NET Proteomic Profiling Reveals New Pathways Potentially Implicated in Dendritic Cell-Mediated Inflammation in DADA2 Patients

Abstract

Purpose: Adenosine deaminase 2 Deficiency (DADA2) is an autoinflammatory disease characterized by systemic vasculopathy, strokes and mild immunodeficiency. Recently NETosis has been implicated in the pathogenesis of Deficiency of Adenosine Deaminase 2. To deep investigate the possible effects of NETs on the immune system we characterized proteomic profile of NETs from DADA2 as compared to HD and Polyarteritis Nodosa (PAN) patients. To determine if NETs contain possibly immunogenic antigens we study functional aspects on Dendritic Cells after in vitro stimulation with NETs.

Methods: Twenty-three DADA2 patients were enrolled. We analyzed NETosis by Imaging Flow Citometry. We evaluated NETs remnants and DNAse in the plasma samples by ELISA assay whereas DNAse activity by DNA digestion. We used quantitative proteomics approach and network analysis to identify NET proteins and pathways in 6 DADA2, 7 PAN and 7 HD. We analyzed circulating and monocyte-derived dendritic cells by flow cytometry.

Results: Neutrophils from DADA2 patients show a significant increased suicidal NETosis. DNAse enzymes were not normal in the level or activity. By proteomic analysis we identified 1356 proteins among which a hundred of proteins were significantly up or down-modulated in DADA2 NETs as compared to normal and disease control NETs in resting condition and after stimulation with PMA, Adenosine and TNFα. DADA2 NETs are significantly more efficient than normal NETs in stimulating patients' monocyte-derived dendritic cells.

Conclusion: We identified different pathways significantly modulated in DADA2 NETs versus PAN/HD NETs. This peculiar protein profile could contribute in activating inflammatory pathways in Dendritic cells in DADA2.

Keywords: DADA2; Inflammation; Neutrophil extracellular traps; PAN; Proteomic.

Fig. 1

DADA2 neutrophils show significant exacerbated NETosis. A Representative image of a normal and NETotic neutrophil analyzed by IFC. Lower Panel: statistical analysis of cells undergoing suicidal NETosis, represented as Round Similarity Ratio between positive cells in unstimulated and PMA/ADE stimulated samples. B Representative image of a vital NETotic neutrophil after PMA/ADE stimulation analyzed by IFC. Lower Panel: statistical analysis of cells undergoing suicidal NETosis, represented as Elongated morphology Ratio between positive cells in unstimulated and PMA/ADE stimulated samples. C Quantification of plasmatic Adenosine, D Histone-associated-DNA-fragments in DADA2 patients, HDs and PAN patients. Bar plots represent means ± SEM. Significant differences (Mann–Whitney test) are indicated. *P <.05, **P <.01, *** P <.005

Fig. 2

Dysregulation in NETs removal mechanisms in DADA2 patients and ROS production is increased DADA2 neutrophils. A Determination of plasmatic Dnase1 in HD, DADA2 and PAN patients. B Determination of DNASE1L3 in HD, DADA2 and PAN plasma patients and frequency of positivity by Fisher test. C DNAse activity in HD, DADA2 and PAN. D MA plot displaying same samples PAN (purple dots), DADA2 (green dots) and HD (pink dots) analyzed for DNase activity with or without protein A sepharose: outlier data beyond the two red lines have a significant variation. (Non-parametric test Friedman for paired data). E Percentage of DHR-123 + positivity gated on cells SSC high/FSC high/; data analyzed by DIVA software and Prism. showing median and range of 30 independent donors, 9 DADA2 and 2 PAN patients

Fig. 3

Analysis of NETs proteins in DADA2, PAN and HDs: VENN Principal component Analysis (PCA) plot, GO Enrichment of biologically relevant pathways significantly modulated in DADA2 and PAN with respect to control groups. A The VENN diagram in the left panel shows the number of identified common and exclusive NET proteins between DADA2, PAN and HDs groups. The VENN diagram in right panel shows NET proteins of DADA2 patients, present at least at 70% in each group, shared or specific for one treatment: NT (untreated), PMA, TNFα, ADE. B Principal component Analysis (PCA) plot shows separation among different biological groups on the basis of NET proteins expression profiles. The percentage of the total variation accounted for the first and second component is shown on the x and y axes, respectively. Green crosses, purple squares, and pink circles refer to DADA2, PAN and HD samples respectively. DADA2 and PAN/HD groups form two well separated clusters on Principal Component 1 (PC1). PAN and HD partially cluster on PC2. C Heatmap representation shows unsupervised hierarchical clustering analysis of significant NET proteins differentially expressed between DADA2, PAN and HDs groups, identified by ANOVA test (S0 = 0.1 and FDR = 0.05) in untreated (NT) conditions. In the heatmap, each row represents a protein and each column corresponds to a sample. Normalized Z-score protein expression levels are indicated by a two-color scale ranging from blue (lowest values) to red (highest values) reported in the horizontal bar at the right of the panel. The purple, pink and green bars below the columns distinguish the PAN, HD and DADA2 group respectively. The tree dendrogram at the top of the plot displays the results of the unsupervised hierarchical clustering analysis, placing similar sample/proteome profile values next to each other. The optimal association between the main clusters of NETs proteins identified (indicated by the coloured bars on the left side) and DADA2, PAN and HD groups of samples are shown. D Heatmap representation after PMA stimulation. E Pathway enrichment analyses and network of the protein clusters #296 (NET proteins significantly upregulated in NT neutrophils of DADA2 group respect to PAN/HD groups) of the heat map shown in panel C. Functional enrichment analysis for GO biological processes were conducted by ShinyGO, and enriched pathways were considered significant if FDR value ≤ 0.05. The graph shows the top enriched terms. F Pathway enrichment analyses of the protein cluster #290 (NET proteins significantly down regulated in NT neutrophils of PAN group respect to DADA2/HD groups) of the heatmap shown in panel C. Functional interaction networks refer to “neg. regulation of blood coagulation”, pathway of cluster #290 enrichment

Fig. 4

Hierarchical Clustering, GO Enrichment of biologically relevant pathways upregulated in DADA2 with respect to control groups with TNFα and ADE stimulation. A In the left panel the heatmap representation shows unsupervised hierarchical clustering analysis of significant NET proteins differentially expressed between DADA2, PAN and HD groups, identified by ANOVA test (S0 = 0.1 and FDR = 0.05) after TNF stimulation. In the right panel is shown the pathway enrichment analyses of the protein cluster #196 (NET proteins of TNF stimulated neutrophils upregulated in DADA2 respect to HD/PAN). Functional interaction network refers to the “cellular response to cytokine stimulus” pathway of cluster #196 enrichment is shown. B In the left panel the heatmap representation shows unsupervised hierarchical clustering analysis of significant NET proteins differentially expressed between DADA2, PAN and HD groups, identified by ANOVA test (S0 = 0.1 and FDR = 0.05) after ADE stimulation. In the right panel is shown the pathway enrichment analyses of the protein cluster #289 (NET proteins of ADE stimulated neutrophils upregulated in DADA2 respect to HD/PAN). Functional interaction network refer to the “Reg. of cysteine-type endopeptidase activity involved in apoptotic processes” pathway of cluster #289 enrichment. In the right panel the biological processes identified in NETs generated after ADE stimulation is shown

Fig. 5

Differentially correlated functional modules extracted by processing the protein expression profiles from DADA2 NETs (vs HD NETs and vs PAN NETs).For each comparison, the most relevant terms (FDR < 0.05) are shown. Connected terms share at least 60% of their genes. A Gene Ontology (GO) Biological Processes. B REACTOME pathways identified by comparing DADA2 NETs vs HD NETs and PAN NETs conditions. C Differentially correlated pathways by comparing samples treated with PMA, TNF or ADE (vs NT) are shown; specifically, in red are highlighted pathways up correlated in PMA, TNF or ADE, while in blue are highlighted those most correlated in NT (FDR < 0.05). D Best ten-ranked hubs/bottlenecks in HD NETs, DADA2 NETs and PAN NETs. Hubs/bottlenecks were selected based on betweenness, centroid and bridging centralities

Fig. 6

Distribution of conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs) in DADA2 patients and effect of DADA2 NETs on maturation and cytokines secretion from MODCs. A Frequency and absolute count of cDCs (upper panel) and pDCs (lower panel), gated on lympho-monocytes. B Expression of activation and maturation markers in MODCs: CD80, CD83, CD86, HLADR. C In vitro production of pro-inflammatory cytokines by MODCs from HDs (blank bars) and DADA2 patients (green bar) stimulated with normal NETs or DADA2 patients derived NETs. D In vitro production of pro-inflammatory cytokines by MODCs from DADA2 patients stimulated with autologous DADA2 NETs or in presence of DNASE1/hrecADA2 or stimulated with PAN NETs. Data shown represent means ± SEMs. Significant differences (Unpaired t test) are indicated. *P <.05, **P <.01, *** P <.005

Fig. 7

Graphical abstract of the work