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

INTRODUCTION: 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 randomised 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%). CONCLUSIONS: 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.

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.

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.

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.

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.

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.

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.

Propionate Ameliorates Alcohol-Induced Liver Injury in Mice via the Gut-Liver Axis: Focus on the Improvement of Intestinal Permeability.

Alcohol-related liver disease (ALD) is a major cause of chronic liver disease worldwide with limited therapeutic options. Here, we first revealed the promising beneficial effect of gut microbiota-derived propionate on alcoholic liver injury in mice. This effect was dependent on the modulation of homeostasis of the gut-liver axis, especially the improvement of intestinal permeability. Dietary supplementation with propionate protected against ethanol-induced loss of hepatic function and hepatic steatosis in mice. Meanwhile, propionate treatment attenuated intestinal epithelial barrier dysfunction, restored the expression of intestinal mucus layer components, suppressed intestinal inflammation, and altered intestinal microbiota dysbiosis, which inhibited the intestinal hyperpermeability and subsequently reduced lipopolysaccharide leakage in ALD mice. Furthermore, as a consequence of endotoxemia amelioration, the liver inflammation-related TLR4-NF-κB pathway was inhibited. Collectively, our results suggested that propionate supplementation may be a promising option for the prevention and treatment of ALD.

A plasma membrane-localized polycystin-1/polycystin-2 complex in endothelial cells elicits vasodilation.

Polycystin-1 (PC-1, PKD1), a receptor-like protein expressed by the Pkd1 gene, is present in a wide variety of cell types, but its cellular location, signaling mechanisms, and physiological functions are poorly understood. Here, by studying tamoxifen-inducible, endothelial cell (EC)-specific Pkd1 knockout (Pkd1 ecKO) mice, we show that flow activates PC-1-mediated, Ca2+-dependent cation currents in ECs. EC-specific PC-1 knockout attenuates flow-mediated arterial hyperpolarization and vasodilation. PC-1-dependent vasodilation occurs over the entire functional shear stress range and via the activation of endothelial nitric oxide synthase (eNOS) and intermediate (IK)- and small (SK)-conductance Ca2+-activated K+ channels. EC-specific PC-1 knockout increases systemic blood pressure without altering kidney anatomy. PC-1 coimmunoprecipitates with polycystin-2 (PC-2, PKD2), a TRP polycystin channel, and clusters of both proteins locate in nanoscale proximity in the EC plasma membrane. Knockout of either PC-1 or PC-2 (Pkd2 ecKO mice) abolishes surface clusters of both PC-1 and PC-2 in ECs. Single knockout of PC-1 or PC-2 or double knockout of PC-1 and PC-2 (Pkd1/Pkd2 ecKO mice) similarly attenuates flow-mediated vasodilation. Flow stimulates nonselective cation currents in ECs that are similarly inhibited by either PC-1 or PC-2 knockout or by interference peptides corresponding to the C-terminus coiled-coil domains present in PC-1 or PC-2. In summary, we show that PC-1 regulates arterial contractility through the formation of an interdependent signaling complex with PC-2 in ECs. Flow stimulates PC-1/PC-2 clusters in the EC plasma membrane, leading to eNOS, IK channel, and SK channel activation, vasodilation, and a reduction in blood pressure.