Introduction: Deficiency of Adenosine deaminase 2 (DADA2) is a monogenic autoinflammatory disorder presenting a broad spectrum of clinical manifestations, including vasculitis, immunodeficiency and hematologic disease. The genetic mutations in ADA2 gene have been associated to an insufficient ADA2 activity leading to reduction in deamination of adenosine to deoxyadenosine, and a consequent accumulation of extracellular adenosine. The pathogenic mechanisms investigated so far have elucidated a skewed polarization from the M2 macrophage subtype to the proinflammatory M1 subtype with an increased production of inflammatory cytokines (TNF-α, Interferon IFN). More recently a chronic neutrophil activation and a dysregulation of NETosis, as process triggered by extracellular Adenosine, inducing TNFα secretion from Macrophages stimulated by NETs, has been implicated in the pathogenesis of DADA2. Objectives: The aim of the project is to dissect NETosis directly in neutrophils isolated from DADA2 patients and healthy controls (HDs), and to quantify suicidal and vital NETosis induced by several stimuli. To determine if NET epitopes can change depending from the inflammatory microenvironment and if protein composition of NETs is disease specific, we used quantitative proteomics approach to characterize NET proteins, released from neutrophils after different stimuli in DADA2 patients, HDs and nongenetic vasculitis. Moreover, we investigated the mechanisms of NETs removal. To verify if NETs can influence maturation or inflammatory phenotype of DCs, we characterized peripheral myeloid Dendritic cells (mDCs) and plasmocytoid DCs (pDC) in DADA2 patients and analyzed in vitro moDC maturation and cytokine production in presence of NETs. Methods: We analyzed and quantified both suicidal and vital NETosis by Imaging Flow Citometry (IFC): neutrophils isolated from peripheral blood from DADA2 patients and HDs were stimulated in vitro with different stimuli (PMA, Adenosine and LPS) to induce NETosis and were analyzed in the ImageStreamXMark II IFC equipped with a MultiMag system. We evaluated also NETs remnants and DNAse in the plasma samples by ELISA assay. We used quantitative proteomics approach to characterize NET proteins, released from neutrophils after different stimuli in DADA2 patients, HDs and nongenetic vasculitis: LC-MS/MS analyses were conducted on Orbitrap Fusion Tribrid mass spectrometer and NET protein quantification was carried out using Label-Free Quantification method. We isolated monocytes from peripheral blood with microbeads and we generated moDC after culture stimulation with LPS/NETs. On the 7th day we analyzed by flow cytometry the phenotype and the markers of differentiation. Quantification of cytokines was performed by flow cytometry bead array. Results: We enrolled 14 patients with Adenosine Deaminase 2 deficiency diagnosis who underwent blood sampling at out Center, collecting also plasma samples from further 6 genetically confirmed DADA2 patients enrolled by other Italian Pediatric Rheumatology Units. Neutrophils from DADA2 show a significant increased suicidal NETosis, identified as nuclear decondensation and Myeloperoxidase (MPO) colocalization with DNA, following PMA stimulation and we observed an increase also with LPS and Adenosine. Then we analyzed vital NETosis, identified as elongated shape of cells, nuclei polarized within the cell and not colocalized with MPO and we found an increased vital NETosis in DADA2 neutrophils. Accordingly, plasmatic levels of circulating nucleosomes (NET remnants) were elevated in patients; DNAse levels were normal but the activity was reduced. We set up experimental conditions for proteomic analysis of NETs, induced by PMA, Adenosine and TNFα, testing two patients, two HDs and two patients with non-genetic vasculitis: in total we identified 1770 proteins among which a hundred of proteins were significantly up or down-modulated in DADA2 NETs compared to controls NETs. DC phenotype in DADA2 patients result concordant with HD, as well as moDCs cytokine production after LPS stimulation. We observed a stimulatory effect of NETs towards induction of TNF alpha, IL-6 production and IP-10 from moDCs in both HD and DADA2. Conclusion: Our findings confirm a dysregulation in NETosis process in DADA2 patients, that can be induced by both LPS and adenosine. An increase of vital NETosis was also identified. Proteomic profile of NETs isolated from DADA2 is different from HD and PAN patients: NETs are qualitatively different between HD and DADA2. NETs in DADA2 may therefore interact differently with innate immunity compartment, stimulating DCs to produce citokines, contributing to the typical inflammatory phenotype.

NETosis Dysregulation in Adenosine Deaminase 2 deficiency (DADA2)

SIGNA, SARA
2022

Abstract

Introduction: Deficiency of Adenosine deaminase 2 (DADA2) is a monogenic autoinflammatory disorder presenting a broad spectrum of clinical manifestations, including vasculitis, immunodeficiency and hematologic disease. The genetic mutations in ADA2 gene have been associated to an insufficient ADA2 activity leading to reduction in deamination of adenosine to deoxyadenosine, and a consequent accumulation of extracellular adenosine. The pathogenic mechanisms investigated so far have elucidated a skewed polarization from the M2 macrophage subtype to the proinflammatory M1 subtype with an increased production of inflammatory cytokines (TNF-α, Interferon IFN). More recently a chronic neutrophil activation and a dysregulation of NETosis, as process triggered by extracellular Adenosine, inducing TNFα secretion from Macrophages stimulated by NETs, has been implicated in the pathogenesis of DADA2. Objectives: The aim of the project is to dissect NETosis directly in neutrophils isolated from DADA2 patients and healthy controls (HDs), and to quantify suicidal and vital NETosis induced by several stimuli. To determine if NET epitopes can change depending from the inflammatory microenvironment and if protein composition of NETs is disease specific, we used quantitative proteomics approach to characterize NET proteins, released from neutrophils after different stimuli in DADA2 patients, HDs and nongenetic vasculitis. Moreover, we investigated the mechanisms of NETs removal. To verify if NETs can influence maturation or inflammatory phenotype of DCs, we characterized peripheral myeloid Dendritic cells (mDCs) and plasmocytoid DCs (pDC) in DADA2 patients and analyzed in vitro moDC maturation and cytokine production in presence of NETs. Methods: We analyzed and quantified both suicidal and vital NETosis by Imaging Flow Citometry (IFC): neutrophils isolated from peripheral blood from DADA2 patients and HDs were stimulated in vitro with different stimuli (PMA, Adenosine and LPS) to induce NETosis and were analyzed in the ImageStreamXMark II IFC equipped with a MultiMag system. We evaluated also NETs remnants and DNAse in the plasma samples by ELISA assay. We used quantitative proteomics approach to characterize NET proteins, released from neutrophils after different stimuli in DADA2 patients, HDs and nongenetic vasculitis: LC-MS/MS analyses were conducted on Orbitrap Fusion Tribrid mass spectrometer and NET protein quantification was carried out using Label-Free Quantification method. We isolated monocytes from peripheral blood with microbeads and we generated moDC after culture stimulation with LPS/NETs. On the 7th day we analyzed by flow cytometry the phenotype and the markers of differentiation. Quantification of cytokines was performed by flow cytometry bead array. Results: We enrolled 14 patients with Adenosine Deaminase 2 deficiency diagnosis who underwent blood sampling at out Center, collecting also plasma samples from further 6 genetically confirmed DADA2 patients enrolled by other Italian Pediatric Rheumatology Units. Neutrophils from DADA2 show a significant increased suicidal NETosis, identified as nuclear decondensation and Myeloperoxidase (MPO) colocalization with DNA, following PMA stimulation and we observed an increase also with LPS and Adenosine. Then we analyzed vital NETosis, identified as elongated shape of cells, nuclei polarized within the cell and not colocalized with MPO and we found an increased vital NETosis in DADA2 neutrophils. Accordingly, plasmatic levels of circulating nucleosomes (NET remnants) were elevated in patients; DNAse levels were normal but the activity was reduced. We set up experimental conditions for proteomic analysis of NETs, induced by PMA, Adenosine and TNFα, testing two patients, two HDs and two patients with non-genetic vasculitis: in total we identified 1770 proteins among which a hundred of proteins were significantly up or down-modulated in DADA2 NETs compared to controls NETs. DC phenotype in DADA2 patients result concordant with HD, as well as moDCs cytokine production after LPS stimulation. We observed a stimulatory effect of NETs towards induction of TNF alpha, IL-6 production and IP-10 from moDCs in both HD and DADA2. Conclusion: Our findings confirm a dysregulation in NETosis process in DADA2 patients, that can be induced by both LPS and adenosine. An increase of vital NETosis was also identified. Proteomic profile of NETs isolated from DADA2 is different from HD and PAN patients: NETs are qualitatively different between HD and DADA2. NETs in DADA2 may therefore interact differently with innate immunity compartment, stimulating DCs to produce citokines, contributing to the typical inflammatory phenotype.
NETosis; NETs; Adenosine deaminase 2 deficiency; DADA2; Dysregulation
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11567/1080132
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