Dysbiosis of oral microbiota and its association with salivary immunological biomarkers in autoimmune liver disease

Abstract

The gut microbiota has recently been recognized to play a role in the pathogenesis of autoimmune liver disease (AILD), mainly primary biliary cholangitis (PBC) and autoimmune hepatitis (AIH). This study aimed to analyze and compare the composition of the oral microbiota of 56 patients with AILD and 15 healthy controls (HCs) and to evaluate its association with salivary immunological biomarkers and gut microbiota. The subjects included 39 patients with PBC and 17 patients with AIH diagnosed at our hospital. The control population comprised 15 matched HCs. Salivary and fecal samples were collected for analysis of the microbiome by terminal restriction fragment length polymorphism of 16S rDNA. Correlations between immunological biomarkers measured by Bio-Plex assay (Bio-Rad) and the oral microbiomes of patients with PBC and AIH were assessed. Patients with AIH showed a significant increase in Veillonella with a concurrent decrease in Streptococcus in the oral microbiota compared with the HCs. Patients with PBC showed significant increases in Eubacterium and Veillonella and a significant decrease in Fusobacterium in the oral microbiota compared with the HCs. Immunological biomarker analysis showed elevated levels of inflammatory cytokines (IL-1β, IFN-γ, TNF-α, IL-8) and immunoglobulin A in the saliva of patients with AILD. The relative abundance of Veillonella was positively correlated with the levels of IL-1β, IL-8 and immunoglobulin A in saliva and the relative abundance of Lactobacillales in feces. Dysbiosis of the oral microbiota is associated with inflammatory responses and reflects changes in the gut microbiota of patients with AILD. Dysbiosis may play an important role in the pathogenesis of AILD.

Fig 1. Analysis of the oral microbiota of the PBC, AIH and HC groups based on the T-RFLP profiles.

(a) Bacterial composition at the phylum level. The relative abundance of the bacterial composition at the phylum level in each sample from the subjects in the AIH, PBC and HC groups is shown on a bar chart. The ID of each subject is tagged to the left of the bar. AIH, autoimmune hepatitis; PBC, primary biliary cholangitis; HC, healthy control. (b) Mean genus abundance in the PBC, AIH and HC groups. Plotted values are the mean abundance of the 8 abundant genera in each group. The results are expressed as the mean ± SD. Differences were compared using the Mann-Whitney U-test; *P<0.05, **P<0.01, ***P<0.0005. (c) Cluster analysis of the bacterial compositions in the saliva samples. The samples from 71 subjects and 8 dominant genera are represented on a double-hierarchical clustering heat map. The blue and red squares represent lower and higher abundances, respectively. The clusters at the bottom indicate similarities among the individual (IDs on the top side) profiles at the genus level. The bacterial compositions are classified into two clusters, Cluster I (n = 32) and Cluster II (n = 39). The clusters on the left side indicate the genera showing similarity in the frequency of identification among samples.

Fig 2. Cytokine levels in the saliva of HCs and patients with PBC or AIH.

The salivary levels of IL-1β (a), IL-8 (b), MIP-1β (c), IgA (d), TNF-α (e), and IFN-γ (f). The results are expressed as the mean ± SD. Differences were compared using the Mann-Whitney U-test; *P<0.05, **P<0.01, ***P<0.005.

Fig 3. Mean genus or order abundance of gut microbiota in the PBC, AIH and HC groups.

The plotted values are the mean abundance of the 8 abundant genera and 1 abundant order in each group. The results are expressed as the mean ± SD. Differences were compared using the Mann-Whitney U-test; *P<0.05, **P<0.01, ***P<0.0005.

Fig 4. Principal component analysis (PCA) of the oral and gut microbiota among the 71 T-RFLP profiles.

(a) The T-RFLP profiles were classified into two clusters by hierarchical cluster analysis (orange circle: Cluster I, green circle: Cluster II). (b) Principal component analysis of the oral microbiota in the AILD (blue circle) and HC groups (red circle). (c) Principal component analysis of the gut microbiota in the AILD (blue circle) and HC groups (red circle). (d) Index (Shannon, y-axis) of genera diversity in oral microbiota, (e) Index (Shannon, y-axis) of genera diversity in gut microbiota.