Supplementary MaterialsSupplemental data jciinsight-4-130508-s264. percentage of traditional Compact disc14++/Compact disc16C, nonclassical Compact disc14+/Compact disc16++, and intermediate Compact disc14++/Compact disc16+ monocyte subsets after LSD versus HSD (Amount 2, A and B). After HSD, CCR2 surface area expression on traditional monocytes strongly elevated (Amount 2C). The result of HS on monocyte phenotype was particular for CCR2, since various other examined chemokine receptors (CCR5, CCR7, CX3CR1), scavenger receptors (Compact disc36, SRA, CD163), and surface proteins associated with monocyte adhesion, migration, and activation (CD11b, CD11c, CD18, CD29, CD49d, CD62L, CD32, CD45RA, CD64, CD86, TLR4, CD200R) were not significantly different between LSD and HSD (Supplemental Number 2; supplemental material available on-line with this short article; https://doi.org/10.1172/jci.insight.130508DS1). CD206 (i.e., mannose receptor C type 1, a marker for antiinflammatory macrophages) Roy-Bz tended to become decreased after HSD (= 0.12 and Roy-Bz = 0.10 for the classical and intermediate subtype, respectively) (Number 2C). Open in a separate window Number 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 storyline (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++/CD16C (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 MFI = (MFI)positive staining C (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 = 11 healthy male volunteers. Data tested using 2-way ANOVA. ***< 0.001. To assess whether HSD affects inflammatory responses, we challenged whole blood with LPS and measured inflammatory cytokine secretion. HSD resulted in an increased secretion of IL-6, but not of IL-8, IL-12, TNF, or MCP-1 (i.e., CCL2, the ligand of CCR2) upon LPS stimulation (Figure 3A). Accordingly, isolated monocytes showed increased IL-6 secretion (< 0.001) when cultured in the presence of HS when compared with normal Roy-Bz medium or medium containing urea as a tonicity control (Figure 3B). Having observed increased monocyte CCR2 expression after HSD, we also assessed the expression of its ligand MCP-1 (CCL2) in the KCTD18 antibody presence of HS. Along with increased IL-6, monocytes tended to secrete more MCP-1 when cultured in the presence of HS in vitro (= 0.09, Figure 3B). Moreover, a HSD significantly increased blood plasma MCP-1 levels (< 0.05, Figure 3C), while we were unable to detect IL-6 in plasma of both LSD and HSD groups. Overall, no carryover effects could be discovered, meaning that diet plan order didn't influence the noticed effects. Open up in another window Shape 3 Aftereffect of sodium on cytokines.(A) Entire blood samples following LSD and HSD were challenged with LPS (24 h with 10 ng/mL) and cytokines were quantified by ELISA. (B) Monocytes of 3rd party healthy donors had been activated for 24 h in RPMI + 10% FCS + 1% PenStrep in the current presence of normal sodium (NS), high sodium (HS), or 80 mM urea as tonicity control. After that, cytokine secretion was quantified by ELISA. (C) Plasma examples after LSD and HSD had been analyzed for MCP-1 and IL-6 by ELISA. IL-6 was below the recognition threshold. For many analyses, ELISA was performed relating.