Inhibition of EETosis with an anti-citrullinated histone antibody: a novel therapeutic approach for eosinophilic inflammatory disorders

Abstract

Eosinophils are a subset of granulocytes that protect the host against fungal and parasitic infection through secretion of their granular contents. In response to specific stimuli, eosinophils also undergo a type of lytic cell death, referred to as eosinophil extracellular trap (EET)-associated cell death (EETosis), where histone citrullination facilitates chromatin decondensation, cell rupture and release of pro-inflammatory, decondensed chromatin into the extracellular environment as EETs. In this study, we show the abundant presence of eosinophils and citrullinated histones in nasal polyp tissue of patients with eosinophilic chronic rhinosinusitis (ECRS). Using live imaging microscopy on purified human eosinophils, we demonstrate that physiologically relevant stimuli induce release of citrullinated EETs and the marker of eosinophil activation galectin-10. While the kinetics of release of EETs and galectin-10 are similar, inhibitors of citrullination block EETosis in a dose dependent manner but fail to inhibit galectin-10 release. The importance of citrullination is further exemplified with CIT-013, a monoclonal antibody specific for citrullinated histones H2A and H4. CIT-013 potently inhibits release of EETs (half-maximal inhibitory concentration of 2.5 nM) without inhibiting other eosinophil functions such as degranulation, adhesion, superoxide production and induction of chemokine expression. Together, this study provides new insights into the requirement of protein arginine deiminase 4 (PAD4) for EETosis, differentiates requirements of EETosis from galectin-10 release, and identifies a novel therapeutic approach for EETosis inhibition by targeting citrullinated histones in eosinophil-driven diseases such as ECRS.

Keywords: EETosis; PAD4; anti-citrullinated histone antibody; citrullination; eosinophil extracellular traps; eosinophilic chronic rhinosinusitis; galectin-10.

Figure 1

EETs rich in citrullination are abundantly present in human ECRS tissue. (A) A representative image of nasal polyp tissue stained with hematoxylin and eosin (H&E). (B) Increased magnification of selected area from A representing H&E stain, DNA (blue), and major basic protein (MBP; red). (C) Immunofluorescence images showing DNA (blue) and citrullinated histone H3 (citH3; red). (D) Immunofluorescence images from the same section as shown in (C) with DNA (blue) and CIT-013 epitope citrullinated histone H2A (citH2A) and citH4 (green). (E) Increased magnification of selected area from (D) representing H&E stain, DNA (blue), and citH2A and citH4 (green). White arrowheads indicate CIT-013 epitope colocalization with diffuse extracellular DNA, while black arrowheads indicate the colocalization of diffused extracellular DNA with the observed presence of eosinophils. Scale bars in A, C and D are 50 µm and in B and E are 10 µm. SL, Sublining; L, Lining.

Figure 2

The kinetic profile of EET release using different EETosis-inducing stimuli visualized and measured by a quantitative immunofluorescence live imaging assay using the impermeable DNA dye Sytox^TM Green. (A) Representative images of EET release at 3 hours post stimulation with 2 µM A23187, 40.5 nM PMA, 0.25 µM platelet activating factor (PAF), and immobilized immune complexes (imICs). Scale bars are 50 µm. (B) Quantification of EET release over time upon stimulation with different stimuli (n = 4-12). (C) Quantification of EET release over time in the presence of 0.25 µM PAF in combination with the indicated concentrations of thrombin (n = 5-12). *P<0.05 and ****P<0.0001, Kruskal-Wallis test and Dunn’s multiple comparison test performed at t = 1 h.

Figure 3

Histone citrullination is required for EETosis. (A) Citrullinated nucleosome detection in EETs harvested 3 hours post stimulation with 2 µM A23187, 40.5 nM PMA, and 0.5 µM platelet activating factor (PAF) without or with 1 U/mL thrombin (n = 2-4). Quantification of A23187- and PMA-induced EET release at t = 3 h in the presence of different concentrations of JBI-589 (B) and AFM-30a (C) (n = 4-7). Data were normalized to A23187- or PMA-induced EET release without inhibitor (set as 100% EET release). ****P<0.0001, ordinary one-way ANOVA with Dunnett’s multiple comparison test.

Figure 4

CIT-013 inhibits EET release regardless of the stimulus. Quantification of EET release in the absence (No Ab) or presence of 169.3 nM of either isotype control antibody (cIgG) or CIT-013 at t = 3 h post stimulation with 2 µM A23187 (n = 18) (A), 40.5 nM PMA (n = 17) (B), 0.25 µM platelet activating factor (PAF) (n = 6) (C), and immobilized immune complexes (imICs) (n = 8) (D). (E) Quantification of EET release induced by A23187, PMA, and 0.5 µM PAF without or with 1 U/mL thrombin at t = 3 h in the presence of different concentrations CIT-013 (n = 3-9). (F) Quantification of NET and EET release at t = 3 h post stimulation with A23187 in the presence of different concentrations CIT-013 (n = 2-9). Data in (F) was normalized to stimulus-induced EET or NET release without CIT-013 (set as 100% EET or NET release). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001, Friedman test with Dunn’s multiple comparison test (A, B), ordinary one-way ANOVA with Dunnett’s multiple comparison test (C), and Kruskal-Wallis test with Dunn’s multiple comparison test (D).

Figure 5

CIT-013 blocks EET release when plasma membrane integrity is compromised. (A) Representative immunofluorescence images of a non-stimulated eosinophil or eosinophils triggered for EETosis with 2 µM A23187 in the presence of HiLyte^TM Fluor 488-conjugated CIT-013 (HL488-CIT-013). The eosinophils activated for EETosis showed chromatin decondensation and a re-organized rounded nucleus in stage 1 of EETosis, nuclear membrane rupture and chromatin spreading within the cytoplasm in stage 2 of EETosis, and plasma membrane rupture and CIT-013 binding to chromatin in stage 3 of the EETosis pathway. Of note, due to technical limitations we were not able to clearly track one specific eosinophil over time and therefore different eosinophils at each stage of the EETosis pathway are shown. (B) More representative immunofluorescence images of eosinophils triggered for EETosis with A23187 in the presence of HiLyte^TM Fluor 488-conjugated CIT-013. (C) Representative immunofluorescence images of eosinophils triggered for EETosis with A23187 in the presence of HiLyte^TM Fluor 488-conjugated isotype control antibody (HL488-cIgG). Scale bars are 10 µm.

Figure 6

CIT-013 does not inhibit eosinophil functions other than EETosis. (A) Binding of HiLyte^TM Fluor 488-conjugated isotype control antibody (HL488-cIgG) or HL488-CIT-013 to healthy eosinophils or eosinophils activated for EETosis (n = 6). (B) Quantification of eosinophil superoxide production induced by 10 ng/mL IL-5 in the absence (No Ab) or presence of 10 µg/mL anti-IL5 receptor antibody (anti-IL-5R), or 169.3 nM of either cIgG or CIT-013 (n = 8). (C) Quantification of EDN in the supernatant of eosinophil culture medium at t = 4 h post stimulation with IL-5 in the absence or presence of anti-IL-5R, cIgG, or CIT-013 (n = 8). (D) Quantification of eosinophil adhesion at t = 4 h post stimulation with IL-5 in the absence or presence of anti-IL-5R, cIgG, or CIT-013 (n = 7). (E) Quantification of MCP-1 gene expression at t = 4 h post stimulation with IL-5 in the absence or presence of anti-IL-5R, cIgG, or CIT-013 (n = 8). **P<0.01 and ***P<0.001, Unpaired two-tailed t test (A), RM one-way ANOVA with Tukey’s multiple comparison test (B, D), Kruskal-Wallis test with Dunn’s multiple comparison test (C, E).

Figure 7

Galectin-10 release is independent of citrullination and chromatin release during EETosis. (A) Quantification of galectin-10 release in the supernatant of eosinophil culture medium at t = 3 h post stimulation of EETosis-inducing stimuli 2 µM A23187, 40.5 nM PMA, and 0.5 µM platelet activating factor PAF (n = 5-8). Combined kinetic profile of EET release and galectin-10 release at t = 0, 1, 2, and 3 h (EETs) or t = 15 min, 1 h, and 3 h (galectin-10) post stimulation with A23187 (n = 7-12) (B), PAF (n = 5-12) (C), and PMA (n = 5-11) (D). (E) Galectin-10 and EDN release in the supernatant of eosinophil culture medium at t = 15 min, 1 h, and 3 h post stimulation of A23187 (relative to the release at t = 3 h (=100%); n = 7-8). (F) Quantification of galectin-10 release in the supernatant of eosinophil culture medium at t = 3 h post stimulation with EETosis-inducing stimuli A23187 in the absence (No inhibitor) or presence of EETosis inhibitors 169.3 nM CIT-013 and 4 µM JBI-589. *P<0.05, **P<0.01, and ***P<0.01, Kruskal-Wallis test with Dunn’s multiple comparison test (A) and two-way ANOVA with Sidak’s multiple comparisons test (E).