“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

Abstract

Background and objectiveThe correlation (Rs > 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 in vivo studies to validate DEspR+ neutrophil-subset as therapeutic target and test efficacy of DEspR-inhibition in acute neutrophilic hyperinflammation models.MethodsWe 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 in vivo in LPS-ALI macaque model and in high-mortality LPS-induced encephalopathy in hypertensive rats.ResultsChipCytometry detected increased BALF total neutrophil and DEspR+CD66b+ neutrophil counts after segmental LPS-challenge compared to baseline (P =0.034), as well as increased peripheral neutrophil counts and neutrophil-lymphocyte ratio (NLR) compared to pre-LPS level (P <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 (P =0.03) and neutrophil influx into BALF (P =0.0001) in LPS-ALI vs vehicle mock-treated LPS-ALI macaques. Ex vivo live cell imaging of macaque neutrophils detected greater “intrinsic adhesion to hard-surface” in DEspR+ vs DEspR[-] neutrophils (P <0.001). Anti-DEspR[hu6g8] antibody abrogated intrinsic high adhesion in DEspR+ neutrophils, but not in DEspR[-] neutrophils (P <0.001). In the LPS-encephalopathy rat model, anti-DEspR[10a3] antibody treatment increased median survival (P =0.0007) and exhibited brain target engagement and bioeffects.ConclusionDetection 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.

Circulating neutrophil extracellular trap (NET)-forming ‘rogue’ neutrophil subset, immunotype [DEspR+CD11b+], mediate multi-organ failure in COVID-19 - an observational study

“…Premised upon preclinical studies detecting a causal role of SARS CoV2 on NETformation and NET-forming neutrophils on capillary-tissue barriers, [8,9] this prospective study of patients with acute COVID-19 identified DEspR+[NET+Ns] as a mediator of multi-organ failure progression. These observations are supported by the causal role of NETs in direct endothelial cell injury demonstrated in ex vivo studies [8][9][10]30], and the causal role of DEspR in extending neutrophil lifespan [20]. We note that while causal mediation analysis has been previously used to evaluate inflammatory of the effect of obesity on risk for mortality in COVID-19 [31], and soluble RAGE receptor levels and angiopoietin-2-levels in sepsis-related ARDS [32,33], here we report causal mediation analysis of NET-forming neutrophil subsets.…”

“…Our recent studies have identified DEspR+CD11b+ neutrophils (DEspR+[Ns] from hereon), as a dysregulated "rogue" neutrophil-subset capable of NET-formation while in the circulation, extended survival, low-clearance, enhanced adhesion, and association with severity measures and mortality in COVID-19 acute respiratory distress syndrome (ARDS) [18][19][20].…”

Circulating neutrophil extracellular trap (NET)-forming ‘rogue’ neutrophil subset, immunotype [DEspR + CD11b +], mediate multi-organ failure in COVID-19—an observational study

“…Premised upon preclinical studies detecting a causal role of SARS CoV2 on NET-formation [ 8 ] and NET-forming neutrophils on capillary-tissue barriers, [ 8 , 9 ]this prospective study of patients with acute COVID-19 identified DEspR + [NET + Ns] as a mediator of multi-organ failure progression. These observations are supported by the causal role of NETs in direct endothelial cell injury demonstrated in ex vivo studies [ 8 – 10 , 30 ], and the causal role of DEspR in extending neutrophil lifespan [ 20 ]. We note that while causal mediation analysis has been previously used to evaluate inflammatory mediators of the effect of obesity on risk for mortality in COVID-19 [ 31 ], and soluble RAGE receptor levels and angiopoietin-2-levels in sepsis-related ARDS [ 32 , 33 ], here we report causal mediation analysis of NET-forming neutrophil subsets.…”

“…Our recent studies have identified DEspR + CD11b + neutrophils (DEspR + [Ns] from hereon), as a dysregulated “rogue” neutrophil-subset capable of NET-formation while in the circulation, extended survival, low-clearance, enhanced adhesion, and association with severity measures and mortality in COVID-19 acute respiratory distress syndrome (ARDS) [ 18 – 20 ]. Here, we test whether the putative “rogue” CD11b + DEspR + NET-forming neutrophil-subset, DEspR + [NET + N], mediates the worsening of early multi-organ dysfunction (measured by the Sequential Organ Failure Assessment [SOFA]-score [ 21 ] at timepoint-1) towards multi-organ failure (measured as higher SOFA-scores at a later timepoint-2), and/or mediates poor clinical outcomes (as measured by intensive care unit free days [ICUFD] by day-28) [ 22 ] in severe COVID-19 using causal mediation analysis.…”

The double‐edged role of neutrophil heterogeneity in inflammatory diseases and cancers

“…195 This phenotype may be induced by CIRP. 196 Carstensen et al 197 demonstrated that inhibition of the DEspR + CD11b + neutrophil subset could alleviate multiple respiratory diseases. 198 While targeting the intervention of CD11b + CD18 + neutrophils would aggravate influenzainduced lung damage, the specific mechanism of which was related to affecting functions of T cells.…”