Salt increases monocyte CCR2 expression and inflammatory responses in humans

Abstract

Inflammation may play a role in the link between high salt intake and its deleterious consequences. However, it is unknown whether salt can induce proinflammatory priming of monocytes and macrophages in humans. We investigated the effects of salt on monocytes and macrophages in vitro and in vivo by performing a randomized crossover trial in which 11 healthy human subjects adhered to a 2-week low-salt and high-salt diet. We demonstrate that salt increases monocyte expression of CCR2, a chemokine receptor that mediates monocyte infiltration in inflammatory diseases. In line with this, we show a salt-induced increase of plasma MCP-1, transendothelial migration of monocytes, and skin macrophage density after high-salt diet. Macrophages demonstrate signs of an increased proinflammatory phenotype after salt exposure, as represented by boosted LPS-induced cytokine secretion of IL-6, TNF, and IL-10 in vitro, and by increased HLA-DR expression and decreased CD206 expression on skin macrophages after high-salt diet. Taken together, our data open up the possibility for inflammatory monocyte and macrophage responses as potential contributors to the deleterious effects of high salt intake.

Keywords: Clinical Trials; Cytokines; Inflammation; Macrophages; Monocytes.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Flow chart for inclusion.

Figure 2. High-salt diet (HSD) induces a proinflammatory monocyte phenotype.

(A) Gating of monocytes and monocyte subtypes. Debris, residual RBC, and granulocytes were first gated out on a FSC/SSC plot (gate i). Gate i was next displayed on a CD14/CD16 plot to select CD14+ and/or CD16+ cells (gate ii). Cells from gate ii were viewed on a CD16/HLA-DR plot to select monocytes (gate iii). This monocyte population was plotted again on a CD14/CD16 plot to gate CD14++/CD16– (classical), CD14++/CD16+ (intermediate), and CD14+/CD16++ (nonclassical) monocyte subsets. (B) Monocyte subsets assessed as described in A, here expressed as a percentage of total monocytes (gate iii) after LSD and HSD. (C) The expression of various surface markers on the monocyte subsets was calculated as ΔMFI = (MFI)positive staining – (MFI)isotype control. CCR2 and CD206 are displayed here; other surface markers are depicted in Supplemental Figure 2. FSC, forward-scattered light. SSC, side-scattered light; LSD, low salt diet; MFI, median fluorescence intensity. Values represent mean ± SEM of n = 11 healthy male volunteers. Data tested using 2-way ANOVA. ***P < 0.001.

Figure 3. Effect of salt on cytokines.

(A) Whole blood samples after LSD and HSD were challenged with LPS (24 h with 10 ng/mL) and cytokines were quantified by ELISA. (B) Monocytes of independent healthy donors were stimulated for 24 h in RPMI + 10% FCS + 1% PenStrep in the presence of normal salt (NS), high salt (HS), or 80 mM urea as tonicity control. Then, cytokine secretion was quantified by ELISA. (C) Plasma samples after LSD and HSD were analyzed for MCP-1 and IL-6 by ELISA. IL-6 was below the detection threshold. For all analyses, ELISA was performed in accordance with the supplier’s protocols (Invitrogen). NS, ([Na+] = 139 mM); HS, ([Na+] = 179 mM). LSD, low-salt diet. HSD, high-salt diet. Values represent mean ± SEM of n = 11 healthy male volunteers (A and C) and n = 3 healthy male donors (B). Data tested using paired t test (A and C) or a 1-way ANOVA (B). *P < 0.05; ***P < 0.001.

Figure 4. High salt increases CCR2 expression and transendothelial migration of monocytes in vitro.

(A and B) Monocytes of independent healthy donors were stimulated for 24 h in RPMI + 10% FCS + 1% PenStrep in the presence of normal salt (NS), high salt (HS), or 80 mM urea as tonicity control. Then, CCR2 gene expression was assessed with qPCR (A) and protein expression with flow cytometry (B). (C and D) After 24-h incubation in NS, HS, or urea, monocytes were added to the monolayer of cultured human arterial endothelial cells. Transmigrated monocytes (red arrows) were distinguished from adhered monocytes by their transitions from bright to black morphology. NS, ([Na+] = 139 mM); HS, ([Na+] = 179 mM). Values represent mean ± SEM of n = 4 (A and B) and n = 5 (C) healthy donors. Data tested using 1-way ANOVA. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 5. HSD increases skin macrophage content with signs of a more proinflammatory and less antiinflammatory phenotype.

(A and B) To assess total skin macrophage content, sections were stained with anti-CD163 (red). Quantification of total macrophage content was expressed as the percentage of the total dermal area of the section that was positively stained for anti-CD163 (A). Anti-CD163 was favored above anti-CD68, since our pilot experiments showed that CD163 proved to be a more consistent macrophage marker than CD68 (Supplemental Figure 1), which is in line with the observations of 2 other research groups (51, 52). (C–H) To assess expression of proinflammatory and antiinflammatory macrophage markers, sections were stained with anti-CD163 (red) and either anti–HLA-DR (blue) (C and D), anti-CD206 (blue) (E and F), or anti-CCR2 (blue) (G and H). Since HLA-DR, CD206, and CCR2 can be expressed by other cells than macrophages, we only took into account the positively stained area of the concerning marker that was also positive for the macrophage marker anti-CD163. Quantification of the total expression of the several markers by macrophages was expressed as the percentage of positively stained area for anti-CD163 that was also positively stained for the concerning marker. (I and J) To assess skin expression of MCP-1, section were stained with anti–MCP-1 (blue) and anti-CD163 (red). Quantification of skin expression of MCP-1 was expressed as the total dermal area of the section that was positively stained for MCP-1 (I). LSD, low-salt diet. HSD, high-salt diet. Scale bar: 200 μm. Values represent mean ± SEM of n = 9–11 healthy male volunteers. Data are tested using a paired t test. *P < 0.05.

Figure 6. High salt (HS) boosts LPS-induced cytokine secretion in macrophages in vitro but does not affect the activation of M(IL-4).

(A) Schematic overview of in vitro LPS stimulation of macrophages. Monocytes of healthy volunteers were cultured in the presence of M-CSF for 7 days to differentiate into mature macrophages and stimulated for 24 h with LPS in the presence of normal salt (NS) concentrations present in IMDM medium (NS; [Na+] = 139 mM), HS ([Na+] = 179 mM; i.e., IMDM medium supplemented with an additional 40 mM NaCl), or 80 mM urea as tonicity control. (B) IL-6, IL-10, IL-12, and TNF were quantified by ELISA. (C) Schematic overview of in vitro IL-4 stimulation of macrophages. (D) Differentially treated macrophages were stained with antibodies against the M2 surface markers CD206 and CD200R, or isotype control, followed by flow cytometric analysis. Representative histogram graphs and corresponding surface expression quantifications (ΔMFI = [MFI]positive staining – [MFI]isotype staining) are presented. (E) Gene expression of IL-4–induced M2 marker genes. The fold inductions of indicated marker genes are shown relative to the expression in untreated macrophages (= 1). M-CSF, macrophage CSF; NS, ([Na+] = 139 mM); HS, ([Na+] = 179 mM); MFI, median fluorescence intensity. Values represent mean ± SEM of at least n = 3 healthy male donors. n = 3 (1 outlier excluded for TNF for B, n = 6 for D, n = 3 for E). Data tested using 1-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001.