High And Low Dietary Salt Intake Induced Dynamics Of Phenotype Change In Human Circulating Monocyte Subsets And Their Potential Mechanisms

Objective:High salt diet (HS) is not only the contributor to hypertension, but also can induce target organ damage (TOD) independent of blood pressure level. Monocyte is one of the main players in inflammatory response which is critical for the initiation and development of TOD. Recent evidence indicates that human circulating monocytes are at least composed of three subsets with distinct physiological functions, including classical (CD14++CD16",[Mon1]), intermediate (CD14++CD16+,[Mon2]), and non-classical (CD14+CD16++,[Mon3]) monocytes. Among them, CD14++CD16++monocytes were considered as "pro-inflammatory" monocytes, participating in a variety of inflammatory diseases, especially in the development of atherosclerosis. Moreover, the formation of monocyte-platelet aggregates (MPA), an sensitive marker for in vivo platelet activation, would further facilitate monocyte adhension to endothelial cells, and consequently, migration to subendotheial region. However, it remains unclear whether HS could have an impact on monocyte subsets phenotype and MPA formation, and thus contributes to the exacerbation of TOD. Therefore, the purpose of present study is to investigate the dynamics of phenotype change in human circulating monocyte subsets induced by high and low dietary salt intake, and exploring their potential mechanisms.Methods:A total of22healthy volunteers were recruited in our dietary intervention trial (10male aged30.10±2.99years old and12female aged29.40±2.65years old). The participants were provided with HS (15g NaCl per day) for7d and LS (5g NaCl per day) for7d. All foods and beverages were prepared by study dietitians and provided by the study staff.24-h urine was collected at baseline,24h after HS,7d after HS,3d after LS and7d after LS. Overnight fasting blood samples were drawn at baseline,24h after HS,7d after HS,24h after LS and7d after LS. Monocyte subsets and their association with platelet aggregates were analyzed using a four-color flow cytometric platform, based on CD86, CD14, CD16and CD41. Circulating monocytes from each participant were further purified by density-gradient centrifugation to harvest peripheral blood mononuclear cells (PBMC), followed by magnetic activated cell sorting (MACS) by positive selection using anti-human CD14coated microbeads at baseline,7d after HS and7d after LS. Total RNA of purified monocytes was extracted using standard TRIzol procedure. Real-time PCR was used to analyze the mRNA expression of related pro-inflammatory and anti-inflammatory molecules, including C-C chemokine ligand5(CCL5), C-C chemokine receptor5(CCR5), monocyte chemotactic protein-1(MCP-1), C-C chemokine receptor2(CCR2), transforming growth factor-β (TGF-β), tumor necrosis factor-a (TNF-a), nuclear factor-KB (NF-κB), C-X3-C chemokine receptor1(CX3CR1), arginase-1(Arg-1) and P-selectin glycoprotein ligand-1(PSGL-1). Radioimmunoassay was used to detect plasma level of plasma renin activity (PRA), AngiotensinⅡ (Ang Ⅱ), aldosterone (ALD), Insulin (Ins) and C-Peptide (C-P). Enzymatic cycling assay was used to detect serum level of homocysteine (Hey).Results:Of the22healthy volunteers,20subjects completed the dietary intervention and related blood and urine sampling.2subjects withdrew either because of intolerance to HS diets, or due to increased ventricular premature beats during HS intervention. The results from the24-h urinary excretions of sodium and creatinine showed excellent compliance with the study diets. During intervention, there were no significant changes in systolic blood pressure (SBP) and diastolic blood pressure (DBP) at any time points (SBP P for trend:0.6677and DBP P for trend:0.4915). Heart rate increased significantly after HS compared with baseline (P<0.05), and regressed to baseline level after LS. Plasma level of Ins, HOMA-IR and QUICKI did not changed by HS and LS intervention significantly(P for trend:0.0655,0.2529,0.5057). C-P and HOMA-P decreased dramatically after HS compared with baseline, and returned after LS (P for trend <0.0001, P for trend0.0008). In addition, plasma PRA and Angll did not change significantly between baseline, HS and LS (P for trend:0.5315,0.1100). Whereas plasma ALD were significantly inhibited by HS and returned to baseline levels after LS (P for trend:0.0436). Total monocyte count and CD14++CD16-monocytes [classical, Monl] showed mild increase at LS24h (P for trend:0.0199,0.0264, respectively). CD14+CD16++monocyte count and percent [non-classical Mon3] increased slightly at LS7d (P for trend:0.0002,0.0122). There was a dramatic increase of CD14++CD16+monocyte count and percent [intermediate, Mon2] one day after HS, and this trend persisted till one day after LS (P for trend:<0.0001), the magnitude of which exhibited statistical significance compared with those of other time-points (P for trend:<0.0001). MPA count and percent in periphery blood increased significantly at HS24h, and lasted until LS24h (P for trend:<0.0001). This trend could be completely normalized after7days of LS diets. MPA in association with Mon2exhibited coordinated change with Mon2(P for trend:<0.0001). Moreover. Mon2subset had the highest binding affinity with platelet compared with other subsets at any time points (32.53±6.68)%,(47.92±14.89)%,(55.12±12.08)%,(64.09±17.88)%,(19.15±7.89)%for serial five time points, respectively. The mRNA expression level of inflammatory cytokines including MCP-1, CCL5, NF-κB, TGF-β and CX3CR1increased after HS7d, and returned to baseline after LS. The mRNA expression of CCL5and NF-κB at HS7d had statistical significance compared with baseline (P<0.05). The expression of Ang-1decreased after HS7d and increased significantly after LS7d (P<0.05). The mRNA expression of PSGL-1at HS7d mildly decreased (P for trend:0.0553). In comparison with baseline and HS7d, the expression of PSGL-1at LS7d decreased significantly (P for trend0.0001, P for trend0.0262).Conclusions:The present study, to the best of our knowledge, for the first time provides human evidence that variation in dietary salt intake could induce coordinated monocyte subsets dynamics and their association with MPA. Specifically, an increase in dietary salt could lead to a rapid and proportional expansion of CD14++CD16+pool, followed by a gradual decrease despite adherence to high salt diet, which could be normalized by low salt intake. In addition, there is a paralleled relationship between MPA dynamics and the change of CD14++CD16+monocytes in response to fluctuation in dietary salt intake. These evidence may have broad clinical implications for high dietary salt intake induced end organ injury, atherosclerotic lesion development, and acute thromboembolic risk.