Reduced peroxisome proliferator-activated receptor-α and bile acid nuclear receptor NR1H4/FXR may affect the hepatic immune microenvironment of biliary atresia.

Background: Biliary atresia (BA) is a childhood liver disease characterized by fibrous obstruction and obstruction of the extrahepatic biliary system and is one of the most common and serious biliary disorders in infants. Significant inflammation and fibrosis of the liver and biliary tract are the most prominent features, regardless of the initial damage to the BA. Abnormalities in innate or adaptive immunity have been found in human patients and mouse models of BA. We previously reported that children with BA had abnormal lipid metabolism, including free serum carnitine. Objective: To study gene and protein expression levels of the hepatic peroxisome proliferator-activated receptor-α (PPARα) signaling pathway and farnesoid X receptor (FXR) in BA and BA fibrosis, and assess their clinical values. Methods: Low expression of PPARα and NR1H4 (FXR) in BA were validated in the Gene Expression Omnibus database. Functional differences were determined by gene set enrichment analysis based on of PPARα and NR1H4 expression. BA patients from GSE46960 were divided into two clusters by using consensus clustering according to PPARα, NR1H4, and SMAD3 expression levels, and immunoinfiltration analysis was performed. Finally, 58 cases treated in our hospital were used for experimental verification. (IHC: 10 Biliary atresia, 10 choledochal cysts; PCR: 10 Biliary atresia, 14 choledochal cysts; WB: 10 Biliary atresia, 4 choledochal cysts). Results: Bioinformatics analysis showed that the expression of PPARα, CYP7A1 and NR1H4 (FXR) in the biliary atresia group was significantly lower than in the control group. More BA-specific pathways, including TGFß signaling pathway, P53 signaling pathway, PI3K-AKT-mTOR signaling pathway, etc., are enriched in BA patients with low PPARα and NR1H4 expression. In addition, low NR1H4 expression is abundant in inflammatory responses, IL6/STAT3 signaling pathways, early estrogen responses, IL2 STAT5 signaling pathways, and TGFß signaling pathways. The TGFß signaling pathway was significant in both groups. According to the expression of PPARα, NR1H4 and SMAD3, a key node in TGFß pathway, BA patients were divided into two clusters using consensus clustering. In cluster 2, SMAD3 expression was high, and PPARα and NR1H4 expression were low. In contrast to cluster 1, immune cell infiltration was higher in cluster 2, which was confirmed by immunohistochemistry. The mRNA and protein levels of PPARα and NR1H4 in BA patients were lower than in the control group by immunohistochemistry, Western blot analysis and real-time PCR. Conclusions: The downregulation of PPARα and NR1H4 (FXR) signaling pathway may be closely related to biliary atresia.

Bile Acids Activate NLRP3 Inflammasome, Promoting Murine Liver Inflammation or Fibrosis in a Cell Type-Specific Manner.

Bile acids (BA) as important signaling molecules are considered crucial in development of cholestatic liver injury, but there is limited understanding on the involved cell types and signaling pathways. The aim of this study was to evaluate the inflammatory and fibrotic potential of key BA and the role of distinct liver cell subsets focusing on the NLRP3 inflammasome. C57BL/6 wild-type (WT) and Nlrp3-/- mice were fed with a diet supplemented with cholic (CA), deoxycholic (DCA) or lithocholic acid (LCA) for 7 days. Additionally, primary hepatocytes, Kupffer cells (KC) and hepatic stellate cells (HSC) from WT and Nlrp3-/- mice were stimulated with aforementioned BA ex vivo. LCA feeding led to strong liver damage and activation of NLRP3 inflammasome. Ex vivo KC were the most affected cells by LCA, resulting in a pro-inflammatory phenotype. Liver damage and primary KC activation was both ameliorated in Nlrp3-deficient mice or cells. DCA feeding induced fibrotic alterations. Primary HSC upregulated the NLRP3 inflammasome and early fibrotic markers when stimulated with DCA, but not LCA. Pro-fibrogenic signals in liver and primary HSC were attenuated in Nlrp3-/- mice or cells. The data shows that distinct BA induce NLRP3 inflammasome activation in HSC or KC, promoting fibrosis or inflammation.

Glycan biomarkers of autoimmunity and bile acid-associated alterations of the human glycome: Primary biliary cirrhosis and primary sclerosing cholangitis-specific glycans.

We have recently introduced multiple reaction monitoring (MRM) mass spectrometry as a novel tool for glycan biomarker research and discovery. Herein, we employ this technique to characterize the site-specific glycan alterations associated with primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). Glycopeptides associated with disease severity were also identified. Multinomial regression modelling was employed to construct and validate multi-analyte diagnostic models capable of accurately distinguishing PBC, PSC, and healthy controls from one another (AUC = 0.93 ± 0.03). Finally, to investigate how disease-relevant environmental factors can influence glycosylation, we characterized the ability of bile acids known to be differentially expressed in PBC to alter glycosylation. We hypothesize that this could be a mechanism by which altered self-antigens are generated and become targets for immune attack. This work demonstrates the utility of the MRM method to identify diagnostic site-specific glycan classifiers capable of distinguishing even related autoimmune diseases from one another.

Bile Acids Activated Receptors in Inflammatory Bowel Disease.

Once known exclusively for their role in nutrients absorption, bile acids have emerged as signaling molecules, generated from cholesterol breakdown, acting on several immune cells by activating a variety of receptors including the G protein-coupled bile acid receptor 1 (GPABR1 or TGR5), the Farnesoid-X-receptor (FXR) and, as recently discovered, the retinoid-related orphan receptors (ROR)γt. GPBAR1, FXR, and RORγt are highly expressed in cells of the innate and adaptive immune system (i.e., dendritic cells (DCs), macrophages, innate lymphoid 3 cells (ILC3s), and T helper 17 (Th17) lymphocytes) and plays an important role in regulating intestinal and liver immunity, highlighting a role for various bile acid species in regulating immune responses to intestinal microbial antigens. While primary bile acids are generated from the cholesterol breakdown secondary bile acids, the GPBAR1 ligands, and oxo-bile acids derivatives, the RORγt ligands, are generated by the intestinal microbiota, highlighting the potential of these bile acids in mediating the chemical communication between the intestinal microbiota and the host. Changes in intestinal microbiota, dysbiosis, alter the composition of the bile acid pool, promoting the activation of the immune system and development of chronic inflammation. In this review, we focus on the molecular mechanisms by which an altered bile acid signaling promotes intestinal inflammation.

The contribution of gut bacterial metabolites in the human immune signaling pathway of non-communicable diseases.

The interaction disorder between gut microbiota and its host has been documented in different non-communicable diseases (NCDs) such as metabolic syndrome, neurodegenerative disease, and autoimmune disease. The majority of these altered interactions arise through metabolic cross-talk between gut microbiota and host immune system, inducing a low-grade chronic inflammation that characterizes all NCDs. In this review, we discuss the contribution of bacterial metabolites to immune signaling pathways involved in NCDs. We then review recent advances that aid to rationally design microbial therapeutics. A deeper understanding of these intersections between host and gut microbiota metabolism using metabolomics-based system biology platform promises to reveal the fundamental mechanisms that drive metabolic predispositions to disease and suggest new avenues to use microbial therapeutic opportunities for NCDs treatment and prevention. Abbreviations: NCDs: non-communicable disease, IBD: inflammatory bowel disease, IL: interleukin, T2D: type 2 diabetes, SCFAs: short-chain fatty acids, HDAC: histone deacetylases, GPCR: G-protein coupled receptors, 5-HT: 5-hydroxytryptamine receptor signaling, DCs: dendritic cells, IECs: intestinal epithelial cells, T-reg: T regulatory cell, NF-κB: nuclear factor κB, TNF-α: tumor necrosis factor alpha, Th: T helper cell, CNS: central nervous system, ECs: enterochromaffin cells, NSAIDs: non-steroidal anti-inflammatory drugs, AhR: aryl hydrocarbon receptor, IDO: indoleamine 2,3-dioxygenase, QUIN: quinolinic acid, PC: phosphatidylcholine, TMA: trimethylamine, TMAO: trimethylamine N-oxide, CVD: cardiovascular disease, NASH: nonalcoholic steatohepatitis, BAs: bile acids, FXR: farnesoid X receptor, CDCA: chenodeoxycholic acid, DCA: deoxycholic acid, LCA: lithocholic acid, UDCA: ursodeoxycholic acid, CB: cannabinoid receptor, COBRA: constraint-based reconstruction and analysis.

Mechanisms of Colonization Resistance Against Clostridioides difficile.

Clostridioides difficile is an urgent antimicrobial-resistant bacterium, causing mild to moderate and sometimes life-threatening disease. Commensal gut microbes are critical for providing colonization resistance against C difficile and can be leveraged as non-antibiotic alternative therapeutics for the prevention and treatment of C difficile infection.

Bile Acid Signaling in Inflammatory Bowel Diseases.

Bile acids are a group of chemically different steroids generated at the host/microbial interface. Indeed, while primary bile acids are the end-product of cholesterol breakdown in the host liver, secondary bile acids are the products of microbial metabolism. Primary and secondary bile acids along with their oxo derivatives have been identified as signaling molecules acting on a family of cell membrane and nuclear receptors collectively known as "bile acid-activated receptors." Members of this group of receptors are highly expressed throughout the gastrointestinal tract and mediate the bilateral communications of the intestinal microbiota with the host immune system. The expression and function of bile acid-activated receptors FXR, GPBAR1, PXR, VDR, and RORγt are highly dependent on the structure of the intestinal microbiota and negatively regulated by intestinal inflammation. Studies from gene ablated mice have demonstrated that FXR and GPBAR1 are essential to maintain a tolerogenic phenotype in the intestine, and their ablation promotes the polarization of intestinal T cells and macrophages toward a pro-inflammatory phenotype. RORγt inhibition by oxo-bile acids is essential to constrain Th17 polarization of intestinal lymphocytes. Gene-wide association studies and functional characterizations suggest a potential role for impaired bile acid signaling in development inflammatory bowel diseases (IBD). In this review, we will focus on how bile acids and their receptors mediate communications of intestinal microbiota with the intestinal immune system, describing dynamic changes of bile acid metabolism in IBD and the potential therapeutic application of targeting bile acid signaling in these disorders.

MAIT cell activation in adolescents is impacted by bile acid concentrations and body weight.

Bile acids (BAs) are produced by liver hepatocytes and were recently shown to exert functions additional to their well-known role in lipid digestion. As yet it is not known whether the mucosal-associated invariant T (MAIT) cells, which represent 10-15% of the hepatic T cell population, are affected by BAs. The focus of the present investigation was on the association of BA serum concentration with MAIT cell function and inflammatory parameters as well as on the relationship of these parameters to body weight. Blood samples from 41 normal weight and 41 overweight children of the Lifestyle Immune System Allergy (LISA) study were analyzed with respect to MAIT cell surface and activation markers [CD107a, CD137, CD69, interferon (IFN)-γ, tumor necrosis factor (TNF)-α] after Escherichia coli stimulation, mRNA expression of promyelocytic leukemia zinc finger protein (PLZF) and major histocompatibility complex class I-related gene protein (MR1), the inflammatory markers C-reactive protein (CRP), interleukin (IL)-8 and macrophage inflammatory protein (MIP)-1α as well as the concentrations of 13 conjugated and unconjugated BAs. Higher body weight was associated with reduced MAIT cell activation and expression of natural killer cell marker (NKp80) and chemokine receptor (CXCR3). BA concentrations were positively associated with the inflammatory parameters CRP, IL-8 and MIP-1α, but were negatively associated with the number of activated MAIT cells and the MAIT cell transcription factor PLZF. These relationships were exclusively found with conjugated BAs. BA-mediated inhibition of MAIT cell activation was confirmed in vitro. Thus, conjugated BAs have the capacity to modulate the balance between pro- and anti-inflammatory immune responses.

Sex-related differences in urinary immune-related metabolic profiling of alopecia areata patients.

INTRODUCTION: Alopecia areata is a well-known autoimmune disease affecting humans. Polyamines are closely associated with proliferation and inflammation, and steroid hormones are involved in immune responses. Additionally, bile acids play roles in immune homeostasis by activating various signaling pathways; however, the roles of these substances and their metabolites in alopecia areata remain unclear. OBJECTIVES: In this study, we aimed to identify differences in metabolite levels in urine samples from patients with alopecia areata and healthy controls. METHODS: To assess polyamine, androgen, and bile acid concentrations, we performed high-performance liquid chromatography-tandem mass spectrometry. RESULTS: Our results showed that spermine and dehydroepiandrosterone levels differed significantly between male patients and controls, whereas ursodeoxycholic acid levels were significantly higher in female patients with alopecia areata than in controls. CONCLUSION: Our findings suggested different urinary polyamine, androgen, and bile acid concentrations between alopecia areata patients and normal controls. Additionally, levels of endogenous substances varied according to sex, and this should be considered when developing appropriate treatments and diagnostic techniques. Our findings improve our understanding of polyamine, androgen, and bile acid profiles in patients with alopecia areata and highlight the need to consider sex-related differences.

Effects of supplemental dietary bile acids on growth, liver function and immunity of juvenile largemouth bass（Micropterus salmoides）fed high-starch diet.

The present study was conducted to investigate the effects of bile acids (BAs) on the growth, liver function and immunity of the largemouth bass fed high-starch diet. The experiment set three isonitrogenous and isoenergetic semi-purified diets, LS: low-starch diet (5%), HS: high-starch diet (19%) and SB: high-starch diet with BAs (350 mg/kg diet). An 8-week feeding trial was conducted in largemouth bass of initial weight 23.69 ± 0.13 g. The results indicated that the weight gain (WG) and protein efficiency ratio (PER) of fish fed LS and SB were significantly higher than HS treatment. The superoxide dismutase (SOD) and catalase (CAT) activities of SB group were significantly increased, while malondialdehyde (MDA) content significantly reduced in liver compared with HS group. The activities of alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and glucose contents in plasma of SB group were significantly lower than HS treatment, whereas the content of triglyceride (TG) and total cholesterol (TC) in plasma were significantly higher than HS treatment. Additionally, the plasma immunoglobulin count, lysozyme activity and the blood leukocyte count (WBC) in SB group were significantly higher than HS group. The results of paraffin section of liver showed the histopathological alterations were significantly reduced in the SB group compared to HS group. All in all, this study revealed that bile acids supplement could significantly improve growth performance, enhance liver function and immune ability, and alleviate stress responses of M. salmoides fed high-starch diet.

Impaired Intestinal Farnesoid X Receptor Signaling in Cystic Fibrosis Mice.

BACKGROUND & AIMS: The bile acid (BA)-activated farnesoid X receptor (FXR) controls hepatic BA synthesis and cell proliferation via the intestinal hormone fibroblast growth factor 19. Because cystic fibrosis (CF) is associated with intestinal dysbiosis, anomalous BA handling, and biliary cirrhosis, we investigated FXR signaling in CF. METHODS: Intestinal and hepatic expression of FXR target genes and inflammation markers was assessed in Cftr null mice and controls. Localization of the apical sodium-dependent BA transporter was assessed, and BAs in gastrointestinal tissues were analyzed. The CF microbiota was characterized and FXR signaling was investigated in intestinal tissue and organoids. RESULTS: Ileal murine fibroblast growth factor 19 ortholog (Fgf15) expression was strongly reduced in CF mice, compared with controls. Luminal BA levels and localization of apical sodium-dependent BA transporter was not affected, and BAs induced Fgf15 up to normal levels in CF ileum, ex vivo, and CF organoids. CF mice showed a dysbiosis that was associated with a marked up-regulation of genes involved in host-microbe interactions, including those involved in mucin glycosylation, antimicrobial defense, and Toll-like receptor signaling. Antibiotic treatment reversed the up-regulation of inflammatory markers and restored intestinal FXR signaling in CF mice. Conversely, FXR-dependent gene induction in ileal tissue and organoids was repressed by bacterial lipopolysaccharide and proinflammatory cytokines, respectively. Loss of intestinal FXR activity was associated with a markedly blunted hepatic trophic response to oral BA supplementation, and with impaired repression of Cyp7a1, the gene encoding the rate-limiting enzyme in BA synthesis. CONCLUSIONS: In CF mice, the gut microbiota represses intestinal FXR activity, and, consequently, FXR-dependent hepatic cell proliferation and feedback control of BA synthesis.

Human Postprandial Nutrient Metabolism and Low-Grade Inflammation: A Narrative Review.

The importance of the postprandial state has been acknowledged, since hyperglycemia and hyperlipidemia are linked with several chronic systemic low-grade inflammation conditions. Humans spend more than 16 h per day in the postprandial state and the postprandial state is acknowledged as a complex interplay between nutrients, hormones and diet-derived metabolites. The purpose of this review is to provide insight into the physiology of the postprandial inflammatory response, the role of different nutrients, the pro-inflammatory effects of metabolic endotoxemia and the anti-inflammatory effects of bile acids. Moreover, we discuss nutritional strategies that may be linked to the described pathways to modulate the inflammatory component of the postprandial response.

Metabolomics analysis of human acute graft-versus-host disease reveals changes in host and microbiota-derived metabolites.

Despite improvement in clinical management, allogeneic hematopoietic stem cell transplantation (HSCT) is still hampered by high morbidity and mortality rates, mainly due to graft versus host disease (GvHD). Recently, it has been demonstrated that the allogeneic immune response might be influenced by external factors such as tissues microenvironment or host microbiota. Here we used high throughput metabolomics to analyze two cohorts of genotypically HLA-identical related recipient and donor pairs. Metabolomic profiles markedly differ between recipients and donors. At the onset of acute GvHD, in addition to host-derived metabolites, we identify significant variation in microbiota-derived metabolites, especially in aryl hydrocarbon receptor (AhR) ligands, bile acids and plasmalogens. Altogether, our findings support that the allogeneic immune response during acute GvHD might be influenced by bile acids and by the decreased production of AhR ligands by microbiota that could limit indoleamine 2,3-dioxygenase induction and influence allogeneic T cell reactivity.

Diet, lipids and colon cancer.

Dietary fat is digested and absorbed in the small intestine and can then be utilized as an energy source and/or as a reservoir for other bioactive lipid species. Excessive dietary fat has been implicated in the induction and/or aggravation of several diseases, including colorectal cancer (CRC). Diets with high fat content have been shown to exacerbate CRC through regulation of intestinal inflammation and proliferation, as well as alteration of bile acid pools, microbiota, and bioactive lipid species. This chapter will investigate the effects of dietary fat on CRC development and pathobiology, and possible mechanisms for specific lipid species in those processes.

Bridging intestinal immunity and gut microbiota by metabolites.

The gastrointestinal tract is the site of nutrient digestion and absorption and is also colonized by diverse, highly mutualistic microbes. The intestinal microbiota has diverse effects on the development and function of the gut-specific immune system, and provides some protection from infectious pathogens. However, interactions between intestinal immunity and microorganisms are very complex, and recent studies have revealed that this intimate crosstalk may depend on the production and sensing abilities of multiple bioactive small molecule metabolites originating from direct produced by the gut microbiota or by the metabolism of dietary components. Here, we review the interplay between the host immune system and the microbiota, how commensal bacteria regulate the production of metabolites, and how these microbiota-derived products influence the function of several major innate and adaptive immune cells involved in modulating host immune homeostasis.

Let's get this pyrin started!

Inflammasomes enable cells to respond to pathogens or biological damage, but the specific signals being used to convey these messages are not always clear. A new paper identifies two potential microbiota-derived metabolites, the bile acid analogues BAA485 and BAA473, as the first small molecules to activate the pyrin inflammasome. These results suggest that microbiota may be able to modulate this inflammatory process which, in turn, may contribute to the maintenance of intestinal homeostasis.

Pruritus secondary to primary biliary cholangitis: a review of the pathophysiology and management with phototherapy.

BACKGROUND: Primary biliary cholangitis (PBC) is an autoimmune hepatobiliary disorder characterized by destruction of liver bile ducts leading to intrahepatic cholestasis. It causes intractable pruritus for which ultraviolet (UV)B phototherapy is an experimental treatment when alternative therapies fail. The pathophysiology of cholestatic itch and the mechanism of action of narrowband UVB in this condition remains poorly understood. OBJECTIVES: To summarize the current literature and propose testable hypotheses for the mechanism of action of phototherapy in attenuating itch. METHODS: A focused PubMed search for articles relating to the pathogenesis of itch in cholestatic disease was performed. A total of 3855 articles were screened and 50 were found suitable for literature review. Evidence from this literature review was combined with author expertise in the area. RESULTS: Formulated hypotheses focus on the role of bile salts, autotaxin and specific receptors including G-protein-coupled bile acid receptor, Gpbar1 (also known as TGR5) and the nuclear transcription factor farnesoid X receptor. CONCLUSIONS: Several testable mechanisms through which phototherapy may exert its effects are discussed in this review. The next steps are to carry out an objective assessment of the efficacy of phototherapy in cholestatic pruritus, gain further knowledge on the underlying pathways, and subsequently trial its use against current licensed therapies. Such studies could lead to increased mechanistic understanding, identification of novel therapeutic targets and the potential to refine phototherapy protocols, leading to improved control of itch and quality of life in patients with PBC. What's already known about this topic? Primary biliary cholangitis (PBC) is frequently associated with intractable pruritus for which current treatment options are often unsuccessful. Phototherapy is used as an experimental treatment for PBC-associated pruritus when alternative better-studied treatments fail. What does this study add? This study reviews the current literature on the pathophysiology and management of cholestatic pruritus, an area which remains poorly understood. We propose testable hypotheses of the mechanisms behind the attenuation of cholestatic pruritus with phototherapy.

Bile acid analogues are activators of pyrin inflammasome.

Bile acids are critical metabolites in the gastrointestinal tract and contribute to maintaining intestinal immune homeostasis through cross-talk with the gut microbiota. The conversion of bile acids by the gut microbiome is now recognized as a factor affecting both host metabolism and immune responses, but its physiological roles remain unclear. We conducted a screen for microbiome metabolites that would function as inflammasome activators and herein report the identification of 12-oxo-lithocholic acid (BAA485), a potential microbiome-derived bile acid metabolite. We demonstrate that the more potent analogue 11-oxo-12S-hydroxylithocholic acid methyl ester (BAA473) can induce secretion of interleukin-18 (IL-18) through activation of the inflammasome in both myeloid and intestinal epithelial cells. Using a genome-wide CRISPR screen with compound induced pyroptosis in THP-1 cells, we identified that inflammasome activation by BAA473 is pyrin-dependent (MEFV). To our knowledge, the bile acid analogues BAA485 and BAA473 are the first small molecule activators of the pyrin inflammasome. We surmise that pyrin inflammasome activation through microbiota-modified bile acid metabolites such as BAA473 and BAA485 plays a role in gut microbiota regulated intestinal immune response. The discovery of these two bioactive compounds may help to further unveil the importance of pyrin in gut homeostasis and autoimmune diseases.

Interactions between gut microbiota and non-alcoholic liver disease: The role of microbiota-derived metabolites.

There is increasing evidence that the intestinal microbiota plays a mechanistic role in the etiology of non-alcoholic fatty liver disease (NAFLD). Animal and human studies have linked small molecule metabolites produced by commensal bacteria in the gut contribute to not only intestinal inflammation, but also to hepatic inflammation. These immunomodulatory metabolites are capable of engaging host cellular receptors, and may mediate the observed association between gut dysbiosis and NAFLD. This review focuses on the effects and potential mechanisms of three specific classes of metabolites that synthesized or modified by gut bacteria: short chain fatty acids, amino acid catabolites, and bile acids. In particular, we discuss their role as ligands for cell surface and nuclear receptors regulating metabolic and inflammatory pathways in the intestine and liver. Studies reveal that the metabolites can both agonize and antagonize their cognate receptors to reduce or exacerbate liver steatosis and inflammation, and that the effects are metabolite- and context-specific. Further studies are warranted to more comprehensively understand bacterial metabolite-mediated gut-liver in NAFLD. This understanding could help identify novel therapeutics and therapeutic targets to intervene in the disease through the gut microbiota.

Unaccounted risk of cardiovascular disease: the role of the microbiome in lipid metabolism.

PURPOSE OF REVIEW: Not all of the risk of cardiovascular disease can be explained by diet and genetics, and the human microbiome, which lies at the interface of these two factors, may help explain some of the unaccounted risk. This review examines some of the well established links between the microbiome and cardiovascular health, and proposes relatively unexplored associations. RECENT FINDINGS: Byproducts of microbial metabolism are associated with health and disease: Trimethylamine N oxide is associated with atherosclerosis; whereas short-chain fatty acids are associated with decreased inflammation and increased energy expenditure. More broadly, a large number of association studies have been conducted to explore the connections between bacterial taxa and metabolic syndrome. In contrast, the relationship between the microbiome and triglycerides levels remains poorly understood. SUMMARY: We suggest that deeper understanding of the molecular mechanisms that drive linkages between the microbiome and disease can be determined by replacing 16S rRNA gene sequencing with shotgun metagenomic sequencing or other functional approaches. Furthermore, to ensure translatability and reproducibility of research findings, a combination of multiple different complementary '-omic' approaches should be employed.