Osteopontin-mediated neutrophilic infiltration and higher liver injury in a female rodent alcoholic steatohepatitis model

Abstract

Females are known to be more susceptible to alcoholic liver disease (ALD), but the precise mechanism behind this increased susceptibility is not well understood. The objective of this study was to identify the molecular mechanism behind the increased susceptibility of females to alcoholic steatohepatitis (ASH). Female rats in ASH model were found to have significantly higher neutrophilic infiltration in the liver as compared to the males. Osteopontin (OPN), a member of the SIBLING family of proteins was also found to be induced in females. Neutralizing OPN antibody experiments provided further evidence that OPN acts as a chemokine in attracting neutrophils into the liver making females more susceptible to ASH. Since neutrophil transmigration in the liver is mediated by intergins, the mechanism for OPN-mediated neutrophil infiltration was tested. Females in ASH had significantly higher expression of α4β1 and α9β1 integrins, and animals treated with neutralizing OPN antibody had significantly lower expression of these integrins, wherein hepatic neutrophilic infiltration was also decreased by 50% compared to the untreated group. Immunoprecipitation experiments suggested the binding of OPN to α4β1 and α9β1 integrins. OPN-mediated neutrophilic infiltration was further confirmed using Boyden chamber assays. Antibodies directed against α4, β1 integrins and SLAYGLR sequence was also found to significantly inhibit neutrophilic migration in vitro, suggesting that higher hepatic neutrophil chemokinesis in the female ASH appears to be mediated through both α4β1 and α9β1 integrin signaling. In addition, higher liver injury and higher expression of OPN in females were also found to be regulated by estrogen in a biphasic pattern; ovariectomy resulted in significantly increased liver injury compared to intact rats. Depending on dose, estradiol supplementation in the ovariectomized rats fed ethanol resulted in both a protective and damaging effect on liver. Besides OPN, several other oxidative stress related proteins like Ferritin H Chain, ER60, HSP60, Peroxiredoxin 6 were identified by proteomics approach. Females in ASH were found to have differentially regulated levels of these proteins as compared to their male counterparts, highlighting the potential novel mechanisms associated with higher liver injury noted in our female rat ASH model.