Whole blood transcriptomic analysis in ANCA-associated vasculitis

Οι ANCA σχετιζόμενες αγγειίτιδες (AAV) είναι μια ομάδα σπάνιων αυτοάνοσων, δυνητικά απειλητικών για τη ζωή νόσων, οι οποίες μπορούν να επηρεάσουν διάφορα όργανα. Χαρακτηρίζονται από νεκρωτική φλεγμονή και καταστροφή κυρίως μικρών αγγείων. Οι ΑΑV συσχετίζονται συνήθως με το ANCA που είναι ειδικό για τη μυελοϋπεροξειδάση (MPO) ή την πρωτεϊνάση 3 (PR3) και υπάρχουν φυλετικές / εθνοτικές και γεωγραφικές επιρροές στον επιπολασμό, στις συχνότητες των οροτύπων και στους κλινικοπαθολογικούς φαινοτύπους. Η παθογένεση των ασθενειών αυτών δεν είναι ακόμη πλήρως κατανοητή και η έλλειψη αποτελεσματικών μοριακών βιολογικών δεικτών συχνά εμποδίζει την ακριβή πρόγνωση και την επιτυχή διαστρωμάτωση των ασθενών. Προκειμένου να βελτιωθούν τα κριτήρια ταξινόμησης και να παρουσιαστούν νέες ιδέες για τους υποκείμενους μοριακούς μηχανισμούς, παρουσιάσαμε το μεταγραφικό προφίλ του περιφερικού αίματος 42 ασθενών με AAV και 11 υγιών άτομων χρησιμοποιώντας εργαλεία ανάλυσης αλληλουχίας RNA και εμπλουτισμού. Δείξαμε ότι η υπογραφή γονιδιακής έκφρασης στις ΑΑV χαρακτηρίζεται από εκτεταμένη απορρύθμιση των σηματοδότοδικών μονοπατιών της IFN και της αποκοκκοποίησης των ουδετερόφιλων. Έχουμε επιπλέον δείξει ότι η θετικότητα ANCA συνοδεύεται από μεταγραφικές παρεκκλίσεις σχετιζόμενες με τη μιτοφαγία που διαμεσολαβείται από το μονοπάτι της pink/parkin, τις οδούς χυμικής ανοσίας και τα γεγονότα σηματοδότησης Wnt εξαρτώμενα από τη b catenin. Οι οδούς σηματοδότησης της IFN τύπου 1 και αποκοκκιοποίησης των ουδετεροφίλων αντιπροσωπεύουν δύο ισχυρά σήματα που χαρακτηρίζουν την ενεργό νόσου. Η γονιδιακή υπογραφή που σχετίζεται με την ύφεση της νόσου απαρτίζεται από γονίδια που ρυθμίζουν τις αποκρίσεις της τύπου 1 IFN και της IFNγ και την σηματοδότηση μέσω Wnt που εξαρτάται από τη b catenin.. Η σύγκριση των μεταγραφικών προφίλ του ολικού αίματος των ασθενών με ΑΑV σε κατάσταση ενεργού νόσου έναντι ύφεσης αποκάλυψε διαφοροποιήσεις στις οδούς σηματοδότησης της IL-10, αν και δεν υπήρχε στατιστική σημαντικότητα. Επιπλέον, ερευνήσαμε εάν συγκεκριμένες γονιδιακές υπογραφές θα μπορούσαν αποτελεσματικά να διακρίνουν υποτύπους ΑΑV από τα υγιή άτομα. Μείωση της έκφρασης των γονιδίων που σχετίζονται με τη σηματοδότηση IFN και IFNγ τύπου 1, την αποκοκκίωση ουδετερόφιλων και με τη διαμεσολαβούμενη από RIK1 κυτταρικό θάνατο διαφοροποιούν μεταγραφικό προφίλ των GPA ασθενών από υγιείς μάρτυρες. Η υπογραφή γονιδιακής έκφρασης της ΜΡΑ συσχετίστηκε με διαταραχές αποκοκκοποίησης ουδετερόφιλων και απορύθμιση των σημείων ελέγχου κυτταρικού κύκλου και των μηχανισμών απόκρισης σε βλάβη του DNA. Τα γονίδια που χαρακτηρίζουν το μεταγραφικό προφίλ του περιφερειακού αίματος της EGPA εμπλουτίστηκαν κυρίως σε μονοπάτια που σχετίζονται με τις αποκρίσεις IFN τύπου 1, τους μηχανισμούς σηματοδότησης μέσω NCAM και τους μηχανισμούς ρύθμισης των Β κυττάρων. Τέλος, η PCA της ενεργού νεφρικής νόσου στην AAV και του ενεργού LN υπέδειξε ότι ξεχωριστοί παθοφυσιολογικοί μηχανισμοί διακρίνουν τις δύο κλινικές οντότητες. Συλλογικά, τα δεδομένα μας εστιάζουν στις εκτεταμένες μεταγραφικές διαταραχές που χαρακτηρίζουν την ΑΑV και δημιουργούν μία χρήσιμη δεξαμενή γονιδίων διαθέσιμα για περαιτέρω πειράματα. Τα πιο αυστηρά κριτήρια διαλογής των ασθενών και η ανάλυση του μεταγραφικου προφίλ κυττάρικών υποτύπων του περιφερικού αίματος μπορεί να ενισχύσουν την ειδικότητα των παρατηρήσεών μας. Τέλος, τα αποτελέσματά μας μπορούν να συμβάλουν στην ανάπτυξη νέων μοριακών βιοδεικτών ή να βελτιώσουν την αποτελεσματικότητα των ήδη υπαρχόντων στο χαρακτηρισμό της διάγνωσης και στην καθοδήγηση της θεραπείας.AAV is a group of rare autoimmune, potentially life-threatening diseases, which can affect several organs. These are characterized by necrotizing inflammation and destruction of predominantly small vessels. AAV are commonly associated with ANCA specific for myeloperoxidase (MPO) or proteinase 3 (PR3) and there are racial/ethnic and geographic influences on the prevalence, serotype frequencies, and clinicopathologic phenotypes. The pathogenesis of these diseases is still not fully understood and a lack of efficient molecular biomarkers often prevents precise prognosis and successful patients’ stratification. To improve classification criteria and bring new insights into underlying molecular mechanisms, whole blood transcriptome profiling of 42 AAV and 11 healthy individuals was performed using mRNA sequencing. Herein, was demonstrated that AAV is characterized by extensive deregulation of IFN signaling and neutrophil degranulation pathways. It was further shown that ANCA positivity is accompanied by transcriptional aberrations related to pink/parkin mediated mitophagy, humoral immunity pathways and Beta catenin dependent Wnt-signaling events. Type 1 IFN signaling and neutrophil degranulation pathways represent two robust signals that characterize active disease status. A remission signature is linked to genes that regulate type 1 IFN and IFNγ responses and Wnt-mediated beta catenin signaling. Comparison of whole blood transcriptional profiles of AAV patients in active versus remission status revealed alterations in IL-10 signaling pathways, although statistical significance lacked. Furthermore, it was shown whether specific gene signatures could efficiently discriminate AAV subtypes from healthy individuals. Downregulation of genes related to type 1 IFN and IFNγ signaling, neutrophil degranulation and RIK1-mediated cell death differentiated GPA patients from healthy controls. MPA subtype was associated with neutrophil degranulation disturbancies and deregulation of cell cycle checkpoints and DNA damage response mechanisms. The gene set defing EGPA was mainly enriched in pathways related to type 1 IFN responses, NCAM signaling and B cell regulation mechanisms. Finally, PCA of AAV active renal disease and active LN implied that distinct pathophysiological mechanisms distinguish the two clinical entities. Collectively, this study brings into focus the extensive transcriptional perturbations characterizing AAV and generated a useful resource available for further hypothesis generation and experimentation. More-strict patient inclusion criteria, deconvolution analysis and cell type specific transcriptional profiling might enhance the specificity of these observations. Finally, these results may contribute to novel molecular biomarkers development or improve the effectiveness of already existing ones in diagnosis characterization and therapy guidance.

TLR5 participates in the TLR4 receptor complex and promotes MyD88- dependent signaling in environmental lung injury

Lung disease causes significant morbidity and mortality, and is exacerbated by environmental injury, for example through lipopolysaccharide (LPS) or ozone (O3). Toll-like receptors (TLRs) orchestrate immune responses to injury by recognizing pathogen- or danger-associated molecular patterns. TLR4, the prototypic receptor for LPS, also mediates inflammation after O3, triggered by endogenous hyaluronan. Regulation of TLR4 signaling is incompletely understood. TLR5, the flagellin receptor, is expressed in alveolar macrophages, and regulates immune responses to environmental injury. Using in vivo animal models of TLR4-mediated inflammations (LPS, O3, hyaluronan), we show that TLR5 impacts the in vivo response to LPS, hyaluronan and O3. We demonstrate that immune cells of human carriers of a dominant negative TLR5 allele have decreased inflammatory response to O3 exposure ex vivo and LPS exposure in vitro. Using primary murine macrophages, we find that TLR5 physically associates with TLR4 and biases TLR4 signaling towards the MyD88 pathway. Our results suggest an updated paradigm for TLR4/TLR5 signaling

Intravascular heavy chain-modification of hyaluronan during endotoxic shock

During inflammation, the covalent linking of the ubiquitous extracellular polysaccharide hyaluronan (HA) with the heavy chains (HC) of the serum protein inter alpha inhibitor (IαI) is exclusively mediated by the enzyme tumor necrosis factor α (TNFα)-stimulated-gene-6 (TSG-6). While significant advances have been made regarding how HC-modified HA (HC-HA) is an important regulator of inflammation, it remains unclear why HC-HA plays a critical role in promoting survival in intraperitoneal lipopolysaccharide (LPS)-induced endotoxemia while exerting only a modest role in the outcomes following intratracheal exposure to LPS. To address this gap, the two models of intraperitoneal LPS-induced endotoxic shock and intratracheal LPS-induced acute lung injury were directly compared in TSG-6 knockout mice and littermate controls. HC-HA formation, endogenous TSG-6 activity, and inflammatory markers were assessed in plasma and lung tissue. TSG-6 knockout mice exhibited accelerated mortality during endotoxic shock. While both intraperitoneal and intratracheal LPS induced HC-HA formation in lung parenchyma, only systemically-induced endotoxemia increased plasma TSG-6 levels and intravascular HC-HA formation. Cultured human lung microvascular endothelial cells secreted TSG-6 in response to both TNFα and IL1β stimulation, indicating that, in addition to inflammatory cells, the endothelium may secrete TSG-6 into circulation during systemic inflammation. These data show for the first time that LPS-induced systemic inflammation is uniquely characterized by significant vascular induction of TSG-6 and HC-HA, which may contribute to improved outcomes of endotoxemia

Hyaluronan Signaling during Ozone-Induced Lung Injury Requires TLR4, MyD88, and TIRAP

Ozone exposure is associated with exacerbation of reactive airways disease. We have previously reported that the damage-associated molecular pattern, hyaluronan, is required for the complete biological response to ambient ozone and that hyaluronan fragments signal through toll-like receptor 4 (TLR4). In this study, we further investigated the role of TLR4 adaptors in ozone–induced airway hyperresponsiveness (AHR) and the direct response to hyaluronan fragments (HA). Using a murine model of AHR, C57BL/6J, TLR4−/−, MyD88−/−, and TIRAP−/− mice were characterized for AHR after exposure to either ozone (1 ppm×3 h) or HA fragments. Animals were characterized for AHR with methacholine challenge, cellular inflammation, lung injury, and production of pro-inflammatory cytokines. Ozone-exposed C57BL/6J mice developed cellular inflammation, lung injury, pro-inflammatory cytokines, and AHR, while mice deficient in TLR4, MyD88 or TIRAP demonstrated both reduced AHR and reduced levels of pro-inflammatory cytokines including TNFα, IL-1β, MCP-1, IL-6 and KC. The level of hyaluronan was increased after inhalation of ozone in each strain of mice. Direct challenge of mice to hyaluronan resulted in AHR in C57BL/6J mice, but not in TLR4−/−, MyD88−/−, or TIRAP−/− mice. HA-induced cytokine production in wild-type mice was significantly reduced in TLR4−/−, MyD88−/−, or TIRAP−/− mice. In conclusion, our findings support that ozone-induced airway hyperresponsiveness is dependent on the HA-TLR4-MyD88-TIRAP signaling pathway

Size Matters: Molecular Weight Specificity of Hyaluronan Effects in Cell Biology

Hyaluronan signaling properties are unique among other biologically active molecules, that they are apparently not influenced by postsynthetic molecular modification, but by hyaluronan fragment size. This review summarizes the current knowledge about the generation of hyaluronan fragments of different size and size-dependent differences in hyaluronan signaling as well as their downstream biological effects

Size Matters: Molecular Weight Specificity of Hyaluronan Effects in Cell Biology

Hyaluronan signaling properties are unique among other biologically active molecules, that they are apparently not influenced by postsynthetic molecular modification, but by hyaluronan fragment size. This review summarizes the current knowledge about the generation of hyaluronan fragments of different size and size-dependent differences in hyaluronan signaling as well as their downstream biological effects

The Environmental Polymorphism Registry: A Unique Resource that Facilitates Translational Research of Environmental Disease

Background: Dissecting complex disease has become more feasible because of the availability of large-scale DNA resources and advances in high-throughput genomic technology. Although these tools help scientists identify potential susceptibility loci, subjects with relevant genotypes are needed for clinical phenotyping and toxicity studies

Impaired Ciliogenesis in differentiating human bronchial epithelia exposed to non-Cytotoxic doses of multi-walled carbon Nanotubes

Abstract Background Multi-walled carbon nanotubes (MWCNTs) are engineered nanomaterials used for a variety of industrial and consumer products. Their high tensile strength, hydrophobicity, and semi-conductive properties have enabled many novel applications, increasing the possibility of accidental nanotube inhalation by either consumers or factory workers. While MWCNT inhalation has been previously shown to cause inflammation and pulmonary fibrosis at high doses, the susceptibility of differentiating bronchial epithelia to MWCNT exposure remains unexplored. In this study, we investigate the effect of MWCNT exposure on cilia development in a differentiating air-liquid interface (ALI) model. Primary bronchial epithelial cells (BECs) were isolated from human donors via bronchoscopy and treated with non-cytotoxic doses of MWCNTs in submerged culture for 24 h. Cultures were then allowed to differentiate in ALI for 28 days in the absence of further MWCNT exposure. At 28 days, mucociliary differentiation endpoints were assessed, including whole-mount immunofluorescent staining, histological, immunohistochemical and ultrastructural analysis, gene expression, and cilia beating analysis. Results We found a reduction in the prevalence and beating of ciliated cells in MWCNT-treated cultures, which appeared to be caused by a disruption of cellular microtubules and cytoskeleton during ciliogenesis and basal body docking. Expression of gene markers of mucociliary differentiation, such as FOXJ1 and MUC5AC/B, were not affected by treatment. Colocalization of basal body marker CEP164 with γ-tubulin during days 1–3 of ciliogenesis, as well as abundance of basal bodies up to day 14, were attenuated by treatment with MWCNTs. Conclusions Our results suggest that a single exposure of bronchial cells to MWCNT during a vulnerable period before differentiation may impair their ability to develop into fully functional ciliated cells