Eosinophilia and reduced STAT3 signaling affect neutrophil cell death in autosomal-dominant Hyper-IgE syndrome

Abstract

The autosomal-dominant hyper-IgE syndrome (HIES), caused by mutations in STAT3, is a rare primary immunodeficiency that predisposes to mucocutaneous candidiasis and staphylococcal skin and lung infections. This infection phenotype is suggestive of defects in neutrophils, but data on neutrophil functions in HIES are inconsistent. This study was undertaken to functionally characterize neutrophils in STAT3-deficient HIES patients and to analyze whether the patients` eosinophilia affects the neutrophil phenotype in S. aureus infection. Neutrophil functions and cell death kinetics were studied in eight STAT3-deficient patients. Moreover, the response of STAT3-deficient neutrophils to S. aureus and the impact of autologous eosinophils on pathogen-induced cell death were analyzed. No specific aberrations in neutrophil functions were detected within this cohort. However, the half-life of STAT3-deficient neutrophils ex vivo was reduced, which was partially attributable to the presence of eosinophils. Increased S. aureus-induced cell lysis, dependent on the staphylococcal virulence controlling accessory gene regulator (agr)-locus, was observed in STAT3-deficient neutrophils and upon addition of eosinophils. Accelerated neutrophil cell death kinetics may underlie the reported variability in neutrophil function testing in HIES. Increased S. aureus-induced lysis of STAT3-deficient neutrophils might affect pathogen control and contribute to tissue destruction during staphylococcal infections in HIES.

Keywords: HIES; S. aureus; STAT3; eosinophils; neutrophils.

Figure 1

Accelerated cell death kinetics of neutrophils from STAT3‐deficient HIES patients. Granulocytes were isolated from STAT3‐deficient HIES patients and concomitant control samples followed by cytokine or vehicle stimulation. At designated time points (four, nine, and fourteen hours), equal amounts of the samples were stained with Annexin V and Dapi and analyzed by flow cytometry (detailed gating strategy see Supporting Fig. 11). (A–C) displays the same dataset highlighting the gating strategy for the identification of the following cell death categories according to the respective Annexin V and Dapi staining: (A) viable (Annexin V⁻ Dapi⁻), (B) early apoptotic (Annexin V⁺ Dapi⁻) and (C) late apoptotic/necrotic (Annexin V⁺ Dapi⁺) neutrophils. Primary necrotic (Annexin V⁻ Dapi⁺) neutrophils are not displayed since no significant differences were found. (D–I) Quantitative data showing the percentages of (D, G) viable, (E, H) early apoptotic and (F, I) late apoptotic/necrotic neutrophils after stimulation with (D–F) vehicle or (G–I) Il‐8. (D–I) Data are shown as mean ± SD and are pooled from four (D–F) and three (G–I) independent experiments, respectively. Each patient was matched to the concomitant daily control. Matched samples are displayed with the same symbol (dark symbols = HIES patients; light symbols = controls). RM (repeated measures) two‐way Anova with Bonferroni‐Analysis for multiple comparison was used for statistical analysis (p > 0.05 = not significant (ns); p ≤ 0.05 = *; p < 0.01 = **; p < 0.001 = ***; p < 0.0001 = ****).

Figure 2

Augmented spontaneous and S. aureus‐induced lytic cell death in STAT3‐deficient HIES patients. Eosinophils and neutrophils were purified by magnetic cell separation from granulocyte suspensions of STAT3‐deficient HIES patients and concomitant healthy controls. LDH release was measured in supernatants five hours after costimulation of various granulocyte fractions (original granulocyte suspension, highly purified neutrophils, purified neutrophils + 20% purified eosinophils) with vcl (vehicle = medium) or S. aureus WT (MOI 5). Due to limitations in cell availability not all conditions could be performed in every experiment. (A–D) Three individual experiments are shown, including cells from four different HIES patients and their concomitant controls. Depicted is the mean with SD of at least two sample replicates. B+D were performed at the same time point and contain the same controls. (E) The effect of supplementation of neutrophils with eosinophils on S. aureus‐induced cell lysis for available samples in A–C is shown. Data are pooled from three independent experiments.

Figure 3

Increased S. aureus‐induced lytic cell death in STAT3‐inhibited cells. Eosinophils and neutrophils were purified by magnetic cell separation from healthy control granulocyte suspensions. The original granulocyte suspension and/or purified neutrophils were incubated for 60 min with 20 μM ST18 (STAT3 Inhibitor XVIII, BP‐1‐102) or DMSO. Subsequently, cells were washed and coincubated with vcl (vehicle = medium), S. aureus WT and/or S. aureus agr ⁻ (both MOI 5) for five hours. (A) LDH release after stimulation of purified STAT3‐inhibited (ST18) and control (DMSO) neutrophils with/without supplementation of 20% eosinophils with vcl or S. aureus WT for five hours is shown. Displayed are pooled results from eight independent experiments with cells from nine different controls. (B) LDH release following stimulation of STAT3‐inhibited (ST18) and control (DMSO) granulocytes with vcl, S. aureus WT, or S. aureus agr ⁻ (both MOI 5) for five hours is shown. Displayed are pooled results from four independent experiments with cells from eight different controls. (C) IL‐8 release of STAT3‐inhibited (ST18) and control (DMSO) granulocytes following stimulation with vcl, S. aureus WT, or agr‐ (both MOI 5) for five hours is shown. Displayed are pooled results from three independent experiments with six different controls (detection limit 3.1 pg/mL). Statistical analysis A–C: Depicted is the mean with SD. RM (repeated measures) two‐way Anova with Bonferroni‐Analysis for multiple comparison was used for statistical analysis for A–C (p > 0.05 = not significant (ns); p ≤ 0.05 = _*; p < 0.01 = _**; p < 0.001 = _***; p < 0.0001 = _****).