Impaired neutrophil extracellular trap formation in β-thalassaemia/HbE

Neutrophil dysfunction contributes to a high susceptibility to severe bacterial infection which is a leading cause of morbidity and mortality in β-thalassaemia/HbE, especially in splenectomised patients. This study demonstrated another abnormality of neutrophil function, namely neutrophil extracellular trap (NET) formation in splenectomised and non-splenectomised β-thalassaemia/HbE patients who had iron overload. A classification system of morphological NET formation using confocal microscopy was developed, and samples were categorized into early and late phases which were subdivided into web-like and non-web structures. At baseline, neutrophils from non-splenectomised patients (58 ± 4%) and splenectomised patients (65 ± 3%) had higher early phase NETs than those from normal subjects (33 ± 1%). As a mimic of iron overload and infection, haemin/PMA/LPS treatment led to a significant reduction of early NETs and an increase of late NETs in neutrophils from normal and non-splenectomised patients. Interestingly, neutrophils from splenectomised patients had impaired development of late NETs. This suggests that during infection bacteria might not be trapped and may spread from the site of infection resulting in higher susceptibility to severe bacterial infection in splenectomised patients.


Patients
Patients who were under treatment with prednisolone or antibiotics had excluded from this study, and none of the patients were hospitalised or transfused within the proceeding 4 weeks. All patients received daily folic acid for 5 mg/day, deferiprone (GPO-L-ONE Ò , the Government Pharmaceutical Organization, Thailand) for 50-100 mg/kg/day and hydroxyurea (Hydrea, Corden Pharma Latina S.p.A., Italy) for 5-10 mg/kg/day.

Analysis of neutrophil extracellular trap formation
Indirect intracellular immunofluorescence of isolated neutrophils from b-thalassaemia/HbE patients and normal subjects was performed to analysis the morphology and the percentages of NETs.
Briefly, attached neutrophils on 13 mm glass coverslips after PMA, LPS and haemin treatments were fixed with 4% paraformaldehyde solution (Sigma-Aldrich). After permeated and blocked neutrophils were incubated with a mouse anti-neutrophil elastase monoclonal antibody (Thermo Scientific, IL, USA) and a rabbit anti-histone H2A polyclonal antibody (Thermo Scientific) and subsequently a FITC-conjugated goat anti-mouse IgG polyclonal antibody (Thermo Scientific), a Cy5-conjugated goat anti-rabbit IgG polyclonal antibody (Thermo Scientific) and 4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Molecular Probes, OR, USA). Fluorescence confocal microscope images were captured with Z-stack mode by using an Olympus confocal laser scanning microscope FV10i-DOC (Olympus Corporation, Tokyo, Japan) equipped with Olympus FluoView software. Illustration was captured and count 100 neutrophils per specimen at 60´ oil lens. Experiments were duplicated.

Statistical analysis
Data were analysed using SPSS version 18.0 (IBM Collaboration, Chicago, IL, USA) and GraphPad PRISM 6.0 (GraphPad Software, San Diego, CA, USA) software. Comparisons between parameters were evaluated with a non-parametric Mann-Whitney U Test. The correlation coefficient between parameters were calculated with Spearman's Rho (r s ). The threshold for statistical significance for all comparisons was P<0.05.

Supplementary Figure 1.
Morphological classification of NETs. Isolated neutrophils from normal subjects were incubated in either RPMI1640 media as baseline control or media containing 100 ng/mL PMA and 100 ng/mL LPS as activated NET formation for 90 min at 37ºC, 5% CO 2 , then, neutrophils were fixed and performed the indirect intracellular immunofluorescent assay using monoclonal antibody specific to neutrophil elastase (NE, green), histone H2A (red) and nuclear DNA (DAPI) (blue). Illustration was captured using confocal microscope. The criteria for NET classification was considered the lobulation, mean fluorescent intensity (MFI) of NE and histone H2A and projecting plasma membrane. (A) Neutrophils with negative NET had 2-5 lobes of nucleus, circular in shape, low level of NE and histone H2A MFI and not present projecting plasma membrane.
(B) Neutrophils with early phase of NET had 2-5 lobes of nucleus, circular or irregular in shape, increased NE and histone H2A MFI and not present projecting plasma membrane. (C) Neutrophils with non-web like late phase of NET had circular in shape of nucleus, no lobulation of nucleus, circular or irregular in shape of cellular morphology, increased NE and histone H2A MFI and not present projecting plasma membrane or the distance of nucleus to limb of plasma membrane with length shorter than 5 µM. (D) Neutrophils with web like late phase of NET had circular in shape of nucleus, no lobulation of nucleus, irregular in shape of cellular morphology, increased NE and histone H2A MFI and present projecting plasma membrane with the distance of nucleus to limb of plasma membrane at length ≥ 5 µM. i-iii; there were 3 different neutrophils, Ai-Aiii; untreated neutrophils, B-D; PMA/LPS-treated neutrophils. DAPI; 4′, 6-diamidino-2-phenylindole, dihydrochloride, LPS; lipopolysaccharides and PMA; phorbol 12-myristate 13-acetate. Figure 2. NET formation in normal subjects. Isolated neutrophils from peripheral blood samples were incubated with absence or present of combined haemin and PMA/LPS for 90 min at 37°C, 5% CO 2 . (Ai-iv) Untreated neutrophils, (Bi-iv) haemin-treated neutrophils and (C-F) combined PMA/LPS and different doses of haemin treated neutrophils were stained with fluorochrome conjugated antibodies specific to neutrophil elastase (NE, green), histone H2A (red) and nuclear DNA (blue) to analyze fluorescent intensity and morphology. i; merge, ii; anti-neutrophil elastase, iii; anti-histone H2A, iv; 4′, 6-diamidino-2phenylindole, dihydrochloride (DAPI), LPS; lipopolysaccharides and PMA; phorbol 12-myristate 13acetate. Figure 3. NET formation in non-splenectomised b-thalassaemia/HbE patients. Isolated neutrophils from peripheral blood samples were incubated with absence or present of combined haemin and PMA/LPS for 90 min at 37°C, 5% CO 2 . (Ai-iv) Untreated neutrophils, (Bi-iv) haemin-treated neutrophils and (C-F) combined PMA/LPS and different doses of haemin treated neutrophils were stained with fluorochrome conjugated antibodies specific to neutrophil elastase (NE, green), histone H2A (red) and nuclear DNA (blue) to analyze fluorescent intensity and morphology. i; merge, ii; anti-neutrophil elastase, iii; antihistone H2A, iv; 4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI), LPS; lipopolysaccharides and PMA; phorbol 12-myristate 13-acetate.