Suppression of neutrophils by sodium exacerbates oxidative stress and arthritis

Abstract

Introduction: Typical Western diet, rich in salt, contributes to autoimmune disease development. However, conflicting reports exist about the effect of salt on neutrophil effector functions, also in the context of arthritis.

Methods: We investigated the effect of sodium chloride (NaCl) on neutrophil viability and functions in vitro, and in vivo employing the murine K/BxN-serum transfer arthritis (STA) model.

Results and discussion: The effects of NaCl and external reactive oxygen species (H₂O₂) were further examined on osteoclasts in vitro. Hypertonic sodium-rich media caused primary/secondary cell necrosis, altered the nuclear morphology, inhibited phagocytosis, degranulation, myeloperoxidase (MPO) peroxidation activity and neutrophil extracellular trap (NET) formation, while increasing total ROS production, mitochondrial ROS production, and neutrophil elastase (NE) activity. High salt diet (HSD) aggravated arthritis by increasing inflammation, bone erosion, and osteoclast differentiation, accompanied by increased NE expression and activity. Osteoclast differentiation was decreased with 25 mM NaCl or 100 nM H₂O₂ addition to isotonic media. In contrast to NaCl, external H₂O₂ had pro-resorptive effects in vitro. We postulate that in arthritis under HSD, increased bone erosion can be attributed to an enhanced oxidative milieu maintained by infiltrating neutrophils, rather than a direct effect of NaCl.

Keywords: K/BxN serum transfer arthritis; neutrophil extracellular traps (NETs); neutrophils; osteoclasts; reactive oxygen species; sodium chloride.

Figure 1

Hypertonic sodium-rich media decrease viability of human neutrophils and induce primary or secondary cell necrosis in vitro. Cell viability was assessed by flow cytometry after four-color death staining with PI, Hoechst33342, Annexin A5-FITC and DilC1(5) in various salt conditions (1) isotonicity 137 mM NaCl, (2) moderate hypertonicity 200 mM NaCl, and (3) high hypertonicity 300 mM NaCl, at various time points. (A) Flowcytometric dot plot analysis showing the gating strategy used to identify viable, apoptotic, and necrotic cell populations; (B) culture without (w/o) serum; (C) culture with (w/) 10% heat-inactivated FCS. Data were obtained from 3-5 healthy individuals and are presented as mean ± SD. Statistical analysis was performed using Mixed Effects Analysis. Table 1 displays all numerical P values. pNecrotic, primary necrotic; sNecrotic, secondary necrotic.

Figure 2

Neutrophils cultured in hypertonic sodium-rich media increase ROS production and NE activity, but reduce degranulation, phagocytosis and MPO peroxidation activity. Analyses of freshly isolated neutrophils in various salt conditions by flow cytometry: (A) ROS production; (B) Mitochondrial ROS production; (C) Phagocytosis; (D) Degranulation; (E) MPO peroxidation activity; (F) MPO chlorination activity, and (G) NE activity. In (A, B, D–G), neutrophils were either not stimulated (DPBS) or stimulated with PMA (100 ng/mL) or Pyocyanin (10 µM or 50 µM). In (C), cells were incubated with microspheres coated with either 2 mg/mL IVIg or 2 mg/mL HSA (baseline). In (E), (F) and (G), RIPA buffer was used for complete cell lysis and equals maximum enzymatic activity. All y-axes display log₂ values, except in (G). Data were obtained from 3-6 healthy individuals and are presented as mean ± SD. Statistical analysis was performed using 2-way ANOVA. OD, optical density; MFI, mean fluorescence intensity.

Figure 3

Hypertonic sodium-rich media inhibit NET formation in vitro and change neutrophils’ nuclear morphology. (A, B) NET formation in various salt conditions without serum unstimulated (DPBS), and after stimulation with PMA (100 ng/mL) or Pyocyanin (10 µM). Data were obtained from at least 4 healthy individuals and are presented as mean ± SD. Statistical analysis was performed using 2-way ANOVA; (C, D) nuclear morphology of unstimulated neutrophils stained with Hoechst33342 in various salt conditions and analysis by morphometry; statistical analysis was performed applying ordinary one-way ANOVA. MFI, mean fluorescence intensity; bars represent 100 µm.

Figure 4

K/BxN mice kept on HSD develop more severe arthritis than mice on NSD. (A) Clinical scoring of front and hind paws in C57BL/6J mice with K/BxN serum transfer arthritis; corresponding plot of area under the curve (AUC). Data are shown from mice kept on either NSD, n=8, or HSD, n=8, and are presented as mean ± SD; (B) Representative images of HE-stained hind paw sections and their histological quantification at day 10. Upper row: paw sections of mice kept on NSD; lower row: paw sections of mice kept on HSD; (C) Representative images of TRAP-stained hind paw sections and their histological quantification. Upper row: left and right paw of a mouse kept on NSD; lower row: left and right paw of a mouse kept on HSD. Data are shown from mice kept on either NSD, n=5, or HSD, n=5, and are presented as mean ± SD. Statistical analysis was performed using 2-way ANOVA (Clinical Score), Unpaired t-test (Infl.Ar./T.Ar.) or Mann-Whitney test (AUC, Inflammation, Osteoclast Count). AUC, area under the curve; Infl.Ar., inflammation area; T.Ar., tissue area; Er.Ar., eroded area; B.Ar., bone area; N.Oc., osteoclast count; bars represent 900 µm. Black arrows indicate infiltrates (B), or eroded bone (C).

Figure 5

K/BxN mice on HSD are characterized by increased NE expression and enzymatic activity within the carpal bone infiltrates. Representative images of hind paw infiltrates (A, C) and their histological quantification (B, D, respectively), after staining consecutive sections with primary antibody sheep anti-mouse NE (R&D, AF4517) or rat anti-mouse Ly6G (Biolegend, 127601) and DAPI. Corresponding controls (wo1st) were stained with secondary antibody only. Red: DAPI, green: NE or Ly6G, respectively. White asterisks indicate single cells. Pictures were taken using the fluorescence scanner (Aperio Versa 8, Leica Biosystems); (E) NE activity measurement in hind paw sections. Data are presented as mean ± SD. Statistical analysis was performed using Mann-Whitney test (NE MFI) or Unpaired t-test (NE activity); outlier marked with asterisk was excluded. MFI, mean fluorescence intensity; pos, positive; bar represents 200 µm.

Figure 6

Hypertonic NaCl, as well as external ROS (H₂O₂) impair the maturation of murine osteoclasts in vitro. H₂O₂ increases osteoclast resorption activity in vitro. (A) TRAP-stained osteoclasts (n=5 per group) in purple (>3 nuclei) and (B) their quantification. Statistical analysis was performed using Kruskal-Wallis test; N. Oc., osteoclast count; bar represents 50 µM; (C) Von Kossa staining of the corresponding area resorbed by the osteoclasts (white) and (D) quantification per osteoclast (%), n=5. Data are presented as mean ± SD. Statistical analysis was performed using Kruskal-Wallis test. N.Oc., osteoclast count; bars represent 500 µM.