Role of Oxidative Stress in Vascular Low-Grade Inflammation Initiation Due to Acute Salt Loading in Young Healthy Individuals

Abstract

This study aimed to investigate the effect of 7-day high-salt (HS) and the specific role of oxidative stress on vascular low-grade inflammation initiation in young salt-resistant healthy individuals. 30 young healthy individuals adhered to a 7-day low-salt (LS) diet (3.5 g salt/day), followed by a 7-day high-salt (HS) diet (~14.7 g salt/day) protocol. Pro- and anti-inflammatory cytokines, frequencies of peripheral blood Th17 and Treg cells, Th17/Treg ratio, enzymes SGK1, and p38/MAP kinase, as well as biomarkers of endothelial activation and oxidative stress, were measured before and after the 7-day HS diet protocol. Short-term HS diet significantly increased serum level of pro-inflammatory cytokines INF-γ, TNF-α, IL-9, and IL-17A levels, but also of anti-inflammatory cytokines IL-10 and TGF-β1. Relative amount of total SGK1 significantly increased, following the 7-day HS diet. Increased oxidative stress level, following HS diet, was negatively associated with the frequency of Treg cells. The increase in relative amount of total SGK1 in peripheral mononuclear cells following 7-day HS diet suggests lymphocyte (re)activation, in response to HS intake, resulting in enhanced production of pro-inflammatory (IL-17, INF-γ), but also anti-inflammatory cytokines (IL-10 and TGF-β1). Increased oxidative stress, due to HS loading, alters immune regulatory mechanisms, presumably via effects on Treg cells.

Keywords: Th17 cells; cell adhesion molecules; cytokines; endothelium; high-salt diet; inflammation; oxidative stress; regulatory T cells.

Figure 1

Gating strategy for the assessment of peripheral blood regulatory T cells (Treg) by flow cytometry. Panel (A) shows representative dot plots illustrating gating strategy, including exclusion of doublets using forward scatter area (FSC-A) versus forward scatter width (FSC-W) analysis (A-i), gating on live cells negative for aminereactive fixable viability dye (A-ii), lymphocytes (A-iii), and CD3⁺ T cells (A-iv). Next, total CD25 and Foxp3, expressing T cells among CD4⁺CD127 low population, were analyzed (Panel (B)). (B-i) shows representative gating strategy, (B-ii) representative samples from LS and HS protocol. FlowLogic software was used for data analysis and illustration.

Figure 2

Gating strategy for the assessment of peripheral blood Th17 population by flow cytometry. Panel (A) shows representative dot plots illustrating gating strategy, including exclusion of doublets using forward scatter area (FSC-A) versus forward scatter width (FSC-W) analysis (A-i), gating on live cells negative for amine-reactive fixable viability dye (A-ii), lymphocytes (A-iii), CD3⁺ T cells (A-iv), and CD3⁺CD4⁺ T helper cells (A-iv). T helper cells were subsequently analyzed for IL-17A and CD196/CCR6 expression. First, total IL-17-secreting T helper cells were analyzed (Panel (B)) where the gate on IL-17⁺ T cells were defined using florescence minus one (FMO) control for IL17 antibody (B-i). A representative sample demonstrating relative frequencies of peripheral CD4⁺IL-17⁺ T helper cells in healthy young individuals are shown at (B-ii). The population of IL-17-secreting T helper cells was further analyzed for CD196/CCR6 expression (panel (C), C-iii), hence two subpopulations were identified—CD4⁺CD196⁺IL17⁺ corresponding to Th17 cells and CD4⁺CD196-IL17⁺ non-Th17 cells accounting for other T helper subpopulations with the capacity to secrete IL-17. FMO controls for antibodies with specificities for IL17 and CCR6 were utilized for the setup of quadrant gates (C-i, C-ii). FlowLogic software was used for data analysis and illustration.

Figure 3

Effects of a 7-day high-salt diet on the frequency of peripheral regulatory T cells (Treg) in healthy young individuals. Relative frequencies of peripheral Treg are presented as box-and-whisker plots 10 to 90 percentiles; Paired t-test, LS vs. HS; p < 0.05 was considered significant.

Figure 4

Effects of a 7-day high-salt diet on the representation of peripheral Th17 population in healthy young individuals. Relative frequencies of total IL-17-secreting peripheral T helper (CD3⁺CD4⁺) cells are shown at panel (A), while the relative frequencies of CCR6⁺IL17⁺ T cells, corresponding to Th17 cells, are demonstrated at Panel (B). Data are presented as box-and-whisker plots 10 to 90 percentiles. Paired t-test, LS vs. HS; p < 0.05 was considered significant.

Figure 5

The effect of a 7-day high-salt diet on relative amount of total serum- and glucocorticoid regulated kinase 1 (SGK1) (A) and total (B-i) and phosphorylated (B-ii) p38 mitogen-activated protein (MAP) kinase (B) in young healthy individuals. N = 30 (15 women and 15 men). LS—low salt; HS—high-salt; N—number of subjects). Data are presented as box-and-whisker plots 10 to 90 percentiles. Paired t-test, * p < 0.05, LS diet vs. HS diet.

Figure 6

Dichotomous, context-dependent fate of T helper cells, when exposed to high-salt conditions. Naïve cell, exposed to hyperosmotic and pro-inflammatory environment, primed under Th17 polarizing cytokines, acquired enhanced Th17 function. This effect was also observed among other T helper cell compartments (i.e., Th1 subpopulation). Interestingly, if the Th17 cells are void of pro-inflammatory milieu during activation in high NaCl environment (and re-activation at the sites of ongoing immune responses against certain pathogens or (auto)antigens), they acquire anti-inflammatory phenotype and function, characterized by the up-regulation of IL-10, TGFβ, Foxp3, CTLA-4, ICOS, and LAG3. Increased oxidative stress may alter immune regulatory mechanisms, potentially via effects on Treg cells.