Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome.

Hundreds of microbiota genes are associated with host biology/disease. Unraveling the causal contribution of a microbiota gene to host biology remains difficult because many are encoded by nonmodel gut commensals and not genetically targetable. A general approach to identify their gene transfer methodology and build their gene manipulation tools would enable mechanistic dissections of their impact on host physiology. We developed a pipeline that identifies the gene transfer methods for multiple nonmodel microbes spanning five phyla, and we demonstrated the utility of their genetic tools by modulating microbiome-derived short-chain fatty acids and bile acids in vitro and in the host. In a proof-of-principle study, by deleting a commensal gene for bile acid synthesis in a complex microbiome, we discovered an intriguing role of this gene in regulating colon inflammation. This technology will enable genetically engineering the nonmodel gut microbiome and facilitate mechanistic dissection of microbiota-host interactions.

Intestinal transgene delivery with native E. coli chassis allows persistent physiological changes.

Live bacterial therapeutics (LBTs) could reverse diseases by engrafting in the gut and providing persistent beneficial functions in the host. However, attempts to functionally manipulate the gut microbiome of conventionally raised (CR) hosts have been unsuccessful because engineered microbial organisms (i.e., chassis) have difficulty in colonizing the hostile luminal environment. In this proof-of-concept study, we use native bacteria as chassis for transgene delivery to impact CR host physiology. Native Escherichia coli bacteria isolated from the stool cultures of CR mice were modified to express functional genes. The reintroduction of these strains induces perpetual engraftment in the intestine. In addition, engineered native E. coli can induce functional changes that affect physiology of and reverse pathology in CR hosts months after administration. Thus, using native bacteria as chassis to "knock in" specific functions allows mechanistic studies of specific microbial activities in the microbiome of CR hosts and enables LBT with curative intent.

Generation and characterization of mature hepatocyte organoids for liver metabolic studies.

Hepatocyte organoids (HOs) generated in vitro are powerful tools for liver regeneration. However, previously reported HOs have mostly been fetal in nature with low expression levels of metabolic genes characteristic of adult liver functions, hampering their application in studies of metabolic regulation and therapeutic testing for liver disorders. Here, we report development of novel culture conditions that combine optimized levels of triiodothyronine (T3) with the removal of growth factors to enable successful generation of mature hepatocyte organoids (MHOs) of both mouse and human origin with metabolic functions characteristic of adult livers. We show that the MHOs can be used to study various metabolic functions including bile and urea production, zonal metabolic gene expression, and metabolic alterations in both alcoholic liver disease and non-alcoholic fatty liver disease, as well as hepatocyte proliferation, injury and cell fate changes. Notably, MHOs derived from human fetal hepatocytes also show improved hepatitis B virus infection. Therefore, these MHOs provide a powerful in vitro model for studies of human liver physiology and diseases. The human MHOs are potentially also a robust research tool for therapeutic development.

Identification of Proteome-Based Immune Subtypes of Early Hepatocellular Carcinoma and Analysis of Potential Metabolic Drivers.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, ranking fourth in frequency. The relationship between metabolic reprogramming and immune infiltration has been identified as having a crucial impact on HCC progression. However, a deeper understanding of the interplay between the immune system and metabolism in the HCC microenvironment is required. In this study, we used a proteomic dataset to identify three immune subtypes (IM1-IM3) in HCC, each of which has distinctive clinical, immune, and metabolic characteristics. Among these subtypes, IM3 was found to have the poorest prognosis, with the highest levels of immune infiltration and T-cell exhaustion. Furthermore, IM3 showed elevated glycolysis and reduced bile acid metabolism, which was strongly correlated with CD8 T cell exhaustion and regulatory T cell accumulation. Our study presents the proteomic immune stratification of HCC, revealing the possible link between immune cells and reprogramming of HCC glycolysis and bile acid metabolism, which may be a viable therapeutic strategy to improve HCC immunotherapy.

Baicalein alleviates intrahepatic cholestasis by regulating bile acid metabolism via an FXR-dependent manner.

Cholestasis is characterized by impaired bile secretion and flow, leading to the accumulation of toxic bile acids in the liver, further causing inflammatory reaction, fibrosis, and ultimately liver transplantation. Although first-line clinical agents such as Ursodeoxycholic acid (UDCA) and Obeticholic acid (OCA) are available, serious side effects still exist. Therefore, pharmacologic treatment of cholestatic liver disease remains challenging. Here, we used a murine model of cholestasis treated with or without intraperitoneal injection of baicalein and found that baicalein could attenuate 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced inflammatory response, ductular reaction, liver fibrosis, and bile acid metabolism disorders. Furthermore, the therapeutic effect of baicalein was hampered in the presence of Guggulsterone (GS), an Farnesoid X receptor (FXR) antagonist. These results indicated that baicalein alleviated DDC diet-induced cholestatic liver injury in an FXR-dependent manner.

CYP8B1 Catalyzes 12alpha-hydroxylation of C27 Bile Acid: In Vitro Conversion of Dihydroxycoprostanic Acid into Trihydroxycoprostanic Acid.

CYP8B1 is the unique P450 enzyme with sterol 12-oxidation activity, playing an exclusive role in 12α-hydroxylating intermediates along the bile acid (BA) synthesis pathway. Despite the long history of BA metabolism studies, it is unclear whether CYP8B1 catalyzes 12α-hydroxylation of C27 BAs, the key intermediates shuttling between mitochondria and peroxisomes. This work provides robust in vitro evidence that both microsomal and recombinant CYP8B1 enzymes catalyze the 12α-hydroxylation of dihydroxycoprostanic acid (DHCA) into trihydroxycoprostanic acid (THCA). On the one hand, DHCA 12α-hydroxylation reactivity is conservatively detected in liver microsomes of both human and preclinical animals. The reactivity of human tissue fractions conforms well with the selectivity of CYP8B1 mRNA expression, while the contribution of P450 enzymes other than CYP8B1 is excluded by reaction phenotyping in commercial recombinant enzymes. On the other hand, we prepared functional recombinant human CYP8B1 proteins according to a recently published protocol. Titration of the purified CYP8B1 proteins with either C4 (7α-hydroxy-4-cholesten-3-one) or DHCA yields expected blue shifts of the heme Soret peak (type I binding). The recombinant CYP8B1 proteins efficiently catalyze 12α-hydroxylation of both DHCA and C4, with Km of 3.0 and 1.9 µM and kcat of 3.2 and 2.6 min-1, respectively. In summary, the confirmed role of CYP8B1 in 12α-hydroxylation of C27 BAs has furnished the forgotten passageway in the BA synthesis pathway. The present finding might have opened a new window to consider the biology of CYP8B1 in glucolipid metabolism and to evaluate CYP8B1 inhibition as a therapeutic approach of crucial interest for metabolic diseases. Significance Statement Academic community has spent about 90 years interpreting the synthesis of bile acids. However, the 12α-hydroxylation of intermediates catalyzed by CYP8B1 is not completely mapped on the classic pathway, particularly for the C27 bile acids, the pivotal intermediates shuttling between mitochondria and peroxisomes. This work discloses the forgotten 12α-hydroxylation pathway from dihydroxycoprostanic acid into trihydroxycoprostanic acid. The present finding may facilitate evaluating CYP8B1 inhibition as a therapeutic approach of crucial interest for metabolic diseases.

Neuroprotective effects of mesenchymal stromal cells in mouse models of Alzheimer's Disease: The Mediating role of gut microbes and their metabolites via the Microbiome-Gut-Brain axis.

The intricacy and multifaceted nature of Alzheimer's disease (AD) necessitate therapies that target multiple aspects of the disease. Mesenchymal stromal cells (MSCs) emerge as potential agents to mitigate AD symptoms; however, whether their therapeutic efficacy involves modulation of gut microbiota and the microbiome-gut-brain axis (MGBA) remains unexplored. In this study, we evaluated the effects of three distinct MSCs types-derived from the umbilical cord (UCMSC), dental pulp (SHED), and adipose tissue (ADSC)-in an APP/PS1 mouse model of AD. In comparison to saline control, MSCs administration resulted in a significant reduction of behavioral disturbances, amyloid plaques, and phosphorylated tau in the hippocampus and frontal cortex, accompanied by an increase in neuronal count and Nissl body density across AD-afflicted brain regions. Through 16S rRNA gene sequencing, we identified partial restoration of gut microbial balance in AD mice post-MSCs treatment, evidenced by the elevation of neuroprotective Akkermansia and reduction of the AD-associated Sphingomonas. To examine whether gut microbiota involved in MSCs efficacy in treating AD, SHED with better anti-inflammatory and gut microbiota recovery effects among three MSCs, and another AD model 5 × FAD mice with earlier and more pathological proteins in brain than APP/PS1, were selected for further studies. Antibiotic-mediated gut microbial inactivation attenuated MSCs efficacy in 5 × FAD mice, implicating the involvement of gut microbiota in the therapeutic mechanism. Functional analysis of altered gut microbiota and targeted bile acid metabolism profiling revealed a significant enhancement in bile acid variety following MSCs therapy. A chief bile acid constituent, taurocholic acid (TCA), was orally administered to AD mice and similarly abated AD symptoms. Nonetheless, the disruption of intestinal neuronal integrity with enterotoxin abrogated the ameliorative impact of both MSCs and TCA treatments. Collectively, our findings substantiate that MSCs confer therapeutic benefits in AD within a paradigm that primarily involves regulation of gut microbiota and their metabolites through the MGBA.

Swertiamarin relieves radiation-induced intestinal injury by limiting DNA damage.

Radiotherapy is the conventional treatment for pelvic abdominal tumors. However, it can cause some damage to the small intestine and colorectal, which are very sensitive to radiation. Radiation-induced intestinal injury (RIII) affects the prognosis of radiotherapy, causing sequelae of loss of function and long-term damage to patients' quality of life. Swertiamarin is a glycoside that has been reported to prevent a variety of diseases including but not limited to diabetes, hypertension, atherosclerosis, arthritis, malaria, and abdominal ulcers. However, its therapeutic effect and mechanism of action on RIII have not been established. We investigated whether swertiamarin has a protective effect against RIII. In this article, we use irradiator to create cellular and mouse models of radiation damage. Preventive administration of swertiamarin could reduce ROS and superoxide anion levels to mitigate the cellular damage caused by radiation. Swertiamarin also attenuated RIII in mice, as evidenced by longer survival, less weight loss and more complete intestinal barrier. We also found an increase in the relative abundance of primary bile acids in irradiated mice, which was reduced by both FXR agonists and swertiamarin, and a reduction in downstream interferon and inflammatory factors via the cGAS-STING pathway to reduce radiation-induced damage.

Environmentally Relevant Concentrations of Microplastic Exposure Cause Cholestasis and Bile Acid Metabolism Dysregulation through a Gut-Liver Loop in Mice.

The massive production of plastics causes the ubiquitous existence of microplastics (MPs) in the biota, therefore, posing exposure risks and potential health concerns to human beings. However, the exact mechanisms of MPs-induced toxicities and abnormalities are largely unknown. In this study, we developed a mouse model of gavage polystyrene microplastics (PS MPs) for 30 days. We found that PS MPs can damage the intestinal barrier, accumulate in the liver tissue, and cause injury. The liver and intestine are both highly associated with bile acid (BA) metabolism. Indeed, we found that PS MPs dysregulate BA synthesis and efflux-related gene expression in the liver, causing cholestasis. Tandemly, PS MPs alter the ratio of primary to secondary BA in the feces by affecting the composition of the intestinal flora. At last, PS MPs alter mice's fecal BA profile, which affects normal BA metabolism. Taken together, the present study provides robust data on the mechanism of toxicity of MPs causing the disturbance of BA metabolism via a 4-step gut-liver loop.

Targeted LC-MS/MS profiling of bile acids reveals primary/secondary bile acid ratio as a novel biomarker for necrotizing enterocolitis.

Bile acids (BAs) are involved in the development of necrotizing enterocolitis (NEC), which mainly occurs in preterm infants. We aim to identify the change of BAs in preterm infants and validate its potential value in the detection of NEC. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to measure the plasma BAs in healthy preterm infants and patients with NEC. By analyzing the level of BAs in healthy preterm infants, we found that the plasma concentrations of BAs were related to sex, gestational/postnatal age, birth weight, mode of birth, and feeding type after birth. The plasma levels of TCA, GCA, TCDCA, GCDCA, primary BAs, and total BAs and the primary/secondary BA ratio were decreased, while DCA, UDCA, and secondary BAs were increased in NEC. The primary/secondary BA ratio (cutoff point 62.9) can effectively differentiate NEC from healthy preterm infants, with an AUC of 0.9, a sensitivity of 94.5%, and a specificity of 78.1%. Combining the ratio with high-risk factors of NEC can better distinguish between NEC and control, with an AUC of 0.95. Importantly, significantly lower levels of primary/secondary BA ratio were found in infants with surgical NEC than in nonsurgical NEC cases. The cutoff point of 28.7 identified surgical NEC from nonsurgical NEC with sensitivity and specificity of 76.9% and 100%. Thus, our study identified that the primary/secondary BA ratio in the plasma can differentiate NEC from healthy preterm infants and effectively differentiate the surgical NEC from nonsurgical NEC. Therefore, LC-MS/MS was expected to be a novel measurement platform used to distinguish infants who are most in need of close monitoring or early surgical intervention.