Gut virome-colonising Orthohepadnavirus genus is associated with ulcerative colitis pathogenesis and induces intestinal inflammation in vivo

Abstract

Objectives: Ulcerative colitis (UC) is a chronic inflammatory disorder of unknown aetiology. Gut virome dysbiosis is fundamental in UC progression, although its role in the early phases of the disease is far from fully understood. Therefore, we sought to investigate the role of a virome-associated protein encoded by the Orthohepadnavirus genus, the hepatitis B virus X protein (HBx), in UC aetiopathogenesis.

Design: HBx positivity of UC patient-derived blood and gut mucosa was assessed by RT-PCR and Sanger sequencing and correlated with clinical characteristics by multivariate analysis. Transcriptomics was performed on HBx-overexpressing endoscopic biopsies from healthy donors.C57BL/6 mice underwent intramucosal injections of liposome-conjugated HBx-encoding plasmids or the control, with or without antibiotic treatment. Multidimensional flow cytometry analysis was performed on colonic samples from HBx-treated and control animals. Transepithelial electrical resistance measurement, proliferation assay, chromatin immunoprecipitation assay with sequencing and RNA-sequencing were performed on in vitro models of the gut barrier. HBx-silencing experiments were performed in vitro and in vivo.

Results: HBx was detected in about 45% of patients with UC and found to induce colonic inflammation in mice, while its silencing reverted the colitis phenotype in vivo. HBx acted as a transcriptional regulator in epithelial cells, provoking barrier leakage and altering both innate and adaptive mucosal immunity ex vivo and in vivo.

Conclusion: This study described HBx as a contributor to the UC pathogenesis and provides a new perspective on the virome as a target for tailored treatments.

Keywords: chronic ulcerative colitis; experimental colitis; inflammation; intestinal microbiology; mucosal immunity.

Figure 1

Hepatitis B virus X protein (HBx) transcript positivity characterises a cohort of patients with UC. (A) Schematic representation of the experimental workflow for HBx transcript detection in patients with UC. (B) Pie charts summarising HBx positivity in genomic DNA or RNA of UC cohort 1 (Humanitas Research and Clinical Institute, Milan, Italy), and RNA in UC cohort 2 (Fondazione Casa Sollievo della Sofferenza, San Giovanni Rotondo, Foggia, Italy). (C) Correlation heatmap showing multivariate analysis results expressed as Pearson’s coefficients (from 1 to −1). X marks non-significant at p<0.05 (adjustment: Holm). (D) Schematic representation of the experimental workflow for assessing HBx positivity in genomic DNA of patients with UC. (E) On the left, the inheritance tree shows the absence of vertical transmission of HBx genomic DNA in UC cohort 3-derived blood samples (Casa Sollievo della Sofferenza). On the right, is a pie chart summarising the results. Icons from Streamline (https://app.streamlinehq.com).

Figure 2

Hepatitis B virus X protein (HBx) induces colitis symptoms in mice and shapes the colonic mucosal immunity. (A) Schematic experimental workflow of in vivo HBx-induced colitis (n=6/group, two independent experiments). (B) Disease Activity Index (DAI) of mice treated with either GFP-carrying or HBx-carrying liposomes. Differences between groups are statistically significant. Statistical analysis was performed with two-way analysis of variance, with Bonferroni’s postcorrection. Results with a p value <0.05 were considered significant. * P<.05; ** P<.01. (C,D) Box plots showing endoscopic score (C) and colon length (D) of HBx-induced colitic mice. (E, F) Relative cell population abundance (E) and t-distributed stochastic neighbour embedding (t-SNE) multidimensional scaling (F) in HBx-induced colitis mice versus the GFP control. Immunophenotyping was performed on n=3 mice/group. Icons from Streamline (https://app.streamlinehq.com).

Figure 3

Hepatitis B virus X protein (HBx) alters epithelial barrier functions. (A) Graph showing cellular growth rate between HBx-transduced and GFP-transduced epithelial cell line. Experiments were performed in triplicates in three independent experiments. Statistical analysis was performed with two-way analysis of variance with Bonferroni’s postcorrection. (B) Box plot showing transepithelial electrical resistance (TEER, expressed as Ω/cm²) measurements on HBx-transduced and GFP-transduced epithelial cell lines. Statistical analysis was performed with Student’s t-test (C) Schematic representation of organoid isolation. (D) After 7 days in culture, organoids become mature and structured, with crypt and villus domains. (E) Immunofluorescence images showing GFP-transduced and HBx-transduced intestinal organoid structures and the positivity for the epithelial marker EpCAM (red) and the GFP (green). (F, G) Box plots showing real-time PCR results for the stem cell (F) and epithelial barrier markers (G) in organoids transduced with either the GFP-carrying or HBx-carrying lentiviruses, expressed as 2-ΔCT (GAPDH was used as the housekeeping genes). Statistical analysis was performed with Student’s t-test. *P<0.05; **p<0.01; ***p<0.005; ****p<0.001. Icons from Streamline (https://app.streamlinehq.com).

Figure 4

Hepatitis B virus X protein (HBx) regulates the expression of ulcerative colitis (UC)-related genes by binding to enhancer regions. (A) Schematic experimental workflow of the chromatin immunoprecipitation assay with sequencing (ChIP-Seq) and RNA-sequencing (RNA-Seq) analyses of HBx-V5-transduced and GFP-transduced cells. (B) Top three representative DNA motifs enriched in HBx peaks. (C) Pie chart showing gene body-centric annotation of all HBx peaks. (D) Correlation heatmap showing ChIP-Seq coverage multivariate analysis results expressed as Pearson’s coefficients (from 1 to −1). (E) Heatmap showing the relative abundance of the different ChIP-Seq signals within the chromatins states found by hidden Markov modelling. (F, G) MA-plot (F) and Gene Ontology plot (G) showing differentially expressed genes and the resulting dysregulated biological processes, respectively, in HBx-expressing versus GFP-expressing Caco-2 cells. (H) Venn diagram showing the intersection between the differentially expressed genes and HBx putative targets. (I, J) Gene expression heatmap (I) showing the 20 genes found to be differentially expressed while directly bound by HBx within their respective enhancers, summarised in the pie chart (J) showing gene body-centric annotation. Icons from Streamline (https://app.streamlinehq.com).

Figure 5

Hepatitis B virus X protein (HBx)-induced phenotype is reversible in vivo. (A) Schematic experimental workflow of in vivo HBx-induced colitis and small interfering RNA (siRNA)-mediated rescue treatment (n=6 mice/group, 2 independent experiments). (B) Disease Activity Index (DAI) of mice treated with either GFP-carrying or HBx-carrying liposomes and HBx-targeting siRNAs or the scramble. The black dotted line indicates the commencement of siRNA administration. Differences between groups are statistically significant. Statistical analysis was performed with two-way analysis of variance, with Bonferroni’s postcorrection. Results with a p value <0.05 were considered significant. (C, D) Box plots showing endoscopic score (C) and colon length (D) of GFP-carrying or HBx-carrying liposomes and HBx-targeting siRNAs or the scramble. Statistical analysis was performed with Student’s t-test. *P<0.05; **p<0.01; ****p<0.001. Icons from Streamline (https://app.streamlinehq.com).