Search

SUMMARY Dietary salt reduction and exercise are lifestyle modifications for salt-sensitive hypertensives. While exercise has prominent metabolic effects, salt has an adverse effect on metabolic syndrome, of which hypertension is a hallmark. We hypothesized that dietary salt impacts metabolism in a salt-sensitive model of hypertension. An untargeted metabolomic approach demonstrates lower circulating levels of the ketone body, beta-hydroxybutyrate (βOHB), in high salt-fed hypertensive rats. Despite the high salt intake, specific rescue of βOHB levels by nutritional supplementation of its precursor, 1,3-butanediol, attenuates hypertension and protects kidney function. This beneficial effect of βOHB was likely independent of gut-microbiotal and Th17-mediated effects of salt and instead facilitated by βOHB inhibiting the renal Nlrp3 inflammasome. The juxtaposed effects of dietary salt and exercise on salt-sensitive hypertension, which decrease and increase βOHB respectively, indicate that nutritional supplementation of a precursor of βOHB provides a similar benefit to salt-sensitive hypertension as exercise., In Brief Chakraborty et al. report a link between dietary salt, a ketone, and experimental hypertension. Intake of a high salt diet lowers the ketone body betahydroxybutyrate (βOHB), produced by the liver, which functions to prevent Nlrp3-mediated kidney inflammation. Rescuing βOHB by nutritional supplementation of its precursor attenuates hypertension., Graphical Abstract

Iron is required to facilitate the redox activity of heme proteins expressed in both immune and non-immune cells. In this study, we investigated whether the chelation of iron by enterobactin (Ent; a prototypical bacterial siderophore) can affect the immune and nitrosative responses of RAW 264.7 and bone marrow-derived macrophages. We observed that Ent dose-dependently inhibited the LPS-induced secretion of cytokines (i.e., serum amyloid A, IL-6, lipocalin 2) and nitrite in the macrophages. The LPS-induced expression of inducible nitric oxide synthase (iNOS; a heme protein) was also inhibited by Ent at both the mRNA and protein levels. To demonstrate the physiological relevance of these findings, we next investigated whether Ent could protect the intracellular pathogen Salmonella enterica serovars typhimurium in the gentamycin protection assay. Ent reduced the nitrite levels in Salmonella-infected macrophages, but upregulated the expression of iNOS (mRNA, protein) and Arginase-1 (mRNA). More importantly, Ent-treated macrophages were impaired in mediating the killing of Salmonella. Ent-sufficient Salmonella also survived better compared with their isogenic Ent-deficient strains, whereas the addition of exogenous Ent substantially improved the survival of both strains. The inhibitory effects of Ent, however, were abrogated when saturated with iron (1:1 ratio), thus indicating that Ent is required to be in its iron-free form to mediate efficient inhibition on the macrophages. Taken together, our findings advance the concept that Ent not only facilitate bacterial iron uptake, but also serve to safeguard bacteria against macrophage immune responses.

Short-chain fatty acids (SCFAs), major microbial fermentation products of dietary fiber in the gut, are shown to improve gut health. We hypothesized that soluble fiber (inulin and pectin) which are more accessible for microbiota fermentation may offer more pronounced mucoprotection than insoluble fiber (cellulose) in a murine model of chronic colitis. WT (BL6) mice receiving dietary cellulose exhibited robust colitis upon IL-10R neutralization as examined by serological, biochemical, histological and immunological parameters. Similar results were observed in colitis-prone toll-like receptor 5 knockout (Tlr5KO) mice. Surprisingly, prebiotic fiber inulin failed to protect against colitis in both WT and Tlr5KO mice. Remarkably, pectin ameliorates colitis development. GC-MS analysis of cecal content revealed that cecal butyrate, an inflammasome (NLRP3) response modulator in the gut, was augmented in inulin-fed colitic mice. Serum cytokine analysis showed that dietary inulin elevated IL-18, but diminished the IL-1 receptor antagonist (sIL-1Ra). On the flip side, dietary pectin reduced IL-18 and augmented sIL-1Ra. More importantly, suppressing butyrate production by metronidazole, which specifically depletes the butyrate producers in the gut, protected the mice from chronic colitis, suggesting that SCFAs-inflammasome axis shapes the beneficial effects of dietary fiber on the gut health. Collectively, mitigation of chronic colitis by dietary pectin can be partly explained by reduced cecal butyrate and elevated IL-1β inhibitor, sIL-1Ra. A detailed mechanistic study on how pectin-derived metabolites modulate inflammasome signaling may yield development of novel therapeutics to treat intestinal inflammation.

Green tea (Camellia sinensis, Theaceae) contains a plethora of antioxidant polyphenols that may promote health. Amongst these polyphenols, the (−)-epigallocatechin-3-gallate (EGCG) is extensively studied for its potential therapeutic properties in models of inflammatory bowel disease (IBD), although its molecular mechanism is not completely understood. Here, we demonstrate that EGCG can inhibit the activity of myeloperoxidase (MPO; a pro-oxidant neutrophil enzyme associated with IBD flares) in a dose-dependent manner in vitro. By using spectral analysis, we show that EGCG prevents the MPO-catalyzed reaction by reverting the reactive peroxidase heme back to its native inactive ferric state. Oral administration of EGCG to dextran sodium sulfate (DSS)-induced colitic mice significantly reduced the colonic MPO activity and alleviated pro-inflammatory mediators associated with gut inflammation, confirming that our findings extend to in vivo conditions as well. In light of previous reports that EGCG can complex with innate immune protein lipocalin-2 and iron, we next tested whether these factors can affect the bioactivity of EGCG. Intriguingly, the presence of lipocalin-2 or iron significantly mitigate the ability of EGCG to inhibit MPO in vitro. Further, the efficacy of EGCG against DSS-induced gut inflammation is diminished when orally co-administered with iron. These findings indicate that EGCG-mediated inhibition of MPO could be one of the mechanisms by which EGCG exerts its mucoprotective effects. However, counter-regulatory factors such as host lipocalin-2 and dietary iron could explain why EGCG does not ameliorate gut inflammation in some IBD patients.

Natural dietary fiber serves as a substrate for gut microbiotal fermentation generating short chain fatty acids, which are thought to offer protection against inflammatory bowel disease (IBD). However, certain fibers are known to cause moderate to severe intestinal side effects in IBD patients. We hypothesize that soluble fibers (inulin & pectin) which are more accessible for microbiotal fermentation may offer pronounced gut protective effects than insoluble fiber (cellulose). To demonstrate this, chronic colitis was induced in dietary cellulose, inulin or pectin fed C57BL/6 mice by administering three weekly injections of α-IL-10R neutralizing antibody. Mice were euthanized two weeks after the last injection and colitis development was examined by serological, biochemical, histological and immunological parameters. WT mice receiving dietary cellulose along with α-IL-10R mAb exhibited robust colitis as characterized by splenomegaly, colomegaly, elevated colonic MPO activity, pro-inflammatory cytokines (Kc, Il-1β & iNos), systemic and fecal Lcn2 levels (an inflammatory marker) and colon histology score when compared to control mice. Similar results were observed in toll-like receptor 5 knockout (Tlr5KO) mice which are prone to develop microbiota-dependent spontaneous colitis. Surprisingly, prebiotic fiber inulin failed to protect against α-IL-10R neutralization-induced chronic colitis in both WT and Tlr5KO mice. Interestingly, pectin ameliorates colitis development, supporting the notion that not all dietary fibers are created equal in modulating host gut inflammation. Further mechanistic study on how pectin dampens gut inflammation may yield development of novel dietary therapeutics to treat human IBD.

The gut microbiota plays a key role in host metabolism. Toll-Like Receptor 5 (TLR5), a flagellin receptor, is required for gut microbiota homeostasis. Accordingly, TLR5 deficient (T5KO) mice are prone to develop microbiota-dependent spontaneous colitis and metabolic syndrome. Herein, we investigated whether metabolic syndrome in T5KO mice correlates with hepatic dyslipidemia and inflammation. T5KO mice displayed elevated neutral lipids with substantial enrichment of C18:1 (n9) relative to wild-type littermates. Oleate enrichment of hepatic lipids was microbiota-dependent. Analyzing cecal contents via 1H NMR-based metabolomics revealed that T5KO mice exhibited elevated short chain fatty acids (SCFA) with a concomitant increase in colonic SCFA receptors and hepatic pro-inflammatory genes and lipogenic enzymes including stearoyl-CoA desaturase1 (SCD1). SCFA treatment further aggravated metabolic syndrome in T5KO mice. Interestingly, deletion of hepatic SCD1 not only prevented hepatic neutral lipid oleate enrichment but also ameliorated hepatic inflammation and metabolic syndrome in T5KO mice. Collectively, these results underscore the key role of the gut microbiota-liver axis in the pathogenesis of metabolic diseases.

Fatty acids in lipid bilayer substantially influence membrane fluidity and thus dictate cellular functions. Stearoyl CoA Desaturase (SCD-1) is a lipogenic enzyme that assists in the de novo biosynthesis of oleate (C18:1, n9), a major fatty acid in the phospholipids of lipid bilayer. Accordingly SCD-1KO mice display substantially reduced oleate in cell membranes. We investigated the colitogenic capacity of effector T-cells from SCD-1KO mice and their WT littermates in adoptive T-cell transfer colitis model. Splenic effector T-cells (CD4+CD25-) from age and gender matched WT and SCD-1KO mice were isolated by cell sorting using magnetic beads and flow cytometry. RAG-1KO (n=5) were administered 5.0x105 CD4+CD25- cells/mouse intraperitoneally and monitored for body weights and colitis development. RAG-1KO mice which received CD4+CD25- cells from SCD-1KO mice displayed colomegaly, splenomegaly, elevated colonic, systemic lipocalin-2, a general inflammatory marker and hallmarks of colitis at histologic level. Interestingly, colonic TNFα and IL-10 gene transcripts were significantly reduced in SCD-1KO CD4+CD25- recipients whereas the opposite is true for iNOS and lipocalin-2. Further, systemic sIL-1Ra was significantly reduced in SCD-1KO CD4+CD25- recipients when compared to WT T cell recipients. Our results demonstrate that effector T-cells from oleate poor environment attain colitogenic properties and may be associated with increased immunoreactivity to gut microbiotal antigens.

During an inflammatory response in the gut, several commensal bacteria such as E.coli not only survive but also thrive and consequently contribute to the disease pathogenesis. The mechanisms by which such opportunistic pathogens bloom despite the hostile pro-inflammatory milieu of the inflamed gut remain largely unknown. We demonstrated that enterobactin (Ent), a siderophore released by E. coli, is a potent inhibitor of a key bactericidal enzyme of the host innate immune system, myeloperoxidase (MPO). The inhibitory activity is specific as glycosylated Ent (salmochelin) and non-catecholate siderophores such as yersiniabactin and ferrichrome failed to inhibit MPO activity. An E. coli ferrienterobactin permease mutant (ΔfepA) overexpressing Ent, but not 3-dehydroquinate synthase (ΔaroB)/ΔfepA double mutant inhibited MPO activity and exhibited enhanced survival in inflamed guts. This survival advantage was counter-regulated by the host siderophore binding protein, lipocalin 2, which rescued MPO from Ent-mediated inhibition. Spectral analysis revealed that Ent interferes with compound I [oxoiron, Fe(IV)=O] and reverts the enzyme back to its native ferric [Fe(III)] state. These findings define a fundamental mechanism by which E. coli surpasses the host innate immune responses during inflammatory gut diseases and gains a distinct survival advantage.

Apolipoprotein E (ApoE), a secreted protein, plays major role in cholesterol metabolism. ApoE is also known to exhibit anti-inflammatory properties. However, its role in the intestine, specifically during inflammation is not very clear. First, we verified upregulation of colonic ApoE transcripts in various models of murine acute and chronic colitis models. Age and gender matched ApoEKO mice and their WT littermates (n=5) were given either one or 4 weekly injections of IL-10 receptor (IL-10R) neutralizing mAb intraperitoneally and colitis development was monitored. ApoEKO mice given single injection of IL-10R mAb displayed acute colitis. Four weekly injections IL-10R mAb resulted in the development of chronic colitis as measured by colomegaly, splenomegaly and elevated colonic pro-inflammatory gene expression (TNFα, IFNγ) and histological score when compared to WT littermates. IL-10R neutralization also induced substantial levels of lipocalin 2, a general inflammatory marker in the feces, colon, and circulation in ApoEKO mice. Ablation of gut microbiota by broad-spectrum antibiotics substantially protected ApoEKO mice against IL-10R neutralization induced chronic colitis. In addition, cohousing ApoEKO mice with IL-10KO mice exaggerated spontaneous colitis (rectal prolapse) in IL-10KO mice when compared to WT suggesting ApoEKO harbor colitogenic microbiota. Collectively, our results demonstrate that ApoE plays a critical role in protecting the gut against inflammation.