Fig 2: Panel (A). Quantification of CitH3 in neutrophils activated towards NETosis using PMA (100 nM) with or without AAT (500 µg/mL) by ELISA assay after 4 h of incubation. Data are represented as mean of three independent replicates ± standard deviation. *** p < 0.01 vs. CTR; ^ p < 0.01 vs. PMA. Panel (B). Confocal microscopy images of neutrophils activated towards NETosis with PMA adding fluorescent AAT (green). DNA was labeled with DAPI and HNE with specific antibody (red). Scale bar = 50 µm.

Fig 3: Panel (A). Western blotting with anti-AAT antibody. Lanes 1–4: COVID-19 samples not showing the complex (80 kDa band) upon addition of exogenous HNE. Lanes 5–8: BOS samples showing the complex (80 kDa band) upon addition of exogenous HNE. Lane 9: complex generated “in vitro” upon incubation of the HNE and AAT standard proteins. Panel (B). Western blotting with anti-AAT and anti-HNE antibodies. Lane 1: free HNE standard protein. Lanes 2–9: different COVID-19 samples showing an intensity increase of the HNE band upon incubation with scalar amounts of exogenous HNE.

Fig 4: Summary Scheme summarizing our main findings (created with Biorender.com). Briefly, our study demonstrated that neutrophils of people with established T1D undergo NET formation at rates similar to those of HC subjects in response to PMA and ionomycin. Furthermore, we showed that the T1D NETomes, induced by these stimuli, had an enrichment in proteins involved in glucose metabolism, with lower abundances in proteins implicated in innate immunity, compared to those of HC. This altered NETome composition was not associated with higher rates of glycolysis or glycolytic capacity compared to HC. The size of the nodules representing the proteins reflects the protein abundance in HC and T1D NETomes. Abbreviations: HC: healthy control; T1D: type 1 diabetes; NOX: nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; Ca2: calcium; PMA: phorbol-myristate acetate; IONO: ionomycin; S100P: protein S100-P; AMBP: protein AMBP; S100A6: protein S100-A6; AZU1: azurocidin; ELANE: neutrophil elastase; PKM: pyruvate kinase; PGK1: phosphoglycerate kinase; ALDOA: fructose-bisphosphate aldolase A; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; UGP2: UTP-glucose-1-phosphate uridylyltransferase.

Fig 5: Quantification of NET proteins from PMA-stimulated HC and T1D neutrophils. (A) Volcano plot based on fold change (log2) and p-value (-log10) of all differentially expressed NET proteins identified in NETing neutrophils of HC (n = 3) and T1D (n = 4) subjects in response to PMA (<1.0% FDR). Th threshold (grey dotted line) was set at p-value < 0.05. Proteins below the threshold are in grey, while proteins with higher expression levels in T1D subjects are in blue, and those in HC are in yellow. Proteins of interest, as well as their fold change values, are indicated. (B) Hierarchical clustering of normalized abundance values (Progenesis) of significantly different NET proteins of HC (yellow) and T1D (blue) subjects after PMA stimulation (<1.0% FDR, p < 0.05, unpaired two-tailed t-test). Clustering was performed on subjects (columns) and proteins (rows). Scale bar represents the normalized abundance values. (C,D) Normalized abundance values of NET proteins enriched in HC (C) and T1D (D) neutrophils in response to PMA. Abbreviations: S100A6: protein S100-A6; ELANE: neutrophil elastase; AZU1: azurocidin; S100P: protein S100-P; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PGK1: phosphoglycerate kinase; ALDOA: fructose-bisphosphate aldolase A; PKM: pyruvate kinase. Error bars represent mean SEM (n = 3 for HC and n = 4 for T1D). Unpaired two-tailed t-test. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001.