Retter, A. | bioRxiv Science

bioRxiv2023

Novel rapid high-throughput method of NETosis Induction and Inhibition with physiological triggers and inhibitors

Neutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil Extracellular Trap (NET) formation in response to pathogens is increasingly recognized as an essential rapid innate immune response, but when dysregulated contributes to pathogenesis of sepsis and immunothrombotic disease. Current models of NETosis are limited, routinely employing non-physiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines, do not reflect the complex biology underlying neutrophil activation and NETosis, that occurs in whole-blood. Here we describe a novel, high-throughput ex-vivo whole blood induced NETosis model using combinatorial pooling of native NETosis inducing factors in a more biologically relevant Synthetic-Sepsis model. We found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and/or magnitude of NETosis. Despite inter-donor variability, similar sets of pro-inflammatory molecules induced consistent responses across donors. We found at least three biological triggers were necessary to induce NETosis in our system including either TNF- or LT-. To our knowledge, we report the first human ex-vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis. These findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially, providing novel strategies for disease intervention and treatment. EssentialsO_LINETosis is a vital immune response, but dysregulation leads to disastrous health outcomes. C_LIO_LICurrent NETosis models dont reflect the complex endogenous signaling that occurs in whole blood C_LIO_LINetosis induction stimuli differs between isolated neutrophils and whole blood. C_LIO_LIA minimum of three physiological factors are required to induce NETosis in whole blood. C_LI

immunology↗

bioRxiv2023

What mycologists should talk about when they are talking about the International Code of Nomenclature for algae, fungi, and plants

Fungal metabarcoding of substrates such as soil, wood, and water are uncovering an unprecedented number of fungal species that do not seem to produce tangible morphological structures and that defy our best attempts at cultivation, thus falling outside of the ambit of the International Code of Nomenclature for algae, fungi, and plants. The present study uses the new, ninth release of the species hypotheses of the UNITE database to show that species discovery through environmental sequencing vastly outpaces traditional, Sanger sequencing-based efforts in a strongly increasing trend over the last five years. Our findings challenge the present stance of the mycological community - that "the code" works fine and that these complications will somehow sort themselves out given enough time and a following wind - and suggest that we should be discussing not whether to allow DNA-based descriptions (typifications) of species and by extension higher ranks of fungi, but what the precise requirements for such DNA-based typifications should be. We submit a tentative list of such criteria for further discussion. However, the present authors fear that no waves of change will be lapping the shores of mycology for the foreseeable future, leaving the overwhelming majority of extant fungi without formal names and thus scientific and environmental agency. It is not clear to us who benefits from that, but neither fungi nor mycology are likely to be on the winning side.

scientific communication and education↗