Distinct Gut Microbial Signature and Host Genetic Variants in Association with Liver Fibrosis Severity in Patients with MASLD.

Gut microbiota might affect the severity and progression of metabolic dysfunction-associated steatotic liver disease (MASLD). We aimed to characterize gut dysbiosis and clinical parameters regarding fibrosis stages assessed by magnetic resonance elastography. This study included 156 patients with MASLD, stratified into no/mild fibrosis (F0-F1) and moderate/severe fibrosis (F2-F4). Fecal specimens were sequenced targeting the V4 region of the 16S rRNA gene and analyzed using bioinformatics. The genotyping of PNPLA3, TM6SF2, and HSD17B13 was assessed by allelic discrimination assays. Our data showed that gut microbial profiles between groups significantly differed in beta-diversity but not in alpha-diversity indices. Enriched Fusobacterium and Escherichia_Shigella, and depleted Lachnospira were found in the F2-F4 group versus the F0-F1 group. Compared to F0-F1, the F2-F4 group had elevated plasma surrogate markers of gut epithelial permeability and bacterial translocation. The bacterial genera, PNPLA3 polymorphisms, old age, and diabetes were independently associated with advanced fibrosis in multivariable analyses. Using the Random Forest classifier, the gut microbial signature of three genera could differentiate the groups with high diagnostic accuracy (AUC of 0.93). These results indicated that the imbalance of enriched pathogenic genera and decreased beneficial bacteria, in association with several clinical and genetic factors, were potential contributors to the pathogenesis and progression of MASLD.

Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease without Type 2 Diabetes: Stratified by Body Mass Index.

The relationship between gut dysbiosis and body mass index (BMI) in non-diabetic patients with non-alcoholic fatty liver disease (NAFLD) is not adequately characterized. This study aimed to assess gut microbiota's signature in non-diabetic individuals with NAFLD stratified by BMI. The 16S ribosomal RNA sequencing was performed for gut microbiota composition in 100 patients with NAFLD and 16 healthy individuals. The differential abundance of bacterial composition between groups was analyzed using the DESeq2 method. The alpha diversity (Chao1, Shannon, and observed feature) and beta diversity of gut microbiota significantly differed between patients with NAFLD and healthy controls. However, significant differences in their diversities were not observed among subgroups of NAFLD. At the phylum level, there was no trend of an elevated Firmicutes/Bacteroidetes ratio according to BMI. At the genus level, patients with lean NAFLD displayed a significant enrichment of Escherichia-Shigella and the depletion of Lachnospira and Subdoligranulum compared to the non-lean subgroups. Combining these bacterial genera could discriminate lean from non-lean NAFLD with high diagnostic accuracy (AUC of 0.82). Non-diabetic patients with lean NAFLD had a significant difference in bacterial composition compared to non-lean individuals. Our results might provide evidence of gut microbiota signatures associated with the pathophysiology and potential targeting therapy in patients with lean NAFLD.

Distinct alterations of gut microbiota between viral- and non-viral-related hepatocellular carcinoma.

Altered gut microbiota has been connected to hepatocellular carcinoma (HCC) occurrence and advancement. This study was conducted to identify a gut microbiota signature in differentiating between viral-related HCC (Viral-HCC) and non-hepatitis B-, non-hepatitis C-related HCC (NBNC-HCC). Fecal specimens were obtained from 16 healthy controls, 33 patients with viral-HCC (17 and 16 cases with hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, respectively), and 18 patients with NBNC-HCC. Compositions of fecal microbiota were assessed by 16S rRNA sequencing. Bioinformatic analysis was performed by the DADA2 pipeline in the R program. Significantly different genera from the top 50 relative abundance were used to classify between subgroups of HCC by the Random Forest algorithm. Our data demonstrated that the HCC group had a significantly decreased alpha-diversity and changed microbial composition in comparison with healthy controls. Within the top 50 relative abundance, there were 11 genera including Faecalibacterium, Agathobacter, and Coprococcus that were significantly enhanced in Viral-HCC, while 5 genera such as Bacteroides, Streptococcus, Ruminococcus gnavus group, Parabacteroides, and Erysipelatoclostridium were enhanced in NBNC-HCC. Compared to Viral-HCC, the NBNC-HCC subgroup significantly reduced various short-chain fatty acid-producing bacteria, as well as declined fecal butyrate but elevated plasma surrogate markers of microbial translocation. Based on the machine learning algorithm, a high diagnostic accuracy to classify HCC subgroups was achieved with an area under the receiver-operating characteristic (ROC) curve (AUC) of 0.94. Collectively, these data revealed that gut dysbiosis was distinct according to etiological factors of HCC, which might play an essential role in hepatocarcinogenesis. These findings underscore the possible use of a gut microbiota signature for the diagnosis and therapeutic approaches regarding different subgroups of HCC. KEY POINTS: • Gut dysbiosis is connected to hepatocarcinogenesis and can be used as a novel biomarker. • Gut microbiota composition is significantly altered in different etiological factors of HCC. • Microbiota-based signature can accurately distinguish between Viral-HCC and NBNC-HCC.

Alterations in gut virome are associated with cognitive function and minimal hepatic encephalopathy cross-sectionally and longitudinally in cirrhosis.

Cognitive dysfunction due to minimal hepatic encephalopathy (MHE) adversely impacts patients with cirrhosis and more precise therapies are needed. Gut-brain axis changes are therapeutic targets, but prior studies have largely focused on bacterial changes. Our aim was to determine linkages between individual cognitive testing results and bacteria with the virome using a cross-sectional and longitudinal approach. We included cross-sectional (n = 138) and longitudinal analyses (n = 36) of patients with cirrhosis tested using three cognitive modalities, which were psychometric hepatic encephalopathy score (PHES), inhibitory control test (ICT), Stroop, and all three. Stool metagenomics with virome and bacteriome were analyzed studied cross-sectionally and in a subset followed for development/reversal of MHE repeated at 6 months (longitudinally only using PHES). Cross-sectional: We found no significant changes in α/ß diversity in viruses or bacteria regardless of cognitive testing. Cognitively impaired patients were more likely to have higher relative abundance of bacteriophages linked with Streptococcus, Faecalibacterium, and Lactobacillus, which were distinct based on modality. These were also linked with cognition on correlation networks. Longitudinally, 27 patients remained stable while 9 changed their MHE status. Similar changes in phages that are linked with Streptococcus, Faecalibacterium, and Lactobacillus were seen. These phages can influence ammonia, lactate, and short-chain fatty acid generation, which are neuro-active. In conclusion, we found linkages between bacteriophages and cognitive function likely due to impact on bacteria that produce neuroactive metabolites cross-sectionally and longitudinally. These findings could help explore bacteriophages as options to influence treatment for MHE in cirrhosis.

Long-term benefit of DAAs on gut dysbiosis and microbial translocation in HCV-infected patients with and without HIV coinfection.

Long-term effect of Direct-acting antivirals (DAAs) on gut microbiota, short-chain fatty acids (SCFAs) and microbial translocation in patients with hepatitis C virus (HCV) infection who achieve sustained virological response (SVR) were limited. A longitudinal study of 50 patients with HCV monoinfection and 19 patients with HCV/HIV coinfection received DAAs were conducted. Fecal specimens collected at baseline and at week 72 after treatment completion (FUw72) were analyzed for 16S rRNA sequencing and the butyryl-CoA:acetateCoA transferase (BCoAT) gene expression using real-time PCR. Plasma lipopolysaccharide binding protein (LBP) and intestinal fatty acid binding protein (I-FABP) were quantified by ELISA assays. SVR rates in mono- and coinfected patients were comparable (94% vs. 100%). The improvement of gut dysbiosis and microbial translocation was found in responders but was not in non-responders. Among responders, significant restoration of alpha-diversity, BCoAT and LBP were observed in HCV patients with low-grade fibrosis (F0-F1), while HCV/HIV patients exhibited partial improvement at FUw72. I-FABP did not decline significantly in responders. Treatment induced microbiota changes with increasing abundance of SCFAs-producing bacteria, including Blautia, Fusicatenibacter, Subdoligranulum and Bifidobacterium. In conclusion, long-term effect of DAAs impacted the restoration of gut dysbiosis and microbial translocation. However, early initiation of DAAs required for an alteration of gut microbiota, enhanced SCFAs-producing bacteria, and could reduce HCV-related complications.

Identification of a novel gut microbiota signature associated with colorectal cancer in Thai population.

Colorectal cancer (CRC) is the third most common cancer worldwide. Dysbiosis of human gut microbiota has been linked to sporadic CRC. This study aimed to compare the gut microbiota profiles of 80 Thai volunteers over 50 years of age among 25 CRC patients, 33 patients with adenomatous polyp, and 22 healthy controls. The 16S rRNA sequencing was utilized to characterize the gut microbiome in both mucosal tissue and stool samples. The results revealed that the luminal microbiota incompletely represented the intestinal bacteria at the mucus layer. The mucosal microbiota in beta diversity differed significantly among the three groups. The stepwise increase of Bacteroides and Parabacteroides according to the adenomas-carcinomas sequence was found. Moreover, linear discriminant analysis effect size showed a higher level of Erysipelatoclostridium ramosum (ER), an opportunistic pathogen in the immunocompromised host, in both sample types of CRC patients. These findings indicated that the imbalance of intestinal microorganisms might involve in CRC tumorigenesis. Additionally, absolute quantitation of bacterial burden by quantitative real-time PCR (qPCR) confirmed the increasing ER levels in both sample types of cancer cases. Using ER as a stool-based biomarker for CRC detection by qPCR could predict CRC in stool samples with a specificity of 72.7% and a sensitivity of 64.7%. These results suggested ER might be a potential noninvasive marker for CRC screening development. However, a larger sample size is required to validate this candidate biomarker in diagnosing CRC.

Litchi-Derived Polyphenol Alleviates Liver Steatosis and Gut Dysbiosis in Patients with Non-Alcoholic Fatty Liver Disease: A Randomized Double-Blinded, Placebo-Controlled Study.

Preclinical data suggest the role of litchi extract in alleviating non-alcoholic fatty liver disease (NAFLD) by modulating gut microbiota. We aimed at investigating whether oligonol, a litchi-derived polyphenol, could improve liver steatosis and gut dysbiosis in patients with NAFLD. Adults with grade ≥2 steatosis, defined by an MRI proton density fat fraction (MRI-PDFF) of ≥11%, were randomly assigned to receive either oligonol or placebo for 24 weeks. The alteration in the MRI-PDFF and gut microbiota composition assessed by 16S ribosomal RNA sequencing were examined. There were 38 patients enrolled (n = 19 in each group). A significant reduction in the MRI-PDFF between week 0 and week 24 was observed in the oligonol group, while there was a non-significant decrease in the placebo group. A significant improvement in alpha-diversity was demonstrated in both of the groups. The oligonol-induced microbiota changes were characterized by reduced abundance of pathogenic bacteria, including Dorea, Romboutsia, Erysipelotrichaceae UCG-003 and Agathobacter, as well as increased abundance of short-chain fatty acids (SCFAs)-producing bacteria, such as Akkermansia, Lachnospira, Dialister and Faecalibacterium. In summary, this study is the first to provide evidence that supports that oligonol improves steatosis through the modulation of gut bacterial composition. Our results also support the beneficial and complementary role of oligonol in treating NAFLD.

Improvement of Gut Diversity and Composition After Direct-Acting Antivirals in Hepatitis C Virus-Infected Patients With or Without Human Immunodeficiency Virus Coinfection.

BACKGROUND: The influence of direct-acting antivirals (DAAs) on the composition of gut microbiota in hepatitis C virus (HCV)-infected patients with or without human immunodeficiency virus (HIV) is unclear. METHODS: We enrolled 62 patients with HCV monoinfection and 24 patients with HCV/HIV coinfection receiving elbasvir-grazoprevir from a clinical trial. Fecal specimens collected before treatment and 12 weeks after treatment were analyzed using amplicon-based 16S ribosomal RNA sequencing. RESULTS: Sustained virological response rates in the monoinfection and coinfection groups were similar (98.4% vs 95.8%). Pretreatment bacterial communities in the patient groups were less diverse and distinct from those of healthy controls. Compared with HCV-monoinfected patients, HCV/HIV-coinfected individuals showed comparable microbial alpha diversity but decreased Firmicutes-Bacteroidetes ratios. The improvement of microbial dysbiosis was observed in responders achieving sustained virological response across fibrosis stages but was not found in nonresponders. Responders with a low degree of fibrosis exhibited a recovery in alpha diversity to levels comparable to those in healthy controls. Reciprocal alterations of increased beneficial bacteria and reduced pathogenic bacteria were also observed in responders. CONCLUSIONS: This study indicates a short-term effect of direct-acting antivirals in restoration of microbial dysbiosis. The favorable changes in gut microbiota profiles after viral eradication might contribute toward the reduction of HCV-related complications among infected individuals.

Original Research: Analysis of hepatic microRNA alterations in response to hepatitis B virus infection and pegylated interferon alpha-2a treatment.

Interferons play important roles in defense mechanisms against viral infection, and thus interferon therapy has been a standard treatment in chronic hepatitis B patients. Interferons signaling pathways promote interferon-inducible genes including microRNAs. In this research, we aimed to determine microRNAs expression profiles in vitro and in vivo For in vitro model, Huh7 cells were transfected with or without hepatitis B virus plasmid for 6 h, and then treated with 100 ng of pegylated-interferon alpha-2a for 24 h. In vivo, we defined microRNAs expression profiles in pair-liver tissues of chronic hepatitis B patients in comparison between before and after treatment of pegylated-interferon alpha-2a for 48 weeks. Cellular small RNAs were extracted followed by library preparation. To determine microRNAs expression profiles, the next-generation sequencing was carried out on MiSeq platform (Illumina®). In vitro analysis demonstrated that microRNAs can be classified into up-regulated and down-regulated microRNAs in response to hepatitis B virus, interferon, and combination of hepatitis B virus and interferon. Moreover, in vivo analysis revealed microRNAs profiles in non-responders, responders without hepatitis B surface antigen clearance, and responders with hepatitis B surface antigen clearance. The target genes of the candidate microRNAs were determined in terms of roles in cellular pathways and immune response, which might be related to treatment in chronic hepatitis B patients. Results revealed that two down-regulated microRNAs including miR-185-5p and miR-186-5p were correlated in both in vitro and in vivo studies. These two microRNAs might be represented as specific hepatic microRNAs responding to hepatitis B virus and pegylated-interferon alpha-2a treatment, which may remarkable and attractive for further study involving in the association of their target genes and prediction of pegylated-interferon alpha-2a response. Interestingly, microRNAs expression patterns might be useful for understanding the response mechanism and serve as biomarkers for prediction of pegylated-interferon alpha-2a treatment response in patients with chronic hepatitis B.

Human microRNAs profiling in response to influenza A viruses (subtypes pH1N1, H3N2, and H5N1).

MicroRNAs (miRNAs) play an important role in regulation of gene silencing and are involved in many cellular processes including inhibition of infected viral replication. This study investigated cellular miRNA expression profiles operating in response to influenza virus in early stage of infection which might be useful for understanding and control of viral infection. A549 cells were infected with different subtypes of influenza virus (pH1N1, H3N2 and H5N1). After 24 h post-infection, miRNAs were extracted and then used for DNA library construction. All DNA libraries with different indexes were pooled together with equal concentration, followed by high-throughput sequencing based on MiSeq platform. The miRNAs were identified and counted from sequencing data by using MiSeq reporter software. The miRNAs expressions were classified into up and downregulated miRNAs compared to those found in non-infected cells. Mostly, each subtype of influenza A virus triggered the upregulated responses in miRNA expression profiles. Hsa-miR-101, hsa-miR-193b, hsa-miR-23b, and hsa-miR-30e* were upregulated when infected with all three subtypes of influenza A virus. Target prediction results showed that virus infection can trigger genes in cellular process, metabolic process, developmental process and biological regulation. This study provided some insights into the cellular miRNA profiling in response to various subtypes of influenza A viruses in circulation and which have caused outbreaks in human population. The regulated miRNAs might be involved in virus-host interaction or host defense mechanism, which should be investigated for effective antiviral therapeutic interventions.