key: cord-0747065-f2rar7jh
authors: Shambat, Srikanth Mairpady; Gómez-Mejia, Alejandro; Schweizer, Tiziano A.; Huemer, Markus; Chang, Chun-Chi; Acevedo, Claudio; Pijuan, Judith Bergada; Vulin, Clement; Miroshnikova, Nataliya; Hofmänner, Daniel A.; Wendel Garcia, Pedro D.; Hilty, Matthias P; Karl, Philipp Bühler; Schüpbach, Reto A.; Brugger, Silvio D.; Zinkernagel, Annelies S.
title: Neutrophil and monocyte dysfunctional effector response towards bacterial challenge in critically-ill COVID-19 patients
date: 2020-12-01
journal: bioRxiv
DOI: 10.1101/2020.12.01.406306
sha: 1d6233e26af7f4352adf278ff35e0e88206a50d9
doc_id: 747065
cord_uid: f2rar7jh
COVID-19 displays diverse disease severities and symptoms. Elevated inflammation mediated by hypercytokinemia induces a detrimental dysregulation of immune cells. However, there is limited understanding of how SARS-CoV-2 pathogenesis impedes innate immune signaling and function against secondary bacterial infections. We assessed the influence of COVID-19 hypercytokinemia on the functional responses of neutrophils and monocytes upon bacterial challenges from acute and corresponding recovery COVID-19 ICU patients. We show that severe hypercytokinemia in COVID-19 patients correlated with bacterial superinfections. Neutrophils and monocytes from acute COVID-19 patients showed severely impaired microbicidal capacity, reflected by abrogated ROS and MPO production as well as reduced NETs upon bacterial challenges. We observed a distinct pattern of cell surface receptor expression on both neutrophils and monocytes leading to a suppressive autocrine and paracrine signaling during bacterial challenges. Our data provide insights into the innate immune status of COVID-19 patients mediated by their hypercytokinemia and its transient effect on immune dysregulation upon subsequent bacterial infections
addition, we showed that these levels decreased upon recovery and were similar to values measured 116 in healthy donors (Fig. S1 ). For monocyte effectors, we found the most significant changes in the 117 levels of fractalkine (CX3CL1), interferon gamma-induced protein 10 (IP10) and monocyte 118 chemotactic protein-1 (MCP-1) (Fig. S1 ).
In a next step, we sought to investigate whether the cytokine levels varied in COVID-19 patients who
Our data show that acute COVID-19 neutrophils and monocytes had impaired bactericidal function 149 with a significant reduction in their ability to clear bacteria as compared to the same cells stimulated 150 with healthy plasma (Fig. 1D-G) . Similarly, stimulation of healthy neutrophils and monocytes with 151 acute plasma showed significantly impaired clearance of intracellular bacteria (Fig. 1D-G) .
Neutrophils from rec-phase patients did not show any impairment in their ability to eliminate 153 intracellular bacteria compared to healthy cells ( Fig. 1D and F ). In contrast, monocytes from rec-154 phase patients still displayed reduced bacterial killing capacity ( Fig. 1E
Recently, it has been proposed that neutrophil extracellular traps (NETs) contribute to the formation 186 of microthrombi in COVID-19 ARDS and that sera from COVID-19 patients triggered NETs release 187 in healthy neutrophils (Middleton et al., 2020; Zuo et al., 2020) . Since NETs formation is a strategy 188 to eliminate extracellular pathogens (Brinkmann et al., 2004) , we tested the hypothesis that bacterial 189 challenge-mediated cell death of neutrophils isolated from acute patients is due to increased NETs
Samples were thawed on ice and prepared according to the manufacturer's instructions using a 402 custom-made 33-plex human cytokine panel (Procartaplex ThermoFisher). In brief, Luminex TM 403 magnetic beads were added to the 96-well plate placed on a magnetic holder and incubated for 404 2min. The plate was washed twice with assay buffer for 30sec. In parallel, provided standards and 405 plasma samples were diluted in assay buffer (cell culture media was used for cell culture 406 supernatants) and added to the plate. The plate was incubated for 2h at RT at 550rpm in a plate 12 orbital shaker. Next, the plate was washed twice with assay buffer and incubated for 30min at 550rpm 408 with detection antibodies. After two washing steps, the plate was incubated with Streptavidin-PE 409 solution for 30min at 550rpm. Finally, the plate was washed, reading buffer was added and incubated 410 for 10min at RT and 550rpm before running the plate. Data acquisition and analysis were performed
Microscopy and NETs quantification 515 Neutrophils were stimulated as described above and placed within wells of a µ-slide (iBidi) and 516 centrifuged at 200 g for 2 min, after which they were challenged with S. aureus for 1.5h. NETs were 517 stained by directly adding SYTOX TM Green and 2µM Hoechst 33342 (ThermoFisher) for 30min at 518 room temperature to the wells. The confocal laser scanning microscopy images were obtained with 519 a Leica TCS SP8 inverted microscope using a 63×/1.4 oil immersion objective. The whole wells were 520 inspected for NETs formation and two to three representative spots per condition were imaged. The 521 15 obtained images were processed using Imaris 9.2.0 software (Bitplane) to obtain tifs for further 522 analysis. Other standard light microscopy images of fixed cells were obtained on a fully automated 523 Olympus IX83 with a 40X objective (UPLFLN40XPH-2) illuminated with a PE-4000 LED system 524 through a quadband filter set (U-IFCBL50). 16 positions per sample were assigned before the 525 sample was prepared to avoid potential experimenter bias. Automated NET quantification was 526 performed as described in SI Fig.4 : after filtering nuclei on DAPI signal (threshold set manually for 527 each 8-samples experiment), extracellular DNA was quantified on Sytox Green signal. Images 528 containing large cell aggregates that could not be resolved were discarded. Nuclei were counted 529 after watershed segmentation on the DAPI mask. Images were processed using ImageJ software
Expression of key surface markers in COVID-19 acute (red), rec (blue) and in healthy donors' (white) 642 classical (A-C) and non-classical (D-F) monocytes (n=9-11). PCA of cell surface phenotype of 643
rec (blue) and in healthy donors (white) of classical monocytes at 644 the basal level without bacterial challenge (COVID-19 status) (G), upon SA infection (H) or SP 645 infection (I)
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