"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 objective: The 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.

Methods: 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 in vivo in LPS-ALI macaque model and in high-mortality LPS-induced encephalopathy in hypertensive rats.

Results: ChipCytometry 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.

Conclusion: 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.

Keywords: DEspR; LPS-acute inflammation tissue injury models; LPS-brain encephalopathy; acute lung injury; neutrophil subset; segmental LPS challenge.

Figure 1

Representative ChipCytometry detection of DEspR+CD66b+ neutrophils. (A) Diagram of ChipCytometry analysis in the segmental LPS-challenge model to obtain segment-specific human BALF cells at baseline (left lower lung), 24-hours after saline (right middle lobe) or LPS-challenge (left lingular lobe). (B-D) Representative ChipCytometry images from human healthy volunteers at baseline (B), in lung segments with saline instillation (C), and in lung segment with LPS-instillation (D). Columns represent: 1] transmitted light, 2] autofluorescence in phycoerythrin (PE) and 3] Alexa-Fluor (AF)488 channels, 4] CD66b-PE immunofluorescence after subtraction of PE-autofluorescence, 5] DEspR-AF488 immunofluorescence after subtraction of AF488-autofluorescence. Transmitted and fluorescence light images of four representative CD66b+ granulocytes are depicted for each. (E, F) Representative low magnification images of ChipCytometry, after subtraction of autofluorescence signals, showing CD66b+ neutrophils (E), and corresponding CD66b+DEspR+ neutrophils, wherein CD66b+ neutrophils are encircled (F). Red boxed region of interest (ROI) shown in panel G in higher magnification. (G) Representative image showing CD66b+ neutrophils (encircled in yellow) that are DEspR[-] red (↓) and DEspR+CD66b+ neutrophils with nuclear expression of DEspR white (↓). (H, I) Analysis of changes between baseline, 24-hours after segmental-saline and segmental-LPS challenge in BALF (H) number (#) of CD66b+DEspR+ neutrophils (Ns) and (I) BALF total # of neutrophils (Ns). (J, K) Analysis of changes between baseline and 24-hours (24h) post-segmental LPS-challenge in (J) peripheral absolute neutrophil (N) counts (K/μL) in HVs and in (K) peripheral neutrophil-to-lymphocyte ratio.

Figure 2

In vivo analysis in LPS-transient ALI rhesus macaque model studies. (A) Diagram of in vivo study along timeline, t-0 (baseline) to 72hrs with measures obtained. LPS [50 μg/kg] was infused intravenously (IV) after a IV single dose of humanized anti-DEspR (hu6g8) antibody (n = 3), or mock-treatment (Tx) saline for placebo LPS-control (n = 2). Non-LPS PBS-only (n = 1) served as negative control. Measures obtained at designated time points: plasma TNF-α, IL-6; body temperature (temp), hypoxemia (% O2-saturation), flow cytometry analysis of % DEspR+CD11b+ neutrophils (Ns) in BALF, bronchoalveolar lavage fluid and whole blood samples, and absolute total number (#) of peripheral DEspR+CD11b+ neutrophils (K/μL) in whole blood samples. BL, baseline levels, NL, normal levels; 0, zero levels; ↓, decrease; + to +++, increased levels with higher +++; h, hrs; Δ*, significant change between treated and saline mock-treated macaques in corresponding parameter. (B) Pharmacokinetic analysis of anti-DEspR hu6g8 in macaques (n = 2) given 3 mg/kg dose IV. Half-life of distribution, t ½ α: 18.6 hours (h), half-life of elimination, t ½ β: 12.1 days (D). Time points for human IgG4 ELISA measurements in another study group of macaques (n = 2): 2-, 4-, 8-, 24-, 72-, 144-, 312-, 480-hours. Time course of averages of different measures per study group plotted (C-N), n = 3 LPS + treatment (LPS-Tx), n = 2 LPS-saline control, n = 1 no-LPS, no treatment control. One-way repeated measures ANOVA, with Holm-Sidak pairwise comparisons performed, significant P values notated respectively. (C) ELISA plasma TNF-α (ng/ml); (D) ELISA plasma interleukin (IL)-6 (ng/ml); (E) body temperature (°C); (F) O2 saturation (SpO2): *, P = 0.03. (G) neutrophil-lymphocyte ratio (NLR), (H) peripheral % DEspR+CD11b+ neutrophils (Ns): 24h (*) P = 0.02; 72h (**) P = 0.03); (I) peripheral total number (#) of DEspR+CD11b+ Ns (K/μL): 8h (*) P = 0.03; 24h (***) P = 0.0001; 72h (*) P = 0.01. (J) BALF % DEspR+CD11b+ Ns: 4h: (*) P = 0.02. (K) Peripheral absolute neutrophil counts (ANC), (L) % DEspR-negative [-] neutrophils (Ns): 24h (**), P = 0.008, and 72h (**) P = 0.007. (M) Peripheral number (#) of DEspR[-] Ns, and (N) BALF % DEspR[-] Ns: 4h (*) P = 0.02.

Figure 3

Analysis of neutrophil adhesion during live cell imaging. (A) Representative live-cell imaging snapshot showing DEspR+ neutrophils (with bound AF568-hu6g8) and unbound DEspR[-] neutrophils/leukocytes adhered onto the microfluidic-chip channels at t-5 min from seeding, before media change. (B) Representative live-cell imaging snapshot immediately after media change t-5.3 min detecting high-adhesion leukocytes (adhesion resists media change) and concomitant loss of leukocytes (low adhesion). (C) Graph showing that majority of high-adhesion neutrophils (solid bars) are DEspR+ (red); majority of low adhesion cells (open bars) are DEspR[-] (grey), P < 0.0001, Fisher’s Exact test. (D) Representative live-cell imaging snapshot at t-30 min with internalized AF568-anti-DEspR hu6g8 moving towards focal z-plane preset at mid-nuclear level during live-cell imaging. (E) Representative snap shot of high-adhesion cells after media change among DEspR+ and DEspR [-] neutrophils/leukocytes, and concomitant loss of low-adhesion cells. (F) Bar graph showing loss of DEspR+ neutrophils at ~t-30.3 min from seeding and after media change. Fisher’s exact test P value = 0.0029. Bar = 10 μm. (G) Representative serial live cell imaging snapshots at t-7h showing DEspR+ neutrophils fluorescing with internalized AF568-anti-DEspR hu6g8 and exhibiting nuclear and cellular budding characteristic of apoptosis within 10min from start of cell budding. (H) Representative serial live cell imaging snapshots at t-19h showing late apoptosis characteristics with nuclear condensation, and increasing cell swelling and permeability marked by increased staining with impermeant Sytox Green DNA-stain over 70 minutes.

Figure 4

DEspR+ neutrophil-subset roles in LPS-induced tissue injury model. (A) Diagram of LPS-induced multi-organ encephalopathy rat model in hsICH-prone rats. Regular salt-challenge (0.4% NaCl regular rat chow) from embryonic day 0.5 (E0.5) in Dahl Salt-sensitive hypertensive rat inducing hypertension-associated neutrophil/endothelial activation. Sub-endotoxic dose of LPS (1.8 mg/kg IV) was infused in study rats after observation of the 1^st intracerebral hemorrhage event in the age-matched rat cohort around 4m of age (signal ICH ~4m). After LPS was infused, treatment (Tx), either anti-DEspR mAb 10a3 or 6g8 as notated, or mock-treatment (mockTx) saline (vehicle) was given. Treated rats with full recovery were monitored until 35 days (35d). (B) Representative post-mortem images of rat brains after intravascular blood volume replaced with 1X PBS. Left, non-LPS-challenged brain; Middle: anti-DEspR (10a3) mAb treated brain after LPS-infusion. Right: LPS-challenge mock-treated control. (C) Kaplan-Meier survival curve of treated (Tx: 10a3, 1 mg/kg IV, n = 8) vs saline mock-treated (mockTx, n = 9) LPS-challenged hsICH rats. Log Rank Mantel-Cox test: P = 0.0007; hazard ratio (Mantel-Haenszel 10.2, 95% CI of ratio: 2.67 – 39.25). (D-G) Analysis of anti-DEspR mAb target engagement and bioeffects. Study groups are designated (+ or -) per agent (left to right): black open bar = control no LPS and no treatments, black bar = reference control LPS + saline, red bar = LPS + 10a3; red open bar = LPS + 6g8 (D) Minimal to no DEspR+ neutrophils in no-LPS rat control. Compared to LPS + saline control (100% reference), anti-DEspR mAbs 10a3 and 6g8 reduce survival of DEspR+ neutrophils (n = 6 replicates/group), concordant with target engagement and bioeffects in peripheral DEspR+ neutrophils ex vivo. (E) Brain membrane-bound protein levels of mouse-specific immunoglobulin (IgG) in 10a3 or 6g8 mAbs showing brain target engagement, 6g8 > 10a3. (F, G) Analyses of brain non-membrane bound proteins by ELISA detect reduced brain levels of (F) neutrophil-derived myeloperoxidase (MPO) in 10a3 and 6g8-treated rat brains compared to LPS + saline mockTx rat brain, and (G) reduced levels of albumin as a marker of edema in the brain. ng/g brain, nanogram per gram brain; μg/g brain, microgram per gram brain.