Synthetic Siglec-9 Agonists Inhibit Neutrophil Activation Associated with COVID-19 - PubMed (original) (raw)
Synthetic Siglec-9 Agonists Inhibit Neutrophil Activation Associated with COVID-19
Corleone S Delaveris et al. ACS Cent Sci. 2021.
Abstract
Severe cases of coronavirus disease 2019 (COVID-19), caused by infection with SARS-CoV-2, are characterized by a hyperinflammatory immune response that leads to numerous complications. Production of proinflammatory neutrophil extracellular traps (NETs) has been suggested to be a key factor in inducing a hyperinflammatory signaling cascade, allegedly causing both pulmonary tissue damage and peripheral inflammation. Accordingly, therapeutic blockage of neutrophil activation and NETosis, the cell death pathway accompanying NET formation, could limit respiratory damage and death from severe COVID-19. Here, we demonstrate that synthetic glycopolymers that activate signaling of the neutrophil checkpoint receptor Siglec-9 suppress NETosis induced by agonists of viral toll-like receptors (TLRs) and plasma from patients with severe COVID-19. Thus, Siglec-9 agonism is a promising therapeutic strategy to curb neutrophilic hyperinflammation in COVID-19.
© 2021 The Authors. Published by American Chemical Society.
Conflict of interest statement
The authors declare the following competing financial interest(s): C.S.D. and C.R.B. are coinventors on a patent application for cis-binding Siglec agonist glycopolymers as immune suppressants (USPTO63046140). C.R.B. is a co-founder and Scientific Advisory Board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), OliLux Inc., and InterVenn Bio, and a member of the Board of Directors of Eli Lily & Company. C.A.B. is a Scientific Advisory Board member of Catamaran Bio.
Figures
Figure 1
Local and peripheral inflammatory stimuli induce NETosis and a subsequent hyperinflammatory cascade in COVID-19. Both local inflammatory stimuli at the site of SARS-CoV-2 infection (e.g., virions) and peripheral inflammatory stimuli (e.g., the proinflammatory cytokines IL-8 and G-CSF) associated with COVID-19 have been shown to induce NETosis in vitro. These factors are suspected to be causative agents of NETosis in vivo as well, initiating a deleterious hyperinflammatory cascade leading to the symptoms of moderate and severe COVID-19. Agonists of the neutrophil-associated checkpoint receptor Siglec-9 could inhibit NETosis in COVID-19.
Figure 2
Synthetic glycopolypeptides bearing high-affinity Siglec-9 ligands engage Siglec-9 and induce clustering and signaling. (a) Membrane-anchored, cis binding glycopolypeptide 1 (pS9L) induces Siglec-9 signaling, while a soluble control polypeptide 2 (pS9L-sol) or a nonbinding but membrane-anchored control polypeptide 3 (pLac) does not. (b) Structures of the polypeptides pS9L, pS9L-sol, and pLac. Polypeptides are all based on an _O_-lactosyl poly serine-_co_-alanine scaffold and in some cases bear terminal Siglec-9-binding sialic acid analogues and/or C-terminal membrane-anchoring lipids.
Figure 3
A cis-binding Siglec-9 agonist (pS9L) inhibits R848-induced NETosis via Siglec-9 and SHP-1. (a–c) Primary neutrophils were cotreated with R848 (10 μM) and glycopolypeptide (500 nM) in IMDM supplemented 0.5% hiFBS containing the membrane impermeable DNA intercalators Cytotox Green or Red (250 nM). Images were acquired by fluorescence microscopy every 15 min for 12 h. The area of all green fluorescent objects >300 μm2 was quantified, and the total area was averaged across three images per well. Relative NETosis was determined by normalizing to the maximal NET area from R848 treatment alone (t = 8 h). (a) Representative phase contrast and fluorescence images from t = 8 h. Scale bars indicate 40 μm. (b) Quantitation of NETosis over time as area under the curve in (c). Error bars represent SD. (c) NET formation and degradation as a function of time. Error bands represent SEM. (d) Treatment of R848-stimulated neutrophils with various glycopolypeptides. Error bars represent SD. (e) HL-60 cells were transfected with siRNAs against SIGLEC9 (encoding Siglec-9), PTPN6 (encoding SHP-1), or a scrambled control and then grown for 2 days. Cells were then cotreated with R848 (10 μM) and vehicle or pS9L (500 nM). Relative NETosis is determined as in (b), except all objects >200 μm2 were quantified and the R848 maximum in dHL-60s was observed at 2.5 h post induction. Error bars represent SD. Statistics were determined by two-way ANOVA (c) or one-way ANOVA (b,d,e). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Figure 4
Siglec-9 agonist pS9L inhibits NETosis of neutrophils induced by COVID-19 plasma. (a, b) Analysis of publicly available single-cell transcriptomics data for SIGLEC9 expression (a) and PADI4 expression (b) on neutrophils in peripheral blood from healthy donors or COVID-19 patients. Error bars represent SD. Statistics were determined using mixed effects model. ** = p < 0.01; *** = p < 0.001 (c, d) Primary neutrophils were cultured in undiluted and citrate anticoagulated plasma from healthy donors or COVID-19 patients for 4 h. Cells were fixed, stained for extracellular myeloperoxidase, and imaged in DAPI imaging media by fluorescence microscopy. Cells were treated in technical triplicate and imaged across multiple fields of view. (c) Proportion of NET-positive cells (%) across all fields of view. Each dot represents and individual plasma sample. (d) Representative images from a COVID-19 patient plasma sample with or without pS9L. Error bars represent SD. Statistics were determined using mixed effects models to account for samples using repeat neutrophil donors. **** = p < 0.0001.
Update of
- Synthetic Siglec-9 Agonists Inhibit Neutrophil Activation Associated with COVID-19.
Delaveris CS, Wilk AJ, Riley NM, Stark JC, Yang SS, Rogers AJ, Ranganath T, Nadeau KC; Stanford COVID-19 Biobank; Blish CA, Bertozzi CR. Delaveris CS, et al. ChemRxiv [Preprint]. 2020 Dec 17. doi: 10.26434/chemrxiv.13378148. ChemRxiv. 2020. PMID: 33469569 Free PMC article. Updated. Preprint.
References
- Barnes B. J.; Adrover J. M.; Baxter-Stoltzfus A.; Borczuk A.; Cools-Lartigue J.; Crawford J. M.; Daßler-Plenker J.; Guerci P.; Huynh C.; Knight J. S.; et al. Targeting Potential Drivers of COVID-19: Neutrophil Extracellular Traps. J. Exp. Med. 2020, 217 (6), e20200652. 10.1084/jem.20200652. -DOI -PMC -PubMed
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