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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.

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Fig. 1: HSD reduces resting CBF and induces endothelial dysfunction, effects reversed by returning to a normal diet.
Fig. 2: HSD induces delayed cognitive dysfunction.
Fig. 3: The NO precursor l-arginine reverses the neurovascular and cognitive dysfunction of HSD.
Fig. 4: HSD increases inhibitory eNOS phosphorylation.
Fig. 5: HSD induces TH17 differentiation in the small intestine and increases IL-17 plasma levels.
Fig. 6: The neurovascular and cognitive effects of HSD are not observed in mice lacking IL-17 or lymphocytes (Rag1−/− mice).
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.
Fig. 8: IL-17 suppresses NO production via ROCK, and ROCK inhibition ameliorates the neurovascular and cognitive dysfunction of HSD.

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Acknowledgements

We gratefully acknowledge support from the Feil Family Foundation. This work was supported by National Institutes of Health grants R37-NS089323 (C.I.) and 1R01-NS095441 (C.I.), by a Scientist Development Grant from the American Heart Association (G.F.) and by a network grant from the Fondation Leducq (Sphingonet) (C.I., J.A., G.F.).

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Authors

Contributions

Study design: C.I., J.A. and G.F. Conducting experiments and acquiring data: G.F., D.B., L.G.B., G.W., G.R., H.C., I.B., M.M.S., S.G.S., K.K., Y.S., M.M. and H.V. Analyzing data: G.F., D.B., G.W., H.C., I.B., M.M.S. and K.K. Writing the manuscript: G.F. and C.I.

Corresponding author

Correspondence to Costantino Iadecola.

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The authors declare no competing financial interests.

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Integrated supplementary information

Supplementary Figure 1 Effect of HSD on body weight, energy intake, systolic blood pressure, hippocampal resting CBF and CBF response to adenosine and whisker stimulation.

A: HSD tends to reduce body weight and increase caloric intake, but the effect is not statistically significant; Diet: p = 0.2668, Time: p < 0.0001; ND/HSD n = 15/20 mice/group (Two-way ANOVA and Tukey’s test). B: HSD does not alter systolic blood pressure measured in awake animals by tail-cuff plethysmography. Diet: p = 0.9417, Time: p = 0.1382; ND/HSD n = 15 mice/group (Repeated two-way ANOVA and Bonferroni's test). C: HSD does not alter the CBF response to the smooth muscle relaxant adenosine; Diet: p = 0.9659, Time: p = 0.0549; 4 weeks: ND/HSD n = 8/5; 8 weeks: ND/HSD n = 10; 12 weeks: ND/HSD n = 10/8; 24 weeks: ND/HSD n = 7 mice/group (Two-way ANOVA and Tukey's test). D: HSD reduces resting CBF in the hippocampus. * p = 0.0245 vs. 0 weeks; 0 weeks n = 8, 12 weeks n = 7, 24 weeks n = 5 mice/group (One way ANOVA and Tukey’s test). E: Temporal profile of CBF increase induced by whisker stimulation after 4, 8 and 24 weeks of ND or HSD; 4 weeks: ND/HSD n = 7/7; 8 weeks: ND/HSD n = 6/7; 12 weeks: ND/HSD n = 9; 24 weeks: ND/HSD n = 5/7 mice/group. The dotted line indicates the baseline CBF whereas the shaded area represents the standard error. F: CBF increase to whisker stimulation assessed as area under the curve shows a diet effect (Diet: p = 0.0013, Time: p = 0.6687), which did not reach statistical significance at the Tukey’s test; 4 weeks: ND/HSD n = 7/7; 8 weeks: ND/HSD n = 6/7; 12 weeks: ND/HSD n = 9; 24 weeks: ND/HSD n = 5/7 mice/group (Two-way ANOVA and Tukey's test). G: Return to normal diet does not alter MAP and CBF responses to whisker stimulation and adenosine. MAP: p = 0.2406; Whisker: p = 0.2340; Adenosine: p = 0.8010, n = 5 mice/group (One-way ANOVA and Tukey’s test). H: Endothelial dysfunction is also observed in mice fed a 4% HSD for 12 weeks. ACh: * p < 0.0376 vs ND; ND/HSD n = 4/5 mice/group (unpaired t-test, two-tailed). I: A 4% HSD impairs performance at the novel object test (p = 0.0350 vs ND, unpaired t-test, two tailed). J: Resting NO production, assessed by DAF-FM, is reduced in pial microvascular preparation from mice fed a HSD. The effect is reversed by administration of the NO precursor L-arginine. * p = 0.0024; microvessels isolated from 8 (ND Veh), 6 (ND L-Arg), 7 (HSD Veh) and 9 (HSD L-Arg) mice/group (One-way ANOVA and Tukey’s test). Data are expressed as mean ± SEM.

Supplementary Figure 2 Effect of HSD on mRNA levels of proinflammatory mediators in cerebral microvessels.

HSD does not induce upregulation of cytokines, adhesion molecules and other proinflammatory molecules in isolated pial microvascular preparations of mice fed a HSD for 12 weeks. IL-6: * p < 0.0411 vs ND, ND/HSD n = 3/5; VCAM1: * p = 0.0102 vs ND, ND/HSD n = 5 mice/group (unpaired t-test, two-tailed). Data are expressed as mean ± SEM.

Supplementary Figure 3 Effect of HSD or systemic administration of IL-17 on mRNA levels of proinflammatory mediators in brain endothelial cells and on the BBB.

A: HSD or systemic administration of IL-17 do not upregulate cytokines, adhesion molecules and other proinflammatory molecules in endothelial cells FACS-sorted from the brain. ND/HSD n = 10; Veh/IL-17 n = 5/4 mice/group (One-way ANOVA and Tukey’s test). B: HSD does not increase BBB permeability to FITC-dextran (3kDa) both in cortex and hippocampus. p = 0.1143 and p = 0.9624 vs ND; n = 5 mice/group (unpaired t-test, two-tailed). Data are expressed as mean ± SEM.

Supplementary Figure 4 Effect of aging or ETA receptor antagonism on cerebrovascular and cognitive dysfunction induced by HSD.

A: HSD (8 weeks) does not affect MAP in aged mice but, at variance with young mice, significantly attenuates the CBF response induced by whisker stimulation; Diet: * p = 0.0010, Age: * p = 0.0003; ND young/old n = 13-8, HSD young/old n = 12/8 mice/group (Two-way ANOVA and Tukey’s test). HSD does not further decrease the CBF response to ACh in aged mice; Diet: * p < 0.0001, Age: * p = 0.0195 ND young/old n = 10-7, HSD young/old n = 10/8 mice/group (Two-way ANOVA and Tukey’s test). B: Worsening of the performance at the novel object recognition task occurs earlier (8 weeks of HSD), in aged mice than in young mice (12 weeks for HSD)(see Fig. 2a). Total Exploration Time, Diet: * p < 0.0001, Age: p = 0.1935; NOR, Diet: * p = 0.048, Age: * p = 0.0096; ND young/old n = 8–10, HSD young/old n = 10/9 mice/group (Two-way ANOVA and Tukey’s test). C: Neocortical superfusion of the ETA receptor antagonist BQ123 fails to reverse the cerebrovascular effects of HSD. ACh: Diet * p < 0.0008, Treatment p = 0.7523; ND Veh/BQ123 n = 4/4, HSD Veh/BQ123 n = 3/3 mice/group (Two-way ANOVA and Tukey’s test). Data are expressed as mean ± SEM.

Supplementary Figure 5 HSD increases mRNA levels of factors required for Th17 polarization.

A: HSD induces upregulation of IL-22, IL-23R, iNOS, SAA1-3 in distal small intestine of mice fed a HSD. IL-22: * p = 0.0073 vs ND; ND/HSD n = 7/8; IL-23R: * p = 0.0057 vs ND; ND/HSD n = 8/7; iNOS: * p = 0.0005 vs ND; ND/HSD n = 8/9; SAA1: * p = 0.0007 vs ND; ND/HSD n = 8/9; SAA2: * p = 0.0005 vs ND; ND/HSD n = 8/9; SAA3: * p < 0.0001 vs ND; ND/HSD n = 8/9 mice/group (unpaired t-test, two-tailed). B: HSD does not increase IL-17A mRNA levels in the colon. mRNA levels are normalized to IL-17A mRNA levels in the distal small intestine of mice fed a ND. Diet: p = 0.1461, ND/HSD n = 3/4 mice/group (Two-way ANOVA and Tukey's test). C: Plasma levels of TNF-α and IL-6 are not increased after HSD. TNF-α: p = 0.6581 vs ND; IL-6: p = 0.8592 vs ND; n = 12 mice/group (unpaired t-test, two-tailed). n.d: not detectable. Data are expressed as mean ± SEM.

Supplementary Figure 6 HSD does not increase Il17a mRNA levels or TH17 cells in both brain and meninges.

A: HSD does not alter number and B: frequency of T helper and TH17 cells in brain and meninges. Brain T helper: p = 0.1671 vs ND and p = 0.9157 vs ND; Meningeal T helper: p = 0.3882 vs ND and p = 0.5669 vs ND; Brain TH17 cells: p = 0.8709 vs ND and p = 0.6491 vs ND; Meningeal TH17 cells: p = 0.7677 vs ND and p = 0.3192 vs ND; n = 5/group, 2 mice/samples (unpaired t-test, two-tailed). C: IL-17 mRNA levels are not increased in the brain or meninges of mice fed a HSD. Meninges: p = 0.6465 vs ND; ND/HSD n = 4/5 mice group (unpaired t-test, two-tailed). Data are expressed as mean ± SEM.

Supplementary Figure 7 MAP and CBF responses in Il17a–/– or Rag1–/– mice, in WT mice treated with IL-17-neutralizing antibodies and in WT mice treated with IL-17.

A-C: HSD does not alter MAP or CBF responses to whisker stimulation and adenosine in IL17-/- and Rag1-/- mice, or in WT mice receiving IL17 neutralizing antibodies. IL17-/-, MAP, Diet: p = 0.1864, Genotype: p = 0.2153; ND WT/IL17-/- n = 5/4, HSD WT/IL17-/- n = 8/6; Whisker, Diet: p = 0.5651, Genotype: p = 0.8474, WT/IL17-/- n = 6/4, HSD WT/IL17-/- n = 7/6; Adenosine, Diet: p = 0.8710, Genotype: p = 0.1652; WT/IL17-/- n = 6/4, HSD WT/IL17-/- n = 7/7 mice/group. Rag1-/-, MAP, Diet: p = 0.0619, Genotype: p = 0.4521; ND WT/Rag1-/- n = 4/8, HSD WT/Rag1-/- n = 8/10; Whisker, Diet: * p = 0.0094, Genotype: p = 0.5461, WT/Rag1-/- n = 5/8, HSD WT/Rag1-/- n = 8/8; Adenosine, Diet: p = 0.6543, Genotype: p = 0.7647; WT/Rag1-/- n = 5/7, HSD WT/Rag1-/- n = 8/9 mice/group (Two-way ANOVA and Tukey’s test). D: Administration of IL-17 in WT mice does not affect MAP or the increase in CBF produced by adenosine; MAP: p = 0.7772 vs Veh; Veh/IL-17 n = 7-8/mice; Adenosine: p = 0.7102 vs Veh; Veh/IL-17 n = 7-8/mice group (unpaired t-test, two-tailed). Data are expressed as mean ± SEM.

Supplementary Figure 8 Effect of depletion of brain perivascular macrophages and of FTY720 or Y27632 on CBF responses in mice fed HSD.

A: PVM depletion by clodronate does not ameliorate the endothelial dysfunction in mice fed a HSD diet; Diet: * p < 0.0001, Treatment: p = 0.8889; ND Veh/ND CLO n = 4, HSD Veh/HSD CLO n = 5 mice/group (Two-way ANOVA and Tukey’s test). B: Clodronate (i.c.v.) depletes brain PVM in the somatosensory cortex of both ND and HSD-fed mice; Diet: p = 0.9780, Treatment: * p < 0.0001; ND Veh/ND CLO n = 4, HSD Veh/HSD CLO n = 5 mice/group (Two-way ANOVA and Tukey’s test). C: FTY720 has no effect on the endothelial dysfunction induced by HSD; Diet: * p = 0.0001, Treatment: p = 0.3789; ND Veh/ND FTY720 n = 6/4, HSD Veh/HSD FTY720 n = 6/5 mice/group (Two-way ANOVA and Tukey’s test). D: FTY720 administration reduces blood T-helper lymphocytes in both ND and HSD-fed mice. Diet: p = 0.7357, Treatment: p < 0.0001; ND Veh/ND FTY720 n = 6/3, HSD Veh/HSD FTY720 n = 5/4 mice/group (Two-way ANOVA and Tukey’s test). E: The CBF responses to whisker stimulation or adenosine are not altered by Y27632; p > 0.05 vs ND; Whisker, Diet: p = 0.5397, Treatment: p = 0.5804; ND Veh/Y27632 n = 6/6, HSD Veh/Y27632 n = 6/8; Adenosine, Diet: p = 0.5712, Treatment: p = 0.4964; ND Veh/Y27632 n = 6/6, HSD Veh/Y27632 n = 6/7 mice/group (Two-way ANOVA and Tukey’s test). Data are expressed as mean ± SEM.

Supplementary Figure 9 Effect of IL-17 administration on eNOS phosphorylation in human cerebral endothelial cells (HBEC.5i).

A: IL-17 (1-10 ng/mL) increases phosphorylation of eNOS on Thr495, an effect abrogated by the administration of the ROCK inhibitor Y27632 (5μM). * p = 0.0026; Vehicle: IL-17 0 ng/ml n = 8; IL-17 1 ng/ml n = 7, IL-17 10 ng/ml n = 5; Y27632: IL-17 0-10 ng/ml n = 4 independent experiments/group (One-way ANOVA and Tukey’s test). B-D: HSD increases the inhibitory phosphorylation of eNOS at Thr495 in isolated pial microvascular preparations isolated from WT mice and mice injected with an IgG control antibody or vehicle. WT: * p = 0.0380 vs ND; ND/HSD n = 3/6; IgG: * p = 0.0272 vs ND; ND/HSD n = 5/6; Veh: * p = 0.0177 vs ND; ND/HSD n = 3/4 mice/group (unpaired t-test, two-tailed). Data are expressed as mean ± SEM. Immunoblots in A, B, C and D are cropped. Full gel pictures for immunoblots are shown in Supplementary Fig 14.

Supplementary Figure 10 Effect of IL-17 and TNF-α administration on mRNA levels of proinflammatory mediators in brain endothelial cells (bEnd.3).

A: IL-17 (10 ng/mL) does not induce upregulation of cytokines, adhesion molecules and other pro-inflammatory molecules in cultures of bEnd.3 cells. p > 0.05 vs 0; n = 4 independent experiments/group (One-way ANOVA and Tukey’s test). B: TNF-α (10 ng/mL) induces upregulation of cytokines and adhesion molecules in cultures of bEnd.3 cells. ICAM1: * p = 0.0124 vs 0; VCAM1: * p < 0.0001 vs 0; CXCL1: * p = 0.0024; CXCL2: * p = 0.0035 vs 0; CXCL5: * p = 0.0372 vs 0; MCP1: * p < 0.0001 vs 0; n = 3 independent experiments/group (One-way ANOVA and Tukey’s test). Data are expressed as mean ± SEM.

Supplementary Figure 11 Cartoon illustrating the mechanisms of neurovascular and cognitive dysfunction induced by HSD.

HSD induces a TH17 response in the distal small intestine by presumably activating serum glucocorticoid-regulated kinase 1 (SGK1). TH17 cells lead to an increase in circulating IL17, which, in turn, acts on cerebral endothelial cells to induce ROCK activation, inhibitory eNOS phosphorylation, and reduction in eNOS catalytic activity. The resulting reduction in endothelial NO leads to cerebral hypoperfusion, vascular dysregulation and cognitive impairment.

Supplementary Figure 12 Full immunoblots image of panels in Figs. 3 and 6.

Membranes were incubated with anti-pThr495 eNOS antibody (1:500) or anti-pSer1177 eNOS antibody (1:1000) overnight. After incubation with secondary antibody, protein bands were visualized with Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) on a Bio Rad ChemiDoc MP Imaging System. After washes with TBS/0.1% Tween-20 (TBST), membranes were incubated with anti-eNOS antibody (1:1000) overnight. eNOS bands were visualized with Clarity Western ECL Substrate (BioRad). Samples from microvessels preparations (≈30 µg of protein/lane).

Supplementary Figure 13 Full immunoblots image of panels in Figs. 7 and 8.

Membranes were incubated with anti-pThr495 eNOS antibody (1:500) overnight. After incubation with secondary antibody, protein bands were visualized with Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) on a Bio Rad ChemiDoc MP Imaging System. After washes with TBS/0.1% Tween-20 (TBST), membranes were incubated with anti-eNOS antibody (1:1000) overnight. eNOS bands were visualized with Clarity Western ECL Substrate (BioRad). Samples from microvessels preparations and cultures of mouse brain endothelial cells (≈30µg of protein/lane).

Supplementary Figure 14 Full immunoblots image of panels in Supplementary Figure 9.

Membranes were incubated with anti-pThr495 eNOS antibody (1:500) overnight. After incubation with secondary antibody, protein bands were visualized with Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) on a Bio Rad ChemiDoc MP Imaging System. After washes with TBS/0.1% Tween-20 (TBST), membranes were incubated with anti-eNOS antibody (1:1000) overnight. eNOS bands were visualized with Clarity Western ECL Substrate (BioRad). Samples from microvessels preparations and cultures of human brain endothelial cells (≈30 µg of protein/lane).

Supplementary Figure 15 Flow cytometry gating strategy for IL-17+ cells.

Cells were discriminated by FSC-SSC gating and then gated for CD45+CD4+TCRγδ-IL17+ (Th17 cells) or CD45+CD4-TCRγδ+IL17+ (IL17+γδ T cells).

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Faraco, G., Brea, D., Garcia-Bonilla, L. et al. Dietary salt promotes neurovascular and cognitive dysfunction through a gut-initiated TH17 response. Nat Neurosci 21, 240–249 (2018). https://doi.org/10.1038/s41593-017-0059-z

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