Application of IL-36 receptor antagonist weakens CCL20 expression and impairs recovery in the late phase of murine acetaminophen-induced liver injury

Abstract

Overdosing of the analgesic acetaminophen (APAP, paracetamol) is a major cause of acute liver injury. Whereas toxicity is initiated by hepatocyte necrosis, course of disease is regulated by mechanisms of innate immunity having the potential to serve in complex manner pathogenic or pro-regenerative functions. Interleukin (IL)-36γ has been identified as novel IL-1-like cytokine produced by and targeting epithelial (-like) tissues. Herein, we investigated IL-36γ in acute liver disease focusing on murine APAP-induced hepatotoxicity. Enhanced expression of hepatic IL-36γ and its prime downstream chemokine target CCL20 was detected upon liver injury. CCL20 expression coincided with the later regeneration phase of intoxication. Primary murine hepatocytes and human Huh7 hepatocellular carcinoma cells indeed displayed enhanced IL-36γ expression when exposed to inflammatory cytokines. Administration of IL-36 receptor antagonist (IL-36Ra) decreased hepatic CCL20 in APAP-treated mice. Unexpectedly, IL-36Ra likewise increased late phase hepatic injury as detected by augmented serum alanine aminotransferase activity and histological necrosis which suggests disturbed tissue recovery upon IL-36 blockage. Finally, we demonstrate induction of IL-36γ in inflamed livers of endotoxemic mice. Observations presented introduce IL-36γ as novel parameter in acute liver injury which may contribute to the decision between unleashed tissue damage and initiation of liver regeneration during late APAP toxicity.

Figure 1. Expression of IL-36γ in murine APAP-induced liver injury and inflamed hepatocytes.

(ab) Mice received PBS (n = 6) or APAP or (where indicated) APAP/IL-22 (6 h (n = 6), 24 h (n = 9)). (a) Hepatic IL-36γ mRNA was determined by realtime PCR. Target mRNA was normalized to that of GAPDH (means ± SEM versus ctrl; **p < 0.01, ***p < 0.001 versus ctrl; #p < 0.05). (b) Hepatic IL-36αβγ mRNAs were determined by realtime PCR. Target mRNA was normalized to that of GAPDH and is shown as raw data (2^−ddCt × 10⁷; means ± SEM; ***p < 0.001 versus ctrl of the same target gene; n.s., not significant). (ab) Statistical analysis, one-way analysis of variance with post hoc Bonferroni correction. (c) Murine primary hepatocytes were kept as unstimulated control (ctrl) or stimulated with IL-1β/TNFα/IFNγ (each 50 ng/ml). IL-36γ mRNA expression was determined by realtime PCR. Target mRNA was normalized to that of GAPDH (means ± SEM versus ctrl; n = 3). (d) Huh7 cells were kept as unstimulated ctrl or stimulated with IL-1β/TNFα/IFNγ (each 50 ng/ml). After 3 h (left panel) or 24 h (right panel), IL-36γ mRNA (left panel) or protein (right panel) was determined by realtime PCR or immunoblotting, respectively. Target mRNA was normalized to that of GAPDH (means ± SD versus ctrl; n = 8). One representative immunoblot of five independently performed experiments is shown. (cd) Statistical analysis, Student's t-test versus ctrl at the respective time point. (e) Representative murine liver immunohistochemistry of IL-1Rrp2 at 24 h after APAP application (left panel: ctrl; right panel: APAP).

Figure 2. CCL20 expression in murine APAP-induced liver injury.

(a) Mice received PBS (n = 6) or APAP (6 h, n = 6; 24 h, n = 9; 48 h, n = 11) for indicated time points. Hepatic CCL20 and IL-36γ mRNA was determined by realtime PCR. Target mRNA was normalized to that of GAPDH (means ± SEM versus ctrl of target mRNA, ***p < 0.001 and ##p < 0.01 versus ctrl at the respective time point). (b) Mice received PBS (n = 6) or APAP (24 h (n = 9) or 48 h (n = 6)) or APAP/IL-22 (24 h (n = 9) or 48 h (n = 6)). Hepatic CCL20 mRNA was determined by realtime PCR. Target mRNA was normalized to that of GAPDH (means ± SEM versus ctrl; ***p < 0.001 versus ctrl, ###p < 0.001). (ab) Statistical analysis, one-way analysis of variance with post hoc Bonferroni correction).

Figure 3. Application of IL-36Ra modulates CCL20 expression in late murine APAP-induced liver injury.

(abc) Mice received APAP (n = 11) or APAP/IL-36Ra (n = 12) and were maintained for 48 h. Thereafter, hepatic CCL20 mRNA (a) and protein (b) was determined by realtime PCR and ELISA, respectively. (a) Target mRNA was normalized to that of GAPDH and is shown as percent of APAP alone (means ± SEM). (b) Liver tissue CCL20 content was determined by ELISA, is depicted as pg/500 μg total protein, and shown as means ± SEM. (c) Hepatic CCL2 and CXCL2 mRNA was determined by realtime PCR. Target mRNA was normalized to that of GAPDH and is shown as percent of APAP alone set as 100% (means ± SEM). Statistical analysis, Student's t-test; n.s., not significant.

Figure 4. Application of IL-36Ra exacerbates tissue damage in late murine APAP-induced liver injury.

(a) Mice received either APAP (n = 16) or APAP/IL-36Ra (n = 16) and were maintained for 48 h. Thereafter, serum ALT activity was determined and is depicted as units/liter (means ± SEM). (b) Mice received either APAP (n = 8) or APAP/IL-36Ra (n = 8). Statistical analysis of necrotic areas in H&E-stained liver sections after 48 h. (ab) Statistical analysis, Student's t-test. (c) Representative liver sections (H&E staining) 48 h after the onset of APAP intoxication.