Sodium chloride drives autoimmune disease by the induction of pathogenic TH17 cells

Abstract

There has been a marked increase in the incidence of autoimmune diseases in the past half-century. Although the underlying genetic basis of this class of diseases has recently been elucidated, implicating predominantly immune-response genes, changes in environmental factors must ultimately be driving this increase. The newly identified population of interleukin (IL)-17-producing CD4(+) helper T cells (TH17 cells) has a pivotal role in autoimmune diseases. Pathogenic IL-23-dependent TH17 cells have been shown to be critical for the development of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, and genetic risk factors associated with multiple sclerosis are related to the IL-23-TH17 pathway. However, little is known about the environmental factors that directly influence TH17 cells. Here we show that increased salt (sodium chloride, NaCl) concentrations found locally under physiological conditions in vivo markedly boost the induction of murine and human TH17 cells. High-salt conditions activate the p38/MAPK pathway involving nuclear factor of activated T cells 5 (NFAT5; also called TONEBP) and serum/glucocorticoid-regulated kinase 1 (SGK1) during cytokine-induced TH17 polarization. Gene silencing or chemical inhibition of p38/MAPK, NFAT5 or SGK1 abrogates the high-salt-induced TH17 cell development. The TH17 cells generated under high-salt conditions display a highly pathogenic and stable phenotype characterized by the upregulation of the pro-inflammatory cytokines GM-CSF, TNF-α and IL-2. Moreover, mice fed with a high-salt diet develop a more severe form of EAE, in line with augmented central nervous system infiltrating and peripherally induced antigen-specific TH17 cells. Thus, increased dietary salt intake might represent an environmental risk factor for the development of autoimmune diseases through the induction of pathogenic TH17 cells.

Figure 1. Sodium Chloride promotes the stable induction of Th17 cells

a, Naïve CD4 cells were differentiated into Th17 cells in the presence (NaCl) or absence (none) of additional 40mM NaCl and analysed by flow cytometry (FACS) for IL-17A (n=20). b, IL-17A expression was measured by qRT-PCR (left panel, n=10) and ELISA (right panel, n=5). c, Cells were stimulated as in a) under the indicated increased NaCl concentrations and analysed by FACS (one representative experiment of five is shown). d, Cells were stimulated as in a) and were rested in the presence of IL-2. After 1 week, cells were re-stimulated as in a) in the presence or absence of NaCl for another week and analysed by FACS (one representative experiment of five is shown).

Figure 2. High-salt induced Th17 cells display a pathogenic phenotype

a, Microarray analysis of naïve CD4 cells differentiated into Th17 cells in the presence (NaCl) or absence (none) of additional 40mM NaCl. Depicted is a selection of 26 up- and down-regulated genes (mean fold change of two independent experiments). b, qRT-PCR analysis of differentially expressed genes in the two groups (n=5–8).

Figure 3. The induction of Th17 cells by sodium chloride depends on p38/MAPK, NFAT5 and SGK1

a, Naive CD4⁺ cells were stimulated in the presence (NaCl) or absence (none) of additional 40 mM NaCl and were analysed by FACS for phosphorylated p38 (p-p38; n = 5). b, Naïve CD4 cells were differentiated into Th17 cells as indicated in the presence or absence of NaCl and SB202190 (p38i) and analysed by qRT-PCR as depicted in the bar graph (n=7) or by FACS (the left row shows cells differentiated in the absence of TGF-β1). c, Naïve CD4 cells were stimulated for 3h in the presence or absence of NaCl and SB202190 and analysed by qRT-PCR for NFAT5 (n=4). d, Cells were transduced with NFAT5 specific (shNFAT5) or control shRNA (control), stimulated like in b) and analysed by FACS. The bar graphs depict qRT-PCR analyses of NFAT5, IL-17A and SLC5A3 (n=5). CCR6 was analysed by FACS (black histogram: control, grey histogram: shNFAT5, one representative experiment of four is shown). e, Cells were stimulated like in c), but analysed by qRT-PCR for SGK1 (n=4). f, Cells were transduced with a shRNA specific for SGK1 (shSGK1) or a control shRNA (control) and activated like in b), and analysed by FACS. Expression of SGK1 and IL-17A was determined by qRT-PCR (n=5). CCR6 was analysed by FACS (black histogram: control, grey histogram: shSGK1, one representative experiment of four is shown). g, Cells were cultured like in b), but in the presence or absence of the SGK1 inhibitor GSK650394 (SGK1i) and analysed by FACS. The bar graph shows qRT-PCR for IL-17A under similar conditions (n=5). FACS and qRT-PCR (relative expression) data depicted in bar graphs were normalised to controls.

Figure 4. High-salt diet induces Th17 cells in vivo and exacerbates experimental autoimmune encephalomyelitis

a, Naïve murine CD4 cells were stimulated with radiated APC, anti-CD3, IL-6 and TGF-β1 in the presence (NaCl) or absence (none) of additional 40mM NaCl and were analysed by FACS (n=3). b, IL-17A secretion (ELISA) of primary splenocytes, stimulated by anti-CD3 in the presence or absence of NaCl (n=6). c, Mean clinical scores of EAE in HSD animals (squares) or controls (dots, pooled data of two independent experiments with 12 animals). Histological analyses show sections of the spinal cord stained with hematoxylin and eosin (HE), anti-CD3 and anti-Mac-3 for control or HSD animals (scale bar=100 μM) and were quantified for CD3 and Mac-3 (bar graphs, n=5–6). d, Spinal cord from EAE animals was analysed by qRT-PCR (n=5–6). e, Splenocytes from EAE animals were analysed by qRT-PCR (n=4–7). f, Splenocytes from EAE animals were re-stimulated with MOG for 2 days and supernatants were analysed for IL-17A and IFN-γ by ELISA (n=7–8) or cells were analysed for IL-17A by FACS (n=4). qRT–PCR data are depicted as relative expression.