Highlights d AMs patrol and clean the alveolar spaces d AMs have directed movement toward inhaled bacteria d AM migration is crucial for bacterial clearance d AM migration is impaired during viral infection

Highlights d P. aeruginosa keratitis infections result in biofilm formation on the cornea d NETs form at the base of the biofilm, triggered by the type-3 secretion system (T3SS) d NETs stop bacterial dissemination into the brain but promote antibiotic resistance d Blocking exopolysaccharide Psl and the T3SS allowed neutrophils to break down the biofilm

No abstract

Paracoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographic habitat, conidia as infecting form, and thermo-dimorphic characteristics. Polymorphonuclear neutrophils (PMNs) are responsible for an important defense response against fungus, releasing Neutrophil Extracellular Traps (NETs), which can wrap and destroy the yeasts. However, it has been described that some pathogens are able to evade from these DNA structures by releasing DNase as an escape mechanism. As different NETs patterns have been identified in PMNs cultures challenged with different isolates of Paracoccidioides brasiliensis, the general objective of this study was to identify if different patterns of NETs released by human PMNs challenged with Pb18 (virulent) and Pb265 (avirulent) isolates would be correlated with fungal ability to produce a DNase-like protein. To this end, PMNs from healthy subjects were isolated and challenged in vitro with both fungal isolates. The production, release, and conformation of NETs in response to the fungi were evaluated by Confocal Microscopy, Scanning Microscopy, and NETs Quantification. The identification of fungal DNase production was assessed by DNase TEST Agar, and the relative gene expression for hypothetical proteins was investigated by RT-qPCR, whose genes had been identified in the fungal genome in the GenBank (PADG_11161 and PADG_08285). It was possible to verify the NETs release by PMNs, showing different NETs formation when in contact with different isolates of the fungus. The Pb18 isolate induced the release of looser, larger, and more looking like degraded NETs compared to the Pb265 isolate, which induced the release of denser and more compact NETs. DNase TEST Agar identified the production of a DNase-like protein, showing that only Pb18 showed the capacity to degrade DNA in these plates. Besides that, we were able to identify that both PADG_08528 and PADG_11161 genes were more expressed during interaction with neutrophil by the virulent isolate, being PADG_08528 highly expressed in these cultures, demonstrating that this gene could have a greater contribution to the production of the protein. Thus, we identified that the virulent isolate is inducing more scattered and loose NETs, probably by releasing a DNase-like protein. This factor could be an important escape mechanism used by the fungus to escape the NETs action.

SARS-CoV-2 enters the respiratory tract where it infects the alveoli epithelial lining. However, patients have sequelae that extend well beyond the alveoli into the pulmonary vasculature and perhaps beyond to brain and other organs. Because of the dynamic events within blood vessels, histology fails to report on platelet and neutrophil dynamics. Because of the rapid non-transcriptional behaviour of these cells neither single-cell RNAseq nor proteomics report robustly on their critical behaviours. We used intravital-microscopy in level-3 containment to examine the pathogenesis of SARS-CoV-2 within three organs in mice expressing human-ACE-2 ubiquitously (CAG-AC-70) or on epithelium (K18-promoter). Using a neon-green-SARS-CoV-2, we observed both epithelium and endothelium infected in AC70 mice but only the epithelium in K18 mice. There was increased neutrophils in the microcirculation but not in the alveoli of the AC70 lungs. Platelets formed large aggregates in the pulmonary capillaries. Despite only neurons being infected within the brain, profound neutrophil adhesion forming the nidus of large platelet aggregates were observed in the brain microcirculation with many non-perfused microvessels. Neutrophils were seen breaching the brain endothelial layer associated with a significant disruption of the blood-brain-barrier. Despite ubiquitous ACE-2 expression, CAG-AC-70 mice had very small increases in blood cytokine, no increase in thrombin, no infected circulating cells and no liver involvement suggesting limited systemic effects. In summary, our imaging of SARS-CoV-2-infected mice gave direct evidence that there is a significant perturbation locally in the lung and brain microcirculation induced by local viral infection leading to increased inflammation and thrombosis in these organs.