Toll-like receptor 1 as a possible target in non-alcoholic fatty liver disease

Abstract

Toll-like receptors (TLRs) in the liver compartment have repeatedly been attributed to the development of non-alcoholic fatty liver disease (NAFLD). Knowledge on TLR expression in blood cells and their relation to intestinal microbiota and NAFLD development is limited. Here, we determined TLR expression patterns in peripheral blood mononuclear cells (PBMCs) of NAFLD patients and controls, their relation to intestinal microbiota and the impact of TLRs found altered in NAFLD development. Markers of intestinal permeability in blood and TLR mRNA expression in PBMCs were determined in 37 NAFLD patients and 15 age-matched healthy controls. Fecal microbiota composition was evaluated in 21 NAFLD patients and 9 controls using 16S rRNA gene amplicon sequencing. Furthermore, TLR1^-/- and C57BL/6 mice (n = 5-6/group) were pair-fed a liquid control or a fat-, fructose- and cholesterol-rich diet. Intestinal microbiota composition and markers of intestinal permeability like zonulin and bacterial endotoxin differed significantly between groups with the latter markers being significantly higher in NAFLD patients. Expression of TLR1-8 and 10 mRNA was detectable in PBMCs; however, only TLR1 expression, being higher in NAFLD patients, were significantly positively correlated with the prevalence of Holdemanella genus while negative correlations were found with Gemmiger and Ruminococcus genera. TLR1^-/- mice were significantly protected from the development of diet-induced NAFLD when compared to wild-type mice. While intestinal microbiota composition and permeability differed significantly between NAFLD patients and healthy subjects, in PBMCs, only TLR1 expression differed between groups. Still, targeting these alterations might be a beneficial approach in the treatment of NAFLD in some patients.

Figure 1

Global bacterial community structure and diversity indexes of intestinal microbiota in NAFLD patients and controls. (A) Venn diagram representing the operational taxonomic units (OTUs)/species of the unfiltered reads, (B) Principal Coordinates Analysis (PCoA) with Elucidian dissimilarity distance, (C) canonical correspondance analysis (CCA) and (D) redundance analysis (RDA), (E) Shannon diversity index, (F) species richness, (G) species evenness and (H) Simpon’s index. Each dot represents the microbial community of each proband. Data are shown as means ± SEM, n = 9 controls, n = 21 NAFLD patients, *p ≤ 0.05. C healthy controls, NAFLD patients with non-alcoholic fatty liver disease.

Figure 2

Microbial composition in feces of NAFLD patients and controls. (A) Relative abundance of bacterial genera in feces of NAFLD patients and controls as well as the average abundances of (B–G) genera and (H, I) species that are statistically different. Data are shown as means ± SEM, n = 9 controls, n = 21 NAFLD patients, *p ≤ 0.05. C healthy controls, NAFLD patients with non-alcoholic fatty liver disease.

Figure 3

Markers of intestinal permeability and TLR expression in human PBMCs of NAFLD patients and controls. (A) Endotoxin, (B) zonulin, (C) citrulline and (D) lipoteichoic acid in plasma as well as (E) TLR1 to TLR5 and (F) TLR6 to TLR10 mRNA expression. Data are shown as means ± SEM, n = 10–14 controls, n = 31–36 NAFLD patients, *p ≤ 0.05. C healthy controls, LTA lipoteichoic acid, NAFLD patients with non-alcoholic fatty liver disease, PBMCs peripheral blood mononuclear cells, TLR toll-like receptor.

Figure 4

Correlation analyses of TLR1 mRNA expression and bacterial taxa or markers of intestinal permeability as well as markers of intestinal permeability stratified to TLR1. (A) Heatmap showing the Pearson correlation between the bacterial taxa and TLR1 and correlation analysis of TLR1 mRNA expression in human PBMCs with (B) endotoxin and (C) LTA concentrations in plasma. (D) TLR1 mRNA expression, concentration of (E) endotoxin, (F) LTA and (G) zonulin stratified according to TLR1. Letters before the name of bacterial communities represent the taxonomical level, g = genus, f = family, o = order. Higher values in red above 0 show a positive correlation, while values lower than 0 in blue indicate a negative correlation. Correlations marked with an X are statistically significant. Line indicates the cut-off of 200% TLR1 mRNA expression. TLR1 expression ≤ C, patients with NAFLD showing a TLR1 mRNA expression in PBMCs ≤ TLR1 mRNA expression in PBMCs of C; TLR1 expression > C, patients with NAFLD showing a TLR1 mRNA expression in PBMCs > TLR1 mRNA expression in PBMCs of C. Data are shown as means ± SEM, n = 7–14 controls, n = 16–31 NAFLD patients, *p ≤ 0.05. C healthy controls, LTA lipoteichoic acid, NAFLD patients with non-alcoholic fatty liver disease, PBMCs peripheral blood mononuclear cells, TLR toll-like receptor.

Figure 5

TLR1 mRNA expression in human PBMCs in healthy probands. Human PBMCs were stimulated ex vivo with 100 ng/ml LPS or 10 µg/ml LTA for 24 or 48 h. Data are shown as means ± SEM, n = 6, *p ≤ 0.05. LPS lipopolysaccharide, LTA lipoteichoic acid, PBMCs peripheral blood mononuclear cells, TLR toll-like receptor, US unstimulated.

Figure 6

Effect of TLR1 on markers of intestinal permeability and indices of liver damage in FFC-fed mice. Tlr1 mRNA expression in (A) PBMCs and (B) liver tissue, (C) endotoxin concentration as well as (D) LTA levels in plasma of C- and FFC-fed wild-type mice. (E) Representative pictures (magnification ×200 and ×400) of hematoxylin and eosin staining and (F) NAFLD activity score (NAS) in liver tissue, (G) mRNA expression of Tnfα in liver tissue and (H) concentration of TBARS in liver tissue of C- and FFC-fed TLR1^−/− and wild-type mice. Data are shown as means ± SEM, n = 5–8, except of (A) as yield of PBMCs was rather low, 2 samples were pooled respectively and for (D): n = 4 in C-fed mice, *p ≤ 0.05 between C- and FFC-fed wild-type mice; ^ap ≤ 0.05 compared to C-fed wild-type mice; ^cp ≤ 0.05 compared to C-fed TLR1^−/− mice; ^dp ≤ 0.05 compared to FFC-fed TLR1^−/− mice. C control diet, FFC fat-, fructose- and cholesterol-rich diet, LTA lipoteichoic acid, PBMCs peripheral blood mononuclear cells, TBARS thiobarbituric acid reactive substances, Tlr toll-like receptor, Tnf tumor necrosis factor.