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. 2022 Jul 7;26:206. doi: 10.1186/s13054-022-04062-5

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© The Author(s) 2022

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 1 — Neutrophils from COVID-19 patients undergoing NETosis express GSDMD. A Single-cell analysis of BALF from patients with COVID-19 across severity status (Healthy control, Moderate, and Severe). UMAP visualization from gene expression data of 66,452 cells, highlighting neutrophil expression cluster in severe COVID-19 patients (red) from bronchoalveolar lavage fluid (BALF) cells. B Pie chart plot representing the proportion of GSDMD expressing neutrophils. C UpSet plot showing the intersection of inflammasome genes expressed in neutrophils, including PYCARD, CASP4, and CASP1, derived from COVID-19 severe patients. The point diagram indicates the intersection among the genes and the bar graph shows the number of GSDMD expressing neutrophils in each intersection (y-axis). D Representative confocal analysis of GSDMD-NT and NETs in the lung tissue sample from autopsies of COVID-19 patients (n = 6 or control n = 3). Immunostaining for DNA (DAPI, blue), myeloperoxidase (MPO, green), and the GSDMD cleaved fraction (GSDMD-NT, red) are shown. The scale bar indicates 50 μm at 630× magnification. E Zoomed images of Fig. 1D inset white square. F The expression of GSDMD-NT was quantified by MFI per field. G Correlation between colocalization GSDMD-NT:DAPI and NETs (MPO:DAPI). H Representative confocal analysis of GSDMD and NETs in the blood neutrophils isolated from COVID-19 patients (n = 5) or controls (n = 5). Cells were stained for DNA (DAPI, blue), MPO (green), and GSDMD-NT (red). Scale bar indicates 50 μm, 4× digital zoom was performed in the inset white square. I Expression of GSDMD-NT was quantified by MFI per field. J Expression of full-length GSDMD (GSDMD-FL) and active GSDMD (GSDMD-NT) by Western blot. Moderate COVID-19 (M, n = 3) severe COVID-19 (S, n = 4), and healthy controls (n = 36). (K) Western blot quantification by densitometry. GSDMD-NT values obtained were normalized to total beta-actin (L) Circulating amounts of MPO/DNA-NETs and M GSDMD from plasma of patients with moderate COVID-19 (n = 15) severe COVID-19 (n = 21), and healthy controls (n = 320). N Correlation between plasmatic levels of MPO/DNA-NETs and GSDMD. The data are expressed as mean ± SEM (*p < 0.05; t test in F and I, Pearson’s correlation in G and M, one-way ANOVA followed by Tukey’s in K and L)