A Butyrate-Yielding Dietary Supplement Prevents Acute Alcoholic Liver Injury by Modulating Nrf2-Mediated Hepatic Oxidative Stress and Gut Microbiota.

Alcoholic liver disease (ALD) is a globally prevalent form of liver disease for which there is no effective treatment. Recent studies have found that a significant decrease in butyrate was closely associated with ALD development. Given the low compliance and delivery efficiency associated with oral-route butyrate administration, a highly effective butyrate-yielding dietary supplement, butyrylated high-amylose maize starch (HAMSB), is a good alternative approach. Here, we synthesized HAMSB, evaluated the effect of HAMSB on acute ALD in mice, compared its effect with that of oral administration of butyrate, and further studied the potential mechanism of action. The results showed HAMSB alleviated acute ALD in mice, as evidenced by the inhibition of hepatic-function impairment and the improvement in liver steatosis and lipid metabolism; in these respects, HAMSB supplementation was superior to oral sodium butyrate administration. These improvements can be attributed to the reduction of oxidative stress though the regulation of Nrf2-mediated antioxidant signaling in the liver and the improvement in the composition and function of microbiota in the intestine. In conclusion, HAMSB is a safe and effective dietary supplement for preventing acute ALD that could be useful as a disease-modifying functional food or candidate medicine.

Dietary therapy of murine primary biliary cholangitis induces hepatocellular steatosis: A cautionary tale.

BACKGROUND AND AIMS: There is increased interest in utilizing dietary interventions to alter the progression of autoimmune diseases. These efforts are driven by associations of gut microbiota/metabolites with levels of short-chain fatty acids (SCFAs). Propionate is a key SCFA that is commonly used as a food preservative and is endogenously generated by bacterial fermentation of non-digestible carbohydrates in the gut. A thesis has suggested that a diet rich in propionate and other SCFAs can successfully modulate autoimmunity. Herein, we investigated the effect of long-term administration of propionylated high-amylose resistant starches (HAMSP) on the course of murine primary biliary cholangitis. MATERIALS AND METHODS: Groups of female ARE-Del mice were fed an HAMSP diet either before or after disease onset. A detailed immunobiological analysis was performed involving autoantibodies and rigorous T-cell phenotyping, including enumeration of T-cell subsets in the spleen, liver, intestinal intraepithelial lymphocytes and lamina propria by flow cytometry. Histopathological scores were used to assess the frequency and severity of liver inflammation and damage to hepatocytes and bile ducts. RESULTS: Our results demonstrate that a long-term propionate-yielding diet re-populated the T-cell pool with decreased naïve and central memory T-cell subsets and an increase in the effector memory T cells in mice. Similarly, long-term HAMSP intake reduced CD4+CD8+ double-positive T cells in intraepithelial lymphocytes and the intestinal lamina propria. Critically, HAMSP consumption led to moderate-to-severe hepatocellular steatosis in ARE-Del mice, independent of the stage of autoimmune cholangitis. CONCLUSIONS: Our data suggest that administration of HAMSP induces both regulatory and effector T cells. Furthermore, HAMSP administration resulted in hepatocellular steatosis. Given the interest in dietary modulation of autoimmunity and because propionate is widely used as a food preservative, these data have significant implications. This study also provides new insights into the immunological and pathological effects of chronic propionate exposure.

The Relationship between Liver Volume, Clinicopathological Characteristics and Survival in Patients Undergoing Resection with Curative Intent for Non-Metastatic Colonic Cancer.

INTRODUCTION: The prognostic value of CT-derived liver volume in terms of cancer outcomes is not clear. The aim of the present study was to examine the relationship between liver area on a single axial CT-slice and the total liver volume in patients with colonic cancer. Furthermore, we examine the relationship between liver volume, determined using this novel method, clinicopathological variables and survival. METHODS: Consecutive patients who underwent potentially curative surgery for colonic cancer were identified from a prospectively maintained database. Maximal liver area on axial CT-slice (cm2) and total volume (cm3), were obtained by the manual segmentation of pre-operative CT-images in a PACS viewer. The maximal liver area was normalized for body height2 to create the liver index (LI) and values, categorized into tertiles. The primary outcome of interest was overall survival (OS). Relationships between LI and clinico-pathological variables were examined using chi-square analysis and binary logistic regression. The relationship between LI and OS was examined using cox proportional hazard regression. RESULTS: A total of 359 patients were included. A total of 51% (n = 182) of patients were male and 73% (n = 261) were aged 65 years or older. 81% (n = 305) of patients were alive 3-years post-operatively. The median maximal liver area on the axial CT slice was 178.7 (163.7-198.4) cm2. The median total liver volume was 1509.13 (857.8-3337.1) cm3. Maximal liver area strongly correlated with total liver volume (R2 = 0.749). The median LI was 66.8 (62.0-71.6) cm2/m2. On multivariate analysis, age (p < 0.001), sex (p < 0.05), BMI (p < 0.001) and T2DM (p < 0.05) remained significantly associated with LI. On univariate analysis, neither LI (continuous) or LI (tertiles) were significantly associated with OS (p = 0.582 and p = 0.290, respectively). CONCLUSIONS: The simple, reliable method proposed in this study for quantifying liver volume using CT-imaging was found to have an excellent correlation between observers and provided results consistent with the contemporary literature. This method may facilitate the further examination of liver volume in future cancer studies.

Indole-3-acetic acid ameliorates dextran sulfate sodium-induced colitis via the ERK signaling pathway.

Microbiota-derived catabolism of nutrients is closely related to ulcerative colitis (UC). The level of indole-3-acetic acid (IAA), a microbiota-dependent metabolite of tryptophan, was decreased significantly in the feces of UC patients. Thus supplementation with IAA could be a potential therapeutic method for ameliorating colitis. In this work, the protective effect of supplementation with IAA on dextran sulfate sodium (DSS)-induced colitis was evaluated, and the underlying mechanism was elucidated. The results indicated that the administration of IAA significantly relieved DSS-induced weight loss, reduced the disease activity index (DAI), restored colon length, alleviated intestinal injury, and improved the intestinal tight junction barrier. Furthermore, IAA inhibited intestinal inflammation by reducing the expression of proinflammatory cytokines and promoting the production of IL-10 and TGF-ß1. In addition, the ERK signaling pathway is an important mediator of various physiological processes including inflammatory responses and is closely associated with the expression of IL-10. Notably, IAA treatment induced the activation of extracellular signal-regulated kinase (ERK), which is involved in the progression of colitis, while the ERK inhibitor U0126 attenuated the beneficial effects of IAA. In summary, IAA could attenuate the clinical symptoms of colitis, and the ERK signaling pathway was involved in the underlying mechanism. Supplementation with IAA could be a potential option for preventing or ameliorating UC.

Influence of angiotensin II on the gut microbiome: modest effects in comparison to experimental factors.

AIMS: Animal models are regularly used to test the role of the gut microbiome in hypertension. Small-scale pre-clinical studies have investigated changes to the gut microbiome in the angiotensin II hypertensive model. However, the gut microbiome is influenced by internal and external experimental factors, which are not regularly considered in the study design. Once these factors are accounted for, it is unclear if microbiome signatures are reproduceable. We aimed to determine the influence of angiotensin II treatment on the gut microbiome using a large and diverse cohort of mice and to quantify the magnitude by which other factors contribute to microbiome variations. METHODS AND RESULTS: We conducted a retrospective study to establish a diverse mouse cohort resembling large human studies. We sequenced the V4 region of the 16S rRNA gene from 538 samples across the gastrointestinal tract of 303 male and female C57BL/6J mice randomized into sham or angiotensin II treatment from different genotypes, diets, animal facilities, and age groups. Analysing over 17 million sequencing reads, we observed that angiotensin II treatment influenced α-diversity (P = 0.0137) and ß-diversity (i.e. composition of the microbiome, P < 0.001). Bacterial abundance analysis revealed patterns consistent with a reduction in short-chain fatty acid producers, microbial metabolites that lower blood pressure. Furthermore, animal facility, genotype, diet, age, sex, intestinal sampling site, and sequencing batch had significant effects on both α- and ß-diversity (all P < 0.001). Sampling site (6.8%) and diet (6%) had the largest impact on the microbiome, while angiotensin II and sex had the smallest effect (each 0.4%). CONCLUSION: Our large-scale data confirmed findings from small-scale studies that angiotensin II impacted the gut microbiome. However, this effect was modest relative to most of the other factors studied. Accounting for these factors in future pre-clinical hypertensive studies will increase the likelihood that microbiome findings are replicable and translatable.

Targeting HDAC6 to treat heart failure with preserved ejection fraction in mice.

Heart failure with preserved ejection fraction (HFpEF) poses therapeutic challenges due to the limited treatment options. Building upon our previous research that demonstrates the efficacy of histone deacetylase 6 (HDAC6) inhibition in a genetic cardiomyopathy model, we investigate HDAC6's role in HFpEF due to their shared mechanisms of inflammation and metabolism. Here, we show that inhibiting HDAC6 with TYA-018 effectively reverses established heart failure and its associated symptoms in male HFpEF mouse models. Additionally, in male mice lacking Hdac6 gene, HFpEF progression is delayed and they are resistant to TYA-018's effects. The efficacy of TYA-018 is comparable to a sodium-glucose cotransporter 2 (SGLT2) inhibitor, and the combination shows enhanced effects. Mechanistically, TYA-018 restores gene expression related to hypertrophy, fibrosis, and mitochondrial energy production in HFpEF heart tissues. Furthermore, TYA-018 also inhibits activation of human cardiac fibroblasts and enhances mitochondrial respiratory capacity in cardiomyocytes. In this work, our findings show that HDAC6 impacts on heart pathophysiology and is a promising target for HFpEF treatment.

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 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.

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.

Human CXCR1 knock-in mice infer functional expression of a murine ortholog.

Targeting CXCR1 and CXCR2 chemokine receptors to block neutrophil migration to sites of inflammation is a promising therapeutic approach for various inflammatory and autoimmune diseases. However, assessing the translational potential of such therapies using mouse models is challenging due to the unclear expression of CXCR1 at the protein level. Although CXCR2 has been well characterized in both mice and humans, the protein-level expression of CXCR1 in mice (mCXCR1) remains controversial. To address this issue, we generated a novel human CXCR1 knock-in (hCXCR1 KI) mouse model in which the transgene is under the control of the native mouse promoter and regulatory elements. Using an anti-human CXCR1 monoclonal antibody (anti-hCXCR1 monoclonal antibody), we found that hCXCR1 was highly expressed on neutrophils in the hCXCR1 KI mice, comparable to levels observed in human neutrophils. This successful expression of hCXCR1 in this mouse model suggests that functional mCXCR1 likely exists. To investigate the functional role of CXCR1, we investigated how antagonizing this receptor using anti-hCXCR1 monoclonal antibody in the arthritis model would affect disease outcomes. Antibody treatment significantly alleviated all signs of joint inflammation. In summary, our newly generated hCXCR1 KI transgenic mice provide a valuable tool to investigate the therapeutic efficacy of small molecules or monoclonal antibodies that antagonize this receptor in neutrophil-mediated pathologies.

Neflamapimod inhibits endothelial cell activation, adhesion molecule expression, leukocyte attachment and vascular inflammation by inhibiting p38 MAPKα and NF-κB signaling.

Neflamapimod, a selective inhibitor of the alpha isoform of p38 mitogen-activated protein kinase (MAPKα), was investigated for its potential to inhibit lipopolysaccharide (LPS)-induced activation of endothelial cells (ECs), adhesion molecule induction, and subsequent leukocyte attachment to EC monolayers. These events are known to contribute to vascular inflammation and cardiovascular dysfunction. Our results demonstrate that LPS treatment of cultured ECs and rats leads to significant upregulation of adhesion molecules, both in vitro and in vivo, which can be effectively inhibited by neflamapimod treatment. Western blotting data further reveals that neflamapimod inhibits LPS-induced phosphorylation of p38 MAPKα and the activation of NF-κB signaling in ECs. Additionally, leukocyte adhesion assays demonstrate a substantial reduction in leukocyte attachment to cultured ECs and the aorta lumen of rats treated with neflamapimod. Consistent with vascular inflammation, LPS-treated rat arteries exhibit significantly diminished vasodilation response to acetylcholine, however, arteries from rats treated with neflamapimod maintain their vasodilation capacity, demonstrating its ability to limit LPS-induced vascular inflammation. Overall, our data demonstrate that neflamapimod effectively inhibits endothelium activation, adhesion molecule expression, and leukocyte attachment, thereby reducing vascular inflammation.

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.

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.

Indole acetylated high-amylose maize starch: Synthesis, characterization and application for amelioration of colitis.

Tryptophan metabolites such as indole-3-acetic acid (IAA) are critical for gut health, through their binding to the aryl hydrocarbon receptor (AhR), and may be useful for treatment of gastrointestinal diseases. Delivery of IAA to the colon is necessary, and one strategy is use of esterified starches which get digested in the colon by gut microbes. High amylose maize starch (HAMS) resists digestion in the upper gastrointestinal tract and is fermented by gut microbiota to release short-chain fatty acids (SCFAs), which are also beneficial to intestinal homeostasis. IAA esterified to HAMS (HAMSIAA) was synthesized with different degrees of substitution (DSs) by controlling the ratio of IAA vs HAMS. Successful incorporation of indole acetyl group was verified by NMR and FTIR spectra. XRD revealed that the crystalline type of HAMSIAA changed from B to V-type. SEM showed the destroyed surface of the starch granules. HAMSIAA with DS ~ 0.3 effectively increased IAA in the colon, to levels unachievable by oral IAA delivery. HAMSIAA increased pathways downstream of AhR activation, including CYP1A1 mRNA expression and IL-22 protein levels, and greatly improved DSS-induced colitis. HAMSIAA could serve as an ideal means for colon-targeted delivery of IAA and a promising nutraceutical for amelioration of inflammatory conditions.

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.

Prebiotic intervention with HAMSAB in untreated essential hypertensive patients assessed in a phase II randomized trial.

Fibers remain undigested until they reach the colon, where some are fermented by gut microbiota, producing metabolites called short-chain fatty acids (SCFAs), such as acetate and butyrate1. SCFAs lower blood pressure in experimental models2-5, but their translational potential is unknown. Here we present the results of a phase II, randomized, placebo-controlled, double-blind cross-over trial (Australian New Zealand Clinical Trials Registry ACTRN12619000916145) using prebiotic acetylated and butyrylated high-amylose maize starch (HAMSAB) supplementation6. Twenty treatment-naive participants with hypertension were randomized to 40 g per day of HAMSAB or placebo, completing each arm for 3 weeks, with a 3-week washout period between them. The primary endpoint was a reduction in ambulatory systolic blood pressure. Secondary endpoints included changes to circulating cytokines, immune markers and gut microbiome modulation. Patients receiving the HAMSAB treatment showed a clinically relevant reduction in 24-hour systolic blood pressure independent of age, sex and body mass index without any adverse effects. HAMSAB increased levels of acetate and butyrate, shifted the microbial ecosystem and expanded the prevalence of SCFA producers. In summary, a prebiotic intervention with HAMSAB could represent a promising option to deliver SCFAs and lower blood pressure in patients with essential hypertension.