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Staphylococcus aureus modulates the host immune response directly by interacting with the immune cells or indirectly by secreting molecules (secretome). Relevant differences in virulence mechanisms have been reported for the secretome produced by different S. aureus strains. The present study investigated the S. aureus secretome impact on peripheral bovine mononuclear cells (PBMCs) by comparing two S. aureus strains with opposite epidemiological behavior, the genotype B (GTB)/sequence type (ST) 8, associated with a high within-herd prevalence, and GTS/ST398, associated with a low within-herd prevalence. PBMCs were incubated with different concentrations (0%, 0.5%, 1%, and 2.5%) of GTB/ST8 and GTS/ST398 secretome for 18 and 48 h, and the viability was assessed. The mRNA levels of pro- (IL1-β and STAT1) and anti-inflammatory (IL-10, STAT6, and TGF-β) genes, and the amount of pro- (miR-155-5p and miR-125b-5p) and anti-inflammatory (miR-146a and miR-145) miRNAs were quantified by RT-qPCR. Results showed that incubation with 2.5% of GTB/ST8 secretome increased the viability of cells. In contrast, incubation with the GTS/ST398 secretome strongly decreased cell viability, preventing any further assays. The GTB/ST8 secretome promoted PBMC polarization towards the pro-inflammatory phenotype inducing the overexpression of IL1-β, STAT1 and miR-155-5p, while the expression of genes involved in the anti-inflammatory response was not affected. In conclusion, the challenge of PBMC to the GTS/ST398 secretome strongly impaired cell viability, while exposure to the GTB/ST8 secretome increased cell viability and enhanced a pro-inflammatory response, further highlighting the different effects exerted on host cells by S. aureus strains with epidemiologically divergent behaviors., (© 2023. The Author(s).)

Coronavirus disease 2019 (COVID-19) is associated with an increased risk of thrombotic events. Ischemic stroke in COVID-19 patients entails high severity and mortality rates. Here we aimed to analyze cerebral thrombi of COVID-19 patients with large vessel occlusion (LVO) acute ischemic stroke to expose molecular evidence for SARS-CoV-2 in the thrombus and to unravel any peculiar immune-thrombotic features. We conducted a systematic pathological analysis of cerebral thrombi retrieved by endovascular thrombectomy in patients with LVO stroke infected with COVID-19 (n = 7 patients) and non-covid LVO controls (n = 23). In thrombi of COVID-19 patients, the SARS-CoV-2 docking receptor ACE2 was mainly expressed in monocytes/macrophages and showed higher expression levels compared to controls. Using polymerase chain reaction and sequencing, we detected SARS-CoV-2 Clade20A, in the thrombus of one COVID-19 patient. Comparing thrombus composition of COVID-19 and control patients, we noted no overt differences in terms of red blood cells, fibrin, neutrophil extracellular traps (NETs), von Willebrand Factor (vWF), platelets and complement complex C5b-9. However, thrombi of COVID-19 patients showed increased neutrophil density (MPO + cells) and a three-fold higher Neutrophil-to-Lymphocyte Ratio (tNLR). In the ROC analysis both neutrophils and tNLR had a good discriminative ability to differentiate thrombi of COVID-19 patients from controls. In summary, cerebral thrombi of COVID-19 patients can harbor SARS-CoV2 and are characterized by an increased neutrophil number and tNLR and higher ACE2 expression. These findings suggest neutrophils as the possible culprit in COVID-19-related thrombosis., (© 2022. The Author(s).)