Deficiency of Interleukin-15 Enhances Susceptibility to Acetaminophen-Induced Liver Injury in Mice

<div><p>Hepatocytes have a direct necrotic role in acetaminophen (APAP)-induced liver injury (AILI), prolonged secondary inflammatory response through innate immune cells and cytokines also significantly contributes to APAP hepatotoxicity. Interleukin 15 (IL-15), a multifunction cytokine, regulates the adaptive immune system and influences development and function of innate immune cells. To better understand the role of IL-15 in liver injury, we treated wild-type (WT) and IL-15-knockout (<em>Il15^−/−</em>) mice with a hepatotoxic dose of APAP to induce AILI and evaluated animal survival, liver damage, APAP metabolism in livers and the inflammatory response. Production of pro-inflammatory cytokines/chemokines was greater in <em>Il15^−/−</em> than WT mice. Subanalysis of hepatic infiltrated monocytes revealed greater neutrophil influx, along with greater hepatic induction of inducible nitric oxide synthase (iNOS), in <em>Il15^−/−</em> than WT mice. In addition, the level of hepatic hemeoxygenase 1 (HO-1) was partially suppressed in <em>Il15^−/−</em> mice, but not in WT mice. Interestingly, elimination of Kupffer cells and neutrophils did not alter the vulnerability to excess APAP in <em>Il15^−/−</em> mice. However, injection of galactosamine, a hepatic transcription inhibitor, significantly reduced the increased APAP sensitivity in <em>Il15^−/−</em> mice but had minor effect on WT mice. We demonstrated that deficiency of IL-15 increased mouse susceptibility to AILI. Moreover, Kupffer cell might affect APAP hepatotoxicity through IL-15.</p> </div

Injection of GalN reduces the enhanced susceptibility to AILI in <i>Il15^−/−</i> and WT mice.

<p>Mice were pre-treated with or without 700 mg/kg of GalN 20 minutes before APAP challenge. (A) ALT levels in serum and (B) liver histopathological changes at 6 hr after APAP (H&E staining; magnification ×4). (C) The quantification results of hepatic necrotic area by software Image-Pro Plus analysis. (D) Hepatic GSH levels at 0.5 hr with or without APAP injection. *<i>P</i><0.05; **<i>P</i><0.01; <i>n.s.</i>, not significant. Data are mean ± SEM from 4∼6 mice per group.</p

IL-15 was elevated in serum and up-regulated in KC-enriched fraction but not in total liver during AILI in WT mice.

<p>(A) IL-15 levels in mouse serum at indicated time points after APAP injection were evaluated by ELISA. Relative mRNA levels of IL-15 in (B) total liver at 0, 2 and 8 hr, and (C) KC-enriched fraction at 0 and 8 hr after APAP injection were analyzed by quantitative PCR. *<i>P</i><0.05; **<i>P</i><0.01. Data are mean ± SEM from 6∼8 mice per group.</p

Inductions of hepatic inflammatory genes are greater in <i>Il15^−/−</i> than WT mice with APAP challenge.

<p>Relative mRNA levels of (A) pro-inflammatory cytokines IL-1β, TNFα and IL-6; (B) vascular adhesion molecules ICAM-1 and VCAM-1; and (C) chemokines MIP-1α, KC/GRO and MIP-2α at indicated times after injection with APAP. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; #<i>P</i><0.05 compared with WT at 0 hr. Data are mean ± SEM from 6∼8 mice per group.</p

The hepatic HO-1 induction was suppressed in <i>Il15^−/−</i> than WT mice with APAP challenge.

<p>(A) Time course of hepatic HO-1 induction after APAP injection in WT mice. HO-1 (B) mRNAs at 0, 2 and 8 hr and (C) proteins-representative and quantitative data at 8 hr post-APAP treatment. HO-1 (D) mRNA levels and (E) protein-representative and quantitative data of hepatocytes at 8 hr with APAP challenge. #<i>P</i><0.05, compared with WT 0 hr; *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001. Data are mean ± SEM from 3∼6 mice per group in (A) and 6∼8 mice per group in (B∼E).</p

The similar activation of hepatic Nrf2-related and ROS detoxification genes in APAP-injected mice.

<p>(A) The hepatic mRNA and (B) protein levels of GCLC at 0 and 8 hr after APAP challenge; representative and quantitative data are in upper and lower panels, respectively. Hepatic mRNA levels of Nrf2-related genes (C) NQO-1, GstPi-1, MRP-2, and MRP-3, and ROS detoxification genes (D) SOD-1, SOD-2 and catalase at 0 and 8 hr after APAP challenge. *<i>P</i><0.05; #<i>P</i><0.05 compared with WT at 0 hr; <i>n.s.</i>, not significant. Data are mean ± SEM from 7∼8 mice per group in (A), and 4∼5 mice per group in (B).</p

Effects of exogenous IL-15 or IL-15 neutralizing antibody administration on AILI in <i>Il15^−/−</i> or WT mice.

<p>Before APAP injection, twenty micrograms of recombinant murine IL-15 were subcutaneously (SC) or intraperatoneally (IP) injected into <i>Il15^−/−</i> mice. (A) Serum ALT levels and (B) representative liver histopathological changes at 8 hr after APAP injection (H&E staining; magnification ×4). One hundred and twenty micrograms of Rat anti-IL-15 neutralizing antibody or Rat IgG2a isotype control antibody were intraperitoneally injected into WT mice. (C) Serum ALT levels and (D) representative liver histopathological changes at 8 hr after APAP injection (H&E staining; magnification ×4). <i>n.s.</i>, not significant. Data represent means ± SEM from 5∼8 mice per group, and 3 mice in IL-15 SC and Rat anti-IL-15 neutralizing antibody only group.</p

Effects of neutrophil and Kupffer cell elimination on AILI in mice.

<p>(A) 1.5 mg/kg vinblastine (Vin) was intravenously injected into mice 5 days before APAP challenge of mice. (B) Mice were intravenously injected with 30 mg/kg GdCl₃ 36∼40 hr before APAP challenge. Serum ALT levels were analyzed at 4, 6 and 8 hr in <i>Il15^−/−</i> mice, and 8 hr in WT mice after APAP injection. (C) The serum IL-15 levels, and (D) the dot plot of serum ALTs and relative IL-15 levels at 8 hr after APAP injection in WT mice with or without GdCl₃ pretreatment. The ratio of ALT/IL-15 level was on the right panel. (E) Hepatic mRNA levels of TNFα and IL-1β and (F) hepatic nitrotyrosine formation (magnification ×20) at 8 hr post-APAP in WT and <i>Il15^−/−</i> mice with GdCl₃ pretreatment or not. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; <i>n.s.</i>, not significant. Data are mean ± SEM from 5∼8 mice per group.</p

Deficiency of IL-15 exacerbates APAP-induced fulminant hepatitis in mice.

<p>(A) Survival of <i>Il15^−/−</i> (n = 8) and WT (n = 10) mice for 5 days after APAP injection. <i>P</i><0.01 for <i>Il15^−/−</i> mice compared to WT controls. Serum levels of (B) ALT and (C) AST at indicated times after treatment with APAP. (D) Serum ALT levels and (E) liver histopathological changes at 8 hr after treatment with 200 mg/kg of APAP (H&E staining; magnification, upper panels, ×4; lower panel, ×10). *<i>P</i><0.05; **<i>P</i><0.001. Data are mean ± SEM from 5∼8 mice per group.</p