Regulation of neutrophil function by selective targeting of glycan epitopes expressed on the integrin CD11b/CD18

Polymorphonuclear neutrophils (PMNs) play a critical role in the innate immune response to invading pathogens. However, dysregulated mucosal trafficking of PMNs and associated epithelial tissue damage is a pathological hallmark of numerous inflammatory conditions including inflammatory bowel disease. The glycoprotein CD11b/CD18 plays a well‐described role in regulating PMN transepithelial migration and PMN inflammatory functions. Previous studies have demonstrated that targeting of the N‐linked glycan Lewis X on CD11b blocks PMN transepithelial migration (TEpM). Given evidence of glycosylation‐dependent regulation of CD11b/CD18 function, we performed MALDI TOF Mass Spectrometry (MS) analyses on CD11b/CD18 purified from human PMNs. Unusual glycan epitopes identified on CD11b/CD18 included high Mannose oligosaccharides recognized by the Galanthus Nivalis lectin and biantennary galactosylated N‐glycans recognized by the Phaseolus Vulgaris erythroagglutinin lectin. Importantly, we show that selective targeting of glycans on CD11b with such lectins results in altered intracellular signaling events that inhibit TEpM and differentially affect key PMN inflammatory functions including phagocytosis, superoxide release and apoptosis. Taken together, these data demonstrate that discrete glycan motifs expressed on CD11b/CD18 such as biantennary galactose could represent novel targets for selective manipulation of CD11b function and reduction of PMN‐associated tissue damage in chronic inflammatory diseases.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154461/1/fsb220152-sup-0003-FigS3.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154461/2/fsb220152_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154461/3/fsb220152-sup-0004-TableS1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154461/4/fsb220152-sup-0001-FigS1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154461/5/fsb220152.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154461/6/fsb220152-sup-0002-FigS2.pd

The EcoChip : a wireless multi-sensor platform for comprehensive environmental monitoring

This paper presents the EcoChip, a new system based on state-of-the-art electro-chemical impedance (EIS) technologies allowing the growth of single strain organisms isolated from northern habitats. This portable system is a complete and autonomous wireless platform designed to monitor and cultivate microorganisms directly sampled from their natural environment, particularly from harsh northern environments. Using 96-well plates, the EcoChip can be used in the field for realtime monitoring of bacterial growth. Manufactured with highquality electronic components, this new EIS monitoring system is designed to function at a low excitation voltage signal to avoid damaging the cultured cells. The high-precision calibration network leads to high-precision results, even in the most limiting contexts. Luminosity, humidity and temperature can also be monitored with the addition of appropriate sensors. Access to robust data storage systems and power supplies is an obvious limitation for northern research. That is why the EcoChip is equipped with a flash memory that can store data over long periods of time. To resolve the power issue, a low-power microcontroller and a power management unit control and supply all electronic building blocks. Data stored in the EcoChip’s flash memory can be transmitted through a transceiver whenever a receiver is located within the functional transmission range. In this paper, we present the measured performance of the system, along with results from laboratory tests in-vitro and from two field tests. The EcoChip has been utilized to collect bio-environemental data in the field from the northern soils and ecosystems of Kuujjuarapik and Puvirnituq, during two expeditions, in 2017 and 2018, respectively. We show that the EcoChip can effectively carry out EIS analyses over an excitation frequency ranging from 750 Hz to 10 kHz with an accuracy of 2.35%. The overall power consumption of the system was 140.4 mW in normal operating mode and 81 µW in sleep mode. The proper development of the isolated bacteria was confirmed through DNA sequencing, indicating that bacteria thrive in the EcoChip’s culture wells while the growing conditions are successfully gathered and stored

Study of the expression and the tissue-specific transcriptional regulation mechanisms of the ST6GAL2 gene

La sialylation est l’une des dernières étapes de la biosynthèse des chaînes glycaniques des glycoprotéines et des glycolipides. La sialylation en a2,6 des structures N-acétyllactosaminiques (Galß1-4GlcNAc) est souvent retrouvée en périphérie des glycannes et est impliquée dans de nombreux mécanismes d’adhésion et de reconnaissance cellule / cellule ou hôte / pathogène. Chez l’Homme, deux sialyltransférases synthétisent ce type d’épitope glycanique : hST6Gal I et hST6Gal II. Elles se distinguent par leur spécificité de substrat accepteur et par leur profil d’expression tissulaire. Alors que le gène ST6GAL1 codant hST6Gal I est exprimé dans la plupart des tissus, ST6GAL2 présente une expression tissulaire plus restreinte, se limitant essentiellement au cerveau embryonnaire et adulte. Par ailleurs, hST6Gal II présente des similitudes en termes de spécificité de substrat et d’expression tissulaire avec la sialyltransférase identifiée chez D. melanogaster et semble avoir conservé certaines propriétés ancestrales essentielles pour le développement du tissu nerveux. Plusieurs études ont montré que l’expression des sialyltransférases est contrôlée au niveau transcriptionnel par l’utilisation de promoteurs tissulaires régulant l’expression de manière tissu-spécifique. Si les données concernant ST6GAL2 sont encore limitées, il apparaît cependant que l’expression de ce gène est finement contrôlée par des mécanismes apparemment conservés au cours de l’évolution. Le projet de thèse que nous avons mené a eu pour but d’identifier les régions 5’-non traduites de ST6GAL2 et de caractériser les régions promotrices associées. A partir d’un modèle cellulaire de neuroblastome en culture, nous avons identifié par 5’ RACE trois types de transcrits qui diffèrent par leur premier exon non traduit. Ces exons, appelés EX, EY et EZ, sont situés à plus de 42 kpb du premier exon commun codant et ne sont séparés que de 124 et 87 pb, respectivement. Par Q-PCR en duplex avec le gène normalisateur HPRT, nous avons montré que les transcrits initiés par l’exon EX et EY étaient prépondérants par rapport aux transcrits contenant EZ, à la fois dans plusieurs lignées cellulaires à caractère neuronal et dans des échantillons de tissu cérébral humain. Nous avons également montré que la protéine hST6Gal II est exprimée dans les différents lobes du cortex cérébral, dans le cervelet et dans l’hippocampe. Nous avons isolé différentes régions génomiques situées en amont et à l’intérieur de la région EX/EY/EZ que nous avons sous cloné en amont du gène de la luciférase pour des tests d’activité. Nous avons défini deux régions promotrices, en amont des exons EX et EY. Des expériences de mutagenèse dirigée couplées à des analyses bioinformatiques nous ont révélé que les facteurs de transcription NF-?B et NRSF sont probablement des répresseurs de la transcription, alors que les facteurs Sox5, SP1, Pura et Olf1 agirait comme des éléments activateurs de la transcription de ST6GAL2. Les facteurs NRSF, Sox5, Pura et Olf1 régulent notamment la transcription de gènes impliqués dans le fonctionnement et le développement neuronal, suggérant un rôle de ST6GAL2 dans les fonctions neuronales. Enfin, nous avons mis en évidence une forte augmentation de l’expression ST6GAL2 au cours de la différentiation en neurones des cellules NT2/D1 sous l’action de l’acide rétinoïque, suggérant un rôle potentiel de cette enzyme au cours de la différentiation neuronale.Sialylation is one of the last step of the biosynthesis of glycan chains carried by glycoproteins and glycolipids. The a2,6-sialylation of N-acetyllactosaminyl (Galß1-4GlcNAc) structures is commonly found at the end of glycan chains and is involved in numerous cell / cell or host / pathogen adhesion and recognition events. In Human, two sialyltransferases synthesise this glycan epitope, namely hST6Gal I and hST6Gal II. They differ from each other in substrate specificity an in tissue-specific pattern of expression. Whereas the gene encoding hST6Gal I, ST6GAL1, is expressed in almost all tissues, ST6GAL2 shows a narrower pattern of tissue expression essentially limited to fetal and adult brain. In addition, hST6Gal II exhibits similarities in terms of substrate specificity and gene expression pattern with the sialyltransferase identified in D. melanogaster and therefore, seems to have conserved ancestral properties required for brain function and growing nervous tissue. Several studies have shown that the expression of sialyltransferases is controlled at the transcriptional level by the use of specific promoters that regulate their expression in a tissue-specific fashion. Data about ST6GAL2 are rather limited; however, it appears the expression of this gene is finely regulated by mechanisms likely conserved through evolution. The aim of this thesis was to identify the 5’ non translated regions of the ST6GAL2 gene and to characterize the associated promoter regions. From a neuroblastoma cultured cell model, we identified by 5’RACE three types of transcripts which are different only in their first non-coding exon. Those exons, named EX, EY and EZ, are located more then 42 kbp upstream of the first common coding exon and are only separated by 124 and 87 bp, respectively. Using Taqman duplex Q-PCR technology we have shown that the transcripts initiated by EX and EY are predominantly expressed compared to EZ both in several cell lines and in human brain tissue samples. We also demonstrated that the hST6Gal II protein is expressed in the different lobes of the human cerebral cortex, the cerebellum and the hippocampus. We isolated different genomic sequences upstream EX and within EX/EY/EZ region and inserted them in a reporter vector for luciferase assays. We could define two promoter sequences upstream EX and ZY. PCR site-directed mutagenesis experiments along with bioinformatics analysis revealed that transcription factors NF-?B and NRSF are likely to act as transcription inhibitors, whereas the Sox5, SP1, Pura and Olf1 factors would be involved in the transcriptional activation of ST6GAL2. The NRSF, Sox5, Pura and Olf1 transcription factors are notably involved in the transcriptional regulation of genes related to neuronal functions and the neuronal development. Eventually, we have shown evidence of a strong increased ST6GAL2 expression during neuronal differentiation of the NT2/D1 cell line under acid retinoic treatment, suggesting of putative role this enzyme in neuronal differentiation

Étude de l'expression et des mécanismes de régulation transcriptionnelle tissu-spécifique du gène ST6GAL2

La sialylation est l une des dernières étapes de la biosynthèse des chaînes glycaniques des glycoprotéines et des glycolipides. La sialylation en a2,6 des structures N-acétyllactosaminiques (Galß1-4GlcNAc) est souvent retrouvée en périphérie des glycannes et est impliquée dans de nombreux mécanismes d adhésion et de reconnaissance cellule / cellule ou hôte / pathogène. Chez l Homme, deux sialyltransférases synthétisent ce type d épitope glycanique : hST6Gal I et hST6Gal II. Elles se distinguent par leur spécificité de substrat accepteur et par leur profil d expression tissulaire. Alors que le gène ST6GAL1 codant hST6Gal I est exprimé dans la plupart des tissus, ST6GAL2 présente une expression tissulaire plus restreinte, se limitant essentiellement au cerveau embryonnaire et adulte. Par ailleurs, hST6Gal II présente des similitudes en termes de spécificité de substrat et d expression tissulaire avec la sialyltransférase identifiée chez D. melanogaster et semble avoir conservé certaines propriétés ancestrales essentielles pour le développement du tissu nerveux. Plusieurs études ont montré que l expression des sialyltransférases est contrôlée au niveau transcriptionnel par l utilisation de promoteurs tissulaires régulant l expression de manière tissu-spécifique. Si les données concernant ST6GAL2 sont encore limitées, il apparaît cependant que l expression de ce gène est finement contrôlée par des mécanismes apparemment conservés au cours de l évolution. Le projet de thèse que nous avons mené a eu pour but d identifier les régions 5 -non traduites de ST6GAL2 et de caractériser les régions promotrices associées. A partir d un modèle cellulaire de neuroblastome en culture, nous avons identifié par 5 RACE trois types de transcrits qui diffèrent par leur premier exon non traduit. Ces exons, appelés EX, EY et EZ, sont situés à plus de 42 kpb du premier exon commun codant et ne sont séparés que de 124 et 87 pb, respectivement. Par Q-PCR en duplex avec le gène normalisateur HPRT, nous avons montré que les transcrits initiés par l exon EX et EY étaient prépondérants par rapport aux transcrits contenant EZ, à la fois dans plusieurs lignées cellulaires à caractère neuronal et dans des échantillons de tissu cérébral humain. Nous avons également montré que la protéine hST6Gal II est exprimée dans les différents lobes du cortex cérébral, dans le cervelet et dans l hippocampe. Nous avons isolé différentes régions génomiques situées en amont et à l intérieur de la région EX/EY/EZ que nous avons sous cloné en amont du gène de la luciférase pour des tests d activité. Nous avons défini deux régions promotrices, en amont des exons EX et EY. Des expériences de mutagenèse dirigée couplées à des analyses bioinformatiques nous ont révélé que les facteurs de transcription NF-?B et NRSF sont probablement des répresseurs de la transcription, alors que les facteurs Sox5, SP1, Pura et Olf1 agirait comme des éléments activateurs de la transcription de ST6GAL2. Les facteurs NRSF, Sox5, Pura et Olf1 régulent notamment la transcription de gènes impliqués dans le fonctionnement et le développement neuronal, suggérant un rôle de ST6GAL2 dans les fonctions neuronales. Enfin, nous avons mis en évidence une forte augmentation de l expression ST6GAL2 au cours de la différentiation en neurones des cellules NT2/D1 sous l action de l acide rétinoïque, suggérant un rôle potentiel de cette enzyme au cours de la différentiation neuronale.Sialylation is one of the last step of the biosynthesis of glycan chains carried by glycoproteins and glycolipids. The a2,6-sialylation of N-acetyllactosaminyl (Galß1-4GlcNAc) structures is commonly found at the end of glycan chains and is involved in numerous cell / cell or host / pathogen adhesion and recognition events. In Human, two sialyltransferases synthesise this glycan epitope, namely hST6Gal I and hST6Gal II. They differ from each other in substrate specificity an in tissue-specific pattern of expression. Whereas the gene encoding hST6Gal I, ST6GAL1, is expressed in almost all tissues, ST6GAL2 shows a narrower pattern of tissue expression essentially limited to fetal and adult brain. In addition, hST6Gal II exhibits similarities in terms of substrate specificity and gene expression pattern with the sialyltransferase identified in D. melanogaster and therefore, seems to have conserved ancestral properties required for brain function and growing nervous tissue. Several studies have shown that the expression of sialyltransferases is controlled at the transcriptional level by the use of specific promoters that regulate their expression in a tissue-specific fashion. Data about ST6GAL2 are rather limited; however, it appears the expression of this gene is finely regulated by mechanisms likely conserved through evolution. The aim of this thesis was to identify the 5 non translated regions of the ST6GAL2 gene and to characterize the associated promoter regions. From a neuroblastoma cultured cell model, we identified by 5 RACE three types of transcripts which are different only in their first non-coding exon. Those exons, named EX, EY and EZ, are located more then 42 kbp upstream of the first common coding exon and are only separated by 124 and 87 bp, respectively. Using Taqman duplex Q-PCR technology we have shown that the transcripts initiated by EX and EY are predominantly expressed compared to EZ both in several cell lines and in human brain tissue samples. We also demonstrated that the hST6Gal II protein is expressed in the different lobes of the human cerebral cortex, the cerebellum and the hippocampus. We isolated different genomic sequences upstream EX and within EX/EY/EZ region and inserted them in a reporter vector for luciferase assays. We could define two promoter sequences upstream EX and ZY. PCR site-directed mutagenesis experiments along with bioinformatics analysis revealed that transcription factors NF-?B and NRSF are likely to act as transcription inhibitors, whereas the Sox5, SP1, Pura and Olf1 factors would be involved in the transcriptional activation of ST6GAL2. The NRSF, Sox5, Pura and Olf1 transcription factors are notably involved in the transcriptional regulation of genes related to neuronal functions and the neuronal development. Eventually, we have shown evidence of a strong increased ST6GAL2 expression during neuronal differentiation of the NT2/D1 cell line under acid retinoic treatment, suggesting of putative role this enzyme in neuronal differentiation.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

La formation professionnelle et technique

La couv. porte en outre: La formation professionnelle et techniqu

Mucin Glycans Signal through the Sensor Kinase RetS to Inhibit Virulence-Associated Traits in Pseudomonas aeruginosa

© 2020 Mucus is a densely populated ecological niche that coats all non-keratinized epithelia, and plays a critical role in protecting the human body from infections. Although traditionally viewed as a physical barrier, emerging evidence suggests that mucus can directly suppress virulence-associated traits in opportunistic pathogens including Pseudomonas aeruginosa. However, the molecular mechanisms by which mucus affords this protection are unclear. Here, we show that mucins, and particularly their associated glycans, signal through the Dismed2 domain of the sensor kinase RetS in P. aeruginosa. We find that this RetS-dependent signaling leads to the direct inhibition of the GacS-GacA two-component system, the activity of which is associated with a chronic infection state. This signaling includes downregulation of the type VI secretion system (T6SS), and prevents T6SS-dependent bacterial killing by P. aeruginosa. Overall, these results shed light on how mucus impacts P. aeruginosa behavior, and may inspire novel approaches for controlling P. aeruginosa infections

Aberrantly glycosylated IgG elicits pathogenic signaling in podocytes and signifies lupus nephritis

Lupus nephritis (LN) is a serious complication occurring in 50% of patients with systemic lupus erythematosus (SLE) for which there is a lack of biomarkers, a lack of specific medications, and a lack of a clear understanding of its pathogenesis. The expression of calcium/calmodulin kinase IV (CaMK4) is increased in podocytes of patients with LN and lupus-prone mice, and its podocyte-targeted inhibition averts the development of nephritis in mice. Nephrin is a key podocyte molecule essential for the maintenance of the glomerular slit diaphragm. Here, we show that the presence of fucose on N-glycans of IgG induces, whereas the presence of galactose ameliorates, podocyte injury through CaMK4 expression. Mechanistically, CaMK4 phosphorylates NF-κB, upregulates the transcriptional repressor SNAIL, and limits the expression of nephrin. In addition, we demonstrate that increased expression of CaMK4 in biopsy specimens and in urine podocytes from people with LN is linked to active kidney disease. Our data shed light on the role of IgG glycosylation in the development of podocyte injury and propose the development of “liquid kidney biopsy” approaches to diagnose LN

Pilot Study Showing Feasibility of Phosphoproteomic Profiling of Pathway-Level Molecular Alterations in Barrett’s Esophagus

(1) Background: Barrett's esophagus is a major risk factor for esophageal adenocarcinoma. In this pilot study, we employed precision mass spectrometry to map global (phospho)protein perturbations in Barrett's esophagus lesions and adjacent normal tissue to glean insights into disease progression. (2) Methods: Biopsies were collected from two small but independent cohorts. Comparative analyses were performed between Barrett's esophagus samples and adjacent matched (normal) tissues from patients with known pathology, while specimens from healthy patients served as additional controls. (3) Results: We identified and quantified 6810 proteins and 6395 phosphosites in the discovery cohort, revealing hundreds of statistically significant differences in protein abundances and phosphorylation states. We identified a robust proteomic signature that accurately classified the disease status of samples from the independent patient cohorts. Pathway-level analysis of the phosphoproteomic profiles revealed the dysregulation of specific cellular processes, including DNA repair, in Barrett's esophagus relative to paired controls. Comparative analysis with previously published transcriptomic profiles provided independent evidence in support of these preliminary findings. (4) Conclusions: This pilot study establishes the feasibility of using unbiased quantitative phosphoproteomics to identify molecular perturbations associated with disease progression in Barrett's esophagus to define potentially clinically actionable targets warranting further assessment

Cosmc controls B cell homing

Migration and homing of B cells to lymph nodes are important for B cell functions, but their regulation is poorly understood. Here, the authors show that B cell-specific deletion of Cosmc results in decreased protein O-glycosylation, loss of B cell homing to both lymphoid and nonlymphoid organs, and altered transendothelial migration implicated in this loss