Introduction:
The liver is continuously exposed to foreign antigens and bacterial products derived from gut microbiota, which is known to be involved in the progression of various liver diseases such as non-alcoholic fatty liver disease, hepatic encephalopathy, as well as viral and alcoholic hepatitis. However, less is known concerning the influence of gut microbiota on immune-mediated liver injury and inflammasome activation. Modulation of the gut microbiome could be important to alleviate immune-mediated liver injury. For that purpose we analyzed the progression of concanavalin A (ConA)-induced hepatitis in germ-free mice as well as mice pre-treated with broad-spectrum antimicrobials.
Materials and Methods:
FIR x tiger mice were treated with broad-spectrum antimicrobials (ATM: Neomycin [5 mg/ml], Bacitracin [5 mg/ml], Pimaricin [1.25 µg/ml]) or water for 7 days prior to ConA [7 mg/kg] challenge. Liver injury induced by ConA was assessed by determination of plasma alanine aminotransferase (ALT) activity. Plasma levels of cytokines (IFNγ, TNFα, IL-6, IL-2) and soluble CD14 (sCD14) were determined by ELISA. Key mediators of inflammation were determined via Western blot (IL-1β) and RT-PCR (il-1β, nlrp3). Immunophenotyping of hepatic lymphocytes was performed by flow cytometry. The composition of the gut microbiome was analyzed by 16S rRNA sequencing of fecal isolates. Gut permeability was assessed by measuring the translocation of FITC-dextran from the gut into the serum.
Results:
Broad-spectrum ATM pre-treatment decreased the amount of operational taxonomic units (OTUs) and ameliorated ConA-induced liver injury, observed in significantly reduced ALT levels. The same effect was shown in germ-free mice. Flow cytometry analysis revealed that ATM pre-treatment led to increased infiltration of anti-inflammatory CD11b CCR2-CX3CR1 macrophages with concomitant decreased presence of inflammatory macrophages (CD11b CCR2 CX3CR- cells) after ConA challenge. ATM treatment further reduced the hepatic expression of NLRP3 and IL-1β. The western blot of cleaved IL-1β confirmed reduced levels of active IL-1β in the liver of ATM pre-treated mice after ConA challenge. Increased gut permeability in ConA treated animals was not observed, serum levels of sCD14 however were increased upon ConA challenge.
Conclusion:
We clearly demonstrated a correlation between the gut microbiota and immune-mediated liver damage. ATM treatment prevents the first hit of sterile liver inflammation, thereby preventing liver injury upon ConA injection. Future experiments are intended to identify a medication-related approach suitable for therapeutic intervention.
