Chronic mineral oil administration increases hepatic inflammation in wild type mice compared to lipocalin 2 null mice

Abstract

Lipocalin 2 (LCN2), an acute-phase protein produced during acute liver injury, plays an important role in the innate immune response against bacterial infection via iron scavenging. LCN2 further influences neutrophil development and physiology leading to increased inflammatory responses. We investigated the roles of LCN2 in chronic inflammation and fibrosis, using repeated carbon tetrachloride (CCl₄) in mineral-oil injection. Surprisingly, mice treated with the mineral oil vehicle alone showed liver inflammation, evidenced by neutrophil and monocyte-macrophage infiltration. Fluorescence-activated cell sorting (FACS) of isolated liver leukocytes showed significantly high CD45⁺ leukocyte concentrations in CCl₄ mice, but no difference of Ly6G⁺ neutrophils between mineral oil and CCl₄ application. Liver CD11b⁺ F4/80⁺ cells counted higher in CCl₄ mice, but the proportions of Gr1^high, an indicator of inflammation, were significantly higher in mineral oil groups. Liver myeloperoxidase (MPO), expressed in neutrophils and monocytes, showed higher levels in wild type mice compared to Lcn2^-/- in both mineral-oil and CCl₄ treated groups. Hepatic and serum LCN2 levels were remarkably higher in the mineral oil-injected wild type group compared to the CCl₄. Wild type animals receiving mineral oil showed significantly higher inflammatory cytokine- and chemokine mRNA levels compared to Lcn2^-/- mice, with no differences in the CCl₄ treated groups. RNA sequencing (RNA-Seq) confirmed significant downregulation of gene sets involved in myeloid cell activation and immune responses in Lcn2 null mice receiving chronic mineral oil versus wild-type. We observed significant upregulation of gene sets and proteins involved in cell cycle DNA replication, with downregulation of collagen-containing extracellular matrix genes in Lcn2^-/- mice receiving CCl_4, compared to the wild type. Consequently, the wild type mice developed slightly more liver fibrosis compared to Lcn2^-/- mice, evidenced by higher levels of collagen type I in the CCl₄ groups and no liver fibrosis in mineral oil-treated mice. Our findings indicate that serum and hepatic LCN2 levels correlate with hepatic inflammation rather than fibrosis.