SAMe prevents the up regulation of toll-like receptor signaling in Mallory-Denk body forming hepatocytes

Abstract

Mallory-Denk body (MDB) formation is a component of alcoholic and non alcoholic hepatitis. In the present study, the role of the toll-like receptor (TLR) signaling pathway was investigated in the mechanism of MDB formation in the DDC-fed mouse model. Microarray analysis data mining, performed on the livers of drug-primed mice refed DDC, showed that TLR2/4 gene expression was significantly up regulated by DDC refeeding. SAMe supplementation prevented this up regulation and prevented the formation of MDBs. qRT-PCR analysis confirmed these results. TLR2/4 activates the adapter protein MyD88. The levels of MyD88 were increased by DDC refeeding. The increase of MyD88 was also prevented by SAMe supplementation. Results showed that MyD88-independent TLR3/4-TRIF-IRF3 pathway was not up regulated in the liver of DDC refed mice. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is the downstream protein recruited by the MyD88/IRAK protein complex, and is involved in the regulation of innate immune responses. Results showed a significant increase in the levels of TRAF-6. TRAF-6 activation leads to activation of NFkB and the mitogen-activated protein kinase (MAPK) cascade. The TRAF-6 increase was ameliorated by SAMe supplementation. These results suggest that DDC induces MDB formation through the TLR2/4 and MyD88-dependent signaling pathway. In conclusion, SAMe blocked the over-expression of TLR2/4, and their downstream signaling components MyD88 and TRAF-6. SAMe prevented the DDC-induced up regulation of the TLR signaling pathways, probably by preventing the up regulation of INF-gamma receptors by DDC feeding. INFgamma stimulates the up regulation of TLR2. The ability of SAMe feeding to prevent TLR signaling up regulation has not been previously described.

Figure 1

Hepatocytes from a mouse refed DDC and a mouse refed DDC + SAMe stained with Antibodies to UbD (green) and Ubiquitin (red). Note that the cytoplasm of MDB forming cells stained positive for UbD (green). The UBD positive MBD forming cells increased in the livers of DDC refed mice. This increase was prevented in the liver of DDC refed mice with SAMe. Tricolor filter ×10.

Figure 2

qRT-PCR analysis showed that TLR4 (A) and TLR4 (B) receptors were up regulated in the livers of mice refed DDC and SAMe prevented it (Mean+SEM, n=3). Western blot analysis (C) confirmed the result of PCR.

Figure 3

The protein level of MyD88 (A) and TRAF-6 (B) were increased by DDC refeeding and SAMe prevented this change for MyD88. SAMe tended to prevent the increase in TRAF6 induced by DDC refeeding (Mean±SEM, n=3).

Figure 4

Mice refed DDC showed up regulation of CD14 expression in their livers, as shown by qRT-PCR. SAMe feeding prevented this up regulation (Mean±SEM, n=3).

Figure 5

DDC refeeding caused a significant increase in IL-1B in liver and SAMe feeding prevented this increase as shown by Western blot (Mean±SEM, n=3).

Figure 6

Western blot analysis of trimethylated histone 3 lysine 24 (H3K27me3) in the liver samples from DDC Refed mice and DDA+SAMe mice.