Dietary salt promotes neurovascular and cognitive dysfunction through a gut-initiated TH17 response

Abstract

A diet rich in salt is linked to an increased risk of cerebrovascular diseases and dementia, but it remains unclear how dietary salt harms the brain. We report that, in mice, excess dietary salt suppresses resting cerebral blood flow and endothelial function, leading to cognitive impairment. The effect depends on expansion of TH17 cells in the small intestine, resulting in a marked increase in plasma interleukin-17 (IL-17). Circulating IL-17, in turn, promotes endothelial dysfunction and cognitive impairment by the Rho kinase-dependent inhibitory phosphorylation of endothelial nitric oxide synthase and reduced nitric oxide production in cerebral endothelial cells. The findings reveal a new gut-brain axis linking dietary habits to cognitive impairment through a gut-initiated adaptive immune response compromising brain function via circulating IL-17. Thus, the TH17 cell-IL-17 pathway is a putative target to counter the deleterious brain effects induced by dietary salt and other diseases associated with TH17 polarization.

Fig.1 |. HSD reduces resting CBF and induces endothelial dysfunction, effects reversed by returning to a normal diet.

a, HSD (NaCI 8%) does not alter MAP (diet: P = 0.08134, time: P = 0.4982; 4 weeks: normal diet (ND) and HSD, n = 7 and 6 mice, respectively; 8 weeks: ND and HSD n = 11 and 21 mice, respectively; 12 weeks: ND and HSD n = 8 and 9 mice, respectively; 24 weeks: ND and HSD n = 7 mice per group; two-way ANOVA and Tukey’s test). b, HSD reduces resting CBF in the neocortex, assessed by arterial spin labeling MRI. The images show CBF after 12 weeks of HSD (^⋆P < 0.0001; 0 weeks n = 13 mice, 8 weeks n = 5, 12 weeks n = 10, 24 weeks n = 5 mice per group; one-way ANOVA and Tukey’s test). c, HSD attenuates the CBF increase induced by neocortical application of ACh (diet: ^⋆P < 0.0001, time: P = 0.2858; 4 weeks: ND and HSD n = 8 and 5 mice, respectively; 8 weeks: ND and HSD n = 10; 12 weeks: ND and HSD n = 9 and 8; 24 weeks: ND and HSD n = 7 and 6 mice per group; two-way ANOVA and Tukey’s test). d, The CBF response to whisker stimulation shows a diet effect (diet: P = 0.0066, time: P = 0.9966), which did not reach statistical significance after Tukey’s test (4 weeks: ND and HSD n = 8 and 5 mice, respectively; 8 weeks: ND and HSD n = 10 mice per group; 12 weeks: ND and HSD n = 9 mice per group; 24 weeks: ND and HSD n = 5 mice per group; two-way ANOVA and Tukey’s test). Dotted line indicates the baseline CBF whereas the shaded area represents the standard error. e, The neurovascular effects of HSD (12 weeks) are reversible by returning dietary sodium intake to normal levels for 4 weeks (NaCl 0.5%; resting CBF: ^⋆P < 0.0231 vs. ND; ND n = 5, HSD and HSD→ND n = 4 mice per group; ACh: ^⋆P = 0.0013 vs. ND; ND, HSD and HSD→ND n = 5 mice per group; one-way ANOVA and Tukey’s test). f, In isolated pial microvascular preparations, exposure to ACh (100 μM) increases NO production in mice fed ND, an effect attenuated in mice fed HSD for 12 weeks (diet: ^⋆P = 0.0009, treatment: ^⋆P < 0.0001; microvessels isolated from 9 ND and 10 HSD mice per group; repeated two-way ANOVA and Bonferroni’s test). Scale bar, 50 μm. RFU, relative fluorescence unit. Data represent the average of 3 independent experiments. For all panels, data are expressed as mean ± s.e.m.

Fig. 2 |. HSD induces delayed cognitive dysfunction.

a, HSD induces deficits in recognition (nonspatial) memory as assessed by the novel object test, first observed after 12 weeks (diet: ^⋆P < 0.0001, time: ^⋆P = 0.0046; 8 weeks: ND and SD n = 12 and 15 mice, respectively; 12 weeks: ND and HSD n = 15 mice per group; 24 weeks: ND and HSD n = 16 mice per group; two-way ANOVA and Tukey’s test). Total exploration time was not affected (P > 0.05). b, The deficit in novel object recognition is rescued by returning to normal diet for 4 weeks; ^⋆P = 0.0137 vs. ND; ND n = 8 mice; HSD and HSD→ND n = 7 mice per group; one-way ANOVA and Tukey’s test). c, HSD (12 weeks) alters spatial memory, as assessed by the Barnes maze test. No differences in the latency to find the escape hole or distance traveled are observed during training (days 1–3). At days 4 and 5 latency and distance traveled are increased in the HSD group, suggesting a deficit in spatial memory (diet: ^⋆P = 0.045, time: ^⋆P < 0.0001; ND and HSD n = 18 and 15 mice, respectively; repeated-measures two-way ANOVA and Tukey’s test). d, The ability to build a nest (nesting behavior), reflecting activities of daily living in rodents, is altered in mice fed HSD for 12 weeks, as indicated by a lower nest score (see Methods) and increased amount of untorn nesting material (nest score: ^⋆P = 0.0267 HSD vs. ND; untorn weight: ^⋆P = 0.0254 HSD vs. ND, n = 15 mice per group; two-tailed Mann–Whitney U test and two-tailed unpaired t test). Data are expressed as mean ± s.e.m.

Fig. 3 |. The NO precursor L-arginine reverses the neurovascular and cognitive dysfunction of HSD.

a, Administration of L-arginine in the drinking water (10 g per L), starting at week 8 of HSD and continued through week 12, did not alter MAP or CBF responses to whisker stimulation, but ameliorated the endothelial dysfunction induced by HSD (diet: ^⋆P = 0.0043; treatment: ^⋆P = 0.0360, ND vehicle (Veh) vs. L-arginine (L-arg) n = 6 and 5 mice per group, HSD Veh vs. L-arg n = 6 and 6 mice per group; two-way ANOVA and Tukey’s test). b, The cognitive deficits induced by HSD were also improved by L-arginine administration (diet: =0.0366, treatment: ^⋆P = 0.0118; ND Veh vs. L-arg n = 10 mice per group, HSD Veh vs. L-arg n = 12 and 10 mice respectively; two-way ANOVA and Tukey’s test). c, L-Arginine administration rescues the NO increase produced by ACh in cerebral microvascular preparations from HSD mice (diet: ^⋆P = 0.0315, treatment: ^⋆P < 0.0001; microvessels isolated from 5 ND and 6 HSD mice per group; repeated-measures two-way ANOVA and Bonferroni’s test). Data are expressed as mean ± s.e.m.

Fig. 4 |. HSD increases inhibitory eNOS phosphorylation.

a, HSD (8 weeks) increases the inhibitory phosphorylation of eNOS at Thr⁴⁹⁵ in isolated pial microvascular preparations (^⋆P = 0.0051 vs. ND; microvessels isolated from 7 ND and 9 HSD mice per group; unpaired t test, two-tailed). b, The activatory phosphorylation of eNOS at Ser¹¹⁷⁷ is not affected by HSD (P = 0.6615 vs. ND; microvessels isolated from 8 ND and 7 HSD mice per group; unpaired t test, two-tailed). Data are derived from 2 independent experiments and expressed as mean ± s.e.m. Immunoblots in a and b are cropped; full gel pictures for immunoblots are shown in Supplementary Fig. 12.

Fig. 5 |. HSD induces TH17 differentiation in the small intestine and increases IL-17 plasma levels.

a, IL-17⁺ cells accumulate in the lamina propria of the small intestine of IL-17-GFP reporter mice fed a HSD for 8 weeks (scale bar, 200 μm). b, Magnification showing localization of IL-17⁺GFP⁺ cells to the lamina propria (scale bar, 50 μm). The experiment was repeated independently twice with similar results. c, HSD increases TH17 lymphocytes in the lamina propria (^⋆P = 0.0004 vs. ND; n = 8 mice per group; unpaired t test, two-tailed), but T-helper lymphocytes are not increased (P > 0.05 vs. ND). d, Representative flow cytometry plot illustrating the increase in CD4⁺IL-17⁺ cells (TH17) induced by HSD. The experiment was repeated independently twice with similar results. e, IL-17⁺ γδ T cells, another source of IL-17, or IFNγ⁺ γδ T cells are not increased after HSD (P = 0.8266 and P = 0.4446 vs. ND; ND and HSD n = 7 and 9 mice per group (unpaired t test, two-tailed). Inset boxes indicate T-helper lymphocytes that are IL-17⁺. f, Regulatory T cell (Treg) lymphocytes are reduced in the lamina propria of the small intestine of mice fed HSD, but TH1 cells are not affected (^⋆P = 0.0088 vs. ND and P = 0.7933; n = 8 mice per group (unpaired t test, two-tailed). g, TH17 cells are slightly increased in lymph nodes and spleen (lymph nodes (LN): ^⋆P = 0.0098 vs. ND, ND and HSD n = 6 and 8 mice, respectively; spleen: ^⋆P = 0.0061, ND and HSD n = 8 mice per group, unpaired t test, two-tailed), but not in blood (P > 0.05 vs. ND). h,i, Il17a mRNA, normalized to levels in the distal small intestine of ND mice, is markedly increased in the distal small intestine but is not increased in blood leukocytes, lymph nodes or spleen (diet: ^⋆P < 0.0001; proximal (prox) and middle (mid) small intestine: ND and HSD n = 5 and 4 mice, respectively; distal (dist) small intestine: ND and HSD n = 6 and 8 mice, respectively; blood: ND and HSD n = 5 mice per group; lymph nodes: ND and HSD n = 4 mice per group; spleen: ND and HSD n = 5 mice per group; two-way ANOVA and Tukey’s test). j, HSD increases plasma IL-17 at 8, 12 and 24 weeks (diet: ^⋆P < 0.0001, time: P = 0.2997; 8 weeks: ND and HSD n = 11 and 15 mice, respectively; 12 weeks: ND and HSD n = 10 and 11 mice, respectively; 24 weeks: ND and HSD n = 9 mice per group; two-way ANOVA and Tukey’s test). Data are expressed as mean ± s.e.m.

Fig. 6 |. The neurovascular and cognitive effects of HSD are not observed in mice lacking IL-17 or lymphocytes (Rag1^−/− mice).

a,b, Il17a mRNA in the distal small intestine and plasma IL-17 are not detectable (n.d.) in Il17a^−/− mice on HSD for 12 weeks (diet: ^⋆P < 0.0001, genotype: ^⋆P < 0.0001, mRNA: wild-type (WT) ND and HSD n = 6 and 8 mice, respectively, Il17a^−/− ND and HSD n = 5 mice per group; plasma: WT ND and HSD n = 12 and 10 mice per group, Il17a^−/− ND and HSD n = 5 mice per group; two-way ANOVA plus Tukey’s test). c,d, The attenuation of the response to ACh and cognitive impairment induced by HSD are ameliorated in Il17a^−/− mice (ACh: diet: ^⋆P = 0.0003, genotype: ^⋆P = 0.0189; WT ND and HSD n = 6 and 8 mice, respectively, Il17a^−/− ND and HSD n = 4 and 7 mice per group; novel object recognition task (NOR): diet: ^⋆P = 0.0007, genotype: ^⋆P = 0.0202; WT ND and HSD n = 15 and 12 mice per group, Il17a^−/− ND and HSD n = 14 and 13 mice per group; two-way ANOVA and Tukey’s test). e, eNOS inhibitory phosphorylation induced by HSD is not present in pial microvascular preparations of Il17a^−/− mice (P = 0.8043 vs. ND; microvessels from 6 ND and 8 HSD mice per group; unpaired t test, two-tailed). f,g, Il17a mRNA and plasma IL-17 are not detectable in Rag1^−/− mice on HSD for 12 weeks (diet: ^⋆P < 0.0001, genotype: ^⋆P < 0.0001, mRNA: WT ND and HSD n = 6 and 8 mice, respectivley, Rag1^−/− ND and HSD n = 5 mice per group; plasma: WT ND and HSD n = 12 and 10 mice, respectively, Rag1^−/− ND and HSD n = 5 mice per group; two-way ANOVA plus Tukey’s test). h,i, The attenuation of the response to ACh and the attendant cognitive impairment induced by HSD are not observed in Rag1^−/− mice (ACh: diet: ^⋆P < 0.0001, genotype: ^⋆P = 0.0035; WT ND and HSD n = 5 and 10 mice, respectively, Rag1^−/− ND and HSD n = 8 mice per group; NOR: diet: ^⋆P = 0.0046, denotype: ^⋆P = 0.0496; WT ND and HSD n = 10 and 11 mice, respectively, Rag1^−/− ND and HSD n = 15 and 14 mice per group; two-way ANOVA and Tukey’s test). j, HSD fails to increase eNOS inhibitory phosphorylation in Rag1^−/− mice (P = 0.2330 vs. ND; microvessels isolated from 4 ND and 5 HSD mice per group; unpaired t test, two-tailed). Data were obtained from 2 independent experiments and are expressed as mean ± s.e.m. Immunoblots in e and j are cropped; full gel pictures for immunoblots are shown in Supplementary Fig. 12.

Fig. 7 |. The neurovascular and the cognitive effects of HSD are prevented by IL-17-neutralizing antibodies and reproduced by IL-17 administration in mice fed a normal diet.

a,b, Systemic (i.p.) administration of IL-17-neutralizing antibodies prevents the endothelial dysfunction and cognitive deficits of chronic (12 weeks) HSD (ACh: diet: ^⋆P = 0.0013, treatment: ^⋆P = 0.0062; ND IgG and anti-IL-17 n = 6 and 5 mice per group, HSD IgG and anti-IL-17 n = 7 mice per group; NOR: diet: ^⋆P = 0.0003, treatment: ^⋆P = 0.0374; ND IgG and anti-IL-17 n = 10 mice per group, HSD IgG and anti-IL-17 n = 8 and 10 mice per group; two-way ANOVA and Tukey’s test). c, eNOS inhibitory phosphorylation is not increased in HSD mice injected with IL-17-neutralizing antibodies (P = 0.3717 vs. ND; microvessels isolated from 6 ND and 8 HSD mice per group; unpaired t test, two-tailed). d, Systemic administration of exogenous IL-17 (i.p.) for 1 week increases plasma IL-17 to the same level as HSD (^⋆P = 0.0137 vs. ND; ND n = 9, HSD n = 7, IL-17 n = 5 mice per group; one-way ANOVA and Tukey’s test). e, IL-17 and attenuates the CBF response to ACh (^⋆P = 0.003 vs. Veh; Veh n = 6, IL-17 n = 8 mice per group; unpaired t test, two-tailed). f, IL-17 attenuates the performance at the novel object (^⋆P = 0.0422 vs. Veh; Veh and IL-17 n = 10 mice per group; unpaired t test, two-tailed). g, IL-17 increases eNOS phosphorylation on Thr⁴⁹⁵ in cerebral blood vessels (^⋆P = 0.0473 vs. ND; microvessels isolated from 4 Veh and IL-17 mice per group; unpaired t test, two-tailed). Data are expressed as mean ± s.e.m. Immunoblots in c and g are cropped; full gel pictures for immunoblots are shown in Supplementary Fig. 13.

Fig. 8 |. IL-17 suppresses NO production via ROCK, and ROCK inhibition ameliorates the neurovascular and cognitive dysfunction of HSD.

a,b, IL-17 induces eNOS phosphorylation at Thr⁴⁹⁵ (^⋆P < 0.0001 vs. vehicle 0 IL-17, n = 3−6 independent experiments; one-way ANOVA and Tukey’s test). c, IL-17 attenuates the increase in NO induced by ACh in mouse brain endothelial cell cultures. The attenuation by IL-17 of the ACh-induced NO increase is prevented by coadministration of Y27632 (5 μM), but not by inhibitors of ERK (PD98059; 10 μM) or PKC (Go6976; 1 μM). NO increase: ^⋆P = 0.0156, treatment: ^⋆P < 0.0001; n = 3−6 independent experiments per group; repeated-measures two-way ANOVA and Tukey’s test). d, Systemic administration of Y27632 does not affect the elevation in plasma IL-17 induced by 12 weeks of HSD (diet: ^⋆P < 0.0001, treatment: P = 0.5559, ND Veh and Y27632 n = 9 and 5 mice per group, HSD Veh and Y27632 n = 7 and 9 mice per group; two-way ANOVA and Tukey’s test). e, MAP is also not affected (ND Veh and Y27632 n = 6 and 5 mice per group, HSD Veh and Y27632 n = 6 and 8 mice per/group). f, Y27632 prevents the attenuation of the CBF response to ACh induced by HSD (diet: ^⋆P < 0.0001, treatment: ^⋆P = 0.0364, ND Veh and Y27632 n = 6 mice per group, HSD Veh and Y27632 n = 6 and 8 mice per group; two-way ANOVA and Tukey’s test). g, Y27632 prevents the behavioral dysfunction induced by HSD (diet: ^⋆P = 0.0486, ND Veh and Y27632 n = 11 and 10 mice per group, HSD Veh and Y27632 n = 11 and 11 mice per group; two-way ANOVA and Tukey’s test). h, Y27632 blocks the increase in eNOS phosphorylation induced by HSD (P = 0.5761 vs. ND; microvessels isolated from 5 ND and 9 HSD mice per group; unpaired t test, two-tailed). Data were derived from 3 independent experiments and are expressed as mean ± s.e.m. Immunoblots in a and h are cropped; full gel pictures for immunoblots are shown in Supplementary Figs. 13 and 14.