Frailty is a multifactorial geriatric condition linked to increased mortality and adverse health outcomes and is associated with gut microbiome features that differ from those observed in healthy ageing. We analyze gut metagenomic profiles in relation to estimated frailty severity and frailty-related clinical outcomes assessed with an internally developed and validated Frailty Mortality Index (FMI) in the SUPERB cohort, comprising 2,081 Swedish women aged 75-80 years. The FMI is a composite measure that integrates functional, physiological and psychological dimensions associated with frailty and mortality risk, and shows stronger associations with mortality compared to the Charlson Comorbidity Index in the SUPERB cohort. The FMI is inversely associated with microbial diversity, gene richness, and predicted functional capacity, which are linked to physical function, mortality and fall-related injuries. A total of 404 bacterial species are significantly associated with FMI, and most show concordant associations in a Chinese cohort of 1,448 older adults. Here we show microbial signatures linked to frailty and mortality across different continents.

Cardiovascular diseases (CVD) remain a major global health challenge. Early markers of disease initiation and progression are urgently needed. We, and others, have previously shown changes in the gut microbiome in association with metabolic and CVD. Here, we demonstrate that gut microbiome-related changes can be detected in association with subclinical variations in heart and kidney function. Markers related to gut microbial metabolism of aromatic amino acids, phenylalanine and tyrosine, associate with circulating pro-atrial natriuretic peptide and estimated glomerular filtration rate in a metabolically healthy European population. Observational and genetic evidence further identify microbiome-related metabolites as mediators of this gut microbiome-kidney axis, with their baseline levels associating with incident CVD in an external Canadian population. Altogether, our work suggests that the gut microbiome interacts with the cardiorenal axis and participates in an interorgan crosstalk affecting host physiology and risk of CVD.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the bile ducts that can lead to biliary cancer and end-stage liver disease. PSC is associated with inflammatory bowel disease and an altered gut microbiota. However, the molecular mechanisms underlying gut-liver interactions in PSC remain poorly characterized. Here we show that the gut microbiota-derived metabolite imidazole propionate (ImP) is a disease driver in PSC. Individuals with PSC have higher circulating ImP levels than individuals with related conditions, and high ImP levels predict reduced survival in PSC. Cholangiocytes exposed to ImP show activated mammalian target of rapamycin complex 1 (mTORC1) signalling and secrete pro-inflammatory and pro-fibrogenic factors. Chronic administration of ImP to mice induces liver inflammation and fibrosis through a p38-dependent mechanism, upstream of mTORC1. We propose that chronic exposure to ImP induces cholangiocyte injury, which alone or in concert with other factors causes clinical worsening of PSC. Therefore, targeting ImP production or signalling may represent therapeutic avenues in PSC.

Intestinal transit time (TT) varies considerably between healthy individuals and affects gut microbiota composition and activity. Whether differences in the gut microbiota composition also affect the TT is not well elucidated. In this study, we conducted two animal experiments to explore causality between the gut microbiota and TT. In the first experiment, we transplanted two groups of female germ-free (GF) Swiss-Webster mice with fecal material from two healthy human donors with fast and slow TT phenotypes. Following transplantation with human feces, we observed a decrease in TT for both groups of GF recipient mice (from 300 min to 167 min, 95% CI: ±45; and from 369 vs 205 min, 95% CI: ±52) corresponding to reductions of approximately 45% in each group, supporting previous findings that the mere presence of a gut microbiota reduces TT. However, we found no differences in TT between the two recipient groups. In the second experiment, we transplanted two groups of female GF C57Bl/6J mice with cecal material from two different conventional C57Bl/6J mouse donor groups treated with the TT-increasing drug loperamide or a saline vehicle. Again, no differences in TT were observed between the two recipient groups. These findings indicate that either the transferred microbiota did not engraft effectively, or that gut microbiota composition itself is not the principal driver of inter-individual TT variation.

Obesity's metabolic heterogeneity is not fully captured by body mass index (BMI). Here we show that deep multi-omics phenotyping of 1,408 individuals defines a metabolome-informed obesity metric (metBMI) that captures adipose tissue-related dysfunction across organ systems. In an external cohort (n = 466), metBMI explained 52% of BMI variance and more accurately reflected adiposity than other omics models. Individuals with higher-than-expected metBMI had 2-5-fold higher odds of fatty liver disease, diabetes, severe visceral fat accumulation and attenuation, insulin resistance, hyperinsulinemia and inflammation and, in bariatric surgery (n = 75), achieved 30% less weight loss. This obesogenic signature aligned with reduced microbiome richness, altered ecology and functional potential. A 66-metabolite panel retained 38.6% explanatory power, with 90% covarying with the microbiome. Mediation analysis revealed a bidirectional, metabolite-centered host-microbiome axis, mediated by lipids, amino acids and diet-derived metabolites. These findings define an adipose-linked, microbiome-connected metabolic signature that outperforms BMI in stratifying cardiometabolic risk and guiding precision interventions.

Context:It has been suggested that consumption of saccharin, a widely used artificial sweetener, decreases insulin sensitivity in rodents and humans, but studies show conflicting results. Objective:To investigate if saccharin affects insulin sensitivity in a proof-of-concept study in humans using hyperinsulinemic-euglycemic clamp. Methods:In an open-label pilot study, we recruited 14 overweight participants without diabetes who were mean 60.5 (SD 4.1) years of age and had a body mass index of 27.6 (SD 0.7). Insulin sensitivity, assessed by hyperinsulinemic-euglycemic clamp, was determined before and after consumption of 5 mg/kg saccharin/day for 3 months. Blood was collected for analysis of diabetes-related biomarkers. Stool samples were collected before, during, and after saccharin consumption for microbiota profiling by 16S rRNA gene sequencing. Results:Thirteen of the 14 participants (6 men, 7 women) completed the study. There was no change in insulin sensitivity (mean M value difference [ΔM] -0.1, P = .85) or body weight (mean difference -0.1 kg, P = .70) after consumption of saccharin. However, the mean glycated hemoglobin decreased from 38.7 mmol/mol (SD 3.0) at visit 1 to 36.8 (SD 3.4) at visit 4 (P = .003). Overall, there was no change in composition or richness of the gut microbiota at the end of the study. Conclusion:This study did not demonstrate an association between saccharin intake and impaired insulin sensitivity in adult, overweight participants without diabetes assessed by hyperinsulinemic-euglycemic clamp.

Secretory cells are major structural and functional constituents of the lung airways. Their heterogeneity, spatial organization and specification mechanisms are partially understood. Here, we analyze secretory lung cell-types at single-cell resolution. In the airway epithelium, we find opposing, partially overlapping gene-expression gradients along the proximal-distal airway axis superimposed on a general gene program encoding detoxification. One graded program is elevated proximally and relates to innate immunity, whereas the other is enriched distally, encoding lipid metabolism and antigen presentation. Intermediately positioned cells express moderate levels of both graded programs creating a differentiation continuum towards each end. Lineage tracing analysis during development reveals the sequential establishment of the gradients in common epithelial progenitors postnatally. We show that Fgfr2b regulates the airway patterning by inducing and maintaining high levels of lipid biosynthesis and vesicle trafficking in distal airways and down-regulating innate-immunity genes in vivo and in airway organoids. Our analysis offers a framework for studying epithelial and lung tissue organization to better understand cellular roles in tissue-level pathology.

Background and Aims The gut microbiota is a modulator of cardiometabolic disease. Circulating imidazole propionate (ImP) is a microbiota-derived proatherogenic amino acid metabolite modulating the inflammatory response of myeloid cells, endothelial function, and glucose metabolism. This study examined the prognostic value of ImP in patients with coronary artery disease (CAD). Methods Circulating ImP levels were measured in independent prospective cohorts of patients with acute coronary syndrome (ACS; Swiss ACS cohort n = 4787, Swiss cardiac magnetic resonance imaging cohort n = 150, German ACS cohort n = 1428) and chronic coronary syndrome (CCS; German CCS cohort n = 701). Major adverse cardiovascular events (MACE), defined as the first occurrence of a composite of death, non-fatal myocardial infarction, or non-fatal stroke after admission, were the primary endpoint. Cox models, accounting for established risk factors including the gut-derived cardiovascular risk factor trimethylamine N-oxide, were used to evaluate the predictive value of ImP. Results Circulating ImP was associated with more advanced CAD and with cardiometabolic characteristics including diabetes and elevated high-sensitivity C-reactive protein. High ImP was an independent predictor of MACE [Swiss ACS cohort: hazard ratio (HR) per log2 increase 1.22, 95% confidence interval (CI) 1.10-1.35, P < .001; German ACS cohort: HR 2.34, 95% CI 1.46-3.76, P < .001; German CCS cohort: HR 1.32, 95% CI 1.13-1.53, P < .001)] and of mortality (Swiss ACS cohort: HR 1.34, 95% CI 1.17-1.54, P < .001; German ACS cohort: HR 2.38, 95% CI 1.48-3.82, P < .001; German CCS cohort: HR 1.50, 95% CI 1.14-1.98, P = .004) after adjustment for established risk factors. Imidazole propionate provided predictive value beyond trimethylamine N-oxide (Swiss ACS cohort: HR 1.30, 95% CI 1.05-1.61, P = .014; German CCS cohort: HR 1.31, 95% CI 1.12-1.53, P = .001). Conclusions Gut microbiota-derived ImP predicted MACE in patients with CAD independently of traditional risk factors and holds promise as a therapeutic target. Imidazole propionate may refine risk stratification for personalized secondary prevention strategies.

Bariatric surgeries, such as Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG), improve obesity and type 2 diabetes (T2D). Both surgeries affect the gut microbiota, but their contribution to T2D remission remains unclear. In this subanalysis (RYGB, n = 39; SG, n = 38) of the randomized controlled Oseberg trial ( NCT01778738 ), in which participants underwent either RYGB or SG surgery, we profiled the faecal microbiome of individuals with obesity and T2D before and 12 months after surgery. We show that both surgeries altered the microbiome in the same direction, but with larger changes after RYGB. The SG-associated altered microbiome composition correlated positively with circulating glucagon-like peptide 1 levels, beta-cell function and 5 year T2D remission. Remission was also linked to increased gene richness and metabolic potential for fermentation, methanogenesis and butyrate production. Notably, these associations persisted after accounting for the extent of weight loss. Our findings indicate that surgery-specific microbial adaptations influence metabolic improvements and may help to explain heterogeneity in T2D remission after bariatric surgery.

Abstract The gut microbiome modulates metabolic and neurovascular processes implicated in Alzheimer’s disease and related dementias (ADRD), but the underlying mechanisms remain unclear. Here, we identify the bacterial metabolite imidazole propionate (ImP) as a modifier of ADRD pathology. In a cohort of 1196 cognitively unimpaired adults, higher plasma ImP levels were associated with lower preclinical cognitive scores and biomarkers of ADRD, both cross-sectionally and longitudinally. Fecal metagenomic analysis linked putative ImP producers to ADRD phenotypes. Genome-wide integrative analysis revealed a locus on chromosome 12 associated with both plasma ImP levels and AD risk in humans, supporting a host genetic contribution to ImP regulation and a causal role of this metabolite in AD. In mice, chronic ImP administration exacerbated AD-like pathology. ImP impaired brain endothelial barrier and promoted tau hyperphosphorylation in primary neurons, an effect blocked by glycogen synthase kinase-3β inhibition. Together, this study links ImP to hallmarks of neurodegeneration and suggests that targeting ImP may represent a potential strategy to modify ADRD risk.

Background: The quantification of coronary artery calcifications (CAC) is a mainstay in radiological assessment of coronary atherosclerosis and cardiovascular risk, but reflect advanced, possibly late-stage changes in the arteries. Increased volume and changes in attenuation of the epicardial adipose tissue (EAT) on computed tomography (CT) have been linked to adverse cardiovascular events, and these changes in the EAT might reflect earlier stages of the processes leading to clinically manifest atherosclerosis. The relationship between EAT and CAC is subject to a knowledge gap, especially in individuals with no previously known coronary artery disease. Methods: Fully automated EAT analysis with an artificial intelligence-based model was performed in a population sample enriched for pre-diabetics, comprising a total of 1,945 individuals aged 50-64 years, where non-contrast CT images, anthropometric and laboratory data was available on established cardiovascular risk factors. Uni- and multivariable linear regression, gradient-boosting and correlation analyses were performed to determine the explanatory value of EAT volume and attenuation data with regards to CAC data. Results: Neither EAT volume nor EAT attenuation was associated with the presence or severity of CAC, when adjusting for established cardiovascular risk factors, and had only weak explanatory value in gradient-boosting and correlation analyses. Age was the strongest predictor of CAC in both sexes. Conclusion: No independent association was found between CAC and total EAT volume or attenuation. Importantly, these findings do not rule out early stage or local effects on coronary atherosclerosis from the EAT immediately surrounding the coronary arteries.

Parkinson's disease (PD) is characterized by the selective degeneration of midbrain dopaminergic neurons and aggregation of α-synuclein. Emerging evidence implicates the gut microbiome in PD, with microbial metabolites proposed as potential pathological mediators. However, the specific microbes and metabolites involved, and whether gut-derived metabolites can reach the brain to directly induce neurodegeneration, remain unclear. Here we show that elevated levels of Streptococcus mutans (S. mutans) and its enzyme urocanate reductase (UrdA), which produces imidazole propionate (ImP), in the gut microbiome of patients with PD, along with increased plasma ImP. Colonization of mice with S. mutans harboring UrdA or Escherichia coli expressing UrdA from S. mutans increases systemic and brain ImP levels, inducing PD-like symptoms including dopaminergic neuronal loss, astrogliosis, microgliosis, and motor impairment. Additionally, S. mutans exacerbates α-synuclein pathology in a mouse model. ImP administration alone recapitulates key PD features, supporting the UrdA-ImP axis as a microbial driver of PD pathology. Mechanistically, mTORC1 activation is crucial for both S. mutans- and ImP-induced PD pathology. Together, these findings identify microbial ImP, produced via UrdA, as a direct pathological mediator of the gut-brain axis in PD.

The Clostridium genus is highly heterogeneous, encompassing numerous species and strains, many of which remain to be isolated and characterized to better understand their relationship to host physiology. This study aimed to isolate and characterize novel bacterial species within the Clostridium genus and explore their potential links to host health. Under microaerophilic conditions, we isolated and characterized three bacterial isolates belonging to a new anaerobic Clostridium species, designating Clostridium sp. DSM 115107 (Clostridium filamentum ETTB3) as the type strain. C. filamentum ETTB isolates are rod- to filament-shaped, Gram-positive bacteria and exhibit poor growth when cultured on rich media such as LYBHI. Genome sequencing and phylogenetic analysis revealed that C. filamentum ETTB belongs to the Clostridium genus and clusters closely with Clostridium saudiense JCC. Interestingly, C. filamentum ETTB has a significantly smaller genome compared to C. saudiense JCC containing a reduced repertoire of genes involved in carbohydrate degradation and amino acid synthesis and a larger number of genes related to cell motility, including an additional copy of the fliC gene. Unlike C. saudiense, C. filamentum ETTB adopted a filamentous morphology when in contact with Caco-2 cells and stimulate the TLR5 pathway in Caco-2 cells. Metagenomics analysis revealed that C. filamentum ETTB is present in both industrialized and non-industrialized populations, although the relative abundance varying considerably between and within individuals. Our study identifies a novel bacterial strain adapted for the human gut that has the potential to influence host immune response by activating TLR5 pathway. ### Competing Interest Statement F.B. receives research support from Biogaia AB and Novo Nordisk A/S, is founder and shareholder of Implexion Pharma AB and Roxbiosens Inc, and is on the scientific advisory board for Bactolife A/S. V.T. is co-founder and shareholder of Roxbiosens Inc.

BACKGROUND:The microbially produced amino acid-derived metabolite imidazole propionate (ImP) contributes to the pathogenesis of type 2 diabetes. However, the effects of ImP on endothelial cell (EC) physiology and its role in atherosclerotic coronary artery disease are unknown. Using both human and animal model studies, we investigated the potential contributory role of ImP in the development of atherosclerosis. METHODS:Plasma levels of ImP were measured in patients undergoing elective cardiac angiography (n=831) by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Odds ratios and corresponding 95% confidence intervals for coronary artery disease were calculated based on the ImP quartiles using both univariable and multivariable logistic regression models. The effects of ImP on functional properties of ECs were assessed using HAECs (human aortic endothelial cells). In a mouse model of carotid artery injury, the impact of ImP on vascular regeneration was examined. Additionally, atheroprone Apoe-/- mice fed a high-fat diet were treated with and without ImP (800 µg), and aortic atherosclerotic lesion area was evaluated after 12 weeks. Next-generation sequencing, Western blot analysis, small interfering RNA-based gene knockdown, and tamoxifen-inducible Cre-loxP experiments were performed to investigate ImP-mediated molecular mechanisms. RESULTS:Plasma ImP levels in subjects undergoing cardiac evaluation were associated with increased risk of prevalent coronary artery disease. We found that ImP dose dependently impaired migratory and angiogenic properties of human ECs and promoted an increased inflammatory response. Long-term exposure to ImP compromised the repair potential of the endothelium after an arterial insult. In atheroprone Apoe-/- (apolipoprotein E-/-) mice, ImP increased atherosclerotic lesion size. Mechanistically, ImP attenuated insulin receptor signaling by suppressing the PI3K (phosphoinositide 3-kinase)/AKT pathway leading to sustained activation of the FOXO1 (forkhead box protein O1) transcription factor. Genetic inactivation of endothelial FOXO1 signaling in ImP-treated mice enhanced the angiogenic activity and preserved the vascular repair capacity of ECs after carotid injury. CONCLUSIONS:Our findings reveal a hitherto unknown role of the microbially produced histidine-derived metabolite ImP in endothelial dysfunction and atherosclerosis, suggesting that ImP metabolism is a potential therapeutic target in atherosclerotic cardiovascular disease.

The human gut microbiome strongly influences host metabolism by fermenting dietary components into metabolites that signal to the host. Our previous work has shown that Intestinimonas butyriciproducens is a prevalent commensal bacterium with the unique ability to convert dietary fructoselysine to butyrate, a well-known signaling molecule with proven health benefits. Dietary fructoselysine is an abundant Amadori product formed in foods during thermal treatment and is part of foods rich in dietary advanced glycation end products which have been associated with cardiometabolic disease. It is therefore of interest to investigate the causal role of this bacterium and fructoselysine metabolism in metabolic disorders. We assessed associations of I. butyriciproducens with metabolic risk biomarkers at both strain and functional levels using a human cohort characterized by fecal metagenomic analysis. We observed that the level of the bacterial strain as well as fructoselysine fermentation genes were negatively associated with BMI, triglycerides, HbA1c, and fasting insulin levels. We also investigated the fructoselysine degradation capacity within the Intestinimonas genus using a culture-dependent approach and found that I. butyriciproducens is a key player in the butyrogenic fructoselysine metabolism in the gut. To investigate the function of I. butyriciproducens in host metabolism, we used the diet-induced obesity mouse model to mimic the human metabolic syndrome. Oral supplementation with I. butyriciproducens counteracted body weight gain, hyperglycemia, and adiposity. In addition, within the inguinal white adipose tissue, bacterial administration reduced inflammation and promoted pathways involved in browning and insulin signaling. The observed effects may be partly attributable to the formation of the short-chain fatty acids butyrate from dietary fructoselysine, as butyrate plasma and cecal levels were significantly increased by the bacterial strain, thereby contributing to the systemic effects of the bacterial treatment. I. butyriciproducens ameliorates host metabolism in the context of obesity and may therefore be a good candidate for new microbiota-therapeutic approaches to prevent or treat metabolic diseases.

Gut bacterial metabolism of dietary flavonoids results in the production of a variety of phenolic acids, whose contributions to health remain poorly understood. Here, we show that supplementation with the commonly consumed flavonoid quercetin impacted gut microbiome composition and resulted in a significant reduction in atherosclerosis burden in conventionally raised (ConvR) Apolipoprotein E (ApoE) knockout (KO) mice but not in germ-free (GF) ApoE KO mice. Metabolomic analysis revealed that consumption of quercetin significantly increased plasma levels of benzoylglutamic acid, 3,4 dihydroxybenzoic acid (3,4-DHBA) and its sulfate-conjugated form in ConvR mice, but not in GF mice supplemented with the flavonoid. Levels of these metabolites were negatively associated with atherosclerosis burden. Furthermore, we show that 3,4-DHBA prevented lipopolysaccharide (LPS)-induced decrease in transendothelial electrical resistance (TEER). These results suggest that the effects of quercetin on atherosclerosis are influenced by gut microbes and are potentially mediated by bacterial metabolites derived from the flavonoid.

Cardiometabolic diseases (CMD) are on the rise globally with one billion people expected to suffer from obesity and 643 million from type 2 diabetes by 2030, of which one-third will likely develop chronic kidney disease and two-thirds will die from cardiovascular disease (CVD). However, the mechanistic and molecular drivers of the transition from health to disease remain elusive. Here, in 275 metabolically healthy individuals recruited to the MetaCardis study, we identify a gut microbiome-kidney-heart axis that is predictive of future cardiovascular events. This axis, as evidenced by the associations between gut microbial metabolism of phenylalanine and tyrosine with variations in both kidney functon(as measured by estimated glomerular filtration rate) and circulating pro-atrial natriuretic peptide concentration, shows a depletion pattern in metabolically unhealthy participants of the MetaCardis study (n = 1,602) indicating a loss of health-sustaining microbiome features with CMD progression. We then validate that microbial compounds from the phenylalanine and tyrosine pathways and their host co-metabolites act as mediators of the gut microbiome-kidney associations. Moreover, Mendelian Randomization analysis adds genetic evidence to suggest that the microbial mediator metabolites regulate host kidney function and vice versa. Finally, we demonstrate that plasma metabolites derived from the microbial metabolism of phenylalanine and tyrosine associate with incident CVD in the Canadian Longitudinal Study on Aging (n = 8,669). Collectively, our results depict the presence of a gut microbiome-kidney-heart axis in metabolically healthy individuals. Major aberrations of the gut microbiome as part of this axis throughout life may increase risk of CVD.