitro studies of the blood-brain barrier.

Melanoma cell adhesion molecule-positive CD8 T lymphocytes mediate central nervous system inflammation.

OBJECTIVE: Although Tc17 lymphocytes are enriched in the central nervous system (CNS) of multiple sclerosis (MS) subjects and of experimental autoimmune encephalomyelitis (EAE) animals, limited information is available about their recruitment into the CNS and their role in neuroinflammation. Identification of adhesion molecules used by autoaggressive CD8(+) T lymphocytes to enter the CNS would allow further characterization of this pathogenic subset and could provide new therapeutic targets in MS. We propose that melanoma cell adhesion molecule (MCAM) is a surface marker and adhesion molecule used by pathogenic CD8(+) T lymphocytes to access the CNS. METHODS: Frequency, phenotype, and function of MCAM(+) CD8(+) T lymphocytes was characterized using a combination of ex vivo, in vitro, in situ, and in vivo approaches in humans and mice, including healthy controls, MS subjects, and EAE animals. RESULTS: Herein, we report that MCAM is expressed by human effector CD8(+) T lymphocytes and it is strikingly upregulated during MS relapses. We further demonstrate that MCAM(+) CD8(+) T lymphocytes express more interleukin 17, interferon γ, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor than MCAM(-) lymphocytes, and exhibit an enhanced killing capacity toward oligodendrocytes. MCAM blockade restricts the transmigration of CD8(+) T lymphocytes across human blood-brain barrier endothelial cells in vitro, and blocking or depleting MCAM in vivo reduces chronic neurological deficits in active, transfer, and spontaneous progressive EAE models. INTERPRETATION: Our data demonstrate that MCAM identifies encephalitogenic CD8(+) T lymphocytes, suggesting that MCAM could represent a biomarker of MS disease activity and a valid target for the treatment of neuroinflammatory conditions.