SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike-ACE2 receptor interaction
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Abstract
Coronavirus disease-2019 (COVID-19) is primarily a respiratory disease, however, an increasing number of reports indicate that SARS-CoV-2 infection can also cause severe neurological manifestations, including precipitating cases of probable Parkinson’s disease. As microglial NLRP3 inflammasome activation is a major driver of neurodegeneration, here we interrogated whether SARS-CoV-2 can promote microglial NLRP3 inflammasome activation utilising a model of human monocyte-derived microglia. We identified that SARS-CoV-2 isolates can bind and enter microglia, triggering inflammasome activation in the absence of viral replication. Mechanistically, microglial NLRP3 could be both primed and activated with SARS-CoV-2 spike glycoprotein in a NF-κB and ACE2-dependent manner. Notably, virus- and spike protein-mediated inflammasome activation in microglia was significantly enhanced in the presence of α-synuclein fibrils, which was entirely ablated by NLRP3-inhibition. These results support a possible mechanism of microglia activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson’s disease in certain COVID-19 infected individuals, and a potential therapeutic avenue for intervention.
SIGNIFICANCE STATEMENT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) principally affects the lungs, however there is evidence that the virus can also reach the brain and lead to chronic neurological symptoms. In this study, we examined the interaction SARS-CoV-2 with brain immune cells, by using an ex-vivo model of human monocyte-derived microglia. We identified robust activation of the innate immune sensor complex, NLRP3 inflammasome, in cells exposed to SARS-CoV-2. This was dependent on spike protein-ACE2 receptor interaction and was potentiated in the presence of α-synuclein. We therefore identify a possible mechanism for SARS-CoV-2 and increased vulnerability to developing neurological dysfunction. These findings support a potential therapeutic avenue for treatment of SARS-CoV-2 driven neurological manifestations, through use of NLRP3 inflammasome or ACE2 inhibitors.
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SciScore for 10.1101/2022.01.11.475947: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Ethics and biological safety: Ethical approval for collecting and utilising human donor blood was obtained from The University of Queensland Human Research Ethics Committee (HREC approval #2020000559). Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication Contamination: All cell lines were verified to be mycoplasma free by first culturing the cells in antibiotic-free media and then subjected to a mycoplasma tested using MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, UK). Table 2: Resources
Antibodies Sentences Resources Blocking buffer was removed, and serial dilutions of Nipah F- or SARS-CoV-2 S … SciScore for 10.1101/2022.01.11.475947: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Ethics and biological safety: Ethical approval for collecting and utilising human donor blood was obtained from The University of Queensland Human Research Ethics Committee (HREC approval #2020000559). Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication Contamination: All cell lines were verified to be mycoplasma free by first culturing the cells in antibiotic-free media and then subjected to a mycoplasma tested using MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, UK). Table 2: Resources
Antibodies Sentences Resources Blocking buffer was removed, and serial dilutions of Nipah F- or SARS-CoV-2 S ectodomain-specific antibodies, 5B3 and CR3022 (81), were added. CR3022suggested: NoneNext, an HRP-conjugated goat anti-human secondary antibody (ThermoFisher) was added, and the plates incubated for 1 hour at 37 °C. anti-human secondarysuggested: NoneAdditionally, low endotoxin levels for the soluble hACE2, and monoclonal antibodies (3E8 and CO5) were also produced and validated by SDS-PAGE and ELISA. CO5suggested: NoneFollowing this, cells were incubated for 2 hrs at room temperature with combinations of the following antibodies: rabbit anti-TMEM (1:100), rabbit anti-ASC (1:250), and mouse anti-Tubulin (1:250). anti-TMEMsuggested: Noneanti-ASCsuggested: Noneanti-Tubulinsuggested: (LSBio (LifeSpan Cat# LS-B4778-250, RRID:AB_10801436)Secondary antibodies used were Donkey anti-rabbit 555 (1:2000) and donkey anti-mouse 488 (1:2000). anti-mousesuggested: NoneSamples were stained using the following fluorescent secondary antibodies: Donkey anti-rabbit Alexa Fluor® 555, anti-rabbitsuggested: NoneExperimental Models: Cell Lines Sentences Resources Cells lines: Vero E6 cells (African green monkey kidney cell clones) and Caco-2 (human colorectal adenocarcinoma cells), and human embryonic kidney 293T cells (HEK293T) were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat inactivated foetal calf serum (FCS) (Bovogen, USA), penicillin (100 U/mL) and streptomycin (100 μg/mL) (P/S) and maintained at 37 °C with 5 % CO2. Caco-2suggested: None293Tsuggested: NoneTo induce the differentiation of MDMi, we incubated monocytes under serum-free conditions using RPMI-1640 Glutamax (Life Technologies) with 1% penicillin/ streptomycin (Lonza) and Fungizone (2.5 μg/ml; Life Technologies) and a mixture of the following human recombinant cytokines: M-CSF (10 ng/ml; Preprotech, 300-25), GM-CSF (10 ng/ml; Preprotech,300-03), NGF-β (10 ng/ml; Preprotech, 450-01), MCP-1(CCL2) (100 ng/ml; Preprotech, 300-04), and IL-34 (100 ng/ml; Preprotech, 200-34-250) under standard humidified culture conditions (37°C, 5% CO2) for up to 14 days. MCP-1suggested: NoneBinding assay: Vero E6 and MDMi cells were inoculated with an MOI=1 of SARS-CoV-2 (D614) for 2 hours at 4 °C and then, cells were washed 8 times with fresh cold media to remove unbound viruses. Vero E6suggested: NoneBriefly, HEK293T cells were co-transfected with the following plasmids: 1 μg of p8.91 (encoding a second-generation lentiviral packaging plasmid for HIV-1 gag-pol), 1.5 μg of pCSFLW (firefly luciferase reporter gene) and, 1 μg of plasmid encoding SARS-CoV2 spike (D614) with C-terminal 18 amino acid deletion using Lipofectamine LTX Plus reagent (Invitrogen, USA) as per manufacturer’s protocol. HEK293Tsuggested: NoneRecombinant DNA Sentences Resources Briefly, HEK293T cells were co-transfected with the following plasmids: 1 μg of p8.91 (encoding a second-generation lentiviral packaging plasmid for HIV-1 gag-pol), 1.5 μg of pCSFLW (firefly luciferase reporter gene) and, 1 μg of plasmid encoding SARS-CoV2 spike (D614) with C-terminal 18 amino acid deletion using Lipofectamine LTX Plus reagent (Invitrogen, USA) as per manufacturer’s protocol. p8.91suggested: NonepCSFLWsuggested: NoneA non-glycoprotein control (NE) was also generated using the same combination of plasmids as above, replacing plasmid encoding SARS-CoV2 spike, with 1 μg of an empty plasmid vector (pcDNA2.1). pcDNA2.1suggested: NoneSoftware and Algorithms Sentences Resources Total number of copies were calculated using a semi-log line regression using GraphPad Prism 8.0.1. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)System settings, camera exposure times, and image brightness and contrast were consistent across all samples and optimised on Imaris 9.1.0 (Bitplane, UK) to create representative images for presentation. Imarissuggested: (Imaris, RRID:SCR_007370)Statistical Analysis: All data were analysed using Prism software (GraphPad 9.1.2). Prismsuggested: (PRISM, RRID:SCR_005375)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 12 and 7. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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