Hypertonic Saline Suppresses NADPH Oxidase-Dependent Neutrophil Extracellular Trap Formation and Promotes Apoptosis

Abstract

Tonicity of saline (NaCl) is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET) formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA), Gram-negative bacterial cell wall component lipopolysaccharide (LPS), calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus), and bacteria (Pseudomonas aeruginosa and Staphylococcus aureus). We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2)-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12), and P. aeruginosa. Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H₂O₂ reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol). This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic solutions do not suppress NOX2-independent NETosis. Although hypertonic saline partially suppresses ionomycin-induced NETosis, it enhances A23187-induced NETosis, and it does not alter S. aureus-induced NETosis. Overall, this study determined that hypertonic saline suppresses NOX2-dependent NETosis induced by several agonists; in contrast, it has variable effects on neutrophil death induced by NOX2-independent NETosis agonists. These findings are important in understanding the regulation of NETosis and apoptosis in neutrophils.

Keywords: Gram-negative and -positive bacteria-induced NETosis; NOX2-dependent NETosis; NaCl; cystic fibrosis; hypertonic saline; lipopolysaccharide-induced NETosis; neutrophil extracellular traps.

Figure 1

Increasing NaCl concentration suppresses phorbol myristate acetate (PMA)-mediated NETosis. NETosis assays were performed on human neutrophils resuspended in different NaCl concentrations (109, 115, 121, 134, 159, 209, 309, and 509 mM) with 5 µM Styox Green dye. These neutrophils were either unstimulated (−ve control) or activated or by PMA (25 nM). The Sytox Green florescence intensities (proxy for DNA release) were recorded by a plate reader every 30 min up to 210 min. (A) % DNA release (NETotic index) show less NETosis in higher salt conditions in unstimulated neutrophils (−ve control). (B) PMA treatment induces NETosis, and NaCl dose-dependently suppresses this form of NETosis. The suppression with NaCl concentrations at 309 mM and 509 mM is significant at several time points (n = 3−5, *p < 0.05; two-way ANOVA with Bonferroni’s multiple comparison post test). (C) Confocal microscopy of neutrophils with media only (−ve control) or PMA-treated in different salt conditions (109, 309, and 509 mM) were performed. Neutrophils were stained for DNA and myeloperoxidase (MPO); colocalization of the two signifies NETosis of neutrophils. Negative control neutrophils show no substantial neutrophil extracellular trap (NET) release, and higher NaCl conditions suppress the background NETosis. Drastic suppression of NETosis can be seen in PMA-treated neutrophils at 509 and 309 mM, but not in 109 mM salt conditions (n = 3−5; MPO-green; DNA-DAPI; scale bar, 22 µm). See Figure S1 in Supplementary Material for IgG isotype antibody and Figure S2 in Supplementary Material for more salt conditions, low magnification images.

Figure 2

Increasing NaCl concentration suppresses phorbol myristate acetate (PMA)-mediated reactive oxygen species (ROS) production and hydrogen peroxide treatment restores NETosis inhibition. Human neutrophils were pretreated with cytosolic ROS indicator dye DHR123 or 2′,7′-dichlorofluorescein diacetate (DCFDA) in different NaCl concentrated media and were either unstimulated (−ve control) or activated by PMA (25 nM). ROS production kinetics was performed by plate reader assays up to 60 min. (A) Neutrophils in −ve control show a significant NaCl dose dependent suppression of background ROS generation. (B) ROS generation kinetics in PMA-treated neutrophils also show significant suppression of ROS in later time points for hypertonic saline conditions (309 and 509 mM) compared to the normotonic condition (109 mM). The suppression was statistically significant at time points 40, 50, and 60 min (n = 3−4; *p < 0.05; two-way ANOVA with Bonferroni’s multiple comparison posttest). (C) Neutrophils were stained for DNA and DHR123 to observe ROS production in cytosolic space. Confocal microscopy of DHR123-treated neutrophils with 109, 121, 209, and 509 mM NaCl, confirms a dose-dependent suppression of ROS production at higher concentrations in PMA-activated cells. R123 fluorescence was detected in both 109, and 121 mM but not in 209 and 509 mM NaCl condition. Unstimulated (−ve control) neutrophils show little or no ROS production (n = 3−4; R123-green; DNA, DAPI blue; scale bar 22 µm). (D) Intracellular ROS production measured using the DCFDA dye showed very minimal ROS production in −ve control, while (E) PMA treatment induced intracellular ROS production, which is dose dependently suppressed with higher NaCl concentration (n = 3; *p < 0.05; two-way ANOVA with Bonferroni’s multiple comparison posttest). See Figure S3 in Supplementary Material for more salt conditions and low magnification images. Furthermore, during NETosis, hydrogen peroxide was added to human neutrophils after 120 min exposure to different NaCl concentrations. (F) Hydrogen peroxide induced a significant amount of NETosis in a range of varying NaCl concentrations (109 – 509 mM). (G) In addition, Hydrogen peroxide rescued the suppression in PMA-induced NETosis in high salt concentrations, bringing the NETosis levels of neutrophils in 509 mM NaCl close to NETosis levels of neutrophils in 109 mM NaCl (n = 3; *p < 0.05; two-way ANOVA with Bonferroni’s multiple comparison post test).

Figure 3

Increasing choline chloride suppresses phorbol myristate acetate (PMA)-mediated reactive oxygen species (ROS) production and neutrophil extracellular traps (NETs) release. Neutrophils were resuspended in equimolar choline chloride (ChlCl) containing RPMI media, substituting different concentrations of NaCl. These neutrophils were used for the Sytox Green NETosis and DHR123-based ROS production kinetics by plate reader assays. (A) −ve control neutrophils treated with ChlCl shows dose-dependent suppression of background % DNA release at 309 and 509 mM ChlCl conditions. (B) PMA-treated neutrophils show significant dose-dependent suppression of NET release at 309 and 509 mM ChlCl conditions at time points 150, 180 and 210 min (n = 3; two-way ANOVA with Bonferroni’s multiple comparison post test). (C) The % ROS production was calculated by considering the PMA-mediated ROS production at normotonic condition as 100% during 60-min time point. At 60 min, background ROS production is suppressed by high ChlCl doses of 309 and 509 mM. (D) PMA-treated neutrophils experience significant suppression of ROS production at the 60-min time point (*p < 0.05; one-sample t-test compared to hypothetical value 100).

Figure 4

Increasing concentrations of d-mannitol suppress phorbol myristate acetate (PMA)-mediated reactive oxygen species (ROS) production and NETosis. Human neutrophils were treated with d-mannitol and d-sorbitol to determine the effects of osmolarity during NETosis. (A) Unstimulated neutrophils treated with d-mannitol showed no suppression in background NETosis. (B) In contrast, PMA-treated cells in different concentrations of d-mannitol showed significantly lower NETosis in a dosage-dependent manner (n = 3, *p < 0.05; two-way ANOVA with Bonferroni’s multiple comparison post test). ROS production was measured by using neutrophils preloaded with DCFDA under different d-mannitol concentrations. (C,D) The ROS production in PMA-activated neutrophils is suppressed by high d-mannitol concentrations (n = 3, *p < 0.05; two-way ANOVA with Bonferroni’s multiple comparison post test). Similar to d-mannitol, d-sorbitol treatment did not alter background NETosis activation in −ve control while it suppresses PMA-induced NETosis. See Figure S4 in Supplementary Material for d-sorbitol data.

Figure 5

Increasing NaCl concentration suppresses lipopolysaccharide (LPS)-mediated reactive oxygen species (ROS) production and NETosis. (A) % total DNA release (NETosis) of LPS-treated neutrophils are significantly suppressed at higher NaCl concentrations. The suppression is significant in high salt concentrations (209, 309, and 509 mM) at various time points (n = 3−5; *p < 0.05; two-way ANOVA with Bonferroni’s multiple comparison post test). (B) Confocal microscopy of neutrophils shows significant LPS-induced NETosis suppression at hypertonic saline concentrations compared to lower salt dose treatments (n = 3−4; myeloperoxidase, green; DNA, DAPI blue; scale bar, 22 µm). (C,D) LPS-induced ROS production measured by DHR123 and DCFDA dyes, using fluorescent plate reader show the ROS suppression in LPS-treated neutrophils at high NaCl concentrations over 60 min (n = 3−5; *p < 0.05; two-way ANOVA with Bonferroni’s multiple comparison post test).

Figure 6

Increasing concentrations of d-mannitol suppress lipopolysaccharide (LPS)-mediated reactive oxygen species (ROS) production and NETosis. (A) The % DNA release NETosis kinetics shows the suppression of LPS-mediated NETosis in increasing concentrations of d-mannitol (n = 3; *p < 0.05; two-way ANOVA with Bonferroni’s multiple comparison post test). (B) Myeloperoxidase-immunostained and confocal images of unstimulated neutrophils (−ve control) and LPS-treated cells in different concentrations of d-mannitol shows the suppression of NETosis at higher concentration (n = 3; MPO-green; DNA-DAPI; scale bar, 22 µm). (C) LPS-induced ROS production measured by DCFDA in neutrophils under different d-mannitol concentrations, show the ROS suppression at high d-mannitol concentrations over 60 min (n = 3; *p < 0.05; two-way ANOVA with Bonferroni’s multiple comparison post test). See Figures S5 and S6 in Supplementary Material for d-sorbitol NETosis and ROS production data.

Figure 7

Increasing NaCl switches phorbol myristate acetate (PMA)-mediated NETosis to apoptosis. Human neutrophils were either unstimulated (−ve control) or activated by PMA, and immunostained with DAPI (DNA) and cCasp-3 in different salt concentration conditions (109, 115, 121, 134, 159, 209, 309, and 509 mM). (A) Confocal microscopy of unstimulated (−ve control) neutrophils show few apoptotic neutrophils at higher NaCl concentrations, but not lower ones. PMA-treated neutrophils with normotonic NaCl treatment undergo complete NETosis. During the hypertonic conditions, NETosis is suppressed, and the cells appear apoptotic (n = 3; cCasp-3; red, DNA; DAPI, scale bar, 22 µm). (B) % total cell count (based on the immunostaining and cellular morphology) data show a clear trend of NETosis suppression during unstimulated and PMA-mediated activation of neutrophils, while apoptosis is promoted with increasing concentration of NaCl. Collectively, data indicate that suppression of NETosis leads to apoptosis at high salt treatments (n = 3; *p < 0.05; one-way ANOVA with Tukey’s multiple comparison post test). See Figure S8 in Supplementary Material for low magnification images.

Figure 8

Increasing NaCl and osmolytes concentration suppresses ionomycin-induced NETosis. Human neutrophils in different osmolytes (NaCl, d-mannitol, and d-sorbitol) were treated with Nox-independent NETosis agonists A23187 and ionomycin. (A) Initial kinetics of the neutrophils under high concentration showed more NETosis. High NaCl concentrations were not able to suppress NETosis in response to A23187; however, this effect was lost by later time points leading to no overall difference by 210 min. (B) In contrast, ionomycin-induced NETosis show some suppression at later time points. (C,D) Neutrophils treated with different d-mannitol concentrations showed NETosis suppression only in ionomycin activated neutrophils. (E,F) Neutrophils treated under d-sorbitol conditions also show the same suppression pattern as of (C,D) (n = 3; Two-way ANOVA with Bonferroni’s multiple comparison post test).

Figure 9

Increasing NaCl concentrations suppresses Pseudomonas aeruginosa-mediated NETosis. Neutrophils in different NaCl and choline chloride concentrations were activated either with P. aeruginosa or S. aureus (multiplicity of infection 20). (A,B) The % total NETosis induced by P. aeruginosa is suppressed during high NaCl or choline chloride concentrations compared to normal treatments (109 mM). (C,D) In contrast to P. aeruginosa-induced NETosis, S. aureus-induced NETosis was not suppressed by NaCl or choline chloride addition over 210 min (n = 3; *p < 0.05; two-way ANOVA with Bonferroni’s multiple comparison post test).