https://orcid.org/0000-0001-8597-4151
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ABSTRACT
Neutrophil extracellular traps (NETs) contribute to the pathophysiology of multiple inflammatory and autoimmune diseases. Targeting the NETosis pathway has demonstrated significant therapeutic potency in various disease models. Here, we describe a first-in-class monoclonal antibody (CIT-013) with high affinity for citrullinated histones H2A and H4, which inhibits NETosis and reduces tissue NET burden in vivo with significant anti-inflammatory consequences. We provide a detailed understanding of the epitope selectivity of CIT-013. Detection of CIT-013 epitopes in rheumatoid arthritis (RA) synovium provides evidence that RA is an autoimmune disease with excessive citrullinated NETs that can be targeted by CIT-013. We show that CIT-013 acts upon the final stage of NETosis, binding to its chromatin epitopes when plasma membrane integrity is compromised to prevent NET release. Bivalency of CIT-013 is necessary for NETosis inhibition. In addition, we show that CIT-013 binding to NETs and netting neutrophils enhance their phagocytosis by macrophages in an Fc-dependent manner. This is confirmed using a murine neutrophilic airway inflammation model where a mouse variant of CIT-013 reduced tissue NET burden with significant anti-inflammatory consequences. CIT-013’s therapeutic activity provides new insights for the development of NET antagonists and indicates the importance of a new emerging therapy for NET-driven diseases with unmet therapeutic needs.
Acknowledgments
This work is dedicated to Dr Jos Raats who unfortunately has passed away unexpectedly in March 2022. Without him, the ACHA program as it is today would not have seen the light.
We thank Dr J. van Rosmalen for designing, cloning, production, and purification of monovalent CIT-013. We also thank Jeroen Cole, Anna Feher, and Peter Drent from Confocal.nl and Pim French and Maurice de Wit from Erasmus Medical Centre for their support with confocal microscopy. We thank Tim van Breemen and Josephine Stein for their contribution to mechanism of action-related experiments.
Disclosure statement
Citryll employees have financial interest.
Author contribution
P.V., M.L., S.K., A.K., S.D., D.M, and R.G.S.C. designed the experimental strategies. M.L., S.K., D.M., M.F., I.R., A.K., S.D., T.B., K.W., R.P., P.Z., L.E., and E.N. performed experiments and analyzed the data. M.L. and D.M. performed statistical analysis. P.V., M.L., S.K., D.M., M.F., E.N. L.E., H.E., E.M., and R.G.S.C. provided scientific input. M.L., S.K., D.M., H.E., E.M., and R.G.S.C. drafted and/or designed the manuscript. R.G.S.C is the senior investigator of this study. All authors critically reviewed the paper.
Data availability statement
All data are available under a material transfer agreement with Citryll B.V.
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2281763
Abbreviations
| A23187 | = | Calcium ionophore |
| ACHA | = | Anti-citrullinated histone antibodies |
| ACPA | = | Anti-citrullinated protein antibodies |
| ALI | = | Acute lung injury |
| APS | = | Antiphospholipid syndrome |
| ARDS | = | Acute respiratory distress syndrome |
| BALF | = | Bronchoalveolar lavage fluid |
| COPD | = | Chronic obstructive pulmonary disease |
| CFA | = | Complete freund’s adjuvant |
| cIgG | = | Isotype control IgG |
| Cyto D | = | Cytochalasin D |
| Dex | = | Dexamethasone |
| DPI | = | Diphenyleneiodonium chloride |
| FBS | = | Fetal bovine serum |
| Fc | = | Fc gamma receptor |
| HDM | = | House dust mite |
| HK | = | Heat-killed |
| HL488 | = | HiLyteTM Fluor 488 |
| HS | = | Hidradenitis suppurativa |
| HSA | = | Human serum albumin |
| ICs | = | Immune complexes |
| IHC | = | Immunohistochemistry |
| KD | = | Equilibrium dissociation constant |
| m-ACHA | = | Mouse anti-citrullinated histone antibody (murine variant of CIT-013) |
| MPO | = | Myeloperoxidase |
| NADPH | = | Nicotinamide adenine dinucleotide phosphate |
| NE | = | Neutrophil elastase |
| NETs | = | Neutrophil extracellular traps |
| PAD4 | = | Peptidyl arginine deiminase 4 |
| PMA | = | Phorbol 12-myristate 13-acetate |
| RA | = | Rheumatoid arthritis |
| RF | = | Rheumatoid factor |
| ROS | = | Reactive oxygen species |
| RT | = | Room temperature |
| SF | = | Synovial fluid |
| SLE | = | Systemic lupus erythematosus |
| SPR | = | Surface Plasmon Resonance |
Correction Statement
This article has been corrected with minor changes. These changes do not impact the academic content of the article.