High salt primes a specific activation state of macrophages, M(Na)

Abstract

High salt is positively associated with the risk of many diseases. However, little is known about the mechanisms. Here we showed that high salt increased proinflammatory molecules, while decreased anti-inflammatory and proendocytic molecules in both human and mouse macrophages. High salt also potentiated lipopolysaccharide-induced macrophage activation and suppressed interleukin 4-induced macrophage activation. High salt induced the proinflammatory aspects by activating p38/cFos and/or Erk1/2/cFos pathways, while inhibited the anti-inflammatory and proendocytic aspects by Erk1/2/signal transducer and activator of transcription 6 pathway. Consistent with the in vitro results, high-salt diet increased proinflammatory gene expression of mouse alveolar macrophages. In mouse models of acute lung injury, high-salt diet aggravated lipopolysaccharide-induced pulmonary macrophage activation and inflammation in lungs. These results identify a novel macrophage activation state, M(Na), and high salt as a potential environmental risk factor for lung inflammation through the induction of M(Na).

Figure 1

High salt induces a specific activation state in human macrophages. Human monocyte-derived macrophages were treated with additional 51 mM NaCl for 24 h. (A) High salt promotes gene expression of proinflammatory molecules as determined by RNA-Seq. mRNA levels of high-salt-treated human macrophages are presented as fold changes relative to those of untreated human macrophages. (B) High salt promotes gene expression of proinflammatory molecules as determined by qRT-PCR. (C) High salt promotes protein expression of proinflammatory molecules. Protein levels of CXCL1 and IL8 in conditioned media from human macrophages were quantified using ELISA. (D) High salt inhibits gene expression of anti-inflammatory and proendocytic molecules as determined by RNA-Seq. (E) High salt inhibits gene expression of anti-inflammatory and proendocytic molecules as determined by qRT-PCR. (F) High salt decreases protein level of CCL22 in conditioned media from human macrophages as determined by ELISA. (G) High salt decreases protein level of MR in human macrophages as determined by western blotting. Tubulin was used as a loading control. Representative results of qRT-PCR, ELISA and western blotting from three independent experiments are shown. ^*P< 0.05; ^**P< 0.01; ^***P< 0.001.

Figure 2

High salt potentiates M(LPS) and suppresses M(IL4) in mouse BMDMs. (A) High salt enhances LPS-induced gene expression. BMDMs were pretreated with or without additional 51 mM NaCl for 12 h, and then treated with or without 100 ng/ml LPS in the presence or absence of additional 51 mM NaCl for the indicated time periods. (B) High salt enhances LPS-induced protein expression. BMDMs were treated with or without additional 51 mM NaCl for 24 h and 100 ng/ml LPS or PBS was added during the latter half of the treatment. Protein expression of COX2 and iNOS was measured and GAPDH was used as a loading control. (C) High salt suppresses IL4-induced gene expression. BMDMs were treated with 10 ng/ml IL4 or 0.1% BSA in the presence or absence of additional 51 mM NaCl for 24 h. (D) High salt suppresses IL4-induced protein expression. BMDMs were treated in the same way as in C. Arrowhead: non-specific bands. For all panels, gene expression was measured using qRT-PCR and protein expression was measured using western blotting. ^*P< 0.05; ^**P< 0.01; ^***P< 0.001.

Figure 3

p38 MAPK mediates the upregulation of proinflammatory genes of M(Na) and high-salt-induced potentiation of M(LPS). (A) High salt activates p38 in macrophages. BMDMs were treated with or without additional 51 mM NaCl for the indicated time periods. p-p38 and total p38 levels were measured and GAPDH was used as a loading control. (B) SB inhibits high-salt-promoted expression of proinflammatory genes. BMDMs were pretreated with 10 μM SB or DMSO for 2 h and then treated with or without additional 51 mM NaCl for 24 h in the presence of 5 μM SB or DMSO. (C) High salt enhances LPS-induced p38 phosphorylation. BMDMs were treated with or without additional 51 mM NaCl for 13 h and 100 ng/ml LPS was added during the last 1 h of the treatment. (D, E) p38 mediates high-salt-enhanced response to LPS. BMDMs were treated by the same procedure as in B, except that 100 ng/ml LPS was added to all groups during the last 12 h of the treatment. Expression of the indicated genes (D) and proteins (E) was quantified. For all panels, gene expression was measured using qRT-PCR and protein expression or phosphorylation was measured using western blotting. ns, not significant; ^*P< 0.05; ^**P < 0.01; ^***P < 0.001.

Figure 4

Erk1/2 MAPK mediates the gene expression of M(Na) as well as high-salt-induced potentiation of M(LPS) and suppression of M(IL4). (A) High salt activates Erk1/2 in macrophages. BMDMs were treated with or without additional 51 mM NaCl for the indicated time periods. p-Erk and total Erk1/2 (Erk) levels were measured and tubulin was used as a loading control. (B, C) PD inhibits high-salt-induced upregulation of proinflammatory gene expression (B) and downregulation of anti-inflammatory and proendocytic gene expression (C). BMDMs were pretreated with 20 μM PD or DMSO for 1 h and then treated with or without additional 51 mM NaCl for 24 h in the continued presence of 20 μM PD or DMSO. (D) High salt enhances LPS-induced Erk1/2 phosphorylation. BMDMs were treated with or without additional 51 mM NaCl for 13 h and 100 ng/ml LPS was added during the last 1 h of the treatment. (E, F) Erk1/2 mediates high-salt-induced potentiation of M(LPS). BMDMs were treated by the same procedure as in B, except that 100 ng/ml LPS was added to all groups during the last 12 h. Expression of the indicated genes (E) and proteins (F) was quantified. (G) High salt increases p-Erk1/2 at baseline and under IL4 treatment. BMDMs were treated with 10 ng/ml IL4 or BSA in the presence or absence of additional 51 mM NaCl for 6 h. (H, I) Erk1/2 mediates high-salt-induced suppression of M(IL4). BMDMs were treated in the same way as in C, except that 10 ng/ml IL4 was added to all groups during the last 24 h. Expression of the indicated genes (H) and proteins (I) was quantified. For all panels, gene expression was measured using qRT-PCR and protein expression or phosphorylation was measured using western blotting. ns, not significant; ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

Figure 5

AP1 mediates the proinflammatory gene expression of M(Na) and high-salt-induced potentiation of M(LPS). (A) High salt promotes cFos phosphorylation in macrophages. BMDMs were treated with or without additional 51 mM NaCl for the indicated time periods. p-cFos and total cFos (cFos) levels were measured and GAPDH was used as a loading control. (B) SR inhibits high-salt-promoted proinflammatory gene expression. BMDMs were pretreated with 10 μM SR or DMSO for 1 h and then treated with or without additional 51 mM NaCl for 24 h in the continued presence of 10 μM SR or DMSO. (C) High salt-induced increase of p-cFos depends on p38 activation. BMDMs were pretreated with 10 μM SB for 2 h and then treated with or without additional 51 mM NaCl for 12 h in the presence of 5 μM SB or DMSO. (D) High salt-induced increase of cFos phosphorylation depends on Erk1/2 activation. BMDMs were pretreated with 20 μM PD for 1 h and then treated with or without additional 51 mM NaCl for 12 h in the presence of 20 μM PD or DMSO. (E) High salt enhances LPS-induced p-cFos. BMDMs were treated with or without additional 51 mM NaCl for 13 h and 100 ng/ml LPS was added during the last 1 h of the treatment. (F, G) AP1 mediates high-salt-enhanced response to LPS. BMDMs were treated by the same procedure as in B, except that 100 ng/ml LPS was added to all groups during the last 12 h of the treatment. Expression of the indicated genes (F) and proteins (G) was quantified. (H) High salt-induced increase of p-cFos in the presence of LPS depends on p38 activation. BMDMs were treated by the same procedure as in C, except that 100 ng/ml LPS was added to all groups during the last 1 h of the treatment. For all panels, gene expression was measured using qRT-PCR and protein expression or phosphorylation was measured using western blotting. ns, not significant; ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

Figure 6

STAT6 mediates the downregulation of anti-inflammatory and proendocytic gene expression of M(Na) as well as high-salt-induced suppression of M(IL4). (A) High salt decreases levels of p-STAT6 and total STAT6. BMDMs were treated with additional 51 mM NaCl for the indicated time periods. Levels of p-STAT6 and STAT6 were measured and actin was used as a loading control. (B) STAT6 mediates high-salt-induced reduction of anti-inflammatory and proendocytic gene expression. RAW264.7 cells with stable overexpression of STAT6 or GFP were treated with or without additional 51 mM NaCl for 6 h. (C) High-salt-induced reduction of p-STAT6 and total STAT6 depends on Erk1/2 activation. BMDMs were pretreated with 20 μM PD for 1 h and then treated with or without additional 51 mM NaCl for 6 h in the presence of 20 μM PD or DMSO. (D) High salt decreases levels of p-STAT6 and STAT6 in the presence of IL4. BMDMs were treated with or without additional 51 mM NaCl in combination with 10 ng/ml IL4 for 6 h. (E) Overexpression of STAT6 attenuates high-salt-induced suppression of M(IL4). RAW264.7 cells with stable overexpression of STAT6 or GFP were treated with or without additional 51 mM NaCl in combination with 10 ng/ml IL4 for 24 h. (F) High-salt-induced reduction of p-STAT6 and total STAT6 depends on Erk1/2 activation in the presence of IL4. Treatment was the same as in C except that 10 ng/ml IL4 was added to all groups. (G) Proposed model of the signaling pathways that mediate the effects of high salt in macrophages. For all panels, gene expression was measured using qRT-PCR and protein expression or phosphorylation was measured using western blotting. ns, not significant; ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

Figure 7

High salt promotes lung inflammation in mice. (A) High salt increases proinflammatory gene expression in alveolar macrophages at baseline (without LPS challenge). Alveolar macrophages were collected via BAL in mice that received chow diet and tap water (CD group) or high-salt diet (HSD) containing 4% NaCl and tap water containing 1% NaCl (HSD group). Expression of the indicated genes was measured using qRT-PCR (n = 4:4). (B) High salt elevates serum levels of CXCL1 and CXCL2 in LPS-challenged mice. Mice were intraperitoneally injected with LPS (10 μg per 25 g mice) and sacrificed 1 h later. Representative results of ELISA (n = 5:6) from three independent experiments are shown. (C) High salt increases expression of proinflammatory genes in lungs from the same mice as in B. Gene expression was quantified using qRT-PCR (n = 16:17). (D) High salt increases expression of proinflammatory genes in macrophages sorted from lungs after LPS challenge. Mice inhaled 1 mg/ml LPS for 35 min and were sacrificed 3 h later. F4/80⁺ macrophages were sorted from the lungs using flow cytometry. Gene expression was quantified using qRT-PCR (n = 6:6). (E, F) High salt increases CD11b⁺ cells in mouse lungs after LPS challenge as determined by immunofluorescence staining. Mice were challenged with LPS in the same way as in D. Representative immunofluorescence staining for CD11b (red) and images merged with DAPI (pink) are shown in E and quantification in F (n = 6:6). (G, H) High salt increases CD11b⁺ cells and monocytes but not neutrophils in mouse lungs after LPS challenge as determined by flow cytometry. Representative flow cytometry analysis of CD11b⁺ cells, monocytes (CD11b⁺/Ly6G⁻/Ly6C⁺), and neutrophils (CD11b⁺/Ly6G⁺/Ly6C⁻) is shown in G and quantification in H (n = 6:6). (I) High salt increases the numbers of leukocytes and monocytes/macrophages but not PMNs in BAL fluid. Mice inhaled 1 mg/ml LPS for 35 min and were sacrificed 6 h later. Leukocytes were counted using a hemocytometer. The percentages of monocytes/macrophages and PMNs were determined using Wright-Giemsa staining (n = 6:6). (J) High salt aggravates lung edema in LPS-challenged mice. Mice were challenged with LPS in the same way as in I. Lung edema was indicated by wet to dry weight ratio (n = 7:10). ns, not significant; ^*P< 0.05; ^**P< 0.01; ^***P< 0.001.