Fig 1.
HS conditions induce rapid Na+ influx in MΦs.
(A) Total Na+ content of RAW264.7 MΦs ± 10 ng/ml LPS under NS or HS (NS + 40 mM NaCl) conditions (mean ± SD; n = 11–12; Student t test ± Welch correction; *p < 0.05). (B) Relative [Na+]i of RAW264.7 MΦs. Traces of RAW264.7 MΦs stimulated with HS, LPS, or both at t = 10 s (mean ± SD; n = 7–9). (C) Relative [Na+]i of RAW264.7 MΦs. Traces of RAW264.7 MΦs stimulated with HS or 80 mM urea at t = 10 s (mean ± SD; n = 5). For numerical raw data, please see S1 Data. HS, high salt; LPS, lipopolysaccharide; MΦ, monocyte/macrophage-like cell; [Na+]i, intracellular Na+ in situ; NS, normal salt.
Fig 2.
NCX inhibitors abrogate HS-boosted NO production of LPS-stimulated MΦs.
Lower panel: nitrite levels of RAW264.7 MΦs pretreated with indicated inhibitors and stimulated ± LPS ± HS or 80 mM urea (mean ± SD; n = 9; Student t test or Mann–Whitney test; *p < 0.05). Upper panel: changes of means in nitrite production upon indicated stimulation and/or treatment (Δ nitrite). For numerical raw data, please see S1 Data. HS, high salt; LPS, lipopolysaccharide; MΦ, monocyte/macrophage-like cell; NCX, Na+/Ca2+ exchanger; NO, nitric oxide; NS, normal salt; n.s., not significant.
Fig 3.
MΦs express a novel Slc8a1 splice variant.
(A) A representative real-time amplification plot of Slc8a1, Slc8a2, and Slc8a3 in BMDMs, RAW264.7 MΦs, and brain tissue out of two experiments. (B) Shown are the alignments of RNA-seq reads of BMDMs and their coverage with annotated (gray; GENCODE: ENSMUST00000086538.9, ENSMUST00000163123.1, ENSMUST00000163680.8), predicted (blue; RefSeq PREDICTED: XM_006523944), and StringTie-assembled (red; StringTie Assembly version 1, StringTie Assembly version 2) Slc8a1 splice variants. For numerical raw data, please see S1 Data. The RNA-seq data can be found in the GEO database (https://www.ncbi.nlm.nih.gov) under accession number GSE136662. BMDM, bone marrow–derived MΦ; GEO, Gene Expression Omnibus; MΦ, monocyte/macrophage-like cell; RNA-seq, RNA sequencing; Slc8, solute carrier family 8.
Fig 4.
HS exposure results in NCX-mediated inward currents.
(A) Current/voltage relationships of MΦ ± LPS ± HS. Whole-cell VC experiments were performed before and after stimulation of BMDMs. Voltage steps were applied, and differential currents (IHS, ILPS, ILPS+HS) were plotted (mean ± 95% CI; n = 10–13). (B) As in (A), but with NiCl2 pretreatment (means ± 95% CI; n = 10). (C) Current/voltage relationships of BMDMs stimulated with LPS ± HS followed by NiCl2 treatment. Whole-cell VR experiments were performed, and Ni-sensitive (i.e., NCX-sensitive) currents (INCX) were determined (means ± 95% CI; n = 9). (D) ErevNCX (means ± SD; n = 9; Mann–Whitney test; *p < 0.05). (E) Resting Vm of BMDMs (means ± SD; n = 36). For numerical raw data, please see S1 Data. BMDM, bone marrow–derived MΦ; ErevNCX, NCX reversal potential; HS, high salt; I, current; LPS, lipopolysaccharide; MΦ, monocyte/macrophage-like cell; NCX, Na+/Ca2+ exchanger; VC, voltage clamp; Vm, membrane potential; VR, voltage ramp.
Fig 5.
(A) Relative [Ca2+]i levels of RAW264.7 MΦs. Traces of Fura-2–loaded RAW264.7 MΦs stimulated ± HS at t = 10 s (mean ± SD; n = 6). Where indicated, Tg was added (means ± SD; n = 2). (B) As in (A), but RAW264.7 MΦs were stimulated with LPS ± HS at t = 10 s (mean ± SD; n = 5). Where indicated, Tg was added (means ± SD; n = 2). (C) As in (A), but relative [Ca2+]i levels were assessed in Fluo-3/Fura-Red–loaded MΦs (means ± SD; n = 6). (D) As in (B), but relative [Ca2+]i levels were assessed in Fluo-3/Fura-Red–loaded MΦs (means ± SD; n = 8). For numerical raw data, please see S1 Data. [Ca2+]i, intracellular Ca2+ in situ; HS, high salt; LPS, lipopolysaccharide; MΦ, monocyte/macrophage-like cell; Tg, thapsigargin.
Fig 6.
Pharmacological inhibition of NCX activity blunts HS-triggered Na+ influx and Ca2+ loss.
(A) Relative [Na+]i levels in RAW264.7 MΦs. Traces of RAW264.7 MΦs stimulated ± HS (at t = 10 s) ± KB-R pretreatment (means ± SD; n = 4–8). (B) Relative [Na+]i levels in RAW264.7 MΦs. Traces of RAW264.7 MΦs stimulated with LPS ± HS (at t = 10 s) ± KB-R pretreatment (means ± SD; n = 5–8). (C) As in (A), but relative [Ca2+]i levels were assessed in Fura-2–loaded MΦs (means ± SD; n = 5–8). (D) As in (B), but relative [Ca2+]i levels were assessed in Fura-2–loaded MΦs (means ± SD; n = 6–8). For numerical raw data, please see S1 Data. [Ca2+]i, intracellular Ca2+ in situ; HS, high salt; LPS, lipopolysaccharide; MΦ, monocyte/macrophage-like cell; KB-R, KB-R7943 mesylate; [Na+]i, intracellular Na+ in situ; NCX, Na+/Ca2+ exchanger.
Fig 7.
Pharmacological inhibition of NCX activity blocks HS-boosted MΦ activity.
(A) Immunoblotting and densitometry of NFAT5 6 h after LPS ± HS in RAW264.7 MΦs ± KB-R pretreatment (n = 3; paired t test; *p < 0.05). (B) Nos2 levels in RAW264.7 MΦs 4 h after LPS ± HS ± KB-R pretreatment (means ± SD; n = 10; Student t test; *p < 0.05). (C) Immunoblotting and densitometry of NFAT5 in RAW264.7 MΦs 4 h after LPS ± HS ± NiCl2 pretreatment (n = 4; paired t tests; *p < 0.05). (D) Nos2 levels in RAW264.7 MΦs 4 h after LPS ± HS ± NiCl2 pretreatment (means ± SD; n = 6; Student t test + Welch correction; *p < 0.05). (E) Immunoblotting and densitometry of NFAT5 in RAW264.7 MΦs 4 h after LPS ± HS ± SEA pretreatment (n = 5; paired t test; *p < 0.05). (F) Nos2 in RAW264.7 MΦs 4 h after LPS ± HS ± SEA pretreatment (means ± SD; n = 10–12; Mann–Whitney test; *p < 0.05). (G) RFP-GFP-mLC3 RAW264.7 MΦs were infected with Escherichia coli ± HS ± SEA pretreatment. Representative images 2 h after infection (RFP: red; GFP: green; scale bar: 10 μm). (H) Relative E. coli load at 2 h after infection of RAW264.7 MΦs ± HS ± SEA pretreatment (means ± SD; n = 12; Student t tests; *p < 0.05). For numerical raw data, please see S1 Data. For raw immunoblots, please see S1 Blots. CFU, colony forming unit; GFP, green fluorescent protein; HS, high salt; KB-R, KB-R7943 mesylate; LPS, lipopolysaccharide; mLC3, microtubule-associated protein 1 light chain 3; MΦ, monocyte/macrophage-like cell; NCX, Na+/Ca2+ exchanger; NFAT5, nuclear factor of activated T cells 5; Nos2, nitric oxide synthase 2; NS, normal salt; n.s., not significant; RFP, red fluorescent protein; SEA, SEA 0400.
Fig 8.
Slc8a1 silencing decreases Na+ influx and Ca2+ efflux.
(A) Slc8a1 and (B) membranous NCX1 expression in LPS-stimulated ns or Slc8a1-specific siRNA–treated BMDMs after 4 h (means ± SD; n = 8; Mann–Whitney test; *p < 0.05). (C) Relative [Na+]i levels of ns siRNA and Slc8a1 siRNA–treated BMDMs exposed to LPS ± HS at t = 10 s (means ± SD; n = 5–7). (D) Relative [Ca2+]i levels in ns siRNA and Slc8a1 siRNA–treated BMDMs exposed to LPS ± HS at t = 10 s (means ± SD; n = 6–11). For numerical raw data, please see S1 Data. For raw immunoblots, please see S1 Blots. BMDM, bone marrow–derived MΦ; [Ca2+]i, intracellular Ca2+ in situ; HS, high salt; LPS, lipopolysaccharide; [Na+]i, intracellular Na+ in situ; ns, nonsilencing; NCX, Na+/Ca2+ exchanger; siRNA, small interfering RNA; Slc8, solute carrier family 8.
Fig 9.
Slc8a1 silencing abrogates HS-augmented MΦ activity.
(A) ns siRNA and Slc8a1 siRNA RAW264.7 were treated with LPS ± HS. Representative NFAT5 and ACTIN immunoblot 4 h after stimulation from two experiments. (B) As in (A), but Nos2 in BMDMs 4 h after LPS ± HS (means ± SD; n = 6; Mann–Whitney test; *p < 0.05). (C) As in (B), but Δ nitriteHS LPS-LPS after 24 h (means ± SD; n = 8; Student t test). (D) ns siRNA and Slc8a1 siRNA–treated RFP-GFP-mLC3 RAW264.7 MΦs were infected with E. coli ± HS. Representative images 2 h after infection from three experiments (RFP: red; GFP: green; scale bar: 10 μm). (E) ns siRNA and Slc8a1 siRNA–treated BMDMs were infected with E. coli ± HS. E. coli load at 2 h infection (means ± SD; n = 12; Student t tests; *p < 0.05). For numerical raw data, please see S1 Data. For raw immunoblots, please see S1 Blots. BMDM, bone marrow–derived MΦ; CFU, colony forming unit; GFP, green fluorescent protein; HS, high salt; LPS, lipopolysaccharide; mLC3, microtubule-associated protein 1 light chain 3; MΦ, monocyte/macrophage-like cell; NFAT5, nuclear factor of activated T cells 5; Nos2, nitric oxide synthase 2; NS, normal salt; ns, nonsilencing; n.s., not significant; RFP, red fluorescent protein; siRNA, small interfering RNA; Slc8, solute carrier family 8.
Fig 10.
NCX1 is required for Na+ entry and Ca2+ loss upon exposure of MΦs to HS conditions. In LPS-stimulated MΦs, NCX1 is required for HS-boosted NFAT5 accumulation and NO production. Moreover, NCX1 is required for enhanced autolysosome formation and bacterial removal upon HS exposure. HS, high salt; LPS, lipopolysaccharide; MΦ, monocyte/macrophage-like cell; NCX, Na+/Ca2+ exchanger; NFAT5, nuclear factor of activated T cells 5; NO, nitric oxide.