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Authors: Sebastian J. Theobald, Alexander Simonis, Christoph Kreer, Matthias Zehner, Julia Fischer, Marie-Christine Albert, Jakob J. Malin, Jessica Gräb, Sandra Winter, Ute Sandaradura de Silva, Boris Böll, Philipp Köhler, Henning Gruell, Isabelle Suàrez, Michael Hallek, Gerd Fätkenheuer, Norma Jung, Oliver Cornely, Clara Lehmann, Hamid Kashkar, Florian Klein, Jan Rybniker

Link to paper: https://www.researchsquare.com/article/rs-30407/v1

Journal/ Pre-Print: Researchsquare

Tags: Immunology/Immunity, Biochemistry, Cell Biology, Inflammation, Molecular Biology

Research Highlights 

1. SARS-Cov2 S-protein primes NLRP3 inflammasome and IL-1b secretion from COVID-19 patient macrophages but not healthy control (naïve) macrophages

2. S-protein increases IL-1b transcript levels in macrophages of COVID-19 patients much more compared to macrophages from healthy individuals

3. S-protein primed and nigericin induced secretion of IL-1b from COVID-19 patient macrophages can be inhibited by MCC950 (selective NLRP3 inhibitor) & Hydroxychloroquine

Summary 

Monocytes from 6 COVID-19 patients and 6 healthy naïve individuals were isolated from PBMCs and differentiated into macrophages. Cells were incubated with purified SARS-CoV2 S-protein and subsequently challenged with inflammasome activator nigericin. Only patient macrophages responded with IL-1b secretion, and this S-priming effect on inflammasome was regulated transcriptionally, as IL-1b mRNA was elevated in these cells. On the contrary, LPS primed inflammasome equally in patient and healthy macrophages. MCC950, a selective NLRP3 inhibitor, blocked IL-1b secretion, and so did hydroxychloroquine. S-protein alone induced IL-8, -6 and TNFa secretion equally in healthy and COVID-19 macrophages. Convalescent macrophages behaved similarly to patient macrophages, although the strength of the S-priming effect on NLRP3 decreased over time. 

Impact for SARS-CoV2/COVID19 research efforts

Understand the immune response to SARS-CoV2/COVID19-

S-protein only primes NLRP3 from COVID-19 patient monocytes by increasing IL-1b mRNA ex vivo, indicating that COVID-19 environment already reprograms these cells in vivo

Treatment of SARS-CoV2/COVID19 positive individuals- findings relevant to vaccine design targeted to S-protein; authors also speculate on IL-1 inhibitors as possible treatment, or inflammasome inhibitors

Study Type

· In vitro study

· Patient Case study

Strengths and limitations of the paper

Novelty: Ability of SARS-CoV2 spike protein to prime the NLRP3 inflammasome in patient but not healthy monocyte derived macrophages

Standing in the field: Agrees with the increase in pro-inflammatory cytokines seen in COVID-19 patients

Appropriate statistics: Yes

Viral model used: Isolated SARS-CoV2 spike protein was used to treat macrophages differentiated from CD14+-selected PBMCs with M-CSF

Translatability: Not directly, indicative of the potential use of NLRP3 inhibitors (such as MCC950) and/or IL-1 inhibitors, to alleviate cytokine storm in patients

Potential role of S-protein in innate immune recognition: S-protein induces pro-inflammatory cytokine secretion potentially acting as a PAMP. This could correlate with results that immune priming in the context of trained immunity may play a role in better disease outcomes (data from Mihai Netea’s group in the Netherlands). However, as identified below, many limitations remain until this becomes a strong indication

Main limitations:

· low n (6 patients, 6 controls), in one experiment with cells from recovered patients n=2

· Could increased priming effect be caused by endotoxin-contaminated spike protein preparation?

· Does priming with S-protein function via NFkB-pathway? No speculation whatsoever on the possible mechanism. Given that the cells come from the blood they could have experienced some soluble factor to influence their response to S-protein in such a way- possible experiment would be to supplement naïve macrophages with patient sera and look whether S-protein could exert similar priming on NLRP3 to the macrophages from COVID-19 patients.

· Also it would be relevant to examine whether repeated stimulation/priming of macrophages from control blood with S-protein would make them secrete IL-1b to the similar levels as patient macrophages or is it something in the COVID-19 in vivo inflammatory environment, maybe even full virus exposure programming these cells to respond differentially to S-protein

· There are now multiple scRNAseq datasets available of patient PBMCs- it would have been nice to explore those for potential mechanism of differential inflammasome and IL-1b regulation in peripheral monocytes

· 5 out of 6 COVID-19 patients had co-morbidities which could be a reason for increased responsiveness towards S-protein (infected controls without co-morbidities should clarify this)

· Only 2 convalescent individuals were used to test the S-priming effect on NLRP3 upon recovery, it would be relevant to examine this “innate-training” paradigm more thoroughly

· Researchers show IL-1b mRNA upregulation in response to S-protein, but is NLRP3 transcription upregulated as well or is priming with S-protein only changing post-translational modifications on NLRP3 itself (see Juliana et al, 2012)

· Are other inflammasomes involved, such as AIM2 or NLRC4? Inhibition of ASC or repetition of the experiments in ASC-KO mice would clarify the involvement of other inflammasome sensors

· In addition to inhibitor MCC950, it would be nice to show NLRP3-specificity of mechanism in NLRP3-KO cells