Toll-like receptor 4 is a key mediator of murine steatotic liver warm ischemia/reperfusion injury

Abstract

Steatotic donors are routinely rejected for transplantation because of their increased rate of primary nonfunction. These grafts are more sensitive to ischemia/reperfusion (I/R) during transplantation. Removal of endotoxin before reperfusion improves liver performance post-I/R. We hypothesize that the main modality of injury in steatotic livers is toll-like receptor 4 (TLR4) signaling. We fed 4-week-old control and TLR4-deficient (TLR4KO) mice a normal diet (ND) or a 60% high-fat diet (HFD) for 4 weeks to induce steatosis. Mice were subjected to total hepatic ischemia (35 minutes) and reperfusion (1 or 24 hours). Survival improved and liver pathology decreased at 24 hours in TLR4KO HFD animals compared to control HFD animals. An investigation of infiltrates showed that neutrophils and CD4+ cells were increased at 24 hours in control HFD animals, whereas TLR4KO HFD animals were similar to ND controls. Messenger RNA levels of interleukin 6 (IL-6), IL-12, and interferon gamma were elevated at 1 hour in control HFD animals, whereas TLR4KO HFD animals were similar to ND controls. IL-10 levels at 1 hour of reperfusion in control HFD and TLR4KO animals were decreased versus control ND animals. In conclusion, these improvements in liver function in TLR4KO HFD animals implicate TLR4 as a mediator of steatotic graft failure after I/R.

Figure 1

Assessment of TLR4 deficiency with respect to the liver phenotype. (A) Control HFD and (B) TLR4KO animals had similar levels of steatosis at baseline, as determined by Oil Red O staining. (C) TLR4 levels were compared via quantitative reverse-transcription polymerase chain reaction. (D) Serum endotoxin levels were measured at baseline and at 1 hour of reperfusion to assess the quantity of liberated gut endotoxin (n = 5–8/group, depending on survival). Abbreviations: HFD, high-fat diet; ND, normal diet; TLR4, toll-like receptor 4; TLR4KO, toll-like receptor 4–deficient.

Figure 2

TLR4 deficiency increases animal survival in steatotic animals following ischemia/reperfusion. TLR4 deficiency significantly increased the 24-hour animal survival of HFD mice following 35 minutes of total hepatic ischemia (*P < 0.05, n = 5–8/group). Abbreviations: HFD, high-fat diet; KO, knockout; TLR4, toll-like receptor 4.

Figure 3

TLR4 deficiency decreases hepatic injury in steatotic animals following ischemia/reperfusion. At 24 hours, there was a significant decrease in hepatic injury in TLR4KO HFD animals as assessed by circulating levels of ALT. Animals were sacrificed, and serum was collected prior to surgery (baseline) or 1 or 24 hours post-reperfusion. Values for each group (IU/L) are expressed as the mean ± the standard error of the mean (*P < 0.05 versus TLR4KO HFD, n = 5–8/group). Abbreviations: ALT, alanine aminotransferase; HFD, high-fat diet; ND, normal diet; TLR4, toll-like receptor 4; TLR4KO, toll-like receptor 4–deficient.

Figure 4

TLR4 deficiency reduces hepatocellular necrosis following ischemia/reperfusion. Prior to surgery and 1 and 24 hours after reperfusion, H&E slides were made from tissue harvested from the left hepatic lobe of each mouse. At 24 hours following reperfusion, no appreciable necrosis was observed in (A) control ND or (B) TLR4KO ND groups, as illustrated by the representative photomicrographs. (C) However, necrosis was marked in the control HFD group, in which large necrotic foci were present around central veins, whereas (D) TLR4 HFD animals showed little appreciable necrosis. (E) Grading of centrilobular necrosis for each animal was conducted on a 0 to 3 scale, scored per high-powered field, and represented graphically. At 24 hours, necrosis was significantly greater in the control HFD livers compared to the TLR4KO HFD livers, whereas it was greater for both groups in comparison with their ND controls. Values are expressed as the mean ± the standard error of the mean (*P < 0.05 versus HFD TLR4KO, n = 5–8/group). Abbreviations: HFD, high-fat diet; ND, normal diet; TLR4, toll-like receptor 4; TLR4KO, toll-like receptor 4–deficient.

Figure 5

TLR4 deficiency decreases cellular infiltration following ischemia/reperfusion. (A) Sections were stained for GR-1, a granulocyte marker for neutrophils. All groups had increased infiltration at baseline (§P < 0.05 versus baseline). Additionally, there was a significant increase in the neutrophil infiltration in control HFD animals versus TLR4KO HFD animals at 1 hour of reperfusion(*P < 0.05, n = 5–8/group). As the ND groups and the TLR4KO HFD groups returned to baseline levels at 24 hours, the control HFD animals retained a significantly elevated level of neutrophil infiltration (*P < 0.05, n = 5–8/group). (B) At baseline, there were fewer CD3+ cells in the control HFD animals versus the control ND animals (ψP < 0.05). This trend continued when the control ND animals were compared with the TLR4KO ND animals at 1 hour (ψP < 0.05). At 24 hours, the control HFD group was significantly increased over both knockout groups (ζP < 0.05). (C) There was a similar trend at baseline in the CD4+ cells, in that the control HFD animals had significantly fewer cells than the control ND animals (*P < 0.05). There were no differences at 1 hour of reperfusion, but by 24 hours, there were more infiltrating cells in the control HFD animals than in the TLR4KO HFD animals (*P < 0.05). Values are represented as the mean number of cells per HPF ± the standard error of the mean. Abbreviations: HFD, high-fat diet; HPF, high-powered field; KO, knockout; ND, normal diet; TLR4, toll-like receptor 4; TLR4KO, toll-like receptor 4–deficient.

Figure 6

Cytokine mRNA levels are altered because of TLR4 deficiency. Real-time reverse-transcriptase polymerase chain reaction was used to compare cytokine mRNA levels between groups, and the results are expressed as the fold change over the baseline. In the proinflammatory T-helper 1 cytokines, there were significant increases in IL-12, IFN-γ, and IL-6 mRNA levels at 1 hour of reperfusion in the control HFD animals versus their TLR4KO counterparts (*P < 0.05, n = 5–8/group). There was also a blunting of anti-inflammatory T-helper 2 cytokine production, as evidenced by the increased levels of IL-10 present in the ND control animals, which were dramatically decreased in the HFD control animals (*P < 0.05, n = 5–8/group). Abbreviations: HFD, high-fat diet; IFN-γ, interferon gamma; IL, interleukin; mRNA, messenger RNA; ND, normal diet; TLR4, toll-like receptor 4; TLR4KO, toll-like receptor 4–deficient.