Patient samples and CD14+ monocyte isolation
Blood samples were obtained from patients with proven SARS-CoV-2 infection at the University Hospital Cologne, Department I of Internal Medicine and from healthy donors. Infections were diagnosed by PCR from respiratory samples. For all samples, written informed consents approved by the ethics committee of Cologne were available in accordance to the Declaration of Helsinki. PBMCs (peripheral blood mononuclear cells) were purified by density gradient centrifugation (Ficoll Plus, GE Healthcare, Chicago, IL, USA). CD14+ cells were isolated from PBMCs by depletion of non- monocytes, using a monocyte isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany). 5×104 CD14+ cells were seeded into 96-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) and cultured for additional 4 days in Roswell Park Memorial Institute (RPMI) 1640 Medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (Thermo Fisher Scientific) and 50 ng/ml M-CSF (Miltenyi Biotec) for macrophage differentiation at 37°C and 5 % CO2.
SARS-CoV-2 spike protein expression and purification
A prefusion S ectodomain coding pαH plasmid 9, was used for recombinant protein expression. The coding sequence comprises the aminoacids 1−1208 of 2019-nCoV S (GenBank: MN908947), has two proline substitutions at residues 986 and 987, a “GSAS” substitution at residues 682–685 to remove the furin cleavage site, a C- terminal T4 fibritin trimerization motif, a Twin-Strep-tag, and an 8xHis-tag at the C- terminus. 1 L of HEK293-6E cells in FreeStyle 293 medium (Thermo Fisher Scientific) at a cell density of 0.8×106 cells/ml were transfected with polyethylenimine (PEI, Sigma-Aldrich) and 1 µg DNA per 1 mL cell culture medium. Cells were incubated for 7 days at 37°C and 5 % CO2. Supernatants were harvested by centrifugation and filter sterilized through 0.45 µm polyethersulfone (PES) Filter (Thermo Fisher Scientific). Recombinant protein was purified by Strep-Tactin affinity chromatography (IBA lifescience, Göttingen, Germany) according to the Strep-Tactin XT manual at 4°C Buffer of pooled elution fractions was exchanged to PBS pH 7.4 (Thermo Fisher Scientific) by filtrating 4 times with a 100 kDa cutoff regenerated cellulose centrifugal filter (Merck).
SARS-CoV-2 spike binding assay
For analysis of IgG interaction with SARS-CoV-2 protein, high binding 96-well ELISA plates (Corning Inc., Corning, NY, USA) were coated with SARS-CoV-2 spike protein (5 µg/ml) at 4°C overnight, washed 3x with PBS and blocked with PBS, containing 5% BSA (Carl Roth, Karlsruhe, Germany) for 60 min at RT. Thereafter, IgGs were tested at 3-fold dilutions (1:2) starting at concentrations of 166 µg/ml in PBS/5 % BSA for 120 min at RT. IgGs were isolated with Protein G Sepharose® 4 Fast Flow (GE Healthcare). The plates were washed 3x and incubated with horseradish peroxidase- conjugated goat anti-human IgG antibody (Jackson ImmunoResearch West Grove, PA, USA; 1:2500 in PBS/5 % BSA) for 60 min at RT. ELISAs were developed using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) solution (ABTS, Thermo Fisher Scientific) and absorbance (OD 415 nm - 695 nm) was measured with absorbance reader (Tecan, Männedorf, Switzerland).
Inhibitor experiments with ex vivo macrophages stimulated with the SARS-CoV- 2 spike protein
Medium of differentiated macrophages was exchanged and macrophages were incubated for 2 h at 37 °C and 5 % CO2 with either DMSO (Sigma-Aldrich, St. Louis, MO, USA), MCC950 (10µM, Sigma-Aldrich), hydroxycholorquinesulfate (HCQ; 10µM and 30µM) (APEXBIO, Houston, TX, USA) for inflammasome inhibition. Afterwards, lipopolysaccharide (0,5 µg/ml, LPS; Sigma-Aldrich) or SARS-CoV-2 spike protein (0,1 µg/ml) were added for additional 4 h in order to prime the inflammasome process. To activate IL-1β secretion nigericin (5 µM, Sigma-Aldrich) was added for 2 h at 37 °C and 5 % CO2. All assays were performed in technical duplicates. Supernatants were frozen down at -80°C for subsequent cytokine analysis. Macrophages seeded in 96-well plates were covered with in total 50 µl RLT buffer (Qiagen, Hilden, Germany) and frozen down at -80 °C for gene expression analysis.
IL-1β ELISA (BioLegend, San Diego, CA, USA) was performed according to manufactures manual. Briefly, supernatant was diluted 1:50 in IL-1β ELISA Kit diluent and incubated for 2h on previously coated 96-well ELISA plates (Thermo Fisher Scientific). All samples were measured in technical duplicates (inhibition assay was performed in technical duplicates) and concentration was determined with a corresponding standard curve provided by IL-1β ELISA kit. OD was determined with Hidex Sense microplate reader (Hidex, Turku, Finland). Data was analysed with Microsoft Excel (Microsoft, Redmond, WA, USA) and GraphPad Prism 8.0.2 software (GraphPad, San Diego, CA, USA).
Single cell suspensions were prepared from PBMCs and surface antigens were stained with fluorescently labelled antibodies: CD14 BV421 (M5E2) (BioLegend), CD16 APC-Cy7 (B73.1) (BioLegend), CD11c FITC (MJ4-27G12) (Miltenyi), HLA-DR PerCP (AC122) (Miltenyi), CD86 PE (IT2.2) (BioLegend), CD206 (19.2) (Becton Dickinson (BD), Franklin Lakes, NJ, USA) (all in a 1:100 dilution). Data was acquired on a MACSQuant 10 flow cytometer (Miltenyi) . FlowJo (v10.6.2, FlowJo, LLC, Ashland, OR, USA) was used for data analysis and presentation.
Cytokine detection assay
Cytokine quantification in EDTA-treated Plasma and supernatants of macrophages stimulation experiments were performed with the Human Inflammatory Cytokine Kit from BD.. Plasma samples and primary cell culture supernatants were diluted (1:2) with assay buffer and incubated with capture beads and PE detection reagent (all BD) for 1.5 h (plasma samples) or 3 h (supernatant samples) according to the manufacturer's instructions. Data was acquired on a MACSQuant 10 flow cytometer (Miltenyi) and analyzed with FlowJo (v10.6.2, FlowJo)) (geometric mean fluorescence intensity (MFI) of each capture bead population). Cytokine concentrations were calculated by Microsoft Excel (Microsoft) with a standard curve of the MFI using provided cytokine standards.
Gene expression analysis
For gene expression analysis, RNA from 1x105 macrophages were isolated using the RNeasy Mini Kit (Qiagen) in accordance to the manufacturer’s instructions. Subsequently, cDNA was generated by reverse transcription with a Quantitect reverse transcription kit (Qiagen).
Quantitative real time PCR was used to measure expression levels of indicated genes. Samples were measured in technical triplicates in a 96 well-plate Multicolor Real-Time PCR Detection System (IQ™5, BIO-Rad) using LightCycler®SYBR-Green I Mix (Roche, Basel, Switzerland). Data analysis was done based on linear regression of the logarithmic fluorescence values/cycle with the pogram LinRegPCR and target gene expression was normalized to the reference gene Actin.
Statistical analysis was performed with GraphPad Prism 8.0.2 software (GraphPad, San Diego, CA, USA). Statistical parameters (value of n, statistical calculation etc.) are stated in the figure legend. P-values less than or equal to 0.05 were considered statistically significant.
Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request. The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files.