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

Erawan Borkham-Kamphorst,Ute Haas,Manuela Pinoé-Schmidt,Ali T Abdallah,Ralf Weiskirchen

doi:10.1038/s41374-021-00672-9

Erawan Borkham-Kamphorst, Ute Haas + Show 3 more

Open Access

https://doi.org/10.1038/s41374-021-00672-9

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Abstract

AbstractLipocalin 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 Gr1high, 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 CCl4, 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.

Highlights

Lipocalin 2 (LCN2) or neutrophil gelatinase-associated lipocalin (NGAL) in human is a 24 kDa secretory glycoprotein, isolated from specific granules of neutrophils[1]
Upon eight week- mineral oil or CCl4 administration, induction of hepatic Lcn[2] mRNA and protein levels was significant in wild type livers (Fig. 1A, B)
The sections were blocked with 5% serum originating from the animal that the 2nd antibody was made of in 1% BSA in PBST for 1 h at room temperature, followed by incubation of primary antibodies at within the tissue after mineral oil administration, while CCl4 livers showed positive LCN2 in both non-parenchymal infiltrating cells and hepatocytes around central veins (Fig. 1C)

Summary

Introduction

Lipocalin 2 (LCN2) or neutrophil gelatinase-associated lipocalin (NGAL) in human is a 24 kDa secretory glycoprotein, isolated from specific granules of neutrophils[1]. LCN2 plays an important role in innate immune responses against bacterial infections by sequestering iron-containing siderophores[2,3]. Lcn[2] knockout (Lcn2−/−) mice are susceptible to infections, especially to siderophore-producing bacteria[4]. LCN2 induces the production of cytokines and chemokines to promote the migration and phagocytosis of macrophages, resulting in stronger inflammatory responses in wild type compared to Lcn2−/− mice[6,7]. In non-pathogen-driven inflammatory conditions such as non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH), LCN2 plays detrimental roles in the liver through neutrophil infiltration[8,9,10]. LCN2 showed protective effects in bacterial infections and animal models of liver injury[11,12,13,14,15]

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