Macrophage-derived IL-1α promotes sterile inflammation in a mouse model of acetaminophen hepatotoxicity

Abstract

The metabolic intermediate of acetaminophen (APAP) can cause severe hepatocyte necrosis, which triggers aberrant immune activation of liver non-parenchymal cells (NPC). Overzealous hepatic inflammation determines the morbidity and mortality of APAP-induced liver injury (AILI). Interleukin-1 receptor (IL-1R) signaling has been shown to play a critical role in various inflammatory conditions, but its precise role and underlying mechanism in AILI remain debatable. Herein, we show that NLRP3 inflammasome activation of IL-1β is dispensable to AILI, whereas IL-1α, the other ligand of IL-1R1, accounts for hepatic injury by a lethal dose of APAP. Furthermore, Kupffer cells function as a major source of activated IL-1α in the liver, which is activated by damaged hepatocytes through TLR4/MyD88 signaling. Finally, IL-1α is able to chemoattract and activate CD11b⁺Gr-1⁺ myeloid cells, mostly neutrophils and inflammatory monocytes, to amplify deteriorated inflammation in the lesion. Therefore, this work identifies that MyD88-dependent activation of IL-1α in Kupffer cells plays a central role in the immunopathogenesis of AILI and implicates that IL-1α is a promising therapeutic target for AILI treatment.

Keywords: IL-α; Kupffer cells; TLR4; acetaminophen; sterile immunity.

Figure 1

Aberrant innate cell infiltration and activation in response to AILI. Wt mice (n=7 for each time point) were injected with 600 mg/kg APAP, i.p. (a) Intrahepatic leukocytes were counted by FACS at the indicated times. (b) Serum PICCs of mice were measured at the indicated times. ^# denotes under the detection limit.

Figure 2

IL-1α, but not IL-1β, exacerbated the immunopathology by APAP, which accounted for the mortality and morbidity of AILI. (a) IL-1α and IL-1β expression before and after APAP injection (600 mg/kg, i.p) in wt mice in liver tissue; PC and NPC were analyzed by qPCR, as indicated, n=7, for each time point. (b) Hepatic caspase-1 expression before and after APAP injection (600 mg/kg, i.p.) in wt mice was detected by immunoblot. Liver samples from LPS-treated mice (250 mg/kg, i.p.) were used as a positive control. (c) Wt, NLRP3^−/− or Caspase1^−/− mice (n⩾10) were i.p. injected with APAP. Measurement of the survival rates and analysis by the Kaplan-Meier method. (d–h) Wt mice were pre-treated with anti-IL-1α, anti-IL-1β or isotype control antibodies (200 μg/mouse, i.v.) 1 h before APAP injection (n=8 for each group, 600 mg/kg, i.p.). (d) Measurement of the survival rates. (e) Representative hepatic necrosis 24 hpi viewed by H&E staining and statistical analysis. Bar=50 μm. Measurement of the (f) serum ALT and AST levels and (g) serum levels of KC and IL-6 at the indicated time post APAP treatment. (h) Absolute numbers of infiltrated neutrophils and monocytes 24 hpi were analyzed by FACS staining. The data are presented as the mean±s.e.m. Student’s t-test. ***P<0.001; **P<0.01; *P<0.05; ns, no significant difference.

Figure 3

Kupffer cells were the main source of activated IL-1α. (a) NPCs isolated from mice (n=6 for each time point) at 0 or 6 h post APAP injection (600 mg/kg, i.p.) were analyzed by FACS for cell types that produced IL-1α. (b–e) Mice were pre-treated with control empty liposomes (PBS, WT), clodronate liposomes (CL2MBP, ΔM), or clodronate liposomes and then reconstituted with macrophages (WT→ΔM) enriched from wt donor mice (2 × 10⁶ cells in 400 μl of PBS for each mouse, i.v.). Subsequently, APAP (550 mg/kg) was i.p. injected into these mice (n=11 for each group). (b) Macrophage depletion and reconstitution efficiencies. Immunofluorescence staining of frozen liver sections by the F4/80 antibody (green). The nuclei were counterstained with DAPI (blue). Bar=50 μm. Survival rates (c), serum ALT (d) and serum cytokines (e) were analyzed 24 hpi. (f and g) Mice depleted of macrophages by clodronate liposomes (200 μl/mouse, 3 d before APAP injection) were adoptively transferred with macrophages enriched from indicated donor mice (2 × 10⁶ cells in 400 μl PBS for each mouse, i.v.). Two days later, APAP (550 mg/kg) was i.p. injected into these mice (n=8 for each group). Measurement of serum ALT (f) and serum levels of KC, IL-6 and IL-1α (g) at the indicated time post APAP treatment. The data were averaged and are presented as the mean±s.e.m. Student’s t-test. **P<0.01; *P<0.05.

Figure 4

IL-1α activation depended on TLR4/MyD88 signaling in macrophages. Mice depleted of macrophages by clodronate liposomes (200 μl/mouse, 3 d before APAP injection) were adoptively transferred with macrophages enriched from indicated donor mice (2 × 10⁶ cells in 400 μl of PBS for each mouse, i.v.). Two days later, APAP (550 mg/kg) was i.p. injected into these mice (n=8 for each group). (a) Representative hepatic necrosis and statistical analysis were performed 24 h after APAP injection. Bar=50 μm. (b) Measurement of serum ALT in macrophage reconstituted mice at the indicated time post APAP treatment. (c) Serum levels of KC, IL-6 and IL-1α at the indicated time post APAP treatment. (d) Wt macrophages were stimulated with DMSO, APAP, conditioned medium from heat-killed hepatocytes (Heat-killed CM), conditioned medium from DMSO-treated hepatocytes (DMSO CM) or conditioned medium from APAP-treated hepatocytes (APAP CM) for 16 h. Indicated cytokines in the supernatants were measured by LUMINEX assays. (e) Macrophages enriched from wt, MyD88, TLR3^−/−, TLR4^−/− or TLR7/9^−/− mice were stimulated with 100 μl of conditioned medium (described in the Methods section) for 16 h. Indicated cytokines in the supernatants were measured by LUMINEX assays. The data are presented as the mean±s.e.m. Student’s t-test. ***P<0.001; **P<0.01; *P<0.05.

Figure 5

Upregulated IL-1R in non-parenchymal cells attributed to AILI. (a) IL-1R1 expression before and after APAP injection (600 mg/kg, i.p) in wt mice in liver tissue; PC and NPC were analyzed by qPCR, as indicated, n=7, for each time point. (b) IL-1R1 expression before and after APAP injection (600 mg/kg, i.p) in wt mice on NPC was analyzed by FACS, n=7 for each time point. The data are presented as the mean±s.e.m. Student’s t-test. **P<0.01; *P<0.05.