Cellular-specific role of toll-like receptor 4 in hepatic ischemia-reperfusion injury in mice

Abstract

Ischemia-reperfusion (I/R) injury is a process whereby an initial hypoxic insult and subsequent return of blood flow leads to the propagation of innate immune responses and organ injury. The necessity of the pattern recognition receptor, Toll-like receptor (TLR)4, for this innate immune response has been previously shown. However, TLR4 is present on various cell types of the liver, both immune and nonimmune cells. Therefore, we sought to determine the role of TLR4 in individual cell populations, specifically, parenchymal hepatocytes (HCs), myeloid cells, including Kupffer cells, and dendritic cells (DCs) subsequent to hepatic I/R. When HC-specific (Alb-TLR4(-/-) ) and myeloid-cell-specific (Lyz-TLR4(-/-) ) TLR4 knockout (KO) mice were subjected to warm hepatic ischemia, there was significant protection in these mice, compared to wild type (WT). However, the protection afforded in these two strains was significantly less than global TLR4 KO (TLR4(-/-) ) mice. DC-specific TLR4(-/-) (CD11c-TLR4(-/-) ) mice had significantly increased hepatocellular damage, compared to WT mice. Circulating levels of high-mobility group box 1 (HMGB1) were significantly reduced in Alb-TLR4(-/-) mice, compared to WT, Lyz-TLR4(-/-) , CD11c-TLR4(-/-) mice and equivalent to global TLR4(-/-) mice, suggesting that TLR4-mediated HMGB1 release from HCs may be a source of HMGB1 after I/R. HCs exposed to hypoxia responded by rapidly phosphorylating the mitogen-activated protein kinases, c-Jun-N-terminal kinase (JNK) and p38, in a TLR4-dependent manner; inhibition of JNK decreased release of HMGB1 after both hypoxia in vitro and I/R in vivo.

Conclusion: These results provide insight into the individual cellular response of TLR4. The parenchymal HC is an active participant in sterile inflammatory response after I/R through TLR4-mediated activation of proinflammatory signaling and release of danger signals, such as HMGB1.

Figure 1. Confirmation of specificity of TLR4 knockout (TLR4^-/-)

(A) RT-PCR was used to determine mRNA levels of TLR4 within either isolated hepatocytes or non-parenchymal cells (NPCs) from WT, Alb-TLR4^-/-, and TLR4^-/- mice. β-actin was used as endogenous control to determine relative expression compared against WT. (B) TLR4 protein levels were assessed by Western blot analysis. (C) NPCs were isolated and stimulated with LPS for 6h and the supernatant was analyzed by ELISA for either TNF-α or IL-6. (D) RT-PCR analysis of peritoneal macrophages to determine TLR4 mRNA expression in Lyz-TLR4^-/-. (E) LPS response was determined in peritoneal macrophages from WT, Lyz-TLR4^-/-, and TLR4^-/- mice. Data represent mean ± SD. N=2 samples per group run in duplicate. Figure is representative of two experiments with similar results.

Figure 2. Cellular specific role of TLR4 on hepatocellular injury after I/R

(A) Serum ALT levels were analyzed in WT, cell specific and global TLR4^-/- mice after either sham laparotomy or 1h of ischemia and 6h of reperfusion. (B) H&E stained liver sections from obtained from mice 6h after reperfusion (original magnification ×100). Images are representative liver sections from six mice per group. Necrotic area is outlined in black dashed line and percent of area that is necrotic is indicated. (C) Serum cytokine analysis after I/R was performed. (D) The number of neutrophils infiltrating into liver tissue after I/R as assessed by IHC of neutrophils per HPF (×200). Data represents mean ± SEM; n≥6 mice per group; *P<0.05 when compared against WT; #P<0.05 when compare against global TLR4^-/-.

Figure 3. Cellular specific role of TLR4 on HMGB1 release after I/R

(A) Serum HMGB1 ELISA after 6h of reperfusion. *P<0.05 when compared against WT. (B) Serum HMGB1 ELISA in WT, Alb-TLR4^-/-, and TLR4^-/- after either 3h or 6h of reperfusion. *P<0.05 when compared against WT. (C) Immunofluorescence of liver tissue after 3h of reperfusion. Red, HMGB1; green, F-actin; blue, nuclei (original magnification ×600). Data represents mean ± SEM; n≥6 mice per group mice per group.

Figure 4. HO-1 and IL-10 mRNA and protein expression is altered in Lyz-TLR4^-/- and CD11c-TLR4^-/- mice after I/R

Real-time PCR was used to determine (A) HO-1 and (C) IL-10 expression in liver tissue after I/R in Lyz-TLR4^-/- and CD11c-TLR4^-/- mice. Protein quantification was determined using Western blot analysis for HO-1 (B) and Cytometric Bead Array assay for IL-10 (D). β-actin was used as endogenous control to determine relative expression compared against WT. *P<0.05 when compared against WT. Figures A and C are representative from triplicate experiments with pooled RNA from 6 animals per group. Figure B shows two representative animals for each group.

Figure 5. Mitogen-activated protein (MAP) kinase activation is decreased in Alb-TLR4^-/- mice after I/R

Western blot of phosphorylated or total MAP kinases from protein isolated from either ischemic lobe or non-ischemic lobe after 1h of ischemia and 3h of reperfusion. Figure is representative from 3 experiments of 4 animals each with similar results.

Figure 6. Hypoxia induced JNK and p38 phosphorylation is TLR4-depedent

(A) Western blot of p-JNK and p-p38 after WT hepatocytes were exposed to hypoxia for the indicated time period. (B) Western blot of phosphorylated and total MAP kinase levels during hypoxia with comparison between WT and TLR4^-/-. (C) Western blot analysis of TLR4 expression after TLR4^-/-hepatocytes were transfected with AdTLR4 or the control AdLacZ. (D) Western blot analysis of p-JNK, and p-p38 after WT, TLR4^-/-, KO+AdLacZ, or KO+AdTLR4 hepatocytes (MOI 100) were exposed to hypoxia for 30min. Figures are representative of three trials with identical results.

Figure 7. HMGB1 release from hepatocytes under hypoxic stress is JNK-dependent

JNK inhibitor (SP600125; 25μM) or negative control (NC) was added to the media of hepatocytes and they were either exposed to hypoxia or normoxia for 30min. (A) Western blot analysis of JNK and c-Jun activation in absence or presence of JNK inhibitor. (B) Western blot of hepatocyte supernatant for HMGB1 after 24h of either normoxia or hypoxia. Western blot of the supernatant was also performed for β-actin and LDH to estimate cellular death. (C) Western blot for in vivo confirmation of JNK inhibitor efficacy after 1h of ischemia and 3h of reperfusion. Representative samples from 2 mice of each group are shown. (D) ELISA of serum HMGB1 levels after either negative control or JNK inhibitor were given prior to I/R. Data represents mean ± SEM; n≥6 mice per group; *P<0.05.

Figure 8

Schematic summary highlighting role TLR4 plays on individual cell populations after hepatic ischemia-reperfusion. (MPT, mitochondrial permeability transition; ROS, reactive oxygen species)