NAFLD causes selective CD4+ T lymphocyte loss and promotes hepatocarcinogenesis

Abstract

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related death. Non-alcoholic fatty liver disease (NAFLD) affects a large proportion of the US population and is considered to be a metabolic predisposition to liver cancer1,2,3,4,5. However, the role of adaptive immune responses in NAFLD-promoted HCC is largely unknown. Here we show, in mouse models and human samples, that dysregulation of lipid metabolism in NAFLD causes a selective loss of intrahepatic CD4+ but not CD8+ T lymphocytes, leading to accelerated hepatocarcinogenesis. We also demonstrate that CD4+ T lymphocytes have greater mitochondrial mass than CD8+ T lymphocytes and generate higher levels of mitochondrially derived reactive oxygen species (ROS). Disruption of mitochondrial function by linoleic acid, a fatty acid accumulated in NAFLD, causes more oxidative damage than other free fatty acids such as palmitic acid, and mediates selective loss of intrahepatic CD4+ T lymphocytes. In vivo blockade of ROS reversed NAFLD-induced hepatic CD4+ T lymphocyte decrease and delayed NAFLD-promoted HCC. Our results provide an unexpected link between lipid dysregulation and impaired anti-tumour surveillance.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: NAFLD induces a selective loss of intrahepatic CD4+ T lymphocytes and promotes HCC.
Figure 2: Depletion of intrahepatic CD4+ T lymphocytes accelerates tumour development in MYC-ON MCD mice.
Figure 3: Lipid-laden hepatocytes cause CD4+ T lymphocyte death through releasing C18:2.
Figure 4: Mitochondrial ROS mediates C18:2-induced CD4+ T lymphocyte death.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Microarray data have been deposited in the Gene Expression Omnibus under accession number GSE67918.

References

  1. 1

    European Association For The Study Of The Liver & European Organisation For Research And Treatment Of Cancer. EASL-EORTC clinical practice guidelines: management of hepatocellular carcinoma. J. Hepatol. 56, 908–943 (2012)

  2. 2

    Sun, B. & Karin, M. Obesity, inflammation, and liver cancer. J. Hepatol. 56, 704–713 (2012)

  3. 3

    Michelotti, G. A., Machado, M. V. & Diehl, A. M. NAFLD, NASH and liver cancer. Nature Rev. Gastroenterol. Hepatol. 10, 656–665 (2013)

  4. 4

    Schuppan, D. & Schattenberg, J. M. Non-alcoholic steatohepatitis: pathogenesis and novel therapeutic approaches. J. Gastroenterol. Hepatol. 28 (suppl. 1), 68–76 (2013)

  5. 5

    Wree, A., Broderick, L., Canbay, A., Hoffman, H. M. & Feldstein, A. E. From NAFLD to NASH to cirrhosis—new insights into disease mechanisms. Nature Rev. Gastroenterol. Hepatol. 10, 627–636 (2013)

  6. 6

    Greten, T. F., Wang, X. W. & Korangy, F. Current concepts of immune based treatments for patients with HCC: from basic science to novel treatment approaches. Gut 64, 842–848 (2015)

  7. 7

    Greten, T. F., Duffy, A. G. & Korangy, F. Hepatocellular carcinoma from an immunologic perspective. Clin. Cancer Res. 19, 6678–6685 (2013)

  8. 8

    Wolf, M. J. et al. Metabolic activation of intrahepatic CD8+ T cells and NKT cells causes nonalcoholic steatohepatitis and liver cancer via cross-talk with hepatocytes. Cancer Cell 26, 549–564 (2014)

  9. 9

    Shachaf, C. M. et al. MYC inactivation uncovers pluripotent differentiation and tumour dormancy in hepatocellular cancer. Nature 431, 1112–1117 (2004)

  10. 10

    Rinella, M. E. et al. Mechanisms of hepatic steatosis in mice fed a lipogenic methionine choline-deficient diet. J. Lipid Res. 49, 1068–1076 (2008)

  11. 11

    Yang, L. et al. Transforming growth factor β signaling in hepatocytes participates in steatohepatitis through regulation of cell death and lipid metabolism in mice. Hepatology 59, 483–495 (2014)

  12. 12

    Park, E. J. et al. Dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing IL-6 and TNF expression. Cell 140, 197–208 (2010)

  13. 13

    Kapanadze, T. et al. Regulation of accumulation and function of myeloid derived suppressor cells in different murine models of hepatocellular carcinoma. J. Hepatol. 59, 1007–1013 (2013)

  14. 14

    Xia, S. et al. Gr-1+ CD11b+ myeloid-derived suppressor cells suppress inflammation and promote insulin sensitivity in obesity. J. Biol. Chem. 286, 23591–23599 (2011)

  15. 15

    Baeck, C. et al. Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury. Gut 61, 416–426 (2012)

  16. 16

    Henning, J. R. et al. Dendritic cells limit fibroinflammatory injury in nonalcoholic steatohepatitis in mice. Hepatology 58, 589–602 (2013)

  17. 17

    Ma, X. et al. A high-fat diet and regulatory T cells influence susceptibility to endotoxin-induced liver injury. Hepatology 46, 1519–1529 (2007)

  18. 18

    Rakhra, K. et al. CD4+ T cells contribute to the remodeling of the microenvironment required for sustained tumor regression upon oncogene inactivation. Cancer Cell 18, 485–498 (2010)

  19. 19

    Kang, T. W. et al. Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature 479, 547–551 (2011)

  20. 20

    Kreiter, S. et al. Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature 520, 692–696 (2015)

  21. 21

    Giesbertz, P. et al. Metabolite profiling in plasma and tissues of ob/ob and db/db mice identifies novel markers of obesity and type 2 diabetes. Diabetologia 58, 2133–2143 (2015)

  22. 22

    Schönfeld, P. & Wojtczak, L. Fatty acids as modulators of the cellular production of reactive oxygen species. Free Radic. Biol. Med. 45, 231–241 (2008)

  23. 23

    Cao, Y., Rathmell, J. C. & Macintyre, A. N. Metabolic reprogramming towards aerobic glycolysis correlates with greater proliferative ability and resistance to metabolic inhibition in CD8 versus CD4 T cells. PLoS ONE 9, e104104 (2014)

  24. 24

    Sumida, Y., Niki, E., Naito, Y. & Yoshikawa, T. Involvement of free radicals and oxidative stress in NAFLD/NASH. Free Radic. Res. 47, 869–880 (2013)

  25. 25

    Porporato, P. E. et al. A mitochondrial switch promotes tumor metastasis. Cell Reports 8, 754–766 (2014)

  26. 26

    Feldstein, A. E. et al. Mass spectrometric profiling of oxidized lipid products in human nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. J. Lipid Res. 51, 3046–3054 (2010)

  27. 27

    Muir, K. et al. Proteomic and lipidomic signatures of lipid metabolism in NASH-associated hepatocellular carcinoma. Cancer Res. 73, 4722–4731 (2013)

  28. 28

    Beraza, N. et al. Pharmacological IKK2 inhibition blocks liver steatosis and initiation of non-alcoholic steatohepatitis. Gut 57, 655–663 (2008)

  29. 29

    Henao-Mejia, J. et al. Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity. Nature 482, 179–185 (2012)

  30. 30

    Miura, K. et al. Toll-like receptor 2 and palmitic acid cooperatively contribute to the development of nonalcoholic steatohepatitis through inflammasome activation in mice. Hepatology 57, 577–589 (2013)

  31. 31

    Andersson, J. et al. CD4+ FoxP3+ regulatory T cells confer infectious tolerance in a TGF-β-dependent manner. J. Exp. Med. 205, 1975–1981 (2008)

  32. 32

    Nakagawa, H. et al. ER stress cooperates with hypernutrition to trigger TNF-dependent spontaneous HCC development. Cancer Cell 26, 331–343 (2014)

  33. 33

    Ikehara, Y. et al. CD4+ Vα14 natural killer T cells are essential for acceptance of rat islet xenografts in mice. J. Clin. Invest. 105, 1761–1767 (2000)

  34. 34

    Hanczko, R. et al. Prevention of hepatocarcinogenesis and increased susceptibility to acetaminophen-induced liver failure in transaldolase-deficient mice by N-acetylcysteine. J. Clin. Invest. 119, 1546–1557 (2009)

  35. 35

    Radaeva, S. et al. Interferon-α activates multiple STAT signals and down-regulates c-Met in primary human hepatocytes. Gastroenterology 122, 1020–1034 (2002)

  36. 36

    Chen, R. F. Removal of fatty acids from serum albumin by charcoal treatment. J. Biol. Chem. 242, 173–181 (1967)

  37. 37

    Huynh, F. K., Green, M. F., Koves, T. R. & Hirschey, M. D. Measurement of fatty acid oxidation rates in animal tissues and cell lines. Methods Enzymol. 542, 391–405 (2014)

  38. 38

    Pelletier, M., Billingham, L. K., Ramaswamy, M. & Siegel, R. M. Extracellular flux analysis to monitor glycolytic rates and mitochondrial oxygen consumption. Methods Enzymol. 542, 125–149 (2014)

  39. 39

    Kleiner, D. E. et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41, 1313–1321 (2005)

  40. 40

    Bedossa, P. & Poynard, T. An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group. Hepatology 24, 289–293 (1996)

  41. 41

    Hoechst, B. et al. A new population of myeloid-derived suppressor cells in hepatocellular carcinoma patients induces CD4+CD25+Foxp3+ T cells. Gastroenterology 135, 234–243 (2008)

Download references

Acknowledgements

We would like to thank J. Berzofsky, W. Stoffel, E. M. Shevach and S. Thorgeirsson for helpful discussion. A.M.T. was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases. J.L. was supported by the Intramural Program Grant ZIABC011303, NIH, National Cancer Institute (NCI). M.H. was supported by a European Research Council starting grant (LiverCancerMechanism), the Stiftung Experimentelle Biomedizin (Hofschneider Stiftung), the Pre-clinical Comprehensive Center (PCCC) and the Helmholtz foundation. A.W. was supported by grants from the Krebsliga Schweiz (Oncosuisse) and the Promedica Stiftung, Switzerland. D.E.K., A.H.K., D.W.M. and T.F.G. were supported by the Intramural Research Program of the NIH, NCI.

Author information

C.M. and A.H.K. performed experiments. C.M., A.H.K., D.W.M. and T.F.G. analysed data. T.E., J.M.-E., D.E.K., P.J., D.F.S., M.T., V.K., M.E., M.H., A.M.T., H.Z., J.L. and D.F.W. assisted with experiments and analysis or provided valuable reagents. A. H.K. and D.W.M. performed and analysed seahorse studies. M.E., A.W. and M.H. collected/provided human specimens and performed/analysed immunohistochemistries of human samples. C.M. and T.F.G. conceived of and designed the project. C.M. and T.F.G. wrote the manuscript and all authors contributed to writing and provided feedback.

Correspondence to Tim F. Greten.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 MCD, CDAA and HF diets induce NAFLD and promote HCC.

a, Representative imagines of Oil Red O staining of MYC-ON mice fed MCD or CTR. Scale bar, 100 μm. b, Serum ALT levels analysis. Data are mean ± s.e.m.; n = 4, *P < 0.05, one-way ANOVA. ce, The effect of the CDAA diet on tumour development in MYC transgenic mice. Experimental setup, representative liver images and liver surface tumour counts are shown. Scale bar, 10 mm. Data are mean ± s.e.m.; n = 6 for CDAA and n = 5 mice for CTR, P = 0.0345, Student’s t-test. fi, The effect of the CDAA and HF diet on liver carcinogenesis in diethylnitrosoamine (DEN)-injected C57BL/6 mice. Experimental setup, representative tumour-free H&E stainings, macroscopic liver images and surface tumour counts are shown. Scale bar, 100 μm. Data are mean ± s.e.m.; n = 13 for CTR, n = 9 for HF, n = 10 for CDAA, *P < 0.05, one-way ANOVA.

Extended Data Figure 2 Immune cell monitoring in NAFLD-HCC.

aj, MYC mice were fed with an MCD diet or CTR diet. a, b, Intrahepatic immune cells were determined by flow cytometry. Composition (a) and absolute numbers (b) of different intrahepatic immune cell subsets in MYC-ON mice, which were kept for 4 weeks on an MCD diet or CTR diet. Data are mean ± s.e.m.; n ≥ 6, *P < 0.05, one-way ANOVA. c, Representative contour plots of intrahepatic CD4+ T lymphocytes. d, Representative dot plots of CD1d-tetramer staining in CD3loCD4+ population. eg, Absolute number of intrahepatic CD4+ T lymphocytes, frequencies of NK T cells and splenic CD4+ T lymphocytes were measured by flow cytometry. Data are mean ± s.e.m.; n = 4, *P < 0.05, two-way ANOVA. hj, Intrahepatic CD4+ and CD8+ T lymphocyte levels in MYC-ON mice fed with a CDAA diet for 16 weeks. Data are mean ± s.e.m.; n = 6 for CDAA and n = 5 for CTR, *P < 0.05, Student’s t-test. k, l, Intrahepatic CD4+ T lymphocyte levels in DEN-injected BL/6 male mice treated with a CDAA diet, HF diet or CTR for 7 months. Data are mean ± s.e.m.; n = 13 for CTR, n = 9 for HF, n = 10 for CDAA, *P < 0.05, one-way ANOVA. mp, Intrahepatic CD4+ and CD8+ T lymphocytes in tumour-free C57BL/6 mice treated with a CDAA diet for 16 weeks. TF, tumour free. Data are mean ± s.e.m.; n = 3 for CTR, n = 5 for CDAA, *P < 0.05, Student’s t-test. qt, Intrahepatic CD4+ and CD8+ T lymphocytes in tumour-free C57BL/6 mice treated with an HF or low-fat (LF) diet for 6 months. Data are mean ± s.e.m.; n = 2 for CTR, n = 5 for LF, n = 5 for HF, *P < 0.05, one-way ANOVA. ux, CD4+ and CD8+ T lymphocytes in 12-week-old male ob/ob or wild-type lean mice. Data are mean ± s.e.m., n = 5, *P < 0.05, Student’s t-test w, x, MYC mice were fed with MCD or CTR. Macrophage and CD11b+Gr1+ populations were measured. Data are mean ± s.e.m.; n ≥ 4, *P < 0.05, two-way ANOVA.

Extended Data Figure 3 Intrahepatic CD4+ lymphocytes are activated in NAFLD, and CD4 depletion enhances HCC.

ak, MYC-ON mice were fed with MCD or CTR for 4 weeks. ad, CD69 and CD44hiCD62Llo subsets in intrahepatic CD4+ T lymphocytes were measured. Data are mean ± s.e.m.; n = 8 for MCD and n = 6 for CTR, *P < 0.05, Student’s t-test. eg. Ex vivo IFN-γ, IL-4 production in intrahepatic CD4+ T lymphocytes were determined. Data are mean ± s.e.m.; n = 8, *P < 0.05, Student’s t-test. h, Ex vivo staining of T-bet, GATA3, ROR-γt and Foxp3 levels in intrahepatic and splenic CD4+ T lymphocytes. Data are mean ± s.e.m.; n = 3, *P < 0.05, two-way ANOVA. i, Ex vivo IL-17 production by intrahepatic CD4+ T lymphocytes. Data are mean ± s.e.m.; n = 5, *P < 0.05, Student’s t-test. j, Representative dot plots of ROR-γt/IL-17 staining in intrahepatic CD4+ T lymphocytes. k, Absolute number of intrahepatic CD4+ lymphocyte subsets. Data are mean ± s.e.m.; n = 3, *P < 0.05, two-way ANOVA. l, Suppressive function assay of isolated hepatic Treg cells from Foxp3–GFP mice kept on MCD or CTR for 4 weeks. m, Detection of AFP-specific CD4+ T lymphocytes in spleen from MYC-MCD mice. n, Selective depletion of intrahepatic CD4+ T lymphocytes but not NK T cells by i.p. injection of 50 μg anti-CD4 antibody (clone GK1.5). o, p, MYC-ON mice on CTR received 50 μg of GK1.5 antibody or isotype control i.p. once per week for 8 weeks. Representative liver imagines and surface tumour counts are shown. Scale bar, 10 mm. Data are mean ± s.e.m., n = 3.

Extended Data Figure 4 Lipid-laden hepatocytes release C18:2 and induce CD4+ T lymphocyte death via apoptosis.

a, Representative contour plots of ex vivo 7AAD/annexin V staining of intrahepatic CD4+ T lymphocytes from MYC-ON mice fed with MCD or CTR. b, Representative phase-contrast images of primary hepatocytes from MYC-ON mice after MCD or CTR treatment. ce, Isolated primary hepatocytes from MYC-ON mice on MCD or CTR were cocultured with isolated CD4+ T lymphocytes or splenocytes. Cell death levels were measured by flow cytometry. Data are mean ± s.e.m.; n = 4, one-way or two-way ANOVA. f, g, BODIPY 493/503 staining of CD4+ T lymphocytes in liver, spleen or blood from MYC-ON mice with MCD or CTR. Data are mean ± s.e.m.; n = 4, *P < 0.05, two-way ANOVA. h, i, Identification of FFAs in hepatocyte conditioned medium by gas chromatography/mass spectrometry (GC/MS). Data are mean ± s.e.m.; n = 3, *P < 0.05, two-way ANOVA. j, Anti-CD3/28 bead-activated splenocytes were treated with different FFAs, and cell death level in CD4+ or CD8+ T lymphocytes was determined. Data are mean ± s.e.m.; n = 4, *P < 0.05, two-way ANOVA. km, Dose–response curve and time course of C18:2-induced cell death in CD4+ or CD8+ T lymphocytes. n, Caspase3/7 activity in CD4+ lymphocytes after C18:2 treatment. Data are mean ± s.e.m.; n = 9, *P < 0.05, Student’s t-test. o, Dose–response curve of H2O2-induced cell death in CD4+ or CD8+ T lymphocytes. p, Uptake of C18:2 by CD4+ and CD8+ T lymphocytes after incubation with 50 μM C18:2 for 2 h. Data are mean ± s.e.m.; n = 6, *P < 0.05, two-way ANOVA.

Extended Data Figure 5 Ingenuity pathway analysis of microarray data.

CD4+ and CD8+ T lymphocytes sorted from C18:2-treated splenocytes were subjected to microarray analysis. Pathway analysis was done by ingenuity pathway analysis (IPA). n = 3. Ratio is the number of changed genes divided by total genes in the pathway.

Extended Data Figure 6 Mitochondrial ROS mediates C18:2-induced CD4+ T lymphocyte death in vitro and in vivo.

a, Real-time polymerase chain reaction (PCR) confirmed the gene changes from microarray. Data are mean ± s.e.m.; n = 3, *P < 0.05, two-way ANOVA. b, Cpt1a mRNA level in Jurkat cells after FFA treatment. Data are mean ± SEM; n = 6, *P < 0.05, one-way ANOVA. c, Expression of CPT1a in wild-type and two knockdown Jurkat cells. NT, none-targeting control. d, e, OCR analysis of activated CD4+ and CD8+ T lymphocytes upon C18:2 or C16:0 incubation. f, ROS levels of CD4+ or CD8+ T lymphocytes in splenocytes treated with C18:2 or C16:0. Data are mean ± s.e.m.; n = 8, *P < 0.05, two-way ANOVA. g, Mitochondrial ROS in wild-type and two CPT1 knockdown Jurkat cells. i, Cell death of CD4+ or CD8+ T lymphocytes in splenocytes treated with C18:2 in the presence of NAC or catalase. Data are mean ± s.e.m.; n = 4, *P < 0.05, two-way ANOVA. i, j, In vivo blocking ROS with NAC in MYC-ON mice treated with MCD. Some mice also received CD4 antibody depletion. Experimental setup and representative H&E liver sections are shown. Scale bar, 200 μm.

Extended Data Figure 7 C18:2 induces cell death in human CD4+ T lymphocytes, and NASH patients have lower intrahepatic CD4+ T lymphocytes.

a, Cell death levels of sorted human CD4+ T lymphocytes treated with different FFAs. Data are mean ± s.e.m.; n = 4, *P < 0.05, one-way ANOVA. b, ROS level of CD4+ or CD8+ T lymphocyte in peripheral blood mononuclear cells (PBMCs) treated with C18:2 or C16:0. Data are mean ± s.e.m.; n = 6, *P < 0.05, two-way ANOVA. c, d, Serum ALT and AST concentration in different patients. e, Intrahepatic CD4+ T lymphocyte count in biopsies. CD4+ T lymphocytes were identified by immunohistochemistry. Data are mean ± s.e.m.; normal = 6, NASH = 16, ASH = 8, HBV/HCV = 16, *P < 0.05, one-way ANOVA.

Extended Data Figure 8 Immunohistochemistry staining of intrahepatic CD4+ or CD8+ T lymphocytes in patient biopsies.

Representative CD4 or CD8 immunohistochemistry images of liver biopsies from healthy individuals, NASH, ASH patients or patients with HBV or HCV. For each condition, two different magnifications are shown. Scale bar, 100 μm.

Extended Data Table 1 Fatty acid composition of hepatic lipids
Extended Data Table 2 Overview of patient cohort for immunohistochemistry analysis

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ma, C., Kesarwala, A., Eggert, T. et al. NAFLD causes selective CD4+ T lymphocyte loss and promotes hepatocarcinogenesis. Nature 531, 253–257 (2016). https://doi.org/10.1038/nature16969

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.