"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.

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.

A targetable 'rogue' neutrophil-subset, [CD11b+DEspR+] immunotype, is associated with severity and mortality in acute respiratory distress syndrome (ARDS) and COVID-19-ARDS.

Neutrophil-mediated secondary tissue injury underlies acute respiratory distress syndrome (ARDS) and progression to multi-organ-failure (MOF) and death, processes linked to COVID-19-ARDS. This secondary tissue injury arises from dysregulated neutrophils and neutrophil extracellular traps (NETs) intended to kill pathogens, but instead cause cell-injury. Insufficiency of pleiotropic therapeutic approaches delineate the need for inhibitors of dysregulated neutrophil-subset(s) that induce subset-specific apoptosis critical for neutrophil function-shutdown. We hypothesized that neutrophils expressing the pro-survival dual endothelin-1/VEGF-signal peptide receptor, DEspR, are apoptosis-resistant like DEspR+ cancer-cells, hence comprise a consequential pathogenic neutrophil-subset in ARDS and COVID-19-ARDS. Here, we report the significant association of increased peripheral DEspR+CD11b+ neutrophil-counts with severity and mortality in ARDS and COVID-19-ARDS, and intravascular NET-formation, in contrast to DEspR[-] neutrophils. We detect DEspR+ neutrophils and monocytes in lung tissue patients in ARDS and COVID-19-ARDS, and increased neutrophil RNA-levels of DEspR ligands and modulators in COVID-19-ARDS scRNA-seq data-files. Unlike DEspR[-] neutrophils, DEspR+CD11b+ neutrophils exhibit delayed apoptosis, which is blocked by humanized anti-DEspR-IgG4S228P antibody, hu6g8, in ex vivo assays. Ex vivo live-cell imaging of Rhesus-derived DEspR+CD11b+ neutrophils showed hu6g8 target-engagement, internalization, and induction of apoptosis. Altogether, data identify DEspR+CD11b+ neutrophils as a targetable 'rogue' neutrophil-subset associated with severity and mortality in ARDS and COVID-19-ARDS.

Inflammatory cytokines can be monitored in exhaled breath particles following segmental and inhalation endotoxin challenge in healthy volunteers.

Particles in exhaled air (PEx) are generated when collapsed small airways reopen during breathing. PEx can be noninvasively collected by particle impaction, allowing the analysis of undiluted epithelial lining fluid (ELF). We used the endotoxin (LPS) challenge model to proof the concept that PEx can be used to monitor inflammatory changes in the lung. In this pilot study PEx were collected from ten healthy nonsmoking subjects using the PExA® instrument twice before and twice after a segmental LPS challenge (5, 21 h). Following a 4-week washout period, PEx were collected during the week before and 5 h after a whole lung LPS inhalation challenge. PEx biomarkers were compared to blood, bronchoalveolar lavage (BAL) following segmental challenge and induced sputum (ISP) following inhalation challenge. A clear LPS-induced inflammatory response was detectable in BAL fluid, ISP and blood. Albumin and surfactant-protein D were detectable in all PEx samples. While most baseline cytokines were close to or below the detection limit, the median (IQR) IL-6 and IL-8 concentrations in PEx increased significantly after segmental (0.04 (0.03; 0.06) fg/ng PEx; 0.10 (0.08; 0.17) fg/ng PEx) and inhalation LPS challenge (0.19 (0.15; 0.23) fg/ng PEx; 0.32 (0.23; 0.42) fg/ng PEx). Using a highly sensitive analysis platform, we were able to detect a cytokine response in PEx during the early phase of LPS-induced inflammation. This will broaden the spectrum of applications for this noninvasive method to monitor inflammatory processes in the lung, including its use in clinical trials for respiratory drug development.Trial registration: The study has been registered on 07.02.2017 at Clinicaltrials.gov (NCT03044327).

Basophil activation test: Assay precision and BI 1002494 SYK inhibition in healthy and mild asthmatics.

BACKGROUND: Application of basophil activation test (BAT) in clinical trials requires assay validity. Whether assay variability differs between healthy and asthmatic subjects is mostly unknown. This study compares basophil stimulation using blood from healthy and asthmatic subjects with or without inhibition of spleen tyrosine kinase (SYK). METHODS: Whole blood of healthy and mild asthmatic subjects was stimulated with anti-dinitrophenyl (DNP) IgE/DNP bovine serum albumin and anti-IgE. Basophil activation was detected by CD63 and CD203c expression. CD63 expression levels were compared with serum IgE levels. Three operators repeated experiments with three subjects each from both groups at 3 days to observe assay precision. The effect of the SYK inhibitor BI 1002494 was assessed in BAT for both healthy and asthmatic subjects. RESULTS: BAT was reproducible in both groups. Acceptance criteria of <25% CV were mostly fulfilled. Stimulation with anti-DNP (p < 0.001, r = -0.80) but not anti-IgE (p = 0.74, r = 0.05) was related to serum IgE with levels > 200 IU/ml limiting anti-DNP stimulation. BI 1002494 IC50 values were 497 nM and 1080 nM in healthy and 287 nM and 683 nM in asthmatics for anti-DNP and anti-IgE stimulation, respectively. CONCLUSION: BAT, performed with blood from healthy or asthmatic subjects, is a robust test for the measurement of a physiological response in clinical trials. Blood from asthmatic donors with serum IgE > 200 IU/ml is less feasible when using anti-DNP stimulation. SYK inhibition was not affected by disease status.

Prostaglandin D2 metabolites activate asthmatic patient-derived type 2 innate lymphoid cells and eosinophils via the DP2 receptor.

BACKGROUND: Prostaglandin D2 (PGD2) signaling via prostaglandin D2 receptor 2 (DP2) contributes to atopic and non-atopic asthma. Inhibiting DP2 has shown therapeutic benefit in certain subsets of asthma patients, improving eosinophilic airway inflammation. PGD2 metabolites prolong the inflammatory response in asthmatic patients via DP2 signaling. The role of PGD2 metabolites on eosinophil and ILC2 activity is not fully understood. METHODS: Eosinophils and ILC2s were isolated from peripheral blood of atopic asthmatic patients. Eosinophil shape change, ILC2 migration and IL-5/IL-13 cytokine secretion were measured after stimulation with seven PGD2 metabolites in presence or absence of the selective DP2 antagonist fevipiprant. RESULTS: Selected metabolites induced eosinophil shape change with similar nanomolar potencies except for 9α,11ß-PGF2. Maximal values in forward scatter of eosinophils were comparable between metabolites. ILC2s migrated dose-dependently in the presence of selected metabolites except for 9α,11ß-PGF2 with EC50 values ranging from 17.4 to 91.7 nM. Compared to PGD2, the absolute cell migration was enhanced in the presence of Δ12-PGD2, 15-deoxy-Δ12,14-PGD2, PGJ2, Δ12-PGJ2 and 15-deoxy-Δ12,14-PGJ2. ILC2 cytokine production was dose dependent as well but with an average sixfold reduced potency compared to cell migration (IL-5 range 108.1 to 526.9 nM, IL-13 range: 125.2 to 788.3 nM). Compared to PGD2, the absolute cytokine secretion was reduced in the presence of most metabolites. Fevipiprant dose-dependently inhibited eosinophil shape change, ILC2 migration and ILC2 cytokine secretion with (sub)-nanomolar potencies. CONCLUSION: Prostaglandin D2 metabolites initiate ILC2 migration and IL-5 and IL-13 cytokine secretion in a DP2 dependent manner. Our data indicate that metabolites may be important for in vivo eosinophil activation and ILC2 migration and to a lesser extent for ILC2 cytokine secretion.

Increased Neutrophil-Subset Associated With Severity/Mortality In ARDS And COVID19-ARDS Expresses The Dual Endothelin-1/VEGFsignal-Peptide Receptor (DEspR): An Actionable Therapeutic Target.

Neutrophil-mediated secondary tissue injury underlies acute respiratory distress syndrome (ARDS) and progression to multi-organ-failure (MOF) and death, processes linked to severe COVID19. This 'innocent bystander' tissue injury arises in dysregulated hyperinflammatory states from neutrophil functions and neutrophil extracellular traps (NETs) intended to kill pathogens, but injure cells instead, causing MOF. Insufficiency of prior therapeutic approaches suggest need to identify dysregulated neutrophil-subset(s) and induce subset-specific apoptosis critical for neutrophil function-shutdown and clearance. We hypothesized that neutrophils expressing the pro-survival dual endothelin-1/signal peptide receptor, DEspR, are apoptosis-resistant just like DEspR+ cancer cells, hence comprise a consequential pathogenic neutrophil-subset in ARDS and COVID19-ARDS. Here, we report correlation of circulating DEspR+CD11b+ activated neutrophils (DESpR+actNs) and NETosing-neutrophils with severity in ARDS and in COVID19-ARDS, increased DEspR+ neutrophils and monocytes in post-mortem ARDS-patient lung sections, and neutrophil DEspR/ET1 receptor/ligand autocrine loops in severe COVID19. Unlike DEspR[-] neutrophils, ARDS patient DEspR+actNs exhibit apoptosis-resistance, which decreased upon ex vivo treatment with humanized anti-DEspR-IgG4S228P antibody, hu6g8. Ex vivo live-cell imaging of non-human primate DEspR+actNs showed hu6g8 target-engagement, internalization, and induction of apoptosis. Altogether, data differentiate DEspR+actNs as a targetable neutrophil-subset associated with ARDS and COVID19-ARDS severity, and suggest DEspR-inhibition as a potential therapeutic paradigm.

Quantitative analysis of endotoxin-induced inflammation in human lung cells by Chipcytometry.

Chipcytometry is a tool that uses iterative staining cycles with multiple antibodies for a detailed characterization of cells. Cell recognition is based on morphological features. Cells fixed on microfluidic chips can be stored and shipped enabling a centralized analysis, which is important for assessments in multi-center clinical trials. The method was initially implemented for the analysis of cells from peripheral blood. We adapted it to more heterogeneous human lung cells from bronchoalveolar lavage (BAL) fluid and induced sputum (IS). We aimed to assess the performance of Chipcytometry to detect and quantify the endotoxin induced inflammatory response in healthy subjects. BAL and IS samples of 10 healthy subjects were collected prior to and following segmental and inhaled endotoxin challenge. Samples were analyzed by Chipcytometry and were compared with flow cytometry, and differential cell count (DCC). Chipcytometry clearly detected the endotoxin induced inflammatory response which was characterized by a massive increase of neutrophils (BAL: 2.5% to 54.7%; IS: 40.5% to 71.1%) and monocytes (BAL: 7.7% to 24.7%; IS: 8.0% to 14.5%). While some differences between detection methods exist, the overall results were comparable. The ability of Chipcytometry to verify fluorescent signals with morphological features improved the precision of rare cell analysis such as of induced sputum lymphocytes. In conclusion, Chipcytometry enables the quantitative analysis of cells from BAL fluid and IS. Advantages over DCC and flow cytometry include the storage of cells on chips, the ability for re-analysis and the mapping of surface marker binding to morphological information. It therefore appears to be a promising method for use in clinical respiratory drug development.