AUTHOR=Carstensen Saskia , Müller Meike , Tan Glaiza L. A. , Pasion Khristine Amber , Hohlfeld Jens M. , Herrera Victoria L. M. , Ruiz-Opazo Nelson 

TITLE=“Rogue” neutrophil-subset [DEspR+CD11b+/CD66b+] immunotype is an actionable therapeutic target for neutrophilic inflammation-mediated tissue injury – studies in human, macaque and rat LPS-inflammation models

JOURNAL=Frontiers in Immunology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2022.1008390

DOI=10.3389/fimmu.2022.1008390

ISSN=1664-3224

ABSTRACT=<sec><title>Background and objective</title><p>The correlation (Rs &gt; 0.7) of neutrophils expressing the dual endothelin1/signal peptide receptor (DEspR+CD11b+/CD66b+) with severity of hypoxemia (SF-ratio) and multi-organ failure (SOFA-score) in patients with acute respiratory distress syndrome (ARDS) suggest the hypothesis that the DEspR+ neutrophil-subset is an actionable therapeutic target in ARDS. To test this hypothesis, we conducted <italic>in vivo</italic> studies to validate DEspR+ neutrophil-subset as therapeutic target and test efficacy of DEspR-inhibition in acute neutrophilic hyperinflammation models.</p></sec><sec><title>Methods</title><p>We performed tests in lipopolysaccharide (LPS)-induced acute neutrophilic inflammation in three species – human, rhesus macaque, rat – with increasing dose-dependent severity. We measured DEspR+CD66b+ neutrophils in bronchoalveolar lavage fluid (BALF) in healthy volunteers (HVs) 24-hours after segmental LPS-challenge by ChipCytometry, and DEspR+CD11b+ neutrophils in whole blood and BALF in an LPS-induced transient acute lung injury (ALI) model in macaques. We determined anti-DEspR antibody efficacy <italic>in vivo</italic> in LPS-ALI macaque model and in high-mortality LPS-induced encephalopathy in hypertensive rats.</p></sec><sec><title>Results</title><p>ChipCytometry detected increased BALF total neutrophil and DEspR+CD66b+ neutrophil counts after segmental LPS-challenge compared to baseline (<italic>P</italic> =0.034), as well as increased peripheral neutrophil counts and neutrophil-lymphocyte ratio (NLR) compared to pre-LPS level (<italic>P &lt;</italic>0.05). In the LPS-ALI macaque model, flow cytometry detected increased DEspR+ and DEspR[-] neutrophils in BALF, which was associated with moderate-severe hypoxemia. After determining pharmacokinetics of single-dose anti-DEspR[hu6g8] antibody, one-time pre-LPS anti-DEspR treatment reduced hypoxemia (<italic>P</italic> =0.03) and neutrophil influx into BALF <italic>(P</italic> =0.0001) in LPS-ALI <italic>vs</italic> vehicle mock-treated LPS-ALI macaques. <italic>Ex vivo</italic> live cell imaging of macaque neutrophils detected greater “intrinsic adhesion to hard-surface” in DEspR+ <italic>vs</italic> DEspR[-] neutrophils (<italic>P &lt;</italic>0.001). Anti-DEspR[hu6g8] antibody abrogated intrinsic high adhesion in DEspR+ neutrophils, but not in DEspR[-] neutrophils (<italic>P &lt;</italic>0.001). In the LPS-encephalopathy rat model, anti-DEspR[10a3] antibody treatment increased median survival (<italic>P</italic> =0.0007) and exhibited brain target engagement and bioeffects.</p></sec><sec><title>Conclusion</title><p>Detection of increased DEspR+ neutrophil-subset in human BALF after segmental LPS-challenge supports the correlation of circulating DEspR+ neutrophil counts with severity measure (SOFA-score) in ARDS. Efficacy and safety of targeted inhibition of DEspR+CD11b+ neutrophil-subset in LPS-induced transient-ALI and high-mortality encephalopathy models identify a potential therapeutic target for neutrophil-mediated secondary tissue injury.</p></sec>