Bone marrow-derived immune cells mediate sensitization to liver injury in a myeloid differentiation factor 88-dependent fashion

Abstract

Toll-like receptors (TLRs) expressed on both immune cells and hepatocytes recognize microbial danger signals and regulate immune responses. Previous studies showed that TLR9 and TLR2 mediate Propionibacterium acnes-induced sensitization to lipopolysaccharide-triggered acute liver injury in mice. Ligand-specific activation of TLR2 and TLR9 are dependent on the common TLR adaptor, myeloid differentiation factor 88 (MyD88). Here, we dissected the role of MyD88 in parenchymal and bone marrow (BM)-derived cells in liver sensitization. Using chimeric mice with green fluorescent protein-expressing BM cells, we identified that P. acnes-induced liver inflammatory foci are of BM origin. Chimeras with MyD88-deficient BM showed no inflammatory foci after P. acnes or TLR2+TLR9 challenge, suggesting that recruitment of inflammatory cells to the liver required MyD88 expression in BM-derived cells. Further, selective MyD88 deficiency in parenchymal cells in mice with wild-type BM failed to prevent inflammatory cell infiltration. These results demonstrate that MyD88 in immune cells rather than in liver parenchymal cells plays an important role in inflammatory cell recruitment and liver injury.

Fig. 1.

Bone marrow-derived cells are recruited into the liver after P. acnes priming. Wild-type mice transplanted either with wild-type (WT/WT) or wild-type GFP-labeled bone marrow (WT/WT-GFP⁺-BM) (3–5 per group) were injected intraperitoneally with 0.2 mL saline or 0.2 mL saline containing 1 mg heat-killed P. acnes. Mice were challenged 7 days later with 0.5 mg/kg body weight LPS administered intraperitoneally and mice were sacrificed 1.5 hours later. Sections of formalin-fixed, paraffin-embedded livers were stained with anti-GFP–rhodamine and DAPI for nuclear material and analyzed for immunofluorescence (IF). (A,B). One representative IF section of WT/WT-GFP⁺-BM after (A) saline plus LPS or (B) P. acnes plus LPS stimulation is shown out of n = 5 with similar results; the nuclei are shown in blue, the GFP is shown in red for color contrast, the arrows point to granulomas. (C) GFP protein expression in liver tissue was analyzed by Western blot analysis (C) and β-actin was used as loading control.

Fig. 2.

Granuloma formation, liver damage, and cytokine induction are dependent on MyD88 expression in BM-derived cells. Wild-type mice transplanted with wild-type or MyD88-deficient bone marrow (WT/WT-BM and WT/MyD88^−/−-BM, respectively) or MyD88-deficient mice transplanted with wild-type bone marrow (MyD88^−/−/WT-BM) were injected intraperitoneally with 0.2 mL saline, 0.2 mL saline containing 1 mg heat-killed P. acnes, or combination of a TLR2 (5 mg/kg lipoteichoic acid) and a TLR9 ligand (2.5 mg/kg CpG DNA), as indicated (n = 3–5 mice in each experimental group). Three days after TLR2+TLR9 ligands or 7 days after P. acnes priming, mice were administered intraperitoneally 0.5 mg/kg LPS and were sacrificed 1.5 hours later. (A) Sections of formalin-fixed, paraffin-embedded livers were stained with H&E; representative histology sections are shown for each experimental group; the arrows point to granulomas. (B,C) Average number of liver granulomas per 20 high-power fields (B) and mean serum ALT levels ± standard error of the mean (C) is shown. (D,E) Serum levels of (D) TNFα and (E) IFNγ were analyzed by enzyme-linked immunosorbent assay. In (B) through (E), asterisk (*) indicates P < 0.05 in saline primed versus P. acnes or TLR2+TLR9 ligands–primed groups; the # indicates P < 0.05 as defined.