Immune-Responsive Gene 1/Itaconate Activates Nuclear Factor Erythroid 2-Related Factor 2 in Hepatocytes to Protect Against Liver Ischemia-Reperfusion Injury - PubMed (original) (raw)

. 2020 Oct;72(4):1394-1411.

doi: 10.1002/hep.31147. Epub 2020 Oct 12.

Meihong Deng 2, Melanie J Scott 2 3, Guang Fu 1 2, Patricia A Loughran 2 4, Zhao Lei 1 2, Shilai Li 2 5, Ping Sun 2 6, Chenxuan Yang 2 7, Wenbo Li 1 2, Hongbo Xu 2, Feizhou Huang 1, Timothy R Billiar 2

Affiliations

Zhongjie Yi et al. Hepatology. 2020 Oct.

Abstract

Background and aims: Itaconate, a metabolite of the tricarboxylic acid cycle, plays anti-inflammatory roles in macrophages during endotoxemia. The mechanisms underlying its anti-inflammatory roles have been shown to be mediated by the modulation of oxidative stress, an important mechanism of hepatic ischemia-reperfusion (I/R) injury. However, the role of itaconate in liver I/R injury is unknown.

Approach and results: We found that deletion of immune-responsive gene 1 (IRG1), encoding for the enzyme producing itaconate, exacerbated liver injury and systemic inflammation. Furthermore, bone marrow adoptive transfer experiments indicated that deletion of IRG1 in both hematopoietic and nonhematopoietic compartments contributes to the protection mediated by IRG1 after I/R. Interestingly, the expression of IRG1 was up-regulated in hepatocytes after I/R and hypoxia/reoxygenation-induced oxidative stress. Modulation of the IRG1 expression levels in hepatocytes regulated hepatocyte cell death. Importantly, addition of 4-octyl itaconate significantly improved liver injury and hepatocyte cell death after I/R. Furthermore, our data indicated that nuclear factor erythroid 2-related factor 2 (Nrf2) is required for the protective effect of IRG1 on mouse and human hepatocytes against oxidative stress-induced injury. Our studies document the important role of IRG1 in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that the IRG1/itaconate pathway activates Nrf2-mediated antioxidative response in hepatocytes to protect liver from I/R injury.

Conclusions: Our data expand on the importance of IRG1/itaconate in nonimmune cells and identify itaconate as a potential therapeutic strategy for this unfavorable postsurgical complication.

© 2020 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of American Association for the Study of Liver Diseases.

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Figures

Figure 1

Figure 1

Deletion of IRG1 exacerbates liver injury after I/R. WT (C57BL/6NJ) and IRG1 KO (IRG1−/−) mice were subjected to liver ischemia for 1 hour and then reperfusion for 0, 6, and 24 hours (I/R 1/0, 1/6, 1 hour/24 hours) and sham surgery (sham) as control. (A) Serum ALT levels in WT and IRG1−/− mice after sham surgery or 1‐hour ischemia and 0‐hour reperfusion; n = 3 in each sham group, n = 6 in each I/R group. (B) Serum ALT levels in WT and IRG1−/− mice after sham surgery or 1‐hour ischemia and 6‐hour reperfusion; n = 3 in each sham group, n = 7‐9 in each I/R group. (C) Serum ALT levels in WT and IRG1−/− mice after sham surgery or 1‐hour ischemia and 24‐hour reperfusion; n = 3 in each sham group, n = 5‐6 in each I/R group. (D) Representative liver H&E staining (original magnification ×20) of WT and IRG1−/− mice after sham surgery or 1‐hour ischemia and 0‐, 6‐, or 24‐hour reperfusion. (E) Dotted lines in liver H&E staining indicate measured areas of necrosis, quantified in bar graph; n = 4 for each group. (F) Liver damages were evaluated by Suzuki’s histological score (I/R 1 hour/6 hours), quantified in bar graph; n = 5 for each group. (G) RT‐PCR for IRG1 mRNA expression in livers from WT mice after sham surgery or 1‐hour ischemia and 6‐hour reperfusion; n = 3 for sham group, n = 4 for I/R group. (H) LC‐MS analysis of itaconate abundance in livers from WT mice after sham surgery or 1‐hour ischemia and 6‐hour reperfusion; n = 4 for sham, n = 5 for I/R group. Data are presented as means ± SD. *P < 0.05, **P < 0.01. Abbreviation: NS, not significant.

Figure 2

Figure 2

Increased systemic inflammation and hepatic neutrophil infiltration in IRG1−/− mice after I/R. (A) ELISA for plasma IL‐6 levels of WT (C57BL/6NJ) and IRG1−/− mice after sham surgery or 1‐hour ischemia and 6‐hour reperfusion; n = 3 in each sham group, n = 6 in each I/R group. (B‐D) RT‐PCR for mRNA expression of IL‐6, MCP‐1, and Ly6G in livers from WT and IRG1−/− mice after sham surgery or 1‐hour ischemia and 6‐hour reperfusion; n = 3 for sham group, n = 5 for I/R group. (E) MPO levels in livers of WT and IRG1−/− mice after sham surgery or 1‐hour ischemia and 6‐hour reperfusion; n = 3 for sham group, n = 5 for I/R group. (F) Immunofluorescence images of livers from WT and IRG1−/− mice after 1‐hour ischemia and 6‐hour reperfusion; liver tissues were stained with Ly6G (white), Hoecsht nuclear stain (blue), and β‐actin (green); images were taken using confocal microscopy (scale bar, 50 µm). (G) Number of infiltrating neutrophils was determined by Ly6G (no.)/total nuclei (no.) and is represented in bar graph; n = 5 for each group. Images are representative of data from multiple mice per experimental group. Data are presented as means ± SD. *P < 0.05. Abbreviation: DAPI, 4´,6‐diamidino‐2‐phenylindole.

Figure 3

Figure 3

Deletion of IRG1 in either hematopoietic or nonhematopoietic compartments increases liver injury. (A) RT‐PCR for IRG1 mRNA expression in hepatocytes and NPCs isolated from WT (C57BL/6NJ) mice after sham surgery (sham) or 1‐hour ischemia and 6‐hour reperfusion (I/R); n = 4 for sham group, n = 5 for I/R group. Chimeric mice were constructed by adoptive transfer of donor bone marrow cells into irradiated recipient animals using the following recipient/donor combinations of WT (C57BL/6NJ) and IRG1−/− (KO) mice: WT/WT, WT/KO, KO/WT, KO/KO. All chimeric mice were monitored 3 times weekly for the first 2 weeks to ensure successful bone marrow engraftment. Then, the chimeric mice underwent hepatic I/R (1 hour/6 hour) after 8‐12 weeks to ensure stable engraftment. (B) Serum ALT levels in each group of chimeric mice after 1‐hour ischemia and 6‐hour reperfusion; n = 5‐6 in each group. (C) Representative liver H&E staining (original magnification ×20) from each group of chimeric mice after 1‐hour ischemia and 6‐hour reperfusion. (D) Dotted lines in liver H&E staining indicate measured areas of necrosis, quantified in bar graph; n = 4 in each group. (E) LC‐MS analysis of itaconate levels in livers from each group of chimeric mice after 1‐hour ischemia and 6‐hour reperfusion; n = 5 for each group. Images are representative of data from multiple mice per experimental group. Data are presented as means ± SD. *P < 0.05, **P < 0.01.

Figure 4

Figure 4

IRG1 protects hepatocytes from oxidative injury in vivo and in vitro. (A) Confocal microscopic images of TMR (red), Hoecsht nuclear stain (blue), and β‐actin (green) in liver sections from WT (C57BL/6NJ) and IRG1−/− mice subjected to sham surgery or liver ischemia for 1 hour and reperfusion for 6 hours (scale bar, 50 µm), (B) Percentage of TMR red–positive cells was quantified and is represented in bar graph; n = 4 per group. (C) Western blot for apoptosis markers (cleaved caspase‐3, cleaved caspase‐7, etc.) in whole‐liver lysates from WT and IRG1−/− mice subjected to sham surgery or liver I/R (1 hour/6 hours). (D) RT‐PCR for IRG1 mRNA expression in WT hepatocytes subjected to normoxia (control) or 10‐hour hypoxia and 0‐hour, 4‐hour, or 10‐hour reoxygenation (10/0, 10/4, 10 hours/10 hours); n = 5 for each group. (E) IRG1 protein level in WT hepatocytes subjected to normoxia (control) or 10‐hour hypoxia and 0‐hour, 4‐hour, or 10‐hour reoxygenation (10/0, 10/4, 10 hours/10 hours); n = 6 for each group. (F) LDH release (lytic cell death) in WT (C57BL/6NJ) and IRG1−/− hepatocytes subjected to normoxia or 10‐hour hypoxia and 10‐hour reoxygenation (H/R); n = 8 for normoxia group, n = 24 for H/R group. (G) Western blot for apoptosis markers (cleaved caspase‐3, cleaved caspase‐7, etc.) in whole‐cell lysates from WT and IRG1−/− hepatocytes after normoxia or 10‐hour hypoxia and 10‐hour reoxygenation (H/R). (H,I) Western blot for Myc‐Tag, cleaved caspase‐3, and caspase‐3 in whole‐cell lysates from IRG1−/− hepatocytes transfected with IRG1‐expressing plasmid (IRG1) or empty vector followed by normoxia or H/R (10 hour/10 hour). (J) LDH release in IRG1−/− hepatocytes transfected with IRG1 plasmid or empty vector followed by H/R (10 hour/10 hour); n = 8 in each group. Images are representative of data from multiple mice per experimental group. Data are presented as means ± SD. *P < 0.05, **P < 0.01. Abbreviations: cl, cleaved; NS, not significant.

Figure 5

Figure 5

Itaconate ameliorates hepatic I/R injury and rescues hepatocytes from H/R. WT (C57BL/6NJ, B6N) and IRG1−/− mice were subjected to ischemia for 1 hour and then reperfusion for 6 hours. 4‐OI or vehicle control (25 mg/kg body weight) was injected intraperitoneally 2 hours before hepatic I/R and at the time of reperfusion. (A) Serum ALT levels in each group of mice after 1‐hour ischemia and 6‐hour reperfusion; n = 7‐8 in each group. (B) Representative liver H&E staining (original magnification ×20) from each group of mice after 1‐hour ischemia and 6‐hour reperfusion. (C) Dotted lines in liver H&E staining indicate measured areas of necrosis, quantified in bar graph; n = 3 in each group. Primary hepatocytes isolated from WT mice were pretreated with 4‐OI or vehicle control 1 hour prior to normoxia or 10‐hour hypoxia and 10‐hour reoxygenation (H/R). (D) Cell death was assessed by LDH release from hepatocytes after normoxia or H/R; n = 8 for each group. (E,F) Western blot for apoptosis markers (cleaved caspase‐3, cleaved caspase‐7, etc.) in whole‐cell lysates from hepatocytes after normoxia or H/R. Primary hepatocytes isolated from IRG1−/− mice were pretreated with 4‐OI (125 µM) or vehicle control 1 hour prior to normoxia or 10‐hour hypoxia and 10‐hour reoxygenation (H/R). (G) Cell death was accessed by LDH release from hepatocytes after H/R; n = 8 for each group. (H) Western blot for cleaved caspase‐3 and caspase‐3 in whole‐cell lysates from hepatocytes after normoxia or H/R. Images are representative of data from multiple mice per experimental group. Data are presented as means ± SD. *P < 0.05, **P < 0.01. Abbreviation: cl, cleaved.

Figure 6

Figure 6

Deficiency of IRG1 suppressed Nrf2 pathway activation in hepatocytes after oxidative stress. (A,B) Western blot for Nrf2 and its downstream mediators (HO‐1 and NQO1) in whole‐liver lysates from WT (C57BL/6NJ) and IRG1−/− mice subjected to sham surgery or liver I/R (1 hour/6 hours). Quantitation of band density was performed across at least three separate blots and is presented in bar graph. (C) Confocal microscopic images of 4‐HNE (red), Hoecsht nuclear stain (blue), and β‐actin (green) in liver sections from WT and IRG1−/− mice subjected to sham surgery or liver ischemia for 1 hour and reperfusion for 6 hours (scale bar, 50 µm). Area of 4‐HNE staining was quantified by total nuclei number and is represented in bar graph; n = 3 for sham group, n = 4 for I/R group. (D) Western blot for Nrf2 protein in whole‐cell lysates from WT (C57BL/6NJ) and IRG1−/− hepatocytes subjected to normoxia or 10‐hour hypoxia and 10‐hour reoxygenation (H/R). (E) Total nuclear protein was extracted from WT and IRG1−/− hepatocytes after normoxia or H/R (10 hours/10 hours); nuclear Nrf2 was assessed by western blot. (F) Western blot for HO‐1 and NQO1 in whole‐cell lysates from WT and IRG1−/− hepatocytes subjected to normoxia or 10‐hour hypoxia and 10‐hour reoxygenation. Quantitation of band density above was performed across at least three separate blots and is presented in bar graph. (G) Cellular ROS was measured by DCFDA mean fluorescence intensity. Results were presented as percentage change of ROS (over respective normoxic controls) in hepatocytes subjected to H/R (10 hours/10 hours); n = 8 for each group. Images are representative of data from multiple mice per experimental group. Data are presented as means ± SD. *P < 0.05, **P < 0.01. Abbreviations: MFI, mean fluorescence intensity; Nu, nuclear.

Figure 7

Figure 7

Itaconate protects hepatocytes against oxidative stress damage in an Nrf2‐dependent manner. Primary hepatocytes isolated from WT (C57BL/6J) mice were pretreated with 4‐OI or vehicle control 1 hour prior to normoxia or 10‐hour hypoxia and 10‐hour reoxygenation (H/R). (A,B) Western blot for Nrf2 and its downstream mediators (HO‐1 and NQO1) in whole‐cell lysates from hepatocytes subjected to normoxia or H/R. (C) Total nuclear protein was extracted from hepatocytes after normoxia or H/R. Nuclear Nrf2 was assessed by western blot. (D) Cellular ROS was measured by DCFDA mean fluorescence intensity. Results were presented as percentage change of ROS (over respective normoxic controls) in hepatocytes subjected to H/R; n = 8 for each group. WT (C57BL/6J, B6J) and Nrf2−/− mice were subjected to ischemia for 1 hour and then reperfusion for 6 hours (I/R). 4‐OI or vehicle control (25 mg/kg body weight) was injected intraperitoneally 2 hours before hepatic I/R and at the time of reperfusion. (E) Serum ALT levels in each group of mice after hepatic I/R; n = 6‐7 in each group. (F) Representative liver H&E staining (original magnification ×20) from each group of mice after hepatic I/R. (G) Dotted lines in liver H&E staining indicate measured areas of necrosis, quantified in bar graph; n = 3 in each group. Primary hepatocytes isolated from WT (C57BL/6J, B6J) and Nrf2−/− mice were pretreated with 4‐OI (125 µM) or vehicle control 1 hour prior to normoxia or H/R (10 hours/10 hours). (H) Cell death was assessed by LDH release from hepatocytes after normoxia or H/R; n = 8 for each group. (I) Western blot for Nrf2 and HO‐1 in whole‐cell lysates from hepatocytes after normoxia or H/R. (J) Western blot for Nrf2 and HO‐1 in whole‐cell lysates from primary human hepatocytes pretreated with 4‐OI (125 µM) or vehicle control 1 hour before normoxia or 10‐hour hypoxia and 10‐hour reoxygenation (H/R). Images are representative of data. Data are presented as means ± SD. *P < 0.05, **P < 0.01. Abbreviations: MFI, mean fluorescence intensity; NS, not significant; Nu, nuclear.

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