The Salivary Microbiota of Patients With Primary Biliary Cholangitis Is Distinctive and Pathogenic

Abstract

The role of host-microbiota interactions in primary biliary cholangitis (PBC) has received increased attention. However, the impact of PBC on the oral microbiota and contribution of the oral microbiota to PBC are unclear. In this study, thirty-nine PBC patients without other diseases and 37 healthy controls (HCs) were enrolled and tested for liver functions and haematological variables. Saliva specimens were collected before and after brushing, microbiota was determined using 16S rDNA sequencing, metabolomics was profiled using Gas Chromatography-Mass Spectrometer (GC-MS), 80 cytokines were assayed using biochips, and inflammation inducibility was evaluated using OKF6 keratinocytes and THP-1 macrophages. Finally, the effect of ultrasonic scaling on PBC was estimated. Compared with HCs, PBC saliva had enriched taxa such as Bacteroidetes, Campylobacter, Prevotella and Veillonella and depleted taxa such as Enterococcaceae, Granulicatella, Rothia and Streptococcus. PBC saliva also had enriched sCD163, enriched metabolites such as 2-aminomalonic acid and 1-dodecanol, and depleted metabolites such as dodecanoic acid and propylene glycol. sCD163, 4-hydroxybenzeneacetic acid and 2-aminomalonic acid were significantly correlated with salivary cytokines, bacteria and metabolites. Salivary Veillonellaceae members, 2-aminomalonic acid, and sCD163 were positively correlated with liver function indicators such as serum alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). PBC salivary microbes induced more soluble interleukin (IL)-6 receptor α (sIL-6Rα), sIL-6Rβ and tumour necrosis factor ligand superfamily (TNFSF)13B from OKF6 keratinocytes, and PBC salivary supernatant induced more IL-6, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), chemokine (C-C motif) ligand (CCL)13, C-X-C motif chemokine (CXC)L1 and CXCL16 from THP-1 macrophages. Toothbrushing significantly reduced the expression of inflammatory cytokines such as IL-1β, IL-8 and TNF-α and harmful metabolites such as cadaverine and putrescine in PBC but not HC saliva after P-value correction. The levels of ALP and bilirubin in PBC serum were decreased after ultrasonic scaling. Together, PBC patients show significant alterations in their salivary microbiota, likely representing one cause and treatment target of oral inflammation and worsening liver functions.

Keywords: autoimmune diseases; immunity; liver; metabolites; microbiota.

Figure 1

The salivary microbiota of PBC patients is distinct from that of HC subjects. (A) Box plot and distribution of the Chao1 index and Shannon index. (B) PCoA plot based on the unweighted UniFrac distance. (C) Phyla, classes, and orders enriched in PBC saliva. (D) Genera and species enriched in PBC saliva. (E) Classes, orders, families, genera and species depleted in PBC saliva. **P < 0.01; ^# P _adj < 0.05; ^## P _adj < 0.01.

Figure 2

Inflammatory cytokines and harmful metabolites were enriched in PBC saliva compared with that in HC saliva. (A) Inflammatory cytokines were distributed differently in PBC saliva and HC saliva. (B) The levels of certain cytokines with nonsignificant differences between HC and PBC saliva were increased significantly in PBC serum. (C) OPLS-DA plots of the metabolome illustrating that PBC and HC subjects were clearly separated. (D) S-plot highlighting four salivary metabolites as potential biomarkers. (E) Seventeen salivary metabolites were different in peak areas and/or detection rates between HC and PBC samples. *P < 0.05.

Figure 3

Associations between PBC-altered salivary cytokines, metabolites, and bacteria and their associations with haematological variables or liver functions. (A) Association of PBC-altered salivary bacteria with metabolites. (B) Association of PBC-altered salivary bacteria with cytokines. (C) Association of PBC-altered salivary metabolites with cytokines. (D) Association of PBC-altered salivary bacteria, cytokines and metabolites with haematological variables or liver functions. Both correlation coefficients (in absolute value) higher than 0.4 and P < 0.01 were used as significance thresholds.

Figure 4

PBC salivary microbes and supernatants induce more inflammatory cytokine secretion than HC salivary microbes from keratinocytes and THP-1 macrophages. (A) Inflammatory cytokine concentrations that were differentially expressed in OKF6 cells after coculture with HC or PBC salivary microbes. (B) Association of the relative abundance of salivary microbes with altered cytokine concentrations after the coculture of OKF6 with HC or PBC salivary microbes. (C) Inflammatory cytokine concentrations that were differentially expressed in THP-1 macrophages after coculture with HC or PBC salivary supernatants. *P < 0.05.

Figure 5

Toothbrushing reduces the number of inflammatory cytokines in PBC saliva more than that in HC saliva. (A) Number of bacteria per millilitre of saliva in PBC patients and healthy controls before and after toothbrushing. (B) and (C) Bacterial taxa with higher relative abundances in PBC saliva after toothbrushing than before toothbrushing. (D) Inflammatory cytokines that were significantly reduced from before to after toothbrushing in PBC saliva. *P < 0.05; **P < 0.01; ***P < 0.001; ^# P _adj < 0.05; ^## P _adj < 0.01.

Figure 6

Toothbrushing reduces the number of metabolites in PBC saliva more than that in HC saliva. (A) OPLS-DA plot of the salivary metabolome profile in PBC and HC saliva before and after toothbrushing. (B) Metabolites that were significantly altered in both PBC and HC saliva from before to after toothbrushing. (C) Metabolites that were significantly altered only in PBC saliva from before to after toothbrushing. *P < 0.05; **P < 0.01 and ***P < 0.001.