The specialized pro-resolving lipid mediator Protectin D1 affects macrophages differentiation and activity in Adult-onset Still’s disease and COVID-19, two hyperinflammatory diseases sharing similar transcriptomic profiles

Figures

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  Figure 1. Transcriptome analysis of peripheral immune cells from HDs, COVID-19 and AOSD patients. (A) PCA plot. PCA shows the sample in the 2D plane spanned by their first two principal components. (B) Pathway analysis by Pantherdb.org. Bar chart of enriched pathways in COVID19 and AOSD patients. (C) Volcano plots. Volcano plot representation of differential expression analysis of genes in COVID-19 vs HD, AOSD vs HD, and COVID-19 vs AOSD. Red and green points mark the genes with significantly increased or decreased expression (FDR<0.05). The x-axis shows log2fold-changes in expression and the y-axis the log odds of a gene being differentially expressed. (D) Transcriptome analysis from 3 HDs, 2 COVID-19 patients and 3 AOSD patients of genes involved in inflammation, lipid catabolism and monocytes. HD, healthy donors; AOSD, Adult-onset Still’s disease.

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  Figure 2. Levels of protectin D1 in HDs, COVID-19 and AOSD patients. (A). Plasma levels of PD1 (ng/ml) in HDs (n=13) and patients affected by COVID-19 (not-ICU COVID-19, n= 11; ICU COVID-19, n=10) and AOSD (n=13) patients. Data are represented as box plots displaying medians, 25th and 75th percentiles as boxes, and 10th and 90th percentiles as whiskers. HD vs AOSD SS≥1, *p= 0.04; AOSD SS=0 vs AOSD SS≥1, *p=0.02; HD vs ICU COVID-19, p= 0.0006; not-ICU COVID-19 vs ICU COVID-19, *p= 0.02, *** p < 0.0001 (B) Correlation analysis between PD1 levels and white blood cells count, neutrophils count, fibrinogen levels and length of hospital stay in patients with COVID-19. Spearman correlation and linear regression coefficients are displayed. ICU, intensive care unit.

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  Figure 3. Effect of PD1 on polarization of macrophages from HDs, COVID-19 and AOSD patients. Expression of surface markers by flow cytometry on M1 and M2 macrophages from COVID-19 patients (A), not-ICU and ICU COVID-19 patients (B), AOSD patients (C) and HDs (D) after treatment with PD1. Data are represented as box plots displaying medians, 25th and 75th percentiles as boxes, and 10th and 90th percentiles as whiskers, and reported as Mean fluorescent intensity (MFI). M2 macrophages from COVID-19 patients, untreated vs PD1 **p = 0.009; M2 macrophages from AOSD patients, untreated vs PD1 *p =0.02. ICU, intensive care unit.

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  Figure 4. Effect of PD1 on secretion of cytokines/chemokines by macrophages from COVID-19 patients. Radar chart summarizing means of cytokines and chemokines profile of M1 (A) and M2 (B) macrophages from COVID-19 patients after in vitro treatment with PD1. Green plots represent untreated cells, while pink plot PD1-treated cells. (C) Multiplex analysis of IL-10 and MIP-1β levels in supernatants of M2 macrophages from COVID-19 patients treated with PD1. Data are reported as pg/mL and represent 12 independent experiments. IL-10, untreated vs PD1, *p= 0.03; MIP-1β, untreated vs PD1, **p= 0.003.

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  Figure 5. Effect of PD1 on secretion of cytokines/chemokines by macrophages from AOSD patients. Radar chart summarizing cytokines and chemokines profile of M1 (A) and M2 (B) macrophages from COVID-19 patients after in vitro treatment with PD1. Green plot represents untreated cells, while red plot PD1-treated cells. (C) Multiplex analysis of IL-10 and MIP-1β levels in supernatants of M2 macrophages from AOSD patients (n = 10) treated with PD1. Data are reported as pg/mL. IL-10, untreated vs PD1, *p= 0.03; MIP-1β, untreated vs PD1, *p= 0.03.

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  Figure 6. Summary of the in vitro experiments. In vitro PD1 induced the differentiation of monocytes from patients with both AOSD and COVID-19 in M2-macrophages and their secretion of IL-10 and MIP-1β (Created with BioRender.com).