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.

Lipoprotein-Associated Phospholipase A2: A Novel Contributor in Sjögren’s Syndrome-Related Lymphoma?

BackgroundB-cell non-Hodgkin’s lymphoma (B-NHL) is one of the major complications of primary Sjögren’s syndrome (SS). Chronic inflammation and macrophages in SS minor salivary glands have been previously suggested as significant predictors for lymphoma development among SS patients. Lipoprotein-associated phospholipase A2 (Lp-PLA2)—a product mainly of tissue macrophages—is found in the circulation associated with lipoproteins and has been previously involved in cardiovascular, autoimmune, and malignant diseases, including lymphoma.ObjectiveThe purpose of the current study was to investigate the contributory role of Lp-PLA2 in B-NHL development in the setting of primary SS.MethodsLp-PLA2 activity in serum samples collected from 50 primary SS patients with no lymphoma (SS-nL), 9 primary SS patients with lymphoma (SS-L), and 42 healthy controls (HC) was determined by detection of [3H]PAF degradation products by liquid scintillation counter. Moreover, additional sera from 50 SS-nL, 28 SS-L, and 32 HC were tested for Lp-PLA2 activity using a commercially available ELISA kit. Lp-PLA2 mRNA, and protein expression in minor salivary gland (MSG) tissue samples derived from SS-nL, SS-L patients, and sicca controls (SC) were analyzed by real-time PCR, Western blot, and immunohistochemistry.ResultsSerum Lp-PLA2 activity was significantly increased in SS-L compared to both SS-nL and HC by two independent methods implemented [mean ± SD (nmol/min/ml): 62.0 ± 13.4 vs 47.6 ± 14.4 vs 50.7 ± 16.6, p-values: 0.003 and 0.04, respectively, and 19.4 ± 4.5 vs 15.2 ± 3.3 vs 14.5 ± 3.0, p-values: <0.0001, in both comparisons]. ROC analysis revealed that the serum Lp-PLA2 activity measured either by radioimmunoassay or ELISA has the potential to distinguish between SS-L and SS-nL patients (area under the curve [AUC]: 0.8022, CI [95%]: 0.64–0.96, p-value: 0.004 for radioimmunoassay, and AUC: 0.7696, CI [95%]: 0.66–0.88, p-value: <0.0001, for ELISA). Lp-PLA2 expression in MSG tissues was also increased in SS-L compared to SS-nL and SC at both mRNA and protein level. ROC analysis revealed that both MSG mRNA and protein Lp-PLA2 have the potential to distinguish between SS-nL and SS-L patients (area under the curve [AUC] values of 0.8490, CI [95%]: 0.71–0.99, p-value: 0.0019 and 0.9444, CI [95%]: 0.79–1.00, p- value: 0.0389 respectively). No significant difference in either serum Lp-PLA2 activity or MSG tissue expression was observed between SS-nL and HC.ConclusionsLp-PLA2 serum activity and MSG tissue mRNA/protein expression could be a new biomarker and possibly a novel therapeutic target for B-cell lymphoproliferation in the setting of SS

The genomic landscape of ANCA-associated vasculitis: Distinct transcriptional signatures, molecular endotypes and comparison with systemic lupus erythematosus

IntroductionAnti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs) present with a complex phenotype and are associated with high mortality and multi-organ involvement. We sought to define the transcriptional landscape and molecular endotypes of AAVs and compare it to systemic lupus erythematosus (SLE).MethodsWe performed whole blood mRNA sequencing from 30 patients with AAV (granulomatosis with polyangiitis/GPA and microscopic polyangiitis/MPA) combined with functional enrichment and network analysis for aberrant pathways. Key genes and pathways were validated in an independent cohort of 18 AAV patients. Co-expression network and hierarchical clustering analysis, identified molecular endotypes. Multi-level transcriptional overlap analysis to SLE was based on our published data from 142 patients.ResultsWe report here that “Pan-vasculitis” signature contained 1,982 differentially expressed genes, enriched in leukocyte differentiation, cytokine signaling, type I and type II IFN signaling and aberrant B-T cell immunity. Active disease was characterized by signatures linked to cell cycle checkpoints and metabolism pathways, whereas ANCA-positive patients exhibited a humoral immunity transcriptional fingerprint. Differential expression analysis of GPA and MPA yielded an IFN-g pathway (in addition to a type I IFN) in the former and aberrant expression of genes related to autophagy and mRNA splicing in the latter. Unsupervised molecular taxonomy analysis revealed four endotypes with neutrophil degranulation, aberrant metabolism and B-cell responses as potential mechanistic drivers. Transcriptional perturbations and molecular heterogeneity were more pronounced in SLE. Molecular analysis and data-driven clustering of AAV uncovered distinct transcriptional pathways that could be exploited for targeted therapy.DiscussionWe conclude that transcriptomic analysis of AAV reveals distinct endotypes and molecular pathways that could be targeted for therapy. The AAV transcriptome is more homogenous and less fragmented compared to the SLE which may account for its superior rates of response to therapy

Gene Expression as a Guide to the Development of Novel Therapies in Primary Glomerular Diseases

Despite improvements in understanding the pathogenic mechanisms of primary glomerular diseases, therapy still remains nonspecific. We sought to identify novel therapies targeting kidney-intrinsic injury of distinct primary glomerulonephritides through computational systems biology approaches. We defined the unique transcriptional landscape within kidneys from patients with focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD), immunoglobulin A nephropathy (IgAN), membranous nephropathy (MN) and thin basement membrane nephropathy (TBMN). Differentially expressed genes were functionally annotated with enrichment analysis, and distinct biological processes and pathways implicated in each primary glomerular disease were uncovered. Finally, we identified novel drugs and small-molecule compounds that may reverse each glomerulonephritis phenotype, suggesting they should be further tested as precise therapy in primary glomerular diseases

High Comorbidity Burden in Patients with SLE: Data from the Community-Based Lupus Registry of Crete

Comorbidities and multimorbidity, often complicating the disease course of patients with chronic inflammatory rheumatic diseases, may be influenced by disease-intrinsic and extrinsic determinants including regional and social factors. We analyzed the frequency and co-segregation of self-reported comorbid diseases in a community-based Mediterranean registry of patients (n = 399) with systemic lupus erythematosus (SLE). Predictors for multimorbidity were identified by multivariable logistic regression, strongly-associated pairs of comorbidities by the Cramer’s V-statistic, and comorbidities clusters by hierarchical agglomerative clustering. Among the most prevalent comorbidities were thyroid (45.6%) and metabolic disorders (hypertension: 24.6%, dyslipidemia: 33.3%, obesity: 35.3%), followed by osteoporosis (22.3%), cardiovascular (20.8%), and allergic (20.6%) disorders. Mental comorbidities were also common, particularly depression (26.7%) and generalized anxiety disorder (10.7%). Notably, 51.0% of patients had ≥3 physical and 33.1% had ≥2 mental comorbidities, with a large fraction (n = 86) displaying multimorbidity from both domains. Sociodemographic (education level, marital status) and clinical (disease severity, neurological involvement) were independently associated with physical or mental comorbidity. Patients were grouped into five distinct clusters of variably prevalent comorbid diseases from different organs and domains, which correlated with SLE severity patterns. Conclusively, our results suggest a high multimorbidity burden in patients with SLE at the community, advocating for integrated care to optimize outcomes

Molecular Taxonomy of Systemic Lupus Erythematosus Through Data-Driven Patient Stratification: Molecular Endotypes and Cluster-Tailored Drugs

Objectives: Treatment of Systemic Lupus Erythematosus (SLE) is characterized by a largely empirical approach and relative paucity of novel compound development. We sought to stratify SLE patients based on their molecular phenotype and identify putative therapeutic compounds for each molecular fingerprint. Methods: By the use of whole blood RNA-seq data from 120 SLE patients, and in a data-driven, clinically unbiased manner, we established modules of commonly regulated genes (molecular endotypes) and re-stratified patients through hierarchical clustering. Disease activity and severity were assessed using SLEDAI-2K and Lupus Severity Index, respectively. Through an in silico drug prediction pipeline, we investigated drugs currently in use, tested in lupus clinical trials, and listed in the iLINCS prediction databases, for their ability to reverse the gene expression signatures in each molecular endotype. Drug repurposing analysis was also performed to identify perturbagens that counteract group-specific SLE signatures. Results: Molecular taxonomy identified five lupus endotypes, each characterized by a unique gene module enrichment pattern. Neutrophilic signature group consisted primarily of patients with active lupus nephritis, while the B-cell expression group included patients with constitutional features. Patients with moderate severity and serologic activity exhibited a signature enriched for metabolic processes. Mild disease was distributed in two groups, exhibiting enhanced basic cellular functions, myelopoiesis, and autophagy. Bortezomib was predicted to reverse disturbances in the “neutrophilic” cluster, azathioprine and ixazomib in the “B-cell” cluster, and fostamatinib in the “metabolic” patient subgroup. Conclusion: The clinical spectrum of SLE encompasses distinct molecular endotypes, each defined by unique pathophysiologic aberrancies potentially reversible by distinct compounds

Restoration of aberrant gene expression of monocytes in systemic lupus erythematosus via a combined transcriptome-reversal and network-based drug repurposing strategy

Abstract Background Monocytes -key regulators of the innate immune response- are actively involved in the pathogenesis of systemic lupus erythematosus (SLE). We sought to identify novel compounds that might serve as monocyte-directed targeted therapies in SLE. Results We performed mRNA sequencing in monocytes from 15 patients with active SLE and 10 healthy individuals. Disease activity was assessed with the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2 K). Leveraging the drug repurposing platforms iLINCS, CLUE and L1000CDS2, we identified perturbagens capable of reversing the SLE monocyte signature. We identified transcription factors and microRNAs (miRNAs) that regulate the transcriptome of SLE monocytes, using the TRRUST and miRWalk databases, respectively. A gene regulatory network, integrating implicated transcription factors and miRNAs was constructed, and drugs targeting central components of the network were retrieved from the DGIDb database. Inhibitors of the NF-κB pathway, compounds targeting the heat shock protein 90 (HSP90), as well as a small molecule disrupting the Pim-1/NFATc1/NLRP3 signaling axis were predicted to efficiently counteract the aberrant monocyte gene signature in SLE. An additional analysis was conducted, to enhance the specificity of our drug repurposing approach on monocytes, using the iLINCS, CLUE and L1000CDS2 platforms on publicly available datasets from circulating B-lymphocytes, CD4+ and CD8+ T-cells, derived from SLE patients. Through this approach we identified, small molecule compounds, that could potentially affect more selectively the transcriptome of SLE monocytes, such as, certain NF-κB pathway inhibitors, Pim-1 and SYK kinase inhibitors. Furthermore, according to our network-based drug repurposing approach, an IL-12/23 inhibitor and an EGFR inhibitor may represent potential drug candidates in SLE. Conclusions Application of two independent - a transcriptome-reversal and a network-based -drug repurposing strategies uncovered novel agents that might remedy transcriptional disturbances of monocytes in SLE