INTEGRATIVE MICROBIOME CHARACTERIZATION IDENTIFIES STOOL DYSBIOSIS CONTRIBUTING TO INTRAHEPATIC MICROBIOME AND ITS ASSOCIATION WITH HOST TRANSCRIPTOME IN HEPATOCELLULAR CARCINOMA

BACKGROUND: Non-alcoholic steatohepatitis (NASH), a leading cause of liver-related morbidity and mortality, is highly prevalent in people living with HIV (PLWH).¹ Microbial translocation (MT) associated with hepatic inflammation and fibrosis may play a critical role in NASH pathogenesis.² Although HIV is associated with increased gut permeability and MT,³ few studies have evaluated the role of the gut-liver axis in NASH development in PLWH. We aimed to determine the associations of HIV and circulating biomarkers of MT on the FibroScan-AST (FAST)⁵ score, a non-invasive surrogate for NASH with advanced fibrosis, in a large US cohort of women with HIV (WWH) and women without HIV.
METHODS: Vibration controlled transient elastography (VCTE) was conducted from 2013-2018 among 1237 women without viral hepatitis (883 WWH, 354 without HIV). Serum and plasma MT biomarkers kynurenine to tryptophan (KT) ratio (a marker of MT), intestinal fatty acid binding protein (I-FABP, a marker of gut epithelial integrity), and the immune activation markers soluble(s) CD14 and sCD163 were tested within 6 months of the VCTE visit. We used linear regression models to evaluate the associations of HIV, demographic, and metabolic covariates with log-transformed FAST score. Next, we individually added KT ratio, I-FABP, and sCD14 and sCD163 to the models. Finally, we determined the mediating effect of MT using the difference method on the association of HIV with FAST.
RESULTS: Median age was 49 years. Most (74.5%) were Non-Hispanic Black and over half were obese (median BMI 30.5 kg/m²). Among WWH, median CD4 count was 651 cells/mm³ and 91% reported taking antiretroviral therapy. MT biomarker levels were higher in WWH (p< 0.001 for each). On multivariable regression, HIV infection was associated with a 49.2% higher FAST score (95%CI 29,73%), as were age (2.1% increase per year, 95%CI 1,3%), and heavy alcohol use (52% increase over no drinking, 95%CI 17,98%). Black race was associated with a 22% lower FAST score (95%CI -38,-2%). Higher levels of MT biomarker were each significantly associated with higher FAST score (Table 1). After adjustment for MT biomarkers, the association of HIV with FAST score was attenuated, and MT mediated 11-28% of the association of HIV with higher FAST (Figure 1).
CONCLUSION: Compared to women without HIV, WWH have higher circulating biomarkers of MT and higher FAST scores. MT biomarkers are strongly associated with higher FAST score and mediate the association of HIV with higher FAST score. Our findings suggest that MT is an important mechanism by which HIV increases the risk of NASH with advanced fibrosis. This study lays the groundwork for future efforts evaluating the relationship between MT, microbial composition, and NASH in PLWH.

Background:
Non-alcoholic fatty liver disease (NAFLD) is highly prevalent, rising globally, and one of the primary contributors to chronic liver disease and liver-related mortality. Although prior studies suggest a link between the gut microbiome and NAFLD, investigations explaining these dysbioses in terms of genomic and molecular function are lacking.
Methods:
To investigate the role of gut microbial communities in NAFLD, we constructed integrated host-microbiome-molecular profiles, including decades of clinical metadata, stool metagenomes/metatranscriptomes, and metabolomes among 211 participants with NAFLD and 502 healthy controls from the Nurses’ Health Study II.
Results:
We found that NAFLD case status explained a small, but significant amount of variance in gut taxonomic (R²=0.003, P=0.001) and metabolic (R²=0.004, P=0.01) profiles. Per-feature testing using linear models adjusting for age, body mass index, physical activity, diabetes, and diet identified 27 differentially abundant species between NAFLD and healthy controls. Veillonella atypica, V. parvula, V. dispar, Rothia dentocariosa, and Clostridium lavalense were more abundant in NAFLD (notably enriched for oral-typical microbes), while Coprococcus comes, Fusicatenibacter saccharivorans, Faecalibacterium prausnitzii, Oscillibacter spp., and other Firmicutes (generally gut-typical fastidious anaerobes) were depleted in NAFLD (Fig. A, B). These alterations corresponded to other feature-level changes in microbial biochemical potential (i.e., MetaCyc pathways) and their related metabolites (e.g, fatty acids, acylcarnitines, and secondary bile acids, such as isoallolithocholic acid). We observed phylogenetic heterogeneity in the expansion of oral-typical microbes when comparing lean vs. non-lean NAFLD, with Streptococcus spp. relatively enriched in non-lean NAFLD, while other oral species (particularly Veillonella) were comparatively increased in lean NAFLD (Fig. C, D). In a pilot sample of 47 individuals with metatranscriptomic data (n=15 with NAFLD, n=32 controls), we observed altered expression of metabolic pathways involved in arginine biosynthesis (L-arginine biosynthesis I and III), which has previously been linked to attenuated host responses to hepatotoxic injuries, and methionine biosynthesis (HSERMETANA-PWY: L-methionine biosynthesis III), which is an important mediator of hepatic lipid accumulation.
Conclusion:
Our work-in-progress represents among the most dense and complete evaluations of NAFLD gut ecology to-date, incorporating functional metagenomics, metatranscriptomics, and metabolomics. We anticipate a significant expansion in the size of our pilot metatranscriptional cohort, empowering new insights into NAFLD gut microbial activity. Further study is needed to elucidate the biological importance of alterations in gut microbial biochemistry to NAFLD risk and severity.

Background
Parenteral nutrition (PN) is an essential therapy for high risk, preterm infants in whom enteral nutrition is contraindicated or inadequate. Many infants are PN dependent for weeks before enteral tolerance is established; some require PN for months. PN associated cholestasis (PNAC) occurs in an estimated 33% of premature infants requiring PN for over fourteen days. Untreated or unresolved PNAC can result in permanent liver damage and is an indication for liver transplantation.

While PN exposure is a significant risk factor for the development of cholestasis, not all premature infants on PN become cholestatic. The gut microbiome is a known crucial component to bile acid metabolism. There is paucity of research into the preterm gut microbiome and cholestasis. We propose that, in premature infants on PN, features of the early gut microbiome moderate the development of neonatal cholestasis.

Methods
A cohort of nineteen preterm infants (mean gestational age 26.0 +/- 1.9 weeks), had serial bilirubin measurements and serial fecal samples collected during their NICU course at University of Chicago. Fecal samples were subjected to 16S rRNA gene sequencing to evaluate gut microbiome composition. Microbiome measurements and conjugated bilirubin (CB) levels were compared using a mixed-effect linear regression model adjusting for week of life, considering PN status as a moderator, and treating patient as a random effect.

Results
Two distinct microbiome community types were found at the family level. Community Type One was dominated by Enterobacteriaceae and Community Type Two was dominated by unclassified bacteria. Community Type Two, only found in infants on PN at the time of sample collection, was significantly associated with higher CB levels, indicating that a shift in overall β-diversity was putatively linked to cholestasis in preterm infants on PN. At the phylum level, relative abundance of Fusobacteriota was positively associated with CB levels in infants both on and off PN, with a stronger effect size in infants off PN (p=0.004 and p<0.0001). At the family level, relative abundance of Propionibacteriaceae was negatively associated with CB in infants on PN (p=0.022). The relative abundance of Lactobacillaceae was positively associated with an increase in CB regardless of PN status (p<0.0001).

Conclusion
This is the first work to explore the fecal microbiome as it relates to cholestasis in preterm infants on PN, identifying changes in overall microbiome β-diversity associated with CB levels, positive correlations between Fusobacteriota and Lactobacillaceae and CB levels, and identifying Propionibacteriaceae as a potential protective agent. Further work will elucidate implications on bile acid metabolism and may offer insight into potential microbiome therapeutics to prevent PNAC in this vulnerable population.

Background: The intrahepatic microbiome and its role in hepatocellular carcinoma (HCC) remain elusive. We performed integrative microbiome profiling in multi-liver tissue biopsies of HCC and non-HCC regions and compared with stool samples of HCC patients and healthy liver donors.
Methods: We collected liver tissues in multiple sites of intra-tumor, tumor boundary, peri-tumor, and adjacent normal tissue (1-3 biopsies/site) and stool sample from 95 HCC patients (cohort 1=38; cohort 2=57). We also collected liver tissues and stool samples from 53 healthy controls. The microbiome profiling and host gene expression were analysed by 16S amplicon sequencing and RNA sequencing. Germ-free (GF) mice were applied to investigate the translocation of candidate bacteria from gut to liver in their contribution to HCC.
Results: Compared to healthy controls, HCC patients exhibited low-diversity of gut microbiome and high-diversity of liver microbiome, both of which were associated with severity of impaired liver function (P<0.05 for both). We demonstrated a stronger gut-liver microbial connection in HCC patients than healthy controls (P<0.01). Notably, we observed the higher the similarity of gut and liver microbiome, the more serious the liver dysfunction, implying that gut dysbiosis was associated with hepatic microbial changes for facilitating HCC. In line with this, bacterial taxa F. nucleatum (Fn) and B. fragilis (Bf) enriched in stool of HCC patients were also significantly enriched in HCC tissues. There were significant associations between the taxa in gut and the taxa in corresponding HCC tissues (P<0.05). This was validated in a second HCC cohort, suggesting hepatic taxa might derive from gut. We compared the taxa among liver sites of intra-tumor, tumor boundary, peri-tumor and adjacent normal, and identified tumor-associated taxa such as tumor-enriched Fn and Bf were highly varied in different sites, indicating intrahepatic microbiome heterogeneity. Association analysis revealed the tumor-enriched taxa positively related to the enhanced host proinflammatory cytokines (e.g., CXCL1) and oncogenes (e.g., ABL1). To validate our finding, GF mice were gavaged with Bf or Fn. Bacteria colonization in gut and in liver of GF mice was confirmed by bacteria fluorescence tracing, FISH assay and living bacteria culture. Such GF mice exhibited impaired gut barrier integrity, liver dysfunction, enhanced hepatic inflammatory chemokines and activated oncogenic pathways, inferring the bacteria from gut could move to liver to facilitate liver carcinogenesis.
Conclusion: We unraveled for the first time the gut-liver microbial connection in HCC patients and demonstrated the heterogeneity of intrahepatic microbiome is associated with the host oncogenic alteration. Our findings provide new insights on the contribution of gut dysbiosis to intrahepatic taxa in hepatocarcinogenesis.