Multi-Omics Analysis of the Gut-Liver Axis Reveals the Mechanism of Liver Injury in Colitis Mice

Introduction

Despite large scale technological and data-driven advances, efficacious and long-lasting treatments for chronic gut illnesses such as IBD, Crohn’s and Ulcerative Colitis (UC) appear to remain out of reach for the millions of sufferers around the globe.

One of the primary factors driving the complexity of investigations into the cause/treatment of intestinal afflictions such as IBD is the cross-organ communication that occurs prior to, and during illnesses originating in the gut. This complexity is further exacerbated by variability in gut microbiota which interacts with both the gut and the surrounding organs at the cellular and genetic level meaning that a singular approach of study often results in skewed data sets.

Metagenomics using 16S DNA sequencing has given researchers an insight into the various demographics and populations of microbiome communities. Transcriptomic techniques such as RNA-Seq has harnessed the power of the transcriptome to give an insight into the driving factors affecting the genetic control of the various taxa. qRT-qPCR (Quantitative Real Time Polymerase Chain Reaction) is a powerful tool for confirming RNA-Seq analysis data by concentrating on gene lists derived from the afore-mentioned techniques. Histological assessment, coupled with visual scoring, give a tissue-level analysis of disease progression and effect of any applied treatment.

Each one of the tools mentioned above are established methodologies and certainly contribute to the knowledge-base but given that each process has a limitation, a combination approach is preferred as this gives a rounded view of disease mechanism and gene control at both the tissue and subcellular level.

A secondary complication caused by intestinal disease is subsequent liver damage, pathogenesis of which has not been fully described as yet. Being directly linked to the gut, the cross-communication of the various proteins and mediators means that any imbalance in the gut will affect the liver i.e. gut homeostasis is directly linked with liver health.

Here, rather than the direct testing/screening of potential drug treatments, a combination of analysis protocols harnessing the positive aspects of each approach to elucidate the disease control pathways is reported. Using the Dextran Sulphate Sodium (DSS)-induced colitis model, with a cohort comprising a healthy control group and a DSS group, 6 mice in each group, contrasts between diseased versus healthy cohorts are drawn through the study. DSS-treatment induces colonic lesions and morphologies in mice akin to those noted in IBD.

Firstly, a disease activity index (DAI) was deployed in the scoring of both groups of mice to measure disease progression. It was noted that DSS-treated mice exhibited significant weight loss, shorter colon lengths and increase DAI when compared to the control group.

Samples were collected at Day 8 of the study with subsequent histologic analysis (colon crypt depth and inflammation), serum cytokine level analysis (IL-6, IL-1β and TNF-α), TEM (Transmission Electron Microscopy) assessment of colon tight junctions (TJ) and microvilli. Serum levels were found to be heightened in DSS-treated mice. Total Cholesterol (TChol), Low-density lipoprotein (LDL) and triglycerides (TG) were found to be elevated in the treated mice.

The function of the intestine was found to be compromised in the DSS-treated group because microvilli length became shorter and the TJs became less abundant and more disorganised when observed by TEM.

Histologic assessment in combination with the DAI scores points to the elicitation of inflammatory responses in the DSS-treated group. Furthermore, impaired liver function was evident due to the elevated cytokine levels in the serum of the DSS-treated group. Subsequent transcriptomic analysis (RNA-Seq) of the liver confirmed a significant increase in immune response-related pathways such as Leukocyte transendothelial migration, IL-17 signalling pathway and Toll-like receptor signalling.

H&E staining showed that the DSS-treated group exhibited loss of colonic crypts combined with shallow crypt depth and inflammatory cell infiltration.

Total RNA was then extracted from colon samples using the Trizol method. This RNA was used as input template material for the qRT-PCR analysis. Β-Actin was deployed as the housekeeping gene for a delta-delta CT assessment against GOI (Genes Of Interest). Eleven GOI were selected from the affected pathways and were deployed in the qRT-qPCR analysis to confirm the findings of the transcriptomic analysis. Seven of the Eleven genes were upregulated (Cxcl2 the most highly upregulated of the gene-set is an immune response-related gene) and four genes were down-regulated. Elovl3, a gene with a primary function in lipid homeostasis regulation, being the most downregulated of the four genes. Further KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis also highlighted Fads2 (known to be mutated in IBD patients to be downregulated.

gDNA (genomic DNA) was extracted from snap-frozen colon samples and used as input material into the 16S-rRNA (Ribosomal RNA) sequencing library preparation for pyrosequencing, a method for precise quantification of sequence variations via a sequence-based detection technology. Data analysis focused on the significant differences at the phylum and genus level of occupying microbiota by statistically measuring operational taxanomic units (OTUs).

RNA-Seq assessment of the mouse liver and colon RNA was conducted on the Illumina Novaseq with PCA’s generated from Fragments Per Kilobase Million (FPKM) and subsequent differential gene expression analysis carried out in the DESeq platform. Analysis selectively focused on the immune response and metabolism-related pathways to draw correlations between the colonic microbiota and GOI.

The DSS-Treated group exhibited a decrease in OTUs. Both groups exhibited a good clustering of diversity, but the structure was distinctly different across control and DSS-treated groups as inferred by the PCoA (principal coordinates analysis). Abundance of microbiota at the phylum level (Firmiculates and Patescibacteria) were significantly lower in the control group compared to the DSS-treated group. Whereas Bacteriodota, Proteobacteria, Campilobacterota and Desulfobacterota were conversely significantly increased in abundance in the DSS-treated group. At the genus-level a marked decrease in abundance was noted of beneficial bacteria such as Faecalibaculum and Lactobacillus and an increase in harmful bacteria such a Helicobacter and Escherichia-Shigella.

Positive gene-bacterial correlations between Helicobacter and Escherichia-Shigella etc with immune response related genes along with negative gene-bacterial correlations in lipid metabolism related genes in liver tissues (opposite trends of association noted in previously mentioned beneficial bacteria) were observed by selecting Differentially Expressed Genes (DEG) from liver and colon tissues and screening against 17 bacteria with >1% abundance to decipher the relative abundance and expression levels in colon and liver. The correlations, both negative and positive, were similar in the colon. These strong correlations suggest a close syntenic relationship between host genes and the DSS-treated gut microbiota, especially the maliciously prevalent actions of Escherichia-Shigella.

Further evidence for the dramatic changes of colonic microbiota induced by DSS-treatment were evident post metabolomic assessment via PICRUST 2 where it was noted that immune response pathways such as MAPK and IL-17 signalling pathways were upregulated and lipid metabolism pathways such as Fatty acid biosynthesis and Glycerophospholipid metabolism were also upregulated in the DSS-treated group which also saw a downregulation of ketone bodies and secondary bile acid biosynthesis.

Given the breadth and granularity of microbiota information yielded by this study, research into almost every microbial niche could be advanced leading to faster IBD, UC and Crohn’s treatment discoveries based on the increased knowledge garnished by the multiplexed approach.

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Utilising proprietary in-house models and assays, Epistem, a CRO based in the UK, deploy all the aforementioned techniques and more in a manner of combinations to characterise the population, virulence driving forces and spatial compartmentalisation of the gut microbiome such that targeted and effective drug treatments can be developed thus adding vital data of the mechanics of IBD, Crohn’s and UC to the knowledge base.