Microbial metabolites indole derivatives sensitize mice to D-GalN/LPS induced-acute liver failure via the Tlr2/NF-κB pathway

Abstract

Introduction: Acute liver failure (ALF) is a clinical condition with many causes, fast progression, and a poor prognosis. Previous research has indicated that microbial factors have a role in ALF, but a clear picture has yet to emerge.

Methods: To investigate the specific involvement of microbial metabolites in ALF development, we pretreated D-GalN/LPS-induced ALF mice with indole derivatives, an influential class of gut microbial metabolites.

Results: Contrary to their typical role as anti-inflammatory agents in the host, indole-3-acetic acid (IAA), indole-3-lactic acid (ILA), and indolepropionic acid (IPA) gavage sensitize mice to D-GalN/LPS-induced-ALF with a rapid rise in serum transaminases and histologic lesion. For a clearer picture, we performed comprehensive analysis for the IAA therapy. IAA markedly amplified inflammatory response and cellular damage. The transcriptome analysis indicated the participation of the TNF-α/NF-κB signaling pathway. The structure of gut microbiota in ileum and the expression of Toll-like receptor 2 (Tlr2) in the liver were also significantly changed.

Discussion: In conclusion, IAA pretreatment can exacerbate D-GalN/LPS-induced ALF via probable Tlr2/NF-κB pathway involvement and ileac dysbiosis characterized by enriched gram-positive genus with potential pathogenesis. Microbial metabolites IAA may aggravate individual susceptibility to D-GalN/LPS-induced ALF. Further investigation of the underlying mechanism is needed, and intervention with indole derivatives and related commensal species should be undertaken with caution.

Keywords: NF-κB; acute liver failure; gut microbiota; indole derivatives; microbial metabolites; toll-like receptor 2.

Figure 1

Pretreatment of indole derivatives sensitize mice to D-GalN/LPS induced-liver injury. (A) Serum levels of ALT and AST in all groups, and AST/ALT ratios in four ALF groups. (B) Representative liver H&E staining (original magnification × 10/40, scale bar = 100 μm/20 μm). (C) Liver pathological modified HAI. The data are presented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001 for the comparison. Veh, Vehicle (saline control); VeAL, Vehicle + D-GalN/LPS; IAAL, IAA + D-GalN/LPS; ILAL, ILA + D-GalN/LPS; IPAL, IPA + D-GalN/LPS. n = 10 in each group.

Figure 2

IAA administration aggravated D-GalN/LPS induced-Liver inflammation and apoptosis. (A) Relative expression of TNF-α, IL-1β, IL-6, and iNOS mRNA in liver. (B) Serum concentration of TNF-α, IL-1β, IL-6 and MCP-1. (C) Representative liver DAPI/TUNEL immunofluorescence staining (original magnification × 40, scale bar = 20 μm). (D) Percentage of TUNEL-positive cells in each group. The data are presented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001 for the comparison. Veh, Vehicle (saline control); VeAL, Vehicle + D-GalN/LPS; IAAL, IAA + D-GalN/LPS. n = 10 in each group.

Figure 3

IAA alters liver transcriptome expression in D-GalN/LPS induced-ALF. (A) Volcano map for differential genes between VeAL and IAAL. (B) KEGG enrichment analyses of DEGs between VeAL and IAAL. (C) Heatmap of the DEGs from the four selected KEGG pathways. (D) Visualization of the four pathways’ network. Veh, Vehicle (saline control); VeAL, Vehicle + D-GalN/LPS; IAAL, IAA + D-GalN/LPS. n = 3 in each group.

Figure 4

IAA enhanced the TNF-α/NF-κB signaling pathway with the up-regulation of Tlr2 instead of Tlr4 signaling in D-GalN/LPS induced-ALF. (A) The transcriptive activity of NF-κB (P65). (B) Relative expression of IκBα, Tnfaip3, Cxcl2 and Cxcl3 mRNA in liver. (C–E) Relative expression of Tlr4, Tlr2, Tlr6 mRNA in liver. The data are presented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001 for the comparison. Veh, Vehicle (saline control); VeAL, Vehicle + D-GalN/LPS; IAAL, IAA + D-GalN/LPS. n = 10 in each group.

Figure 5

IAA altered the composition of gut microbiota and induced dysbiosis of ileum microbiota in D-GalN/LPS Induced-ALF. (A) Unweighted UniFrac PCoA and PCA plots. (B) Relative abundance of the 10 most abundant taxa at the phylum and genus levels. (C) LDA effect size plots. (D) T-tests between VeAL and IAAL in phylum, genus and species levels. *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001 for the comparison. Veh, Vehicle (saline control); VeAL, Vehicle + D-GalN/LPS; IAAL, IAA + D-GalN/LPS. n = 10 in each group.

Figure 6

Alteration of certain microbial species in ileac dysbiosis was correlated with liver injury in D-GalN/LPS induced-ALF. Correlation analysis of differentially 10 most abundant microbial genera and liver aminotransferase. Correlation analysis was conducted by the Spearman rank correlation test, and red (positive correlation) and blue (negative correlation) colors represent different correlation coefficients. *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001 for the comparison. Veh, Vehicle (saline control); VeAL, Vehicle + D-GalN/LPS; IAAL, IAA + D-GalN/LPS. n = 10 in each group.

Figure 7

Oral gavage of IAA activated liver AHR signaling and the activation of AHR signaling had no impact on D-GalN/LPS induced-ALI. (A) Relative expression of CYP1A1 mRNA in liver. (B) Serum levels of ALT and AST. The data are presented as the mean ± SEM. **p < 0.01; ***p < 0.001 and ****p < 0.0001 for the comparison. Veh, Vehicle (saline control); VeAL, Vehicle + D-GalN/LPS; IAAL, IAA + D-GalN/LPS; FiAL, Ficz+ D-GalN/LPS. n = 10 in each group.