Metabolite-Sensing G Protein-Coupled Receptors-Facilitators of Diet-Related Immune Regulation.

Nutrition and the gut microbiome regulate many systems, including the immune, metabolic, and nervous systems. We propose that the host responds to deficiency (or sufficiency) of dietary and bacterial metabolites in a dynamic way, to optimize responses and survival. A family of G protein-coupled receptors (GPCRs) termed the metabolite-sensing GPCRs bind to various metabolites and transmit signals that are important for proper immune and metabolic functions. Members of this family include GPR43, GPR41, GPR109A, GPR120, GPR40, GPR84, GPR35, and GPR91. In addition, bile acid receptors such as GPR131 (TGR5) and proton-sensing receptors such as GPR65 show similar features. A consistent feature of this family of GPCRs is that they provide anti-inflammatory signals; many also regulate metabolism and gut homeostasis. These receptors represent one of the main mechanisms whereby the gut microbiome affects vertebrate physiology, and they also provide a link between the immune and metabolic systems. Insufficient signaling through one or more of these metabolite-sensing GPCRs likely contributes to human diseases such as asthma, food allergies, type 1 and type 2 diabetes, hepatic steatosis, cardiovascular disease, and inflammatory bowel diseases.

Homeostatic IL-13 in healthy skin directs dendritic cell differentiation to promote TH2 and inhibit TH17 cell polarization.

The signals driving the adaptation of type 2 dendritic cells (DC2s) to diverse peripheral environments remain mostly undefined. We show that differentiation of CD11blo migratory DC2s-a DC2 population unique to the dermis-required IL-13 signaling dependent on the transcription factors STAT6 and KLF4, whereas DC2s in lung and small intestine were STAT6-independent. Similarly, human DC2s in skin expressed an IL-4 and IL-13 gene signature that was not found in blood, spleen and lung DCs. In mice, IL-13 was secreted homeostatically by dermal innate lymphoid cells and was independent of microbiota, TSLP or IL-33. In the absence of IL-13 signaling, dermal DC2s were stable in number but remained CD11bhi and showed defective activation in response to allergens, with diminished ability to support the development of IL-4+GATA3+ helper T cells (TH), whereas antifungal IL-17+RORγt+ TH cells were increased. Therefore, homeostatic IL-13 fosters a noninflammatory skin environment that supports allergic sensitization.

Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes.

Gut dysbiosis might underlie the pathogenesis of type 1 diabetes. In mice of the non-obese diabetic (NOD) strain, we found that key features of disease correlated inversely with blood and fecal concentrations of the microbial metabolites acetate and butyrate. We therefore fed NOD mice specialized diets designed to release large amounts of acetate or butyrate after bacterial fermentation in the colon. Each diet provided a high degree of protection from diabetes, even when administered after breakdown of immunotolerance. Feeding mice a combined acetate- and butyrate-yielding diet provided complete protection, which suggested that acetate and butyrate might operate through distinct mechanisms. Acetate markedly decreased the frequency of autoreactive T cells in lymphoid tissues, through effects on B cells and their ability to expand populations of autoreactive T cells. A diet containing butyrate boosted the number and function of regulatory T cells, whereas acetate- and butyrate-yielding diets enhanced gut integrity and decreased serum concentration of diabetogenic cytokines such as IL-21. Medicinal foods or metabolites might represent an effective and natural approach for countering the numerous immunological defects that contribute to T cell-dependent autoimmune diseases.

Erratum: Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes.

This corrects the article DOI: 10.1038/ni.3713.

A major mechanism for immunomodulation: Dietary fibres and acid metabolites.

Diet and the gut microbiota have a profound influence on physiology and health, however, mechanisms are still emerging. Here we outline several pathways that gut microbiota products, particularly short-chain fatty acids (SCFAs), use to maintain gut and immune homeostasis. Dietary fibre is fermented by the gut microbiota in the colon, and large quantities of SCFAs such as acetate, propionate, and butyrate are produced. Dietary fibre and SCFAs enhance epithelial integrity and thereby limit systemic endotoxemia. Moreover, SCFAs inhibit histone deacetylases (HDAC), and thereby affect gene transcription. SCFAs also bind to 'metabolite-sensing' G-protein coupled receptors (GPCRs) such as GPR43, which promotes immune homeostasis. The enormous amounts of SCFAs produced in the colon are sufficient to lower pH, which affects the function of proton sensors such as GPR65 expressed on the gut epithelium and immune cells. GPR65 is an anti-inflammatory Gαs-coupled receptor, which leads to the inhibition of inflammatory cytokines. The importance of GPR65 in inflammatory diseases is underscored by genetics associated with the missense variant I231L (rs3742704), which is associated with human inflammatory bowel disease, atopic dermatitis, and asthma. There is enormous scope to manipulate these pathways using specialized diets that release very high amounts of specific SCFAs in the gut, and we believe that therapies that rely on chemically modified foods is a promising approach. Such an approach includes high SCFA-producing diets, which we have shown to decrease numerous inflammatory western diseases in mouse models. These diets operate at many levels - increased gut integrity, changes to the gut microbiome, and promotion of immune homeostasis, which represents a new and highly promising way to prevent or treat human disease.

Vasodilators mobilize SK3 channels in endothelial cells to produce arterial relaxation.

Endothelial cells (ECs) line the lumen of all blood vessels and regulate functions, including contractility. Physiological stimuli, such as acetylcholine (ACh) and intravascular flow, activate transient receptor potential vanilloid 4 (TRPV4) channels, which stimulate small (SK3)- and intermediate (IK)-conductance Ca2+-activated potassium channels in ECs to produce vasodilation. Whether physiological vasodilators also modulate the surface abundance of these ion channels in ECs to elicit functional responses is unclear. Here, we show that ACh and intravascular flow stimulate rapid anterograde trafficking of an intracellular pool of SK3 channels in ECs of resistance-size arteries, which increases surface SK3 protein more than two-fold. In contrast, ACh and flow do not alter the surface abundance of IK or TRPV4 channels. ACh triggers SK3 channel trafficking by activating TRPV4-mediated Ca2+ influx, which stimulates Rab11A, a Rab GTPase associated with recycling endosomes. Superresolution microscopy data demonstrate that SK3 trafficking specifically increases the size of surface SK3 clusters which overlap with TRPV4 clusters. We also show that Rab11A-dependent trafficking of SK3 channels is an essential contributor to vasodilator-induced SK current activation in ECs and vasorelaxation. In summary, our data demonstrate that vasodilators activate Rab11A, which rapidly delivers an intracellular pool of SK3 channels to the vicinity of surface TRPV4 channels in ECs. This trafficking mechanism increases surface SK3 cluster size, elevates SK3 current density, and produces vasodilation. These data also demonstrate that SK3 and IK channels are differentially regulated by trafficking-dependent and -independent signaling mechanisms in endothelial cells.

Genetic Coding Variant in GPR65 Alters Lysosomal pH and Links Lysosomal Dysfunction with Colitis Risk.

Although numerous polymorphisms have been associated with inflammatory bowel disease (IBD), identifying the function of these genetic factors has proved challenging. Here we identified a role for nine genes in IBD susceptibility loci in antibacterial autophagy and characterized a role for one of these genes, GPR65, in maintaining lysosome function. Mice lacking Gpr65, a proton-sensing G protein-coupled receptor, showed increased susceptibly to bacteria-induced colitis. Epithelial cells and macrophages lacking GPR65 exhibited impaired clearance of intracellular bacteria and accumulation of aberrant lysosomes. Similarly, IBD patient cells and epithelial cells expressing an IBD-associated missense variant, GPR65 I231L, displayed aberrant lysosomal pH resulting in lysosomal dysfunction, impaired bacterial restriction, and altered lipid droplet formation. The GPR65 I231L polymorphism was sufficient to confer decreased GPR65 signaling. Collectively, these data establish a role for GPR65 in IBD susceptibility and identify lysosomal dysfunction as a potentially causative element in IBD pathogenesis with effects on cellular homeostasis and defense.

Immunomodulatory Effects of Microbiota-Derived Short-Chain Fatty Acids in Autoimmune Liver Diseases.

Nonpathogenic commensal microbiota and their metabolites and components are essential to maintain a tolerogenic environment and promote beneficial health effects. The metabolic environment critically impacts the outcome of immune responses and likely impacts autoimmune and allergic responses. Short-chain fatty acids (SCFAs) are the main metabolites produced by microbial fermentation in the gut. Given the high concentration of SCFAs in the gut and portal vein and their broad immune regulatory functions, SCFAs significantly influence immune tolerance and gut-liver immunity. Alterations of SCFA-producing bacteria and SCFAs have been identified in a multitude of inflammatory diseases. These data have particular significance in primary biliary cholangitis, primary sclerosing cholangitis, and autoimmune hepatitis because of the close proximity of the liver to the gut. In this focused review, we provide an update on the immunologic consequences of SCFA-producing microbiota and in particular on three dominant SCFAs in autoimmune liver diseases.

Dietary Fiber and Microbiota Metabolite Receptors Enhance Cognition and Alleviate Disease in the 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder with poorly understood etiology. AD has several similarities with other "Western lifestyle" inflammatory diseases, where the gut microbiome and immune pathways have been associated. Previously, we and others have noted the involvement of metabolite-sensing GPCRs and their ligands, short-chain fatty acids (SCFAs), in protection of numerous Western diseases in mouse models, such as Type I diabetes and hypertension. Depletion of GPR43, GPR41, or GPR109A accelerates disease, whereas high SCFA yielding diets protect in mouse models. Here, we extended the concept that metabolite-sensing receptors and SCFAs may be a more common protective mechanism against Western diseases by studying their role in AD pathogenesis in the 5xFAD mouse model. Both male and female mice were included. Depletion of GPR41 and GPR43 accelerated cognitive decline and impaired adult hippocampal neurogenesis in 5xFAD and WT mice. Lack of fiber/SCFAs accelerated a memory deficit, whereas diets supplemented with high acetate and butyrate (HAMSAB) delayed cognitive decline in 5xFAD mice. Fiber intake impacted on microglial morphology in WT mice and microglial clustering phenotype in 5xFAD mice. Lack of fiber impaired adult hippocampal neurogenesis in both W and AD mice. Finally, maternal dietary fiber intake significantly affects offspring's cognitive functions in 5xFAD mice and microglial transcriptome in both WT and 5xFAD mice, suggesting that SCFAs may exert their effect during pregnancy and lactation. Together, metabolite-sensing GPCRs and SCFAs are essential for protection against AD, and reveal a new strategy for disease prevention.Significance Statement Alzheimer's disease (AD) is one of the most common neurodegenerative diseases; currently, there is no cure for AD. In our study, short-chain fatty acids and metabolite receptors play an important role in cognitive function and pathology in AD mouse model as well as in WT mice. SCFAs also impact on microglia transcriptome, and immune cell recruitment. Out study indicates the potential of specialized diets (supplemented with high acetate and butyrate) releasing high amounts of SCFAs to protect against disease.

GPR109A expressed on medullary thymic epithelial cells affects thymic Treg development.

Regulatory T cells (Treg) maintain immune homeostasis due to their anti-inflammatory functions. They can be generated either centrally in the thymus or in peripheral organs. Metabolites such as short-chain fatty acids produced by intestinal microbiota can induce peripheral Treg differentiation, by activating G-protein-coupled-receptors like GPR109A. In this study, we identified a novel role for GPR109A in thymic Treg development. We found that Gpr109a-/- mice had increased Treg under basal conditions in multiple organs compared with WT mice. GPR109A was not expressed on T cells but on medullary thymic epithelial cells (mTECs), as revealed by single-cell RNA sequencing in both mice and humans and confirmed by flow cytometry in mice. mTECs isolated from Gpr109a-/- mice had higher expression of autoimmune regulator (AIRE), the key regulator of Treg development, while the subset of mTECs that did not express Gpr109a in the WT displayed increased Aire expression and also enhanced signaling related to mTEC functionality. Increased thymic Treg in Gpr109a-/- mice was associated with protection from experimental autoimmune encephalomyelitis, with ameliorated clinical signs and reduced inflammation. This work identifies a novel role for GPR109A and possibly the gut microbiota, on thymic Treg development via its regulation of mTECs.

Dietary fiber and SCFAs in the regulation of mucosal immunity.

Gut bacterial metabolites such as short-chain fatty acids (SCFAs) have important effects on immune cells and the gut. SCFAs derive from the fermentation of dietary fiber by gut commensal bacteria. Insufficient fiber intake thus compromises SCFA production and, as a consequence, the host's physiology (particularly immune functions). We propose that many Western diseases, including those associated with impaired mucosal responses such as food allergy and asthma, may be affected by insufficient fiber intake and reduced SCFA levels in the gut and blood. Insufficient fiber intake is 1 alternative, or contributor, on top of the "hygiene hypothesis" to the rise of Western lifestyle diseases, and the 2 ideas need to be reconciled. The mechanisms by which SCFAs influence immunity and gut homeostasis are varied; they include stimulation of G protein-coupled receptors (GPCRs), such as GPR43 or GPR41; inhibition of histone deacetylases (and hence, gene transcription changes); and induction of intracellular metabolic changes. SCFAs modulate at many different levels to alter mucosal homeostasis, including changes to gut epithelial integrity, increases in regulatory T-cell numbers and function, and decreased expression of numerous inflammatory cytokines. There is scope for preventing and/or treating diseases by using diets that alter SCFA levels.

Diet, gut microbiota and immune responses.

The fields of immunology, microbiology, nutrition and metabolism are rapidly converging. Here we expand on a diet-microbiota model as the basis for the greater incidence of asthma and autoimmunity in developed countries.

Diet, metabolites, and "western-lifestyle" inflammatory diseases.

One explanation for the increased incidence of allergies, asthma, and even some autoimmune diseases has been the hygiene hypothesis. However, recent studies also highlight an important role for diet and bacterial metabolites in controlling various immune pathways, including gut and immune homeostasis, regulatory T cell biology, and inflammation. Dietary-related metabolites engage "metabolite-sensing" G-protein-coupled receptors, such as GPR43, GPR41, GPR109A, GPR120, and GPR35. These receptors are expressed on immune cells and some gut epithelial cells and generally mediate a direct anti-inflammatory effect. Insufficient intake of "healthy foodstuffs" adversely affects the production of bacterial metabolites. These metabolites and those derived directly from food drive beneficial downstream effects on immune pathways. We propose that insufficient exposure to dietary and bacterial metabolites might underlie the development of inflammatory disorders in Western countries. This review highlights what is currently known about diet, metabolites, and their associated immune pathways in relation to the development of inflammatory disease.

pH and Proton Sensor GPR65 Determine Susceptibility to Atopic Dermatitis.

pH sensing by GPR65 regulates various inflammatory conditions, but its role in skin remains unknown. In this study, we performed a phenome-wide association study and report that the T allele of GPR65-intronic single-nucleotide polymorphism rs8005161, which reduces GPR65 signaling, showed a significant association with atopic dermatitis, in addition to inflammatory bowel diseases and asthma, as previously reported. Consistent with this genetic association in humans, we show that deficiency of GPR65 in mice resulted in markedly exacerbated disease in the MC903 experimental model of atopic dermatitis. Deficiency of GPR65 also increased neutrophil migration in vitro. Moreover, GPR65 deficiency in mice resulted in higher expression of the inflammatory cytokine TNF-α by T cells. In humans, CD4+ T cells from rs8005161 heterozygous individuals expressed higher levels of TNF-α after PMA/ionomycin stimulation, particularly under pH 6 conditions. pH sensing by GPR65 appears to be important for regulating the pathogenesis of atopic dermatitis.

Dietary Fiber Drives IL-1ß-Dependent Peritonitis Induced by Bacteroides fragilis via Activation of the NLRP3 Inflammasome.

Intestinal barrier is essential for dietary products and microbiota compartmentalization and therefore gut homeostasis. When this barrier is broken, cecal content overflows into the peritoneal cavity, leading to local and systemic robust inflammatory response, characterizing peritonitis and sepsis. It has been shown that IL-1ß contributes with inflammatory storm during peritonitis and sepsis and its inhibition has beneficial effects to the host. Therefore, we investigated the mechanisms underlying IL-1ß secretion using a widely adopted murine model of experimental peritonitis. The combined injection of sterile cecal content (SCC) and the gut commensal bacteria Bacteroides fragilis leads to IL-1ß-dependent peritonitis, which was mitigated in mice deficient in NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome components. Typically acting as a damage signal, SCC, but not B. fragilis, activates canonical pathway of NLRP3 promoting IL-1ß secretion in vitro and in vivo. Strikingly, absence of fiber in the SCC drastically reduces IL-1ß production, whereas high-fiber SCC conversely increases this response in an NLRP3-dependent manner. In addition, NLRP3 was also required for IL-1ß production induced by purified dietary fiber in primed macrophages. Extending to the in vivo context, IL-1ß-dependent peritonitis was worsened in mice injected with B. fragilis and high-fiber SCC, whereas zero-fiber SCC ameliorates the pathology. Corroborating with the proinflammatory role of dietary fiber, IL-1R-deficient mice were protected from peritonitis induced by B. fragilis and particulate bran. Overall, our study highlights a function, previously unknown, for dietary fibers in fueling peritonitis through NLRP3 activation and IL-1ß secretion outside the gut.

Circulating precursor CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells indicate Tfh cell activity and promote antibody responses upon antigen reexposure.

Follicular B helper T (Tfh) cells support high affinity and long-term antibody responses. Here we found that within circulating CXCR5⁺ CD4⁺ T cells in humans and mice, the CCR7(lo)PD-1(hi) subset has a partial Tfh effector phenotype, whereas CCR7(hi)PD-1(lo) cells have a resting phenotype. The circulating CCR7(lo)PD-1(hi) subset was indicative of active Tfh differentiation in lymphoid organs and correlated with clinical indices in autoimmune diseases. Thus the CCR7(lo)PD-1(hi) subset provides a biomarker to monitor protective antibody responses during infection or vaccination and pathogenic antibody responses in autoimmune diseases. Differentiation of both CCR7(hi)PD-1(lo) and CCR7(lo)PD-1(hi) subsets required ICOS and BCL6, but not SAP, suggesting that circulating CXCR5⁺ helper T cells are primarily generated before germinal centers. Upon antigen reencounter, CCR7(lo)PD-1(hi) CXCR5⁺ precursors rapidly differentiate into mature Tfh cells to promote antibody responses. Therefore, circulating CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells are generated during active Tfh differentiation and represent a new mechanism of immunological early memory.

SUMO1 modification of PKD2 channels regulates arterial contractility.

PKD2 (polycystin-2, TRPP1) channels are expressed in a wide variety of cell types and can regulate functions, including cell division and contraction. Whether posttranslational modification of PKD2 modifies channel properties is unclear. Similarly uncertain are signaling mechanisms that regulate PKD2 channels in arterial smooth muscle cells (myocytes). Here, by studying inducible, cell-specific Pkd2 knockout mice, we discovered that PKD2 channels are modified by SUMO1 (small ubiquitin-like modifier 1) protein in myocytes of resistance-size arteries. At physiological intravascular pressures, PKD2 exists in approximately equal proportions as either nonsumoylated (PKD2) or triple SUMO1-modifed (SUMO-PKD2) proteins. SUMO-PKD2 recycles, whereas unmodified PKD2 is surface-resident. Intravascular pressure activates voltage-dependent Ca2+ influx that stimulates the return of internalized SUMO-PKD2 channels to the plasma membrane. In contrast, a reduction in intravascular pressure, membrane hyperpolarization, or inhibition of Ca2+ influx leads to lysosomal degradation of internalized SUMO-PKD2 protein, which reduces surface channel abundance. Through this sumoylation-dependent mechanism, intravascular pressure regulates the surface density of SUMO-PKD2-mediated Na+ currents (INa) in myocytes to control arterial contractility. We also demonstrate that intravascular pressure activates SUMO-PKD2, not PKD2, channels, as desumoylation leads to loss of INa activation in myocytes and vasodilation. In summary, this study reveals that PKD2 channels undergo posttranslational modification by SUMO1, which enables physiological regulation of their surface abundance and pressure-mediated activation in myocytes and thus control of arterial contractility.

Deficiency of Prebiotic Fiber and Insufficient Signaling Through Gut Metabolite-Sensing Receptors Leads to Cardiovascular Disease.

BACKGROUND: High blood pressure (BP) continues to be a major, poorly controlled but modifiable risk factor for cardiovascular death. Among key Western lifestyle factors, a diet poor in fiber is associated with prevalence of high BP. The impact of lack of prebiotic fiber and the associated mechanisms that lead to higher BP are unknown. Here we show that lack of prebiotic dietary fiber leads to the development of a hypertensinogenic gut microbiota, hypertension and its complications, and demonstrate a role for G-protein coupled-receptors (GPCRs) that sense gut metabolites. METHODS: One hundred seventy-nine mice including C57BL/6J, gnotobiotic C57BL/6J, and knockout strains for GPR41, GPR43, GPR109A, and GPR43/109A were included. C57BL/6J mice were implanted with minipumps containing saline or a slow-pressor dose of angiotensin II (0.25 mg·kg-1·d-1). Mice were fed diets lacking prebiotic fiber with or without addition of gut metabolites called short-chain fatty acids ([SCFA)] produced during fermentation of prebiotic fiber in the large intestine), or high prebiotic fiber diets. Cardiac histology and function, BP, sodium and potassium excretion, gut microbiome, flow cytometry, catecholamines and methylation-wide changes were determined. RESULTS: Lack of prebiotic fiber predisposed mice to hypertension in the presence of a mild hypertensive stimulus, with resultant pathological cardiac remodeling. Transfer of a hypertensinogenic microbiota to gnotobiotic mice recapitulated the prebiotic-deprived hypertensive phenotype, including cardiac manifestations. Reintroduction of SCFAs to fiber-depleted mice had protective effects on the development of hypertension, cardiac hypertrophy, and fibrosis. The cardioprotective effect of SCFAs were mediated via the cognate SCFA receptors GPR43/GPR109A, and modulated L-3,4-dihydroxyphenylalanine levels and the abundance of T regulatory cells regulated by DNA methylation. CONCLUSIONS: The detrimental effects of low fiber Westernized diets may underlie hypertension, through deficient SCFA production and GPR43/109A signaling. Maintaining a healthy, SCFA-producing microbiota is important for cardiovascular health.