Fig 1: CD14+HLA-DRneg/low monocytes from patients with AMI are not immunosuppressive but exhibit an inflammatory phenotype.(A) May-Grünwald Giemsa stained cytospin preparations of CD14+HLA-DRneg/low and CD14+HLA-DRhigh monocytes. (B) Relative RNA expression of S100A9 and IL1R1 in CD14+HLA-DRneg/low versus CD14+HLA-DRhigh monocytes. (C) T-cell proliferation in the presence of CD14+HLA-DRneg/low or CD14+HLA-DRhigh monocytes assessed by CellTrace Violet dilution after 96 hr of co-culture. (D) Macrophages differentiated from CD14+HLA-DRneg/low monocytes and (E) CD14+HLA-DRhigh cells by 4-day culture with M-CSF. (F) TNF-a, IL-6, and IL-1ß in supernatants of macrophage cultures upon stimulation with IFN-?. Mb=baseline. CD14+HLA-DRneg/low/CD14+HLA-DRhigh cells were isolated by flow-cytometric sorting from patients with AMI (n=5–6). Data are presented as mean ± SEM. *p<0.05.
Fig 2: Multiple intercorrelations among CD10neg neutrophils, HLA-DRneg/low monocytes and immune-inflammation markers.Spearman-correlation matrix of CD16+CD66b+CD10neg neutrophils (%), CD14+HLA-DRneg/low monocytes (%) and circulating levels of MMP-9, S100A9/S100A8, IL-6, IL-1ß, TNF-a, MPO, and NGAL (levels). UA (n=11), NSTEMI (n=10), and STEMI (n=26). Each circle illustrates a significant correlation between different couples of parameters (p<0.05). The correlation coefficient is colored and sized up according to the value; square leaved blank indicates not significant correlation.
Fig 3: Calprotectin Is the Most Abundant Immune Mediator Detected in the Plasma of Patients with Severe COVID-19Shown are plasma levels of calprotectin (S100A8/S100A9), interferon alpha (IFNα2a), and 40 cytokines and chemokines in blood samples collected from 84 patients (controls, 40; mild disease, 18; moderate or severe disease, 25).(A) Volcano plot of cytokine levels in patients with mild COVID-19 compared with controls; IFNα2a is shown in orange.(B) Volcano plot of cytokine levels in patients with severe COVID-19 compared with control patients; IFNα2a is shown in orange. Calprotectin, CXCL11, CXCL13, and CX3CL1, shown in red, are most significantly associated with the severe forms.(C) Circulating levels of CXCL8, IFNα2a, calprotectin, and IL-6 in individual samples in each group.(D) Effect of comorbidities (Table S6) on calprotectin plasma levels in each group.(E) Volcano plot of cytokine levels in patients with severe disease with and without comorbidities.(F) Effect of bacterial infection on calprotectin plasma levels in each group.(G–I) Spearman correlations between calprotectin plasma levels and neutrophil count (G), fibrinogen (H), and D dimers (I).(J–L) Spearman correlations between IL-6 plasma levels and neutrophil count (J), fibrinogen (K), and D dimers (L).Wilcoxon rank-sum test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig 4: Changes in Innate Immune Cell Phenotype Are Detected in Patients with Moderate COVID-19, Related to Figure 7 and Tables S6 and S7A. Bar plots representing the percentage of B cells, CD4+ T cells, CD8+ T cells, NK cells, total monocytes, CD169+, HLA-DRLow and CD141+ classical monocyte subsets, total neutrophils among CD45+ cells, and CD10LowCD101- and CD10LowCD16Low neutrophil subset among all neutrophils in individual patients from each group, with the moderate category (6 patients) highlighted. B. Plasma concentration of IFNα in moderate COVID-19 patients compared to the three other groups. C. ROC analysis showing performance of a diagnostic test using percentage of non-classical monocytes among total monocytes to distinguish controls and mild COVID patients from moderate and severe COVID patients; D. Monocyte subset analysis in the peripheral blood of 2 severe patients, before (left panels) and after (right panels) treatment with the indicated anti-IL-6 antibodies; E. Percentage of HLA-DRlow classical monocytes among classical monocytes in a cohort of 22 patients and 17 controls grouped into 4 clinical categories; F. Correlation between the percentage of HLA-DRlow classical monocytes and non-classical monocytes; G. Percentage of CD16low neutrophils among neutrophils in control and COVID-19 patients of the learning cohort described in Figure 7. H. ROC curves evaluating the discriminating significance of calprotectin plasma level (yellow), nonclassical monocyte fraction (red), CD16low circulating neutrophils (blue) and IFNα2a plasma level (white) between controls/mild patients and moderate/severe patients. AUC, Area Under the Curve; Mann Whitney test; I. Heatmap of blood and bronchoalveolar lavage fluid scRNaseq cells integrated defining the 5 regions of cell populations; J. Pathway analysis (Cytoscape and ClueGo) of DEGs expressed at a higher level in bronchoalveolar monocytes/macrophages from mild versus severe patients. K. UMAP analysis of neutrophils with S100A8 (left panel) and S100A9 (right panel) gene expression level projection (low expression = gray dots; high expression = dark blue dots). Kruskal-Wallis test, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
Fig 5: Immature CD10neg neutrophils from patients with AMI express high amounts of MMP-9 and S100A9 and display resistance to apoptosis.(A) May-Grünwald Giemsa stained cytospin preparations of CD16+CD66b+CD10neg (CD10neg) and CD16+CD66b+CD10pos (CD10pos) neutrophils. (B) Linear regression analysis between the percentages of CD16+CD66b+CD10neg neutrophils and circulating levels of G-CSF in patients with acute coronary syndrome (n=71). (C) Mean fluorescence intensity (MFI) of CD11b, CD101, CD114, and CD177 on CD10neg versus CD10pos neutrophils (n=25). (D) Relative RNA expression of MMP-9 and S100A9 in CD10neg versus CD10pos neutrophils. (E) Percentage of apoptotic neutrophils assessed by flow cytometry using Vybrant DyeCycle Violet stain and SYTOX AADvanced stain. CD10neg/CD10pos neutrophils were isolated by flow-cytometric sorting from patients with AMI (n=4–5). *p<0.05.
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