IL-33 expression in response to SARS-CoV-2 correlates with seropositivity in COVID-19 convalescent individuals

Our understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still developing. We perform an observational study to investigate seroprevalence and immune responses in subjects professionally exposed to SARS-CoV-2 and their family members (155 individuals; ages 5–79 years). Seropositivity for SARS-CoV-2 Spike glycoprotein aligns with PCR results that confirm the previous infection. Anti-Spike IgG/IgM titers remain high 60 days post-infection and do not strongly associate with symptoms, except for fever. We analyze PBMCs from a subset of seropositive and seronegative adults. TLR7 agonist-activation reveals an increased population of IL-6+TNF-IL-1β+ monocytes, while SARS-CoV-2 peptide stimulation elicits IL-33, IL-6, IFNa2, and IL-23 expression in seropositive individuals. IL-33 correlates with CD4+ T cell activation in PBMCs from convalescent subjects and is likely due to T cell-mediated effects on IL-33-producing cells. IL-33 is associated with pulmonary infection and chronic diseases like asthma and COPD, but its role in COVID-19 is unknown. Analysis of published scRNAseq data of bronchoalveolar lavage fluid (BALF) from patients with mild to severe COVID-19 reveals a population of IL-33-producing cells that increases with the disease. Together these findings show that IL-33 production is linked to SARS-CoV-2 infection and warrant further investigation of IL-33 in COVID-19 pathogenesis and immunity.


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Antibodies
Antibodies used SARS-CoV-2-exposed subjects (n=155) were recruited with a standardized procedure based on assessment of inclusion and exclusion criteria. Inclusion criteria were contact to SARS-CoV-2-infected individuals, age of at least 5 years and the ability to provide a written informed consent. The exclusion criterium was the presence of symptoms of an acute SARS-CoV-2 infection (fever, cough, malaise) within the last 14 days prior to blood collection. Study participants gave their written informed consent prior to study enrollment. For subjects <18 years of age, written informed consent was provided by one parent. Sample size was not determined using statical criteria, rather it was a result of the nature of the study. This is a monocentric observational exploratory study of individuals professionally exposed to SARS-CoV-2 and their families.
No data was excluded.
Internal controls were included in all experiments to guarantee technical quality of techniques used. Stimulation experiments were repeated 3-4 times. All attempts at replication were successful. Serum measurements were performed only once in each participant, as individual samples were measured at multiple dilution factors, and we observed in all cases that signals were dependent on the sample concentration, thus increasing confidence in the result.
Samples were processed in batches as they were collected. No preference was given to serological groups (as this was unknown during sample collection). Because this was an observational study, there was no subject allocation to any experimental intervention groups that would require randomization.
Data collection, in vitro experiments and data analysis were all performed blind, with serological groups identifiable only after data analysis during the visualization stage. Serological status and infection history of subjects was not known to scientists collecting samples or performing measurements. All antibodies were commercial in source and as such validated by the manufacturer. All flow cytometry antibodies from Biolegend and eBioscience were subjected to standard procedures for flow cytometry validation by the manufacturers. All newly developed clones at BioLegend undergo validation testing for multiple applications. This serves as a cross-check for specificity and provides clarity for research uses. Typically, antibodies are tested by two or more of the below methods (flow cytometry, western blot, chromatin immunoprecipitation, immunofluorescence, immunohistochemistry, biofunctional assays). Thus, the clone cross-validates itself, by demonstrating functionality across orthogonal testing methods. Additionally, the biological induction of the expression further validates the specificity of the antibody. Knockout or knockdown of gene expression, such as with siRNA, is also an excellent tool for target validation. More information regarding validation and reproducibility can be found online on the manufacturer's website: https://www.biolegend.com/en-us/reproducibility https://www.thermofisher.com/antibody/product/IL-1-beta-Antibody-clone-CRM56-Monoclonal/11-7018-42 https://www.thermofisher.com/antibody/product/TNF-alpha-Antibody-clone-TN3-19-12-Monoclonal/25-7423-82 https://www.thermofisher.com/antibody/product/IL-6-Antibody-clone-MQ2-13A5-Monoclonal/48-7069-42 The IL-33 Antibody (clone: MA5-16242) was validated by the manufacturer for flow cytometry and western blot by staining of Jurkat cells, HUVEC cells and with a partial recombinant IL-33 control. The PE-conjugated monoclonal anti-IL-33 antibody (Clone 002, Abcam) has been validated with the Abpromise guarantee approach of the manufacturer for use in flow cytometry with HUVEC cells used as a positive control. Abcam validation statement: Reproducibility is key to advancing scientific discovery and accelerating scientists' next breakthrough. Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility. We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee. In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for. We are also updating the applications & species that this product has been "predicted to work with," however this information is not covered by our Abpromise guarantee. Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee. Further information on validation can be found on the manufacturers' websites: https://www.abcam.com/pe-il33-antibody-002-ab275607.html https://www.thermofisher.com/antibody/product/IL-33-Antibody-clone-6H617-Monoclonal/MA5-16242 Expi293F cells were purchased from ThermoFisher Scientific.