Carnitine palmitoyltransferase gene upregulation by linoleic acid induces CD4+ T cell apoptosis promoting HCC development - PubMed (original) (raw)
doi: 10.1038/s41419-018-0687-6.
Qiong Fu 1, Chi Ma 1, Michael Kruhlak 2, Haibo Zhang 3, Ji Luo 3, Bernd Heinrich 1, Su Jong Yu 1 4, Qianfei Zhang 1, Andrew Wilson 1, Zhen-Dan Shi 5, Rolf Swenson 5, Tim F Greten 6
Affiliations
- PMID: 29795111
- PMCID: PMC5966464
- DOI: 10.1038/s41419-018-0687-6
Carnitine palmitoyltransferase gene upregulation by linoleic acid induces CD4+ T cell apoptosis promoting HCC development
Zachary J Brown et al. Cell Death Dis. 2018.
Abstract
Hepatocellular carcinoma (HCC) is a common cause of cancer-related death worldwide. As obesity and diabetes become more prevalent, the contribution of non-alcoholic fatty liver disease (NAFLD) to HCC is rising. Recently, we reported intrahepatic CD4+ T cells are critical for anti-tumor surveillance in NAFLD. Lipid accumulation in the liver is the hallmark of NAFLD, which may perturb T cell function. We sought to investigate how the lipid-rich liver environment influences CD4+ T cells by focusing on carnitine palmitoyltransferase (CPT) family members, which control the mitochondrial β-oxidation of fatty acids and act as key molecules in lipid catabolism. Linoleic acid (C18:2) co-localized within the mitochondria along with a corresponding increase in CPT gene upregulation. This CPT upregulation can be recapitulated by feeding mice with a high-C18:2 diet or the NAFLD promoting methionine-choline-deficient (MCD) diet. Using an agonist and antagonist, the induction of CPT genes was found to be mediated by peroxisome proliferator-activated receptor alpha (PPAR-α). CPT gene upregulation increased mitochondrial reactive oxygen species (ROS) and led to cell apoptosis. In vivo, using liver-specific inducible MYC transgenic mice fed MCD diet, blocking CPT with the pharmacological inhibitor perhexiline decreased apoptosis of intrahepatic CD4+ T cells and inhibited HCC tumor formation. These results provide useful information for potentially targeting the CPT family to rescue intrahepatic CD4+ T cells and to aid immunotherapy for NAFLD-promoted HCC.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Figures
Fig. 1. Linoleic acid probes co-localize with the mitochondria.
a Schematic structure of C18:2-BODIPY probe 1 (LAP1) and probe 2 (LAP2). b Mouse 3T3 fibroblast cells were incubated with 2 µM C18:2-BODIPY Probe 1 or Probe 2 together with 10 nM MitoTracker Deep Red for 30 min. Live images were taken at 1 h or 24 h after staining. Scale bar: 20 µm
Fig. 2. Linoleic acid upregulates CPT gene expression in murine and human lymphocytes.
a Mouse splenic CD4+ T cells were isolated and treated with 100 µM fatty acids in vitro for 24 h. The CPT gene (CPT1a/CPT1b/CPT2) mRNA levels were measured by RT-qPCR. b Mouse splenic CD8+ T cells were isolated and treated as above with 100 µM fatty acids in vitro for 24 h. The CPT gene mRNA levels were measured by RT-qPCR. c Human CD4+ T cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) and treated with 100 µM fatty acids in vitro for 24 h. The CPT gene mRNA levels were measured by RT-qPCR. d Human CD8+ T cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) and treated with 100 µM fatty acids in vitro for 24 h. The CPT gene mRNA levels were measured by RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Fig. 3. NAFLD induces the expression of CPT genes in intrahepatic CD4+ T cells.
a 8-week-old female C57BL/6 mice were fed with either low C18:2 (2%, w/w) or high C18:2 (12%, w/w) diet for 8 weeks. CD4+ T cells were isolated from liver and spleen. CPT levels were measured by RT-qPCR. b 8-week-old female C57BL/6 mice were fed with MCD diet or control diet (CTR) for 4 weeks. CD4+ T cells were isolated from liver and spleen. CPT levels were measured by RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Fig. 4. C18:2 upregulates CPT gene expression through PPAR-α.
a Jurkat cells were cultured with various concentrations of the PPAR-α agonist bezafibrate in vitro for 24 h and CPT mRNA levels were measured by RT-qPCR. b Jurkat cells were treated with 200 µM C18:2 in the absence or presence of 1 µg/mL PPAR-α antagonist GW6471 in vitro for 24 h and CPT mRNA levels were measured by RT-qPCR. c Mouse splenic CD4+ T cells were isolated and treated with 100 µM C18:2 in the absence or presence of 0.25 µg/mL GW6471 in vitro for 24 h. CPT mRNA levels were measured by RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Fig. 5. CPT upregulation induces apoptosis through reactive oxygen species.
a Mitochondrial ROS level was measured by mitoSOX in Jurkat cells treated with various concentrations of the PPAR-α agonist bezafibrate in vitro for 24 h. b Jurkat cells were treated with various concentrations of bezafibrate in vitro for 24 h and apoptosis was measured by 7AAD and AnnexinV. c Jurkat cells were treated with 400 µM bezafibrate and/or 1000 U/mL catalase in vitro for 24 h and apoptosis was measured by 7AAD and AnnexinV. d Murine CD4+ T cells were treated with 100 µM C18:2 in the absence or presence of 0.25 µg/mL GW6471 in vitro for 24 h and mitochondrial ROS levels were measured by mitoSOX. e Murine CD4+ T cells and treated with 100 µM C18:2 in the absence or presence of 0.25 µg/mL GW6471 in vitro for 24 h and apoptosis was measured by 7AAD and AnnexinV. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Fig. 6. CPT inhibitor perhexiline reduces HCC incidence in MYC-ON mice fed with MCD diet.
MYC-ON mice were fed with MCD diet and injected with perhexiline or DMSO control three times per week for 5 weeks. The schematic timeline is shown in a. Liver tissue was fixed for H&E staining and the microscopic tumor number was counted. Mice were randomized to each group after doxycycline removed from drinking water (MYC-ON). Representative images of H&E staining (b) and microscopic tumor counts (c) of fixed liver tissue were shown here. d In vivo effect of perhexiline treatment on CD4+ T lymphocytes cell apoptosis. *P < 0.05
Fig. 7. Schematic picture showing CPT genes are upregulated through PPAR-α by C18:2.
C18:2 activates the lipid receptor and transcription activator PPAR-α, which translocated to nucleus and upregulates the expression of CPT genes (CPT1 and CPT2). Therefore, increased level of CPT enzymes on mitochondrial membrane promotes the mitochondrial uptake of C18:2, resulting in elevated ROS and cell apoptosis
References
- GBD 2015 Mortality and Causes of Death Collaborators. Global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 2016;388:1459–1544. doi: 10.1016/S0140-6736(16)31012-1. - DOI - PMC - PubMed
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