Interleukin-10 disrupts liver repair in acetaminophen-induced acute liver failure

Abstract

Introduction: Systemic levels of the anti-inflammatory cytokine interleukin 10 (IL-10) are highest in acetaminophen (APAP)-induced acute liver failure (ALF) patients with the poorest prognosis. The mechanistic basis for this counterintuitive finding is not known, as induction of IL-10 is hypothesized to temper the pathological effects of immune cell activation. Aberrant production of IL-10 after severe liver injury could conceivably interfere with the beneficial, pro-reparative actions of immune cells, such as monocytes.

Methods: To test this possibility, we determined whether IL-10 levels are dysregulated in mice with APAP-induced ALF and further evaluated whether aberrant production of IL-10 prevents monocyte recruitment and/or the resolution of necrotic lesions by these cells.

Results: Our studies demonstrate that in mice challenged with 300 mg/kg acetaminophen (APAP), a hepatotoxic dose of APAP that fails to produce ALF (i.e., APAP-induced acute liver injury; AALI), Ly6C^hi monocytes were recruited to the liver and infiltrated the necrotic lesions by 48 hours coincident with the clearance of dead cell debris. At 72 hours, IL-10 was upregulated, culminating in the resolution of hepatic inflammation. By contrast, in mice treated with 600 mg/kg APAP, a dose that produces clinical features of ALF (i.e., APAP-induced ALF; AALF), IL-10 levels were markedly elevated by 24 hours. Early induction of IL-10 was associated with a reduction in the hepatic numbers of Ly6C^hi monocytes resulting in the persistence of dead cell debris. Inhibition of IL-10 in AALF mice, beginning at 24 hours after APAP treatment, increased the hepatic numbers of monocytes which coincided with a reduction in the necrotic area. Moreover, pharmacologic elevation of systemic IL-10 levels in AALI mice reduced hepatic myeloid cell numbers and increased the area of necrosis.

Discussion: Collectively, these results indicate that during ALF, aberrant production of IL-10 disrupts the hepatic recruitment of monocytes, which prevents the clearance of dead cell debris. These are the first studies to document a mechanistic basis for the link between high IL-10 levels and poor outcome in patients with ALF.

Keywords: acetaminophen; acute liver failure; inflammation; interleukin-10; monocytes.

Figure 1

Liver injury and inflammation in APAP-treated mice. Mice were treated with either 300 mg/kg APAP (AALI) or 600 mg/kg APAP (AALF) for the indicated time. (A) Photomicrographs of H&E-stained liver sections. White arrows indicate areas of necrosis occurring in centrilobular regions. Higher power image of necrotic region in inset in 24 hour images. Arrows indicate evidence of inflammatory infiltrates. (B) Area of necrosis was quantified in sections of liver. *Significantly different from mice treated with 300 mg/kg APAP. All data are expressed as mean ± SEM; n = 5-10 mice per group.

Figure 2

Accumulation of myeloid cells in the livers of mice treated with APAP. Mice were treated with either 300 mg/kg APAP (AALI) or 600 mg/kg APAP (AALF)for the indicated time. (A) Photomicrographs of CD68 immunofluorescent staining in liver sections. Positive staining appears red. Arrows indicate centrilobular regions. (B) The area of CD68 immunofluorescent staining was quantified in sections of liver. *Significantly different from mice treated with 300 mg/kg APAP. Data are expressed as mean ± SEM; n = 5 mice per group.

Figure 3

Hepatic myeloid cell accumulation in APAP-treated mice. Mice were treated with either 300 mg/kg APAP (AALI) or 600 mg/kg APAP (AALF)for the indicated time. (A) Gating for flow cytometry. (B) At 24 hours after APAP treatment, flow cytometry was used to detect F4/80⁺, Ly6C⁺, and Ly6G⁺ cells in the liver. Boxes indicate the positive gate. (C) Absolute cell counts and the percentage of each myeloid cell population within the larger CD45⁺ population by flow cytometry. *Significantly different from mice treated with 300 mg/kg APAP. All data are expressed as mean ± SEM; n = 5-10 mice per group.

Figure 4

Hepatic and systemic levels of IL-10 in APAP-treated mice. Mice were treated with either 300 mg/kg APAP (AALI) or 600 mg/kg APAP (AALF). (A) At the indicated time, IL-10 mRNA levels were measured in the liver. (B) IL-10 protein was measured in serum at 72 hours after APAP treatment. (C) F4/80⁺ and Ly6C⁺ myeloid cells were isolated from the liver at 24 hours after treatment of mice with either 300 mg/kg APAP (AALI) or 600 mg/kg APAP (AALF), and IL-10 mRNA levels were measured. (D) Nonparenchymal cells were isolated from the livers of IL-10 reporter mice treated 24 hours earlier with 300 mg/kg APAP (AALI) or 600 mg/kg APAP (AALF). Flow cytometry was used to identify IL-10 expressing F4/80⁺ cells. Gate for F4/80⁺ cells indicated in the density plots. Representative histogram of IL-10 expression (i.e., GFP⁺) in F4/80⁺ cells. (E) Quantification of the number of F4/80⁺ cells expressing IL-10 in the liver from flow cytometry. *Significantly different from mice treated with 300 mg/kg APAP. Data are expressed as mean ± SEM; n = 4-5 mice per group.

Figure 5

IL-10 expressing (GFP⁺) cells in the livers of mice with AALF. (A, B) Representative dot plots of the indicated cell types expressing IL-10. (C) Percentage of each immune cell type expressing IL-10 in AALF.

Figure 6

Immune suppressive phenotype of myeloid cells in APAP induced ALF. (A) Mice were treated with 600 mg/kg APAP. After 24 hours, flow cytometry was used to detect IL-10 expressing F4/80⁺ cells, indicated by the blue box. Representative histograms of IL-10⁺ F4/80⁺ cells expressing the indicated marker (x-axis). Positive staining indicated by the blue shaded box. (B) Quantification of the flow cytometry in (A) Data are expressed as mean ± SEM; n = 4 mice per group. (C) Analysis of GEO dataset accession number GSE8075, containing gene array analysis of monocytes isolated from patients with APAP-induced ALF that survived and patients with APAP induced ALF that died. Fold change represents ALF patients that died relative to those that survived (e.g., IL-10 mRNA levels 2.43-fold higher in patients that died; indicated with a green arrow and highlighted green). (D) Ingenuity pathway analysis was used to identify upstream regulators in the GSE8075 GEO dataset. Regulators associated with MDSCs are highlighted in red. (n=6 patient samples per group).

Figure 7

Impact of IL-10 neutralization in mice with APAP-induced ALF. Mice were treated with 600 mg/kg APAP (AALF) followed by treatment with control IgG or anti-IL-10 antibody 24 hours later. Livers were collected 72 hours after APAP treatment. (A-D) mRNA levels of the indicated protein were quantified in the liver by real-time PCR. (E) Serum activity of ALT. (F) The number of PCNA positive cells was quantified in sections of liver. Representative photomicrographs of immunohistochemistry staining for PCNA in liver sections from AALF mice treated with (G) isotype control IgG or (H) anti-IL-10 antibody. Positive staining appears dark brown. *Significantly different from mice treated with 600 mg/kg APAP and control IgG. All data are expressed as mean ± SEM; n = 5 mice per group.

Figure 8

Impact of IL-10 on inflammation in mice treated with APAP. (A) Mice were treated with 600 mg/kg APAP (AALF) followed by treatment with control IgG or anti-IL-10 antibody 24 hours later. Livers were collected 72 hours after APAP treatment. CD68 was detected by immunofluorescence (red staining) in sections of liver. Arrows indicate centrilobular regions. (B) Mice were treated with 300 mg/kg APAP (AALI) followed by treatment with either vehicle or 5 mg recombinant IL-10 24 hours later. Livers were collected 48 hours after APAP treatment. CD68 was detected by immunofluorescence (red staining) in sections of liver. Arrows indicate centrilobular regions. (A, B) Representative photomicrographs from an n = 5 mice per group. (C, D) The area of CD68 staining was quantified in whole liver sections. n = 5 mice per group. *Significantly different at p<0.05.

Figure 9

Impact of IL-10 on liver histology in mice treated with APAP. (A–C) Mice were treated with 600 mg/kg APAP (AALF) followed by treatment with control IgG or anti-IL-10 antibody 24 hours later. Livers were collected 72 hours after APAP treatment. (D–F) Mice were treated with 300 mg/kg APAP (AALI) followed by treatment with vehicle or 5 mg recombinant IL-10 24 hours later. Livers were collected 48 hours after APAP treatment. Representative photomicrographs of hematoxylin and eosin-stained liver sections from mice treated with (A) 600 mg/kg APAP (AALF) and control IgG, (B) 600 mg/kg APAP (AALF) and anti-IL-10 antibody, (D) 300 mg/kg APAP (AALI) and vehicle or (E) 300 mg/kg APAP (AALI) and recombinant IL-10. (C, F) The area of necrosis was quantified in the indicated liver sections. n = 5 mice per group. *Significantly different at p<0.05.