Bacteria Are Required for Non-Alcoholic Steatohepatits (NASH)

Purpose: To determine if gnotobiotic (germ-free) mice develop diet-induced nonalcoholic steatohepatits (NASH). Methods: 4 - 6 week old germ-free (GF) or wild-type (WT) male C57BL/6 mice were fed either a high fat (60% milk fat, MD) diet or control (13% fat, CD) diet for 16 weeks, a time sufficient to produce a NASH phenotype in wild-type mice. At the end of the feeding, mice were sacrificed and markers of steatosis and NASH were examined. Liver steatosis was quantified using Oil Red O staining and histopathology by hematoxylin and eosin (H & E) staining (NASH Index). NASH development was further evaluated by neutrophil infiltration and real-time polymerase chain reaction (RT-PCR) of mRNA for inflammation modifying cytokines tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 10 (IL-10), and chemokine C-C motif ligand 2 (CCL2) and liver damage (serum ALT). Results: Animal and liver weights are higher in MD GF mice than CD GF mice. This increased weight correlates with increased hepatic steatosis, as evidenced by increased Oil Red O staining in GF MD mice. These results are similar to WT mice. Proinflammatory cytokine mRNA of IL-1β, CCL2, and TNF-α were inhibited by 353%, 325%, and 219%, respectively, in MD GF compared to MD WT. CD GF and MD GF were not significantly different in for any cytokine except TNF-α which was increased by 215% in MD GF compared to CD GF. The anti-inflammatory cytokine IL-10 is also lower in GF than WT animals. The increased inflammatory cytokine mRNA corresponds with H & E stained liver slides showing increased hepatocyte ballooning and neutrophil infiltration in MD WT compared to MD GF. ALT is significantly higher in MD than CD in both WT and GF, but while CD WT is similar to CD GF, MD WT ALT levels are twice as high compared to MD GF. Conclusion: Bacteria are required for inflammation, but not hepatic steatosis, of NASH development in mice.