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Micro-RNAs of neutrophils in renal ANCA-associated vasculitis

Periodic Reporting for period 1 - MINERVA (Micro-RNAs of neutrophils in renal ANCA-associated vasculitis)

Reporting period: 2016-04-01 to 2018-03-31

Activation of neutrophils by ANCA (“Anti-Neutrophil Cytoplasm Antibodies”) and subsequent microvascular endothelial cell damage is the main feature of ANCA-associated vasculitis (AAV), a severe autoimmune disease that often targets the kidney. There is no specific treatment for AAV to date and 25% of patients with renal damage evolve towards end-stage renal disease, requiring dialysis and kidney transplantation. In addition, there is no reliable biological marker of the disease activity available, which makes the diagnostic, follow-up and treatment of patients difficult. Therefore, the identification of new therapeutic targets and non-invasive biomarkers constitutes a major clinical challenge to improve AAV patients care and to ameliorate their renal outcome.
Micro-RNAs (miRNA) are well established as critical regulators of major biological responses in various cell types, and have been shown to be involved in the pathophysiology of many diseases, including autoimmune diseases. In addition, miRNAs are actively secreted by cells within extracellular vesicles (EV) and are thus detected in the circulation and other biological fluids. Therefore, secreted miRNAs have been proposed as promising non-invasive biomarkers for a variety of human pathologies. Besides, accumulating data indicate that secreted miRNAs actively participate to intercellular cross-talks in various physiological and pathological situations. Indeed, miRNAs released by a cell within EVs can be transferred to another “recipient” cell, where they function to regulate target genes and subsequent biological responses. Recent studies have shown that neutrophils activated by ANCA release EVs that can induce EC activation and damage. This strongly suggests a pathogenic role for neutrophil-derived EVs in the development of vascular endothelial lesions during AAV. However, the mechanisms by which these EVs can induce EC damage and the pathophysiological role of miRNAs they may contain are still completely unknown. More generally, there is still very little data available about the expression and function of miRNAs in association with AAV.
The overall objective of the MINERVA project was to identify miRNAs deregulated in ANCA-activated neutrophils and released in the circulation within EVs, which participate in the development of EC activation and damage over the course of AAV.
The first objective was to identify miRNAs that are differentially expressed in ANCA-activated neutrophils and in EVs they release over the course of AAV.
The second objective was to determine if neutrophils exposed to ANCA communicate with EC through the secretion of miRNA-containing EVs.
The third objective was to analyse the function of identified miRNAs in the regulation of EC responses (activation, lesion/repair).
While miRNAs are emerging as new therapeutic tools and biomarkers for several diseases, this translational study provides the first data regarding the expression and function of neutrophil miRNAs in AAV and may therefore pave the way for novel promising miRNA-targeted therapeutic options in these patients.
During the course of this funding, we studied whether miRNA-loaded extracellular vesicles (EVs) released by neutrophils upon their activation with ANCA can target and be internalized by microvascular endothelial cells (ECs). In an in vitro model, EVs released by activated neutrophils were taken up very efficiently by ECs. This resulted in the transfer of three mature miRNAs to ECs, whose expression is normally very low or absent in quiescent ECs. The overexpression of two of these miRNAs in ECs resulted in profound endothelial damage, especially in inflammatory conditions, characterized by the induction of cell death, the inhibition of cell proliferation, and as a result, impairment of EC repair. The marked deleterious effect of one of these miRNAs on EC survival and repair could be explained in part by its capacity to inhibit the activation of several critical intracellular signaling pathways, as well as the expression of genes involved in cell migration and angiogenesis. In addition, we demonstrated that overexpression of these two miRNAs in ECs also resulted in induction of the expression of proinflammatory molecules.
Altogether, results from this work suggest a model whereby ANCA-activated neutrophils release EV that carry three miRNAs to microvascular EC, where their expression is normally very low or absent in resting state. The consequences of the induced expression of two of these miRNAs involve the induction of a pro-inflammatory response and direct microvascular damage, notably though the induction of EC death and the inhibition of endothelial repair, especially in inflammatory conditions. Thus, these results suggest that inhibition of these two miRNAs in EC may have a promising therapeutic potential for the protection of EC from neutrophil-mediated damage in AAV.
These results were presented through an oral presentation at the American Society of Nephrology Kidney Week in November 2017 (New Orleans, LA, USA), and through a poster presentation at the International Vascular Biology Meeting in June 2018 (Helsinki, Finland). A manuscript regrouping these data is currently under preparation.
During this fellowship, we also analysed miRNA expression profiles by TaqMan Low Density Arrays in neutrophils activated with ANCA in vitro. This led to the identification of 9 miRNAs that were systematically significantly deregulated upon ANCA activation. We then analysed the expression levels of these 9 miRNAs in neutrophils isolated from the blood of 10 patients with AAV either at the initial inflammatory phase at the time of diagnosis, or during the remission phase. This was done thanks to the set-up of a biocollection of samples from AAV patients, collected longitudinally throughout the evolution of their disease, in collaboration with the Internal Medicine Department at CHU de Nantes (NALVANCA biocollection). We thus identified two specific miRNAs that were increased at the time of diagnosis vs. remission. To achieve this work, we are currently enrolling additional patients and implementing the NALVANCA biocollection, in order to determine whether the expression of these miRNAs is increased in neutrophils at the time of a relapse and to increase the number of samples analysed to strengthen our results and perform statistical analyses. Eventually, this work should lead to another publication and a patent on identified miRNAs.
Results from this project are the first to identify miRNAs as major mediators of neutrophil-induced endothelial inflammation and damage associated with ANCA-vasculitis. This suggest that the therapeutic targeting of these miRNAs in EC will have important implications for the treatment of AAV. We are currently developing a mouse model of AAV to test the efficacy of such miRNA-targeted therapeutic approaches in vivo. Besides, these results have implications for the understanding of inflammatory processes in general, as we have found that the activation of neutrophils with other inflammatory stimuli also induces the release of EVs loaded with miRNAs that can be taken up by EC. Therefore, it appears that the mechanisms of neutrophil-induced EC damage identified in this work are not strictly specific to the pathophysiological mechanisms of AAV, but may also be important features of other inflammatory diseases.
Confocal microscopy image of endothelial cells that have internalized extracellular vesicles