EK39-eposter-herrera-victoria - Increased DEspR+CD11b+ neutrophil subset associated with high mortality in patients with ARDS, COVID19-ARDS, and intracerebral hemorrhagic stroke: a targetable nodal switch to decrease neutrophil-driven secondary tissue injury
Increased DEspR+CD11b+ neutrophil subset associated with high mortality in patients with ARDS, COVID19-ARDS, and intracerebral hemorrhagic stroke: a targetable nodal switch to decrease neutrophil-driven secondary tissue injury Victoria L. M. Herrera, Courtney Takahashi, Christopher M. Gromisch, Mai Nguyen, Kristine A. Pasion, Glaiza L. A. Tan, Bakr Jundi, Matthew S. Gromisch, Julie Y. Mosaddhegi, Angela Shih, Joshua Goldstein, W. Taylor Kimberly, Bruce D. Levy, David M. Greer, Allan J. Walkey, Nelson Ruiz-Opazo 1 Boston University School of Medicine, Boston Medical Center 2 Mass General Brigham, Harvard Medical School [BJ, AS, JG, WTK, BDL] Background: With bacteria-killing and matrix-degrading enzymes, dysregulated neutrophils comprise direct effectors of secondary tissue injury that arises from excessive innate-immunity response to a primary injury trigger. “Vicious cycles” of neutrophil secondary tissue injury remains without therapy, and contributes to high mortality associated with perihematomal edema (PHE) in spontaneous intracerebral hemorrhagic stroke (sICH), and multi-organ failure (MOF) with acute respiratory distress syndrome (ARDS). The rapid progression to MOF-ARDS in COVID19 further exposes the urgency of finding therapies that decrease or block neutrophil secondary tissue injury. Using an inter-disciplinary approach, we reasoned that finding pathogenic commonality in sICH, ARDS and COVID19-ARDS could elucidate a targetable nodal switch in neutrophil-driven secondary tissue injury. Based on our cancer xenograft model studies showing that inhibition of the dual endothelin-1/signal peptide receptor (DEspR) on cancer stem-like cells (CSCs) decreases pro-survival Mcl1 and induces CSC-apoptosis, we tested the unifying hypothesis that DEspR+ neutrophils are increased and associated with severity and mortality from neutrophil-driven secondary tissue injury in sICH, ARDS and COVID19-ARDS, and comprise actionable therapeutic targets to re-engage constitutive apoptosis in dysregulated DEspR+ neutrophils. Methods: We investigated the association of circulating DESpR+CD11b+ neutrophils and NETosing neutrophil levels with mortality and different clinical parameters in patients with sICH-PHE, ARDS-MOF and COVID19-ARDS-MOF by immunophenotyping flow cytometry (FCM). As a corollary, we studied whether DEspR-inhibition via murine and humanized anti-DEspR antibodies with minimal effector functions would improve clinically-relevant outcomes in vivo in different animal models of secondary tissue injury: a spontaneous ICH rat model, an LPS-TLR4-activated hemorrhagic encephalopathy rat model, and an LPS-TLR4-activated transient acute lung injury model in non-human primates (NHPs). To advance translational profiling, we tested humanized anti-DEspR IgG4[S228P] antibody target engagement and apoptosis bioeffects in vitro using peripheral blood neutrophils from patients with ARDS, LPS-challenged rats, and NHPs with high baseline DEspR+ neutrophils. Results: We detected significantly increased numbers (K/mL) of DEspR+CD11b+ neutrophils (DEspR+ Ns) by flow cytometry (FCM) analysis of whole blood within 1-hour of sampling comparing non-survivors (nonS) vs survivors (S) in patients with a) ARDS [nonS (mean, 95% CI: 8.06 K/mL, 1.73-14.39), S: (0.80, 0.29 – 1.31)], non-parametric two-tailed p < 0.0001; and with b) sICH [nonS (4.4 K/ul, 2.2 – 6.7) vs S (1.8 K/ul, 0.45 – 3.2) p = 0.0242, n=11). FCM-analysis of total intracellular and cell-surface DEspR+ expression in neutrophils in patients with COVID19-ARDS (disinfected PFA-fixed blood samples) also detected significantly increased number of DEspR+Ns in non-survivors (13.81, 1.17 – 26.44) vs survivors (3.849, 1.08 – 6.62), p = 0.0424, n = 11). Concordantly, the number of DEspR+Ns correlate with clinical severity scores: sequential organ failure assessment (SOFA) score: patients with ARDS: rs 0.7, p = 0.003), in COVID19-ARDS: rS 0.6, p = 0.053; PsO2/FiO2 ratio in COVID-ARDS (rS -0.6, p = 0.04), and with ICH score, Glasgow Coma Scale, 90d-mRS, hematoma and PHE-volume in sICH (rS 0.6 – 0.9, p < 0.05 to < 0.001). Analysis of post-mortem lung sections from patients with ARDS (n = 6), sICH (n = 2), and sICH rat model brains (n = 2) detected both DEspR+ and DEspR[-] neutrophil infiltrates and in the vascular lumen. Semi-quantitative immunofluorescence detects circulating DEspR+CD11b+ NETosing neutrophils, cytoplasts, and NET-DNA-strand networks concordant with microvascular occlusion or low flow ischemia observed in MOF in ARDS, and especially in severe COVID19-MOF. The number of DEspR+ NETosing neutrophils correlated with SOFA scores in patients with COVID19-ARDS (rS 0.7 P = 0.019) and was elevated in non-survivors vs survivors [nonS: (mean 11.57, 95% CI 2.3 – 20.8) vs S: (3.51, 1.7 – 5.3), P = 0.0242). DEspR+ rogue-Ns from ARDS patients exhibit enhanced survival ex vivo: 17-20 hrs 37 °C in whole blood, which was inhibited by humanized anti-DEspR IgG4[S228P]-recombinant antibody, 6g8-humab (% DEspR+ Ns by 25% to 75% of total non-Tx DEspR+ Ns), without further increasing terminal complement complex levels in Tx samples compared to patient-matched non-Tx samples (n = 2). Hu6g8 reduced DEspR+ rat neutrophil survival ex vivo in HBSS buffer, IC50 = 7 nM. Live cell imaging demonstrated hu6g8 antibody target engagement, internalization and time-dependent induction of apoptosis in NHP-DEspR+ neutrophils within 24 hours. Hu6g8 increased apoptotic cell changes 2.5-fold by 12-hours: Tx: 44 apoptotic DEspR+ neutrophils/High Power Field (HPF) vs nonTx: 16 cells/HPF, p < 0.0001, with 18 HPFs/group). In vivo, murine and humanized anti-DEspR increased median overall survival in acute sICH rat model (Kaplan-Meier survival curve analysis Tx (n = 11) vs nonTx (n = 14), p < 0.0001; and in the LPS-induced hemorrhagic encephalopathy rat model with full recovery in 50% of treated rats in contrast to 100% mortality in non-treated rats with hypertension-associated stroke prone risk factor. Similarly, in the LPS-challenged transient acute lung injury NHP-model, 6g8-humab pre-treatment prevented hypoxemia (Tx-NHPs: PsO2 86 – 97%, n = 3; vs nonTx NHP control < 80%, n = 2) and decreased DEspR+CD11b+ neutrophil levels in BAL-fluid at 4 hrs post-LPS infusion (nonTx: 70% DEspR+ Ns, Tx: < 35%) for up to 72 hours, without inducing systemic neutropenia. Implications: The pathogenic commonality of increased DEspR+CD11b+ neutrophil subset in sICH, ARDS and COVID19-ARDS, and observed preclinical efficacy of DEspR inhibition in animal models, collectively support the unifying hypothesis that DEspR+CD11b+ neutrophils comprise a targetable nodal switch with the potential to decrease neutrophil-driven secondary tissue injury, even if relevant to only ARDS, COVID-ARDS, and sICH brain edema. Data differentiate hu6g8 as a candidate antibody therapeutic, and validate the therapeutic paradigm that normalization of neutrophil constitutive apoptosis provides a turnkey to neutrophil function-shutdown, efferocytosis and initiation of active resolution.