Role of Kupffer cells and toll-like receptor 4 in acetaminophen-induced acute liver failure

Abstract

Background: Significant morbidity associated with acute liver failure (ALF) is from the systemic inflammatory response syndrome (SIRS). Toll-like receptor 4 (TLR4) has been shown to play an integral role in the modulation of SIRS. However, little is known about the mechanistic role of TLR4 in ALF. Also, no cell type has been identified as the key mediator of the TLR4 pathway in ALF. This study examines the role of TLR4 and Kupffer cells (KCs) in the development of the SIRS following acetaminophen (APAP)-induced ALF.

Materials and methods: Five groups of mice were established: untreated wild-type, E5564-treated (a TLR4 antagonist), gadolinium chloride -treated (KC-depleted), clodronate-treated (KC-depleted), and TLR4-mutant. Following APAP administration, 72-h survival, biochemical and histologic liver injury, extent of lung injury and edema, and proinflammatory gene expression were studied. Additionally, TLR4 expression was determined in livers of wild-type and KC-depleted mice.

Results: Following APAP administration, wild-type, TLR4-mutant, E5564-treated, and KC-depleted mice had significant liver injury. However, wild-type mice had markedly worse survival compared with the other four treatment groups. TLR4-mutant, E5564-treated, and KC-depleted mice had less lung inflammation and edema than wild-type mice. Selected proinflammatory gene expression (interleukin 1β, interleukin 6, tumor necrosis factor) in TLR4-mutant, E5564-treated, and KC-depleted mice was significantly lower compared with wild-type mice after acute liver injury.

Conclusion: This study demonstrates that survival in APAP-induced ALF potentially correlates with the level of proinflammatory gene expression. This study points to a link between TLR4 and KCs in the APAP model of ALF and, more importantly, demonstrates benefits of TLR4 antagonism in ALF.

Fig. 1. Liver injury and survival following APAP injection (N = 10 mice per group)

(A) Wild-type, E5564-treated, mutant, GdCl₃-treated, and clodronate-treated mice received APAP to induce acute liver injury. The clodronate-treated mice had the highest serum ALT (22222 ± 4700 U/L, *p<0.05). The E5564-treated (7755 ± 3254 U/L), mutant (9266 ± 3358 U/L), and GdCl₃-treated mice (4784 ± 2859 U/L) had significantly lower ALT compared to wild-type mice (**p<0.05). (B) Amount of liver necrosis was similar for E5564-treated (40 ± 7%), mutant (40 ± 12%), and clodronate-treated mice (65 ± 13%) when compared to wild-type mice (54 ± 18%). GdCl₃-treated mice (24 ± 12%) had significantly less liver necrosis compared to the wild-type group (p<0.05). (C) Percentage survival was determined over 72h. All untreated wild-type mice met their mortality equivalent by 28h. At 72h, 90% of mutant mice were alive, 80% of both groups of Kupffer cell-depleted mice were alive, and 70% of E5564-treated mice were alive.

Fig. 2. TLR4 agonist activity and TLR4 expression at baseline and after APAP

(A) In order to determine if the presence of TLR4 agonists were similar in wild-type mice and mutant mice after APAP exposure, 10 wild-type mice and 10 mutant mice were euthanized 12h after administration of APAP. Using a SEAP reporter assay, TLR4 agonist activity was indirectly determined and compared to saline control wild-type mice (N = 10 mice). Wild-type and mutant mice had significantly higher endogenous serum TLR4 activity 12h following APAP compared to wild-type mice at baseline (**p<0.05) (N = 10 mice per group). (B) Liver cells were isolated for baseline TLR4 mRNA expression. The NPC/KC-rich fraction had the highest expression of TLR4 followed by isolated KCs. TLR4 expression was significantly different between all groups (**p<0.05). (C) At baseline, wild-type liver had significantly higher relative TLR4 expression than GdCl₃-treated and clodronate-treated liver (*p<0.05). At 6h after APAP, wild-type liver had higher relative TLR4 expression than GdCl₃-treated and clodronate-treated liver; however, this difference was not significant**p=NS).

Fig. 3. Lung histology, inflammation, and edema 12h after APAP (N = 10 mice per group)

Wild-type, E5564-treated, mutant, GdCl₃-treated, and clodronate-treated mice received APAP to induce acute liver injury. These mice had their lungs excised 12h after APAP to determine the effect liver injury had on lung inflammation. (A) The wild-type group (2.5 ± 0.7) had a significantly higher extent of lung inflammation when individually compared to the other 4 groups (p<0.05). The E5564 (1.5 ± 0.5), mutant (1.4 ± 0.7), and Kupffer cell-depleted groups (GdCl₃ 1.3 ± 0.5, Clodronate 1.6 ± 0.5) were statistically similar in the extent of lung inflammation. Extent was grade 0–3. 0=<5% of section; 1=5-<30%; 2=30-<50%; 3=50% or more. (B) Lung edema was determined by weighing lung tissue immediately after euthanization and repeating lung weights after 72h of drying. The wild-type group (4.75 ± 0.27) had a significantly higher amount of lung edema compared to the other 4 groups (E5564 4.39 ± 0.07, mutant 4.37 ± 0.15, GdCl₃ 4.42 ± 0.19, Clodronate 4.47 ± 0.07) after 12h of APAP (**p<0.05).

Fig. 4. Fold change in gene expression of liver tissue after 6h of APAP (N = 10 mice per group)

Wild-type, E5564-treated, mutant, GdCl₃-treated, and clodronate-treated mice received APAP to induce acute liver injury. These mice were euthanized at 6h after APAP. Their livers were excised and used for pro-inflammatory cytokine gene expression analysis. Fold change in gene expression was measured for il1b, il6, and tnf from 6h liver tissue samples standardized to 0h baseline liver tissue samples. The wild-type group had significantly higher gene expression of the 3 pro-inflammatory cytokines compared to the other 4 groups (** p<0.05).