Impact of combined sodium chloride and saturated long-chain fatty acid challenge on the differentiation of T helper cells in neuroinflammation

Abstract

Background: There has been a marked increase in the incidence of autoimmune diseases like multiple sclerosis (MS) in the last decades which is most likely driven by a change in environmental factors. Here, growing evidence suggests that ingredients of a Western diet like high intake of sodium chloride (NaCl) or saturated fatty acids may impact systemic immune responses, thus increasing disease susceptibility. Recently, we have shown that high dietary salt or long-chain fatty acid (LCFA) intake indeed aggravates T helper (Th) cell responses and neuroinflammation.

Methods: Naïve CD4⁺ T cells were treated with an excess of 40 mM NaCl and/or 250 μM lauric acid (LA) in vitro to analyze effects on Th cell differentiation, cytokine secretion, and gene expression. We employed ex vivo analyses of the model disease murine experimental autoimmune encephalomyelitis (EAE) to investigate whether salt and LCFA may affect disease severity and T cell activation in vivo.

Results: LCFA, like LA, together with NaCl enhance the differentiation of Th1 and Th17 cells as well as pro-inflammatory cytokine and gene expression in vitro. In cell culture, we observed an additive effect of LA and hypertonic extracellular NaCl (NaCl + LA) in Th17 differentiation assays as well as on IL-17, GM-CSF, and IL-2 gene expression. In contrast, NaCl + LA reduced Th2 frequencies. We employed EAE as a model of Th1/Th17 cell-mediated autoimmunity and show that the combination of a NaCl- and LA-rich diet aggravated the disease course and increased T cell infiltration into the central nervous system (CNS) to the same extent as dietary NaCl.

Conclusions: Our findings demonstrate a partially additive effect of NaCl and LA on Th cell polarization in vitro and on Th cell responses in autoimmune neuroinflammation. These data may help to better understand the pathophysiology of autoimmune diseases such as MS.

Keywords: Diet; EAE; Inflammation; Multiple sclerosis; Salt; Saturated fatty acids; T helper cell.

Fig. 1

Combined NaCl + LA challenge promotes polarization of naïve T cells towards Th17 cells in vitro. a Addition of either 40 mM NaCl, 250 μM LA, or both to CD4⁺ T cell differentiation culture under Th1-polarizing conditions (data pooled from five independent experiments, *p < 0.05, ***p < 0.001). b IFNγ production of Th1 cells treated with either 40 mM NaCl, 250 μM LA, or both (data pooled from four to five independent experiments). c Addition of either 40 mM NaCl, 250 μM LA, or both to CD4⁺ T cell differentiation culture under Th17-polarizing conditions (data pooled from three independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001). d IL-17 production of Th17 cells treated with either 40 mM NaCl, 250 μM LA, or both (three to four independent experiments are shown). e Addition of either 40 mM NaCl, 250 μM LA, or both to CD4⁺ T cell differentiation culture under Th2-polarizing conditions (data pooled from three independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 2

NaCl and LA exert distinct effects on Th1/Th17 cell gene expression profiles in vitro. a–c Gene expression analysis of tbx21 (T-bet) (a), csf2 (GM-CSF) (b), and il6 (c) in Th1 differentiation assays (data pooled from four preparations, *p < 0.05, **p < 0.01, ***p < 0.001). d–l Gene expression analysis of il17 (d), rorc (RORγ) (e), csf2 (GM-CSF) (f), il6 (g), il23a (h), tnf (i), il2 (j), ahr (k), and sgk1 (l) in Th17 differentiation assays (two out of four independent preparations are shown, *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 3

Combined high-fat and high-salt diet has no additional effect on EAE severity and immune cell activation. a Clinical course of MOG_35–55 EAE. Mice were fed either a NaCl-rich or a NaCl + LA-rich diet for 4 weeks prior to immunization. Data are shown on a 5-point score scale (n = 15–20 mice per group, data pooled from four independent experiments, *p < 0.05). b, c Ex vivo flow cytometry analysis of Th1 (b) and Th17 (c) cell frequencies in the spinal cord under a NaCl-rich or NaCl + LA-rich diet versus the control diet on day 21 of MOG_35–55 EAE (n = 9–13 mice per group, data pooled from three independent experiments, *p < 0.05). d–f Ex vivo flow cytometry analysis of Th1 (d), Th17 (e), and Treg (f) cell frequencies in the spleen under a NaCl-rich or NaCl + LA-rich diet versus the control diet on day 10 of MOG_35–55 EAE (n = 4–5 mice per group, *p < 0.05, **p < 0.01). g, h Cytokine analyses in splenocyte culture after ex vivo recall with MOG_35–55 (splenocytes harvested on day 10 p.i. of MOG_35–55 EAE, n = 4–5 per group, *p < 0.05, **p < 0.01, ***p < 0.001)