Intersection of salt- and immune-mediated mechanisms of hypertension in the gut microbiome

Hypertension is a leading contributor to cardiovascular morbidity and mortality worldwide, and expert guidelines now recommend lifestyle interventions including dietary sodium restriction in otherwise healthy individuals with blood pressure as low as 120/80 mm Hg. Both high-salt intake and proinflammatory immune cells have been implicated in the pathogenesis of hypertension, with recent studies suggesting a complex interplay between what were previously considered distinct mechanisms. In preclinical studies, proinflammatory Th17 lymphocytes drive blood pressure elevation through effects of the cytokine interleukin-17 to promote sodium reabsorption at several sites along the nephron.¹ Conversely, high-salt concentrations can promote differentiation of naïve T lymphocytes toward the proinflammatory Th17 lineage,² setting up a pathogenic feedback loop in which Th17 cells promote salt retention, which in turn drives recruitment of additional Th17 lymphocytes. In a new study published in Nature, Wilck et al. demonstrate a role for the gut microbiome in mediating the effects of a high-salt diet on Th17 cell differentiation, with potential implications for the pathogenesis and therapy of high blood pressure.³

The authors first evaluated the impact of high-salt intake on the composition of the gut microbiome in mice. Fecal pellets from mice fed a high-salt diet (4% NaCl) were compared with those from mice fed a normal salt diet (0.5% NaCl) by 16s ribosomal RNA gene sequencing, a commonly used approach to identify and classify bacteria in biologic samples. Analysis of the gene sequence data using conventional methods did not detect any reproducible differences at the taxonomic level; however, a concurrent analysis of fecal metabolites demonstrated clear differences between the 2 groups of animals. Because the observed differences in metabolite generation suggested a relevant shift in the gut microbial composition, the investigators used a sensitive machine-learning approach to identify subtle changes in the gut microbiome in response to 14 days of high-salt feeding. This approach identified 8 clusters of closely related bacteria that were most impacted, including a member of the genus Lactobacillus, which was depleted in mice fed a high-salt diet. The reduction in abundance of this organism, which was identified as L murinus, was confirmed by quantitative polymerase chain reaction. The authors then evaluated the impact of a high-salt environment on Lactobacillus isolates in vitro. Increasing concentrations of NaCl within the range observed in the feces of mice fed a high-salt diet inhibited the growth of L murinus and several human Lactobacillus isolates, but had little impact on the growth of control species including Escherichia coli.

Next, the authors considered the hypothesis that salt-induced changes in the gut microbiome mediate the impact of a high-salt diet on the immune system, in particular the polarization of T lymphocytes toward the proinflammatory Th17 phenotype that has been implicated in both hypertension and autoimmune disease.^2,4 In an established model of autoimmune encephalomyelitis, mice fed a high-salt diet developed more severe neurologic disease, which was ameliorated in mice receiving both a high-salt diet and Lactobacillus supplementation by gavage. Characterization of the immune cell population in small bowel tissue by flow cytometry demonstrated increased frequency of Th17 lymphocytes in diseased mice fed a high-salt diet; the frequency was lower in mice who received both a high-salt diet and Lactobacillus treatment. Interestingly, dietary salt intake did not impact the Th17 lymphocyte population in germ-free mice, suggesting that the mechanism may involve interactions between salt-sensitive Lactobacillus species and other gut flora. Exploration of potential mechanisms suggested a role for bacterial indoles, which are normally produced by Lactobacillus and other commensal bacteria and which have been shown to reduce the severity of experimental encephalomyelitis and to prolong life and promote healthy aging in host organisms.⁵ Fecal indoles were significantly reduced in mice fed a high-salt diet but not in animals receiving concomitant treatment with Lactobacillus. In vitro, addition of indole-3-lactic acid to culture media reduced Th17 polarization of T lymphocytes.

The authors next considered the impact of Lactobacillus treatment on salt-sensitive hypertension. Mice fed a high-salt diet had an increase in blood pressure, which was reduced in animals receiving concomitant treatment with Lactobacillus. Analysis of the T lymphocyte population in intestinal and splenic tissue demonstrated an increased frequency of Th17 lymphocytes in animals fed a high-salt diet, which was reduced in animals receiving concomitant Lactobacillus. Neither the high-salt diet nor Lactobacillus treatment altered the frequency of other T lymphocyte subsets.

Finally, the authors conducted a small pilot study in healthy male volunteers who supplemented their regular diet with approximately 2.4 g of additional sodium per day for 14 days. In a subset of participants who underwent ambulatory nocturnal blood pressure monitoring, blood pressure increased significantly from baseline to day 13. There was also an increase in the frequency of Th17 lymphocytes in peripheral blood. Analysis of stool specimens identified 10 populations of Lactobacillus species in 5 of 12 participants at baseline, only one of which was still detectable after the high-salt challenge.

These studies identify alterations in the gut microbiome as a potential mediator of the proinflammatory response to a high-salt diet in experimental models of autoimmune disease and hypertension. In particular, Lactobacillus species were depleted in the setting of very high-salt intake, with a corresponding decrease in the generation of indoles and an increase in the frequency of proinflammatory Th17 lymphocytes. Concomitant treatment with Lactobacillus counteracted some of the deleterious effects of the high-salt diet in mice, with accompanying reduction in the frequency of Th17 cells.³ Although the current study describes an indirect effect of high-salt diet on T cell phenotype mediated by alterations in the gut microbiome, other studies have demonstrated a more direct effect of high-salt intake on the proinflammatory polarization of T lymphocytes through a salt-sensitive kinase expressed in T cells (Figure 1).^2,4 Future studies should therefore consider the relative contribution and potential interaction of these salt-sensitive mechanisms, and whether depleting Lactobacillus species from the gut promotes salt-sensitive hypertension via previously documented effects of Th17 cells to enhance sodium transport in the nephron or to induce endothelial dysfunction.^1,6 Because a low salt diet is sometimes prescribed to combat salt-sensitive hypertension, further investigations might also explore the effects of a low salt diet on the gut microbial composition and attempt to correlate any observed changes in the gut flora with suppression of Th17 cell polarization or induction of “anti-hypertensive” lymphocyte populations such as T regulatory cells.⁷ Moreover, because a high-salt diet enhances mucosal antimicrobial defenses in preclinical studies,⁸ it is tempting to speculate that each individual may have an ideal level of sodium intake that supports the optimal gut microbial composition to balance protection from invading microbes with avoidance of autoimmune activation and hypertension.

Figure 1 |. Proposed mechanisms of salt-sensitive hypertension.

Both high-salt intake and proinflammatory immune cells have been implicated in the pathogenesis of hypertension. Proinflammatory Th17 lymphocytes drive blood pressure elevation through the effects of the cytokine interleukin-17 to induce endothelial dysfunction and promote sodium reabsorption at several sites along the nephron. High-salt concentrations also promote the differentiation of naïve T lymphocytes toward the Th17 lineage, setting up a pathogenic feedback loop. In a new study published in Nature, Wilck et al.³ describe a novel indirect effect of a high-salt diet on T cell phenotype mediated by alterations in the gut microbiome, including depletion of Lactobacillus species and reduced generation of bacterial indoles. Future studies will determine the contribution of these different mechanisms to the development of salt-sensitive hypertension. BP, blood pressure; IL-17, interleukin 17; NaCl, sodium chloride; T, T lymphocyte; Th17, pro-inflammatory Th17 lymphocyte.

Dietary salt consumption in the murine models far exceeded that observed in humans consuming a high-salt diet; nonetheless, exploratory studies suggest that the mechanisms described in the animal model may also be relevant in humans exposed to more modest increases in dietary salt intake. An increase in dietary salt intake of slightly more than 2 g/d promoted an increase in blood pressure and in the frequency of peripheral Th17 lymphocytes, while at the same time depleting Lactobacillus species in the gut of healthy human volunteers. Fewer than one-half of the participants had detectable Lactobacillus species at baseline, and it is interesting to speculate whether variability in microbial composition could contribute to the variability in salt sensitivity that is observed in humans. Perhaps, in the future, microbiome analysis of a stool sample collected at the initiation of antihypertensive therapy will predict salt sensitivity or even efficacy of certain diuretic classes. In follow-up visits, a stool sample might indicate the level of adherence with a low salt diet in salt-sensitive patients. Although the small pilot study did not evaluate the impact of Lactobacillus supplementation on blood pressure and Th17 response in humans, a previous metaanalysis of randomized controlled trials demonstrated a modest but statistically significant reduction in blood pressure with the use of probiotic supplements, most of which contained Lactobacillus species.⁹ The current study provides additional rationale for future studies of Lactobacillus-containing probiotic supplements as adjunctive therapy to prevent or treat high blood pressure, particularly given the challenges of adhering to a low salt diet.