Scoary2: rapid association of phenotypic multi-omics data with microbial pan-genomes.

Unraveling bacterial gene function drives progress in various areas, such as food production, pharmacology, and ecology. While omics technologies capture high-dimensional phenotypic data, linking them to genomic data is challenging, leaving 40-60% of bacterial genes undescribed. To address this bottleneck, we introduce Scoary2, an ultra-fast microbial genome-wide association studies (mGWAS) software. With its data exploration app and improved performance, Scoary2 is the first tool to enable the study of large phenotypic datasets using mGWAS. As proof of concept, we explore the metabolome of yogurts, each produced with a different Propionibacterium reichii strain and discover two genes affecting carnitine metabolism.

Prevalence and characteristics of genetic disease in adult kidney stone formers.

BACKGROUND: Molecular mechanisms of kidney stone formation remain unknown in most patients. Previous studies showed high a heritability of nephrolithiasis, but data on prevalence and characteristics of genetic disease in unselected adults with nephrolithiasis are lacking. This study was conducted to fill this important knowledge gap. METHODS: We performed whole exome sequencing in 787 participants of the Bern Kidney Stone Registry, an unselected cohort of adults with ≥ 1 past kidney stone episode (KSF), and 114 non-stone-forming individuals (NKSF). An exome-based panel of 34 established nephrolithiasis genes was analyzed and variants assessed according to ACMG criteria. Pathogenic (P) or likely pathogenic (LP) variants were considered diagnostic. RESULTS: Mean age of KSF was 47±15 years, and 18% were first time KSF. A Mendelian kidney stone disease was present in 2.9% (23 of 787) of KSF. The most common genetic diagnoses were cystinuria (SLC3A1, SLC7A9; n=13), Vitamin D-24 hydroxylase deficiency (CYP24A1; n=5) and primary hyperoxaluria (AGXT, GRHPR, HOGA1; n=3). 8.1% (64 of 787) of KSF were monoallelic for LP/P variants predisposing to nephrolithiasis, most frequently in SLC34A1/A3 or SLC9A3R1 (n=37), CLDN16 (n=8) and CYP24A1 (n=8). KSF with Mendelian disease had a lower age at the first stone event (30±14 years vs. 36±14 years, p=0.003), were more likely to have cystine stones (23.4% vs. 1.4%) and less likely to have calcium oxalate monohydrates stones (31.9% vs. 52.5%) compared to KSF without genetic diagnosis. The phenotype of KSF with variants predisposing to nephrolithiasis was subtle and showed significant overlap with KSF without diagnostic variants. In NKSF, no Mendelian disease was detected, and LP/P variants were significantly less prevalent compared to KSF (1.8% vs. 8.1%). CONCLUSION: Mendelian disease is uncommon in unselected adult KSF, yet variants predisposing to nephrolithiasis are significantly enriched in adult KSF.

Functional characterization of Francisella tularensis subspecies holarctica genotypes during tick cell and macrophage infections using a proteogenomic approach.

Tularemia is a vector-borne disease caused by the Gram-negative bacterium Francisella tularensis. Known hosts and vectors in Europe are hare and ticks. F. tularensis is transmitted from ticks and animals, but also from the hydrotelluric environment and the consumption of contaminated water or food. A changing climate expands the range in which ticks can live and consequently might contribute to increasing case numbers of tularemia. Two subspecies of F. tularensis are human pathogenic. Francisella tularensis tularensis (Ftt) is endemic in North America, while Francisella tularensis holarctica (Fth) is the only subspecies causing tularemia in Europe. Ft is classified as a category A bioterrorism agent due to its low infectious dose, multiple modes of transmission, high infectivity and potential for airborne transmission and has become a global public health concern. In line with the European survey and previous phylogenetic studies, Switzerland shows the co-distribution of B.6 and B.12 strains with different geographical distribution and prevalence within the country. To establish itself in different host environments of ticks and mammals, F. tularensis presumably undergoes substantial changes on the transcriptomics and proteomic level. Here we investigate the transcriptomic and proteomic differences of five strains of Fth upon infection of rabbit macrophages and tick cells.

Bacterial tolerance to host-exuded specialized metabolites structures the maize root microbiome.

Plants exude specialized metabolites from their roots, and these compounds are known to structure the root microbiome. However, the underlying mechanisms are poorly understood. We established a representative collection of maize root bacteria and tested their tolerance against benzoxazinoids (BXs), the dominant specialized and bioactive metabolites in the root exudates of maize plants. In vitro experiments revealed that BXs inhibited bacterial growth in a strain- and compound-dependent manner. Tolerance against these selective antimicrobial compounds depended on bacterial cell wall structure. Further, we found that native root bacteria isolated from maize tolerated the BXs better compared to nonhost Arabidopsis bacteria. This finding suggests the adaptation of the root bacteria to the specialized metabolites of their host plant. Bacterial tolerance to 6-methoxy-benzoxazolin-2-one (MBOA), the most abundant and selective antimicrobial metabolite in the maize rhizosphere, correlated significantly with the abundance of these bacteria on BX-exuding maize roots. Thus, strain-dependent tolerance to BXs largely explained the abundance pattern of bacteria on maize roots. Abundant bacteria generally tolerated MBOA, while low abundant root microbiome members were sensitive to this compound. Our findings reveal that tolerance to plant specialized metabolites is an important competence determinant for root colonization. We propose that bacterial tolerance to root-derived antimicrobial compounds is an underlying mechanism determining the structure of host-specific microbial communities.

Cycles of fusion and fission enabled rapid parallel adaptive radiations in African cichlids.

Although some lineages of animals and plants have made impressive adaptive radiations when provided with ecological opportunity, the propensities to radiate vary profoundly among lineages for unknown reasons. In Africa's Lake Victoria region, one cichlid lineage radiated in every lake, with the largest radiation taking place in a lake less than 16,000 years old. We show that all of its ecological guilds evolved in situ. Cycles of lineage fusion through admixture and lineage fission through speciation characterize the history of the radiation. It was jump-started when several swamp-dwelling refugial populations, each of which were of older hybrid descent, met in the newly forming lake, where they fused into a single population, resuspending old admixture variation. Each population contributed a different set of ancient alleles from which a new adaptive radiation assembled in record time, involving additional fusion-fission cycles. We argue that repeated fusion-fission cycles in the history of a lineage make adaptive radiation fast and predictable.

Targeted removal of macrophage-secreted interleukin-1 receptor antagonist protects against lethal Candida albicans sepsis.

Invasive fungal infections are associated with high mortality rates, and the lack of efficient treatment options emphasizes an urgency to identify underlying disease mechanisms. We report that disseminated Candida albicans infection is facilitated by interleukin-1 receptor antagonist (IL-1Ra) secreted from macrophages in two temporally and spatially distinct waves. Splenic CD169+ macrophages release IL-1Ra into the bloodstream, impeding early neutrophil recruitment. IL-1Ra secreted by monocyte-derived tissue macrophages further impairs pathogen containment. Therapeutic IL-1Ra neutralization restored the functional competence of neutrophils, corrected maladapted hyper-inflammation, and eradicated the otherwise lethal infection. Conversely, augmentation of macrophage-secreted IL-1Ra by type I interferon severely aggravated disease mortality. Our study uncovers how a fundamental immunoregulatory mechanism mediates the high disease susceptibility to invasive candidiasis. Furthermore, interferon-stimulated IL-1Ra secretion may exacerbate fungal dissemination in human patients with secondary candidemia. Macrophage-secreted IL-1Ra should be considered as an additional biomarker and potential therapeutic target in severe systemic candidiasis.

Memory phase-specific genes in the Mushroom Bodies identified using CrebB-target DamID.

The formation of long-term memories requires changes in the transcriptional program and de novo protein synthesis. One of the critical regulators for long-term memory (LTM) formation and maintenance is the transcription factor CREB. Genetic studies have dissected the requirement of CREB activity within memory circuits, however less is known about the genetic mechanisms acting downstream of CREB and how they may contribute defining LTM phases. To better understand the downstream mechanisms, we here used a targeted DamID approach (TaDa). We generated a CREB-Dam fusion protein using the fruit fly Drosophila melanogaster as model. Expressing CREB-Dam in the mushroom bodies (MBs), a brain center implicated in olfactory memory formation, we identified genes that are differentially expressed between paired and unpaired appetitive training paradigm. Of those genes we selected candidates for an RNAi screen in which we identified genes causing increased or decreased LTM.

Parallels between oncogene-driven cardiac hyperplasia and heart regeneration in zebrafish.

The human heart is poorly regenerative and cardiac tumors are extremely rare. Whether the adult zebrafish myocardium is responsive to oncogene overexpression and how this condition affects its intrinsic regenerative capacity remains unknown. Here, we have established a strategy of inducible and reversible expression of HRASG12V in zebrafish cardiomyocytes. This approach stimulated a hyperplastic cardiac enlargement within 16 days. The phenotype was suppressed by rapamycin-mediated inhibition of TOR signaling. As TOR signaling is also required for heart restoration after cryoinjury, we compared transcriptomes of hyperplastic and regenerating ventricles. Both conditions were associated with upregulation of cardiomyocyte dedifferentiation and proliferation factors, as well as with similar microenvironmental responses, such as deposition of nonfibrillar Collagen XII and recruitment of immune cells. Among the differentially expressed genes, many proteasome and cell-cycle regulators were upregulated only in oncogene-expressing hearts. Preconditioning of the heart with short-term oncogene expression accelerated cardiac regeneration after cryoinjury, revealing a beneficial synergism between both programs. Identification of the molecular bases underlying the interplay between detrimental hyperplasia and advantageous regeneration provides new insights into cardiac plasticity in adult zebrafish.

The regeneration-responsive element careg monitors activation of Müller glia after MNU-induced damage of photoreceptors in the zebrafish retina.

In contrast to mammals, zebrafish can regenerate their damaged photoreceptors. This capacity depends on the intrinsic plasticity of Müller glia (MG). Here, we identified that the transgenic reporter careg, a marker of regenerating fin and heart, also participates in retina restoration in zebrafish. After methylnitrosourea (MNU) treatment, the retina became deteriorated and contained damaged cell types including rods, UV-sensitive cones and the outer plexiform layer. This phenotype was associated with the induction of careg expression in a subset of MG until the reconstruction of the photoreceptor synaptic layer. Single-cell RNA sequencing (scRNAseq) analysis of regenerating retinas revealed a population of immature rods, defined by high expression of rhodopsin and the ciliogenesis gene meig1, but low expression of phototransduction genes. Furthermore, cones displayed deregulation of metabolic and visual perception genes in response to retina injury. Comparison between careg:EGFP expressing and non-expressing MG demonstrated that these two subpopulations are characterized by distinct molecular signatures, suggesting their heterogenous responsiveness to the regenerative program. Dynamics of ribosomal protein S6 phosphorylation showed that TOR signaling became progressively switched from MG to progenitors. Inhibition of TOR with rapamycin reduced the cell cycle activity, but neither affected careg:EGFP expression in MG, nor prevented restoration of the retina structure. This indicates that MG reprogramming, and progenitor cell proliferation might be regulated by distinct mechanisms. In conclusion, the careg reporter detects activated MG, and provides a common marker of regeneration-competent cells in diverse zebrafish organs, including the retina.

Phylogeography of Francisella tularensis subspecies holarctica and epidemiology of tularemia in Switzerland.

Tularemia, an endemic disease that mainly affects wild animals and humans, is caused by Francisella tularensis subsp. holarctica (Fth) in Switzerland. The Swiss Fth population consist of multiple different subclades which are distributed throughout the country. The aim of this study is to characterize the genetic diversity of Fth in Switzerland and to describe the phylogeographic relationship of isolates by single nucleotide polymorphism (SNP) analysis. This analysis is combined with human surveillance data from reported cases over the last 10 years and in vitro and in silico antibiotic resistance tests to provide insight into the epidemiology of tularemia in Switzerland. We sequenced the whole genomes of 52 Fth strains of human or tick origin collected in Switzerland between 2009 and 2022 and analyzed together with all publicly available sequencing data of Swiss and European Fth. Next, we performed a preliminary classification with the established canonical single nucleotide polymorphism nomenclature. Furthermore, we tested 20 isolates from all main Swiss clades for antimicrobial susceptibility against a panel of antimicrobial agents. All 52 sequenced isolates from Switzerland belong to major clade B.6, specifically subclades B.45 and B.46, previously described in Western Europe. We were able to accurately reconstruct the population structure according to the global phylogenetic framework. No resistance to clinically recommended antibiotics could be identified in vitro or in silico in the western B.6 strains.

Molecular characterization of cell types in the squid Loligo vulgaris.

Cephalopods are set apart from other mollusks by their advanced behavioral abilities and the complexity of their nervous systems. Because of the great evolutionary distance that separates vertebrates from cephalopods, it is evident that higher cognitive features have evolved separately in these clades despite the similarities that they share. Alongside their complex behavioral abilities, cephalopods have evolved specialized cells and tissues, such as the chromatophores for camouflage or suckers to grasp prey. Despite significant progress in genome and transcriptome sequencing, the molecular identities of cell types in cephalopods remain largely unknown. We here combine single-cell transcriptomics with in situ gene expression analysis to uncover cell type diversity in the European squid Loligo vulgaris. We describe cell types that are conserved with other phyla such as neurons, muscles, or connective tissues but also cephalopod-specific cells, such as chromatophores or sucker cells. Moreover, we investigate major components of the squid nervous system including progenitor and developing cells, differentiated cells of the brain and optic lobes, as well as sensory systems of the head. Our study provides a molecular assessment for conserved and novel cell types in cephalopods and a framework for mapping the nervous system of L. vulgaris.

OpenGenomeBrowser: a versatile, dataset-independent and scalable web platform for genome data management and comparative genomics.

BACKGROUND: As the amount of genomic data continues to grow, there is an increasing need for systematic ways to organize, explore, compare, analyze and share this data. Despite this, there is a lack of suitable platforms to meet this need. RESULTS: OpenGenomeBrowser is a self-hostable, open-source platform to manage access to genomic data and drastically simplifying comparative genomics analyses. It enables users to interactively generate phylogenetic trees, compare gene loci, browse biochemical pathways, perform gene trait matching, create dot plots, execute BLAST searches, and access the data. It features a flexible user management system, and its modular folder structure enables the organization of genomic data and metadata, and to automate analyses. We tested OpenGenomeBrowser with bacterial, archaeal and yeast genomes. We provide a docker container to make installation and hosting simple. The source code, documentation, tutorials for OpenGenomeBrowser are available at opengenomebrowser.github.io and a demo server is freely accessible at opengenomebrowser.bioinformatics.unibe.ch . CONCLUSIONS: To our knowledge, OpenGenomeBrowser is the first self-hostable, database-independent comparative genome browser. It drastically simplifies commonly used bioinformatics workflows and enables convenient as well as fast data exploration.

2022 taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Monocyte biology conserved across species: Functional insights from cattle.

Similar to human monocytes, bovine monocytes can be split into CD14highCD16- classical, CD14highCD16high intermediate and CD14-/dimCD16high nonclassical monocytes (cM, intM, and ncM, respectively). Here, we present an in-depth analysis of their steady-state bulk- and single-cell transcriptomes, highlighting both pronounced functional specializations and transcriptomic relatedness. Bulk gene transcription indicates pro-inflammatory and antibacterial roles of cM, while ncM and intM appear to be specialized in regulatory/anti-inflammatory functions and tissue repair, as well as antiviral responses and T-cell immunomodulation. Notably, intM stood out by high expression of several genes associated with antigen presentation. Anti-inflammatory and antiviral functions of ncM are further supported by dominant oxidative phosphorylation and selective strong responses to TLR7/8 ligands, respectively. Moreover, single-cell RNA-seq revealed previously unappreciated heterogeneity within cM and proposes intM as a transient differentiation intermediate between cM and ncM.

Extensive diversity and rapid turnover of phage defense repertoires in cheese-associated bacterial communities.

BACKGROUND: Phages are key drivers of genomic diversity in bacterial populations as they impose strong selective pressure on the evolution of bacterial defense mechanisms across closely related strains. The pan-immunity model suggests that such diversity is maintained because the effective immune system of a bacterial species is the one distributed across all strains present in the community. However, only few studies have analyzed the distribution of bacterial defense systems at the community-level, mostly focusing on CRISPR and comparing samples from complex environments. Here, we studied 2778 bacterial genomes and 188 metagenomes from cheese-associated communities, which are dominated by a few bacterial taxa and occur in relatively stable environments. RESULTS: We corroborate previous laboratory findings that in cheese-associated communities nearly identical strains contain diverse and highly variable arsenals of innate and adaptive (i.e., CRISPR-Cas) immunity systems suggesting rapid turnover. CRISPR spacer abundance correlated with the abundance of matching target sequences across the metagenomes providing evidence that the identified defense repertoires are functional and under selection. While these characteristics align with the pan-immunity model, the detected CRISPR spacers only covered a subset of the phages previously identified in cheese, providing evidence that CRISPR does not enable complete immunity against all phages, and that the innate immune mechanisms may have complementary roles. CONCLUSIONS: Our findings show that the evolution of bacterial defense mechanisms is a highly dynamic process and highlight that experimentally tractable, low complexity communities such as those found in cheese, can help to understand ecological and molecular processes underlying phage-defense system relationships. These findings can have implications for the design of robust synthetic communities used in biotechnology and the food industry. Video Abstract.

Targeting lactate dehydrogenase B-dependent mitochondrial metabolism affects tumor initiating cells and inhibits tumorigenesis of non-small cell lung cancer by inducing mtDNA damage.

Once considered a waste product of anaerobic cellular metabolism, lactate has been identified as a critical regulator of tumorigenesis, maintenance, and progression. The putative primary function of lactate dehydrogenase B (LDHB) is to catalyze the conversion of lactate to pyruvate; however, its role in regulating metabolism during tumorigenesis is largely unknown. To determine whether LDHB plays a pivotal role in tumorigenesis, we performed 2D and 3D in vitro experiments, utilized a conventional xenograft tumor model, and developed a novel genetically engineered mouse model (GEMM) of non-small cell lung cancer (NSCLC), in which we combined an LDHB deletion allele with an inducible model of lung adenocarcinoma driven by the concomitant loss of p53 (also known as Trp53) and expression of oncogenic KRAS (G12D) (KP). Here, we show that epithelial-like, tumor-initiating NSCLC cells feature oxidative phosphorylation (OXPHOS) phenotype that is regulated by LDHB-mediated lactate metabolism. We show that silencing of LDHB induces persistent mitochondrial DNA damage, decreases mitochondrial respiratory complex activity and OXPHOS, resulting in reduced levels of mitochondria-dependent metabolites, e.g., TCA intermediates, amino acids, and nucleotides. Inhibition of LDHB dramatically reduced the survival of tumor-initiating cells and sphere formation in vitro, which can be partially restored by nucleotide supplementation. In addition, LDHB silencing reduced tumor initiation and growth of xenograft tumors. Furthermore, we report for the first time that homozygous deletion of LDHB significantly reduced lung tumorigenesis upon the concomitant loss of Tp53 and expression of oncogenic KRAS without considerably affecting the animal's health status, thereby identifying LDHB as a potential target for NSCLC therapy. In conclusion, our study shows for the first time that LDHB is essential for the maintenance of mitochondrial metabolism, especially nucleotide metabolism, demonstrating that LDHB is crucial for the survival and proliferation of NSCLC tumor-initiating cells and tumorigenesis.

Microbial uptake in oral mucosa-draining lymph nodes leads to rapid release of cytotoxic CD8+ T cells lacking a gut-homing phenotype.

The gastrointestinal (GI) tract constitutes an essential barrier against ingested microbes, including potential pathogens. Although immune reactions are well studied in the lower GI tract, it remains unclear how adaptive immune responses are initiated during microbial challenge of the oral mucosa (OM), the primary site of microbial encounter in the upper GI tract. Here, we identify mandibular lymph nodes (mandLNs) as sentinel lymphoid organs that intercept ingested Listeria monocytogenes (Lm). Oral Lm uptake led to local activation and release of antigen-specific CD8+ T cells that constituted most of the early circulating effector T cell (TEFF) pool. MandLN-primed TEFF disseminated to lymphoid organs, lung, and OM and contributed substantially to rapid elimination of target cells. In contrast to CD8+ TEFF generated in mesenteric LN (MLN) during intragastric infection, mandLN-primed TEFF lacked a gut-seeking phenotype, which correlated with low expression of enzymes required for gut-homing imprinting by mandLN stromal and dendritic cells. Accordingly, mandLN-primed TEFF decreased Lm burden in spleen but not MLN after intestinal infection. Our findings extend the concept of regional specialization of immune responses along the length of the GI tract, with CD8+ TEFF generated in the upper GI tract displaying homing profiles that differ from those imprinted by lymphoid tissue of the lower GI tract.

Biparatopic sybodies neutralize SARS-CoV-2 variants of concern and mitigate drug resistance.

The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS-CoV-2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo-EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri-bispecific fusion constructs that exhibit up to 100- and 1,000-fold increase in neutralization potency, respectively. Cryo-EM of the sybody-spike complex additionally reveals a novel up-out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS-CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants.

Whole-genome analyses reveal a novel prophage and cgSNPs-derived sublineages of Brachyspira hyodysenteriae ST196.

BACKGROUND: Brachyspira (B.) hyodysenteriae is a fastidious anaerobe spirochete that can cause swine dysentery, a severe mucohaemorragic colitis that affects pig production and animal welfare worldwide. In Switzerland, the population of B. hyodysenteriae is characterized by the predominance of macrolide-lincosamide-resistant B. hyodysenteriae isolates of sequence type (ST) ST196, prompting us to obtain deeper insights into the genomic structure and variability of ST196 using pangenome and whole genome variant analyses. RESULTS: The draft genome of 14 B. hyodysenteriae isolates of ST196, sampled during a 7-year period from geographically distant pig herds, was obtained by whole-genome sequencing (WGS) and compared to the complete genome of the B. hyodysenteriae isolate Bh743-7 of ST196 used as reference. Variability results revealed the existence of 30 to 52 single nucleotide polymorphisms (SNPs), resulting in eight sublineages of ST196. The pangenome analysis led to the identification of a novel prophage, pphBhCH20, of the Siphoviridae family in a single isolate of ST196, which suggests that horizontal gene transfer events may drive changes in genomic structure. CONCLUSIONS: This study contributes to the catalogue of publicly available genomes and provides relevant bioinformatic tools and information for further comparative genomic analyses for B. hyodysenteriae. It reveals that Swiss B. hyodysenteriae isolates of the same ST may have evolved independently over time by point mutations and acquisition of larger genetic elements. In line with this, the third type of mobile genetic element described so far in B. hyodysenteriae, the novel prophage pphBhCH20, has been identified in a single isolate of B. hyodysenteriae of ST196.

ACKR1 favors transcellular over paracellular T-cell diapedesis across the blood-brain barrier in neuroinflammation in vitro.

The migration of CD4+ effector/memory T cells across the blood-brain barrier (BBB) is a critical step in MS or its animal model, EAE. T-cell diapedesis across the BBB can occur paracellular, via the complex BBB tight junctions or transcellular via a pore through the brain endothelial cell body. Making use of primary mouse brain microvascular endothelial cells (pMBMECs) as in vitro model of the BBB, we here directly compared the transcriptome profile of pMBMECs favoring transcellular or paracellular T-cell diapedesis by RNA sequencing (RNA-seq). We identified the atypical chemokine receptor 1 (Ackr1) as one of the main candidate genes upregulated in pMBMECs favoring transcellular T-cell diapedesis. We confirmed upregulation of ACKR1 protein in pMBMECs promoting transcellular T-cell diapedesis and in venular endothelial cells in the CNS during EAE. Lack of endothelial ACKR1 reduced transcellular T-cell diapedesis across pMBMECs under physiological flow in vitro. Combining our previous observation that endothelial ACKR1 contributes to EAE pathogenesis by shuttling chemokines across the BBB, the present data support that ACKR1 mediated chemokine shuttling enhances transcellular T-cell diapedesis across the BBB during autoimmune neuroinflammation.