The PNPLA3 I148M variant is associated with immune cell infiltration and advanced fibrosis in MASLD: a prospective genotype–phenotype study (original) (raw)

Abstract

Background

Increasing evidence reveals that immune cells significantly contribute to metabolic dysfunction-associated steatotic liver disease (MASLD) progression. The patatin-like phospholipase domain-containing protein 3 (PNPLA3) I148M variant has been linked to hepatic inflammation and fibrosis; however, its role in immune cell infiltration and activation within the liver remains unclear.

Methods

Seventy patients with MASLD were prospectively enrolled. Genomic DNA was extracted from buccal swabs or liver biopsy samples, followed by single nucleotide polymorphism genotyping to determine the rs738409 SNP genotype at codon 148 of PNPLA3. Immunohistochemistry was conducted using CD3 and CD68 antibodies to quantify T cell and macrophage infiltration, respectively. Total RNA extracted from biopsy specimens was used for quantitative reverse transcription polymerase chain reaction to assess the expression of specific markers associated with immune cell activation.

Results

Among the 70 patients with MASLD, 34 had the GG genotype, whereas 21 and 15 had the GC and CC genotypes, respectively. The GG genotype group showed a higher proportion of advanced fibrosis (F3 or F4) than the GC + CC group (P = 0.051). GG genotype carriers exhibited significantly higher CD3+ and CD68+ cell counts in the periportal region than the GC/CC carriers (P < 0.05). The transcriptomic analysis revealed elevated expression of markers associated with chronic antigen stimulation and immune cell activation (CD8A, GZMB, CCL2, and TIMP1) in GG carriers compared with those of GC and CC (P < 0.05). Furthermore, correlations among various markers, including inflammatory, steatosis-associated, and fibrosis-associated markers, exhibited consistent positive correlations.

Conclusions

Our findings revealed that the PNPLA3 I148M variant and increased immune cell infiltration and activation were significantly correlated within the MASLD liver. Further studies are needed to elucidate the mechanistic links between this genetic variant and liver inflammation.

Access this article

Log in via an institution

Subscribe and save

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

Abbreviations

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

BMI:

Body mass index

CAP:

Controlled attenuation parameter

CCL2:

C-C motif chemokine ligand 2

CC:

Wild-type genotype for PNPLA3 I148M variant

CD160:

Cluster of differentiation 160

CD3:

Cluster of differentiation 3 (T cell marker)

CD68:

Cluster of differentiation 68 (macrophage marker)

CD8A:

Cluster of differentiation 8A (cytotoxic T cell marker)

CXCR6:

C-X-C motif chemokine receptor 6

DM:

Diabetes mellitus

FIB-4:

Fibrosis-4 index

GC:

Heterozygous genotype for PNPLA3 I148M variant

GG:

Homozygous genotype for PNPLA3 I148M variant

GGT:

Gamma-glutamyl transpeptidase

GZMB:

Granzyme B

Hb:

Haemoglobin

HDL-C:

High-density lipoprotein cholesterol

HTN:

Hypertension

IFNγ:

Interferon gamma

IL1β:

Interleukin 1 beta

INR:

International normalized ratio

KLRG1:

Killer cell lectin-like receptor subfamily G member 1

LDL-C:

Low-density lipoprotein cholesterol

LSM:

Liver stiffness measurement

MASLD:

Metabolic dysfunction-associated steatotic liver disease

NAS:

NAFLD activity score

NAFLD:

Nonalcoholic fatty liver disease

PDCD1:

Programmed cell death protein 1

PNPLA3:

Patatin-like phospholipase domain-containing protein 3

PPARγ:

Peroxisome proliferator-activated receptor gamma

PT:

Prothrombin time

SD:

Standard deviation

TB:

Total bilirubin

TIGIT:

T cell immunoreceptor with Ig and ITIM domains

TIMP1:

Tissue inhibitor of metalloproteinases 1

TNFα:

Tumor necrosis factor alpha

TOX:

Thymocyte selection-associated high mobility group box

WBC:

White blood cell

References

  1. EASL-EASD-EASO Clinical Practice Guidelines on the management of metabolic dysfunction-associated steatotic liver disease (MASLD). Obes Facts. 2024;17:374–444.
  2. Kim DY. Changing etiology and epidemiology of hepatocellular carcinoma: Asia and worldwide. J Liver Cancer. 2024;24:62–70.
    Article PubMed PubMed Central Google Scholar
  3. Han JW, Sohn W, Choi GH, et al. Evolving trends in treatment patterns for hepatocellular carcinoma in Korea from 2008 to 2022: a nationwide population-based study. J Liver Cancer. 2024;24:274–85.
    Article PubMed PubMed Central Google Scholar
  4. Esler WP, Cohen DE. Pharmacologic inhibition of lipogenesis for the treatment of NAFLD. J Hepatol. 2024;80:362–77.
    Article CAS PubMed Google Scholar
  5. Iwaki M, Fujii H, Hayashi H, et al. Prognosis of biopsy-confirmed metabolic dysfunction-associated steatotic liver disease: a sub-analysis of the CLIONE study. Clin Mol Hepatol. 2024;30:225–34.
    Article PubMed PubMed Central Google Scholar
  6. Harrison SA, Bedossa P, Guy CD, et al. A phase 3, randomized, controlled trial of resmetirom in NASH with liver fibrosis. N Engl J Med. 2024;390:497–509.
    Article PubMed Google Scholar
  7. An J, Sohn JH. Pharmacological advances in the treatment of nonalcoholic fatty liver diseases: focused on global results of randomized controlled trials. Clin Mol Hepatol. 2023;29:S268–75.
    Article PubMed Google Scholar
  8. Puengel T, Tacke F. Pharmacotherapeutic options for metabolic dysfunction-associated steatotic liver disease: where are we today? Expert Opin Pharmacother. 2024;25:1249–63.
    Article CAS PubMed Google Scholar
  9. Johnson SM, Bao H, McMahon CE, et al. PNPLA3 is a triglyceride lipase that mobilizes polyunsaturated fatty acids to facilitate hepatic secretion of large-sized very low-density lipoprotein. Nat Commun. 2024;15:4847.
    Article CAS PubMed PubMed Central Google Scholar
  10. Valenti L, Al-Serri A, Daly AK, et al. Homozygosity for the patatin-like phospholipase-3/adiponutrin I148M polymorphism influences liver fibrosis in patients with nonalcoholic fatty liver disease. Hepatology. 2010;51:1209–17.
    Article CAS PubMed Google Scholar
  11. Liu YL, Patman GL, Leathart JB, et al. Carriage of the PNPLA3 rs738409 C >G polymorphism confers an increased risk of non-alcoholic fatty liver disease associated hepatocellular carcinoma. J Hepatol. 2014;61:75–81.
    Article CAS PubMed Google Scholar
  12. Rosso C, Caviglia GP, Birolo G, et al. Impact of PNPLA3 rs738409 polymorphism on the development of liver-related events in patients with nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol. 2023;21:3314-21.e3.
    Article CAS PubMed Google Scholar
  13. Petta S, Armandi A, Bugianesi E. Impact of PNPLA3 I148M on clinical outcomes in patients with MASLD. Liver Int. 2024;45(3):e16133.
    Article PubMed PubMed Central Google Scholar
  14. Sookoian S, Pirola CJ. Genetics in non-alcoholic fatty liver disease: the role of risk alleles through the lens of immune response. Clin Mol Hepatol. 2023;29:S184–95.
    Article PubMed Google Scholar
  15. BasuRay S, Smagris E, Cohen JC, et al. The PNPLA3 variant associated with fatty liver disease (I148M) accumulates on lipid droplets by evading ubiquitylation. Hepatology. 2017;66:1111–24.
    Article CAS PubMed Google Scholar
  16. Vilar-Gomez E, Pirola CJ, Sookoian S, et al. PNPLA3 rs738409 and risk of fibrosis in NAFLD: exploring mediation pathways through intermediate histological features. Hepatology. 2022;76:1482–94.
    Article CAS PubMed Google Scholar
  17. Li Z, Wang S, Xu Q, et al. The double roles of T cell-mediated immune response in the progression of MASLD. Biomed Pharmacother. 2024;173:116333.
    Article CAS PubMed Google Scholar
  18. Huby T, Gautier EL. Immune cell-mediated features of non-alcoholic steatohepatitis. Nat Rev Immunol. 2022;22:429–43.
    Article CAS PubMed Google Scholar
  19. Mao T, Yang R, Luo Y, et al. Crucial role of T cells in NAFLD-related disease: a review and prospect. Front Endocrinol (Lausanne). 2022;13:1051076.
    Article PubMed Google Scholar
  20. Seth RK, Das S, Kumar A, et al. CYP2E1-dependent and leptin-mediated hepatic CD57 expression on CD8+ T cells aid progression of environment-linked nonalcoholic steatohepatitis. Toxicol Appl Pharmacol. 2014;274:42–54.
    Article CAS PubMed Google Scholar
  21. Kazankov K, Jørgensen SMD, Thomsen KL, et al. The role of macrophages in nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Nat Rev Gastroenterol Hepatol. 2019;16:145–59.
    Article CAS PubMed Google Scholar
  22. Sung PS. Crosstalk between tumor-associated macrophages and neighboring cells in hepatocellular carcinoma. Clin Mol Hepatol. 2022;28:333–50.
    Article PubMed Google Scholar
  23. Sung PS, Park DJ, Roh PR, et al. Intrahepatic inflammatory IgA(+)PD-L1(high) monocytes in hepatocellular carcinoma development and immunotherapy. J Immunother Cancer. 2022;10(5):e003618.
    Article PubMed PubMed Central Google Scholar
  24. Carpino G, Del Ben M, Pastori D, et al. Increased liver localization of lipopolysaccharides in human and experimental NAFLD. Hepatology. 2020;72:470–85.
    Article CAS PubMed Google Scholar
  25. Krenkel O, Tacke F. Liver macrophages in tissue homeostasis and disease. Nat Rev Immunol. 2017;17:306–21.
    Article CAS PubMed Google Scholar
  26. EASL-EASD-EASO Clinical Practice Guidelines on the management of metabolic dysfunction-associated steatotic liver disease (MASLD). J Hepatol. 2024;81:492–542.
  27. Cha JH, Park NR, Cho SW, et al. Chitinase 1: a novel therapeutic target in metabolic dysfunction-associated steatohepatitis. Front Immunol. 2024;15:1444100.
    Article CAS PubMed PubMed Central Google Scholar
  28. Sterling RK, Lissen E, Clumeck N, et al. Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection. Hepatology. 2006;43:1317–25.
    Article CAS PubMed Google Scholar
  29. Brunt EM, Kleiner DE, Wilson LA, et al. Nonalcoholic fatty liver disease (NAFLD) activity score and the histopathologic diagnosis in NAFLD: distinct clinicopathologic meanings. Hepatology. 2011;53:810–20.
    Article CAS PubMed Google Scholar
  30. Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41:1313–21.
    Article PubMed Google Scholar
  31. Xu R, Tao A, Zhang S, et al. Association between patatin-like phospholipase domain containing 3 gene (PNPLA3) polymorphisms and nonalcoholic fatty liver disease: a HuGE review and meta-analysis. Sci Rep. 2015;5:9284.
    Article CAS PubMed PubMed Central Google Scholar
  32. Armisen J, Rauschecker M, Sarv J, et al. AZD2693, a PNPLA3 antisense oligonucleotide, for the treatment of MASH in 148M homozygous participants: two randomized phase I trials. J Hepatol. 2025;83(1):31–42.
    Article CAS PubMed Google Scholar
  33. Boeckmans J, Gatzios A, Schattenberg JM, et al. PNPLA3 I148M and response to treatment for hepatic steatosis: a systematic review. Liver Int. 2023;43:975–88.
    Article CAS PubMed Google Scholar
  34. Park J, Zhao Y, Zhang F, et al. IL-6/STAT3 axis dictates the PNPLA3-mediated susceptibility to non-alcoholic fatty liver disease. J Hepatol. 2023;78:45–56.
    Article CAS PubMed Google Scholar
  35. Sutti S, Albano E. Adaptive immunity: an emerging player in the progression of NAFLD. Nat Rev Gastroenterol Hepatol. 2020;17:81–92.
    Article CAS PubMed Google Scholar
  36. Yeh MM, Brunt EM. Pathological features of fatty liver disease. Gastroenterology. 2014;147:754–64.
    Article CAS PubMed Google Scholar
  37. Bruzzì S, Sutti S, Giudici G, et al. B2-Lymphocyte responses to oxidative stress-derived antigens contribute to the evolution of nonalcoholic fatty liver disease (NAFLD). Free Radic Biol Med. 2018;124:249–59.
    Article PubMed Google Scholar
  38. Grohmann M, Wiede F, Dodd GT, et al. Obesity drives STAT-1-dependent NASH and STAT-3-dependent HCC. Cell. 2018;175:1289-306.e20.
    Article CAS PubMed PubMed Central Google Scholar
  39. Ghazarian M, Revelo XS, Nøhr MK, et al. Type I interferon responses drive intrahepatic T cells to promote metabolic syndrome. Sci Immunol. 2017;2(10):eaai7616.
    Article PubMed PubMed Central Google Scholar
  40. Sutti S, Jindal A, Locatelli I, et al. Adaptive immune responses triggered by oxidative stress contribute to hepatic inflammation in NASH. Hepatology. 2014;59:886–97.
    Article CAS PubMed Google Scholar
  41. Breuer DA, Pacheco MC, Washington MK, et al. CD8(+) T cells regulate liver injury in obesity-related nonalcoholic fatty liver disease. Am J Physiol Gastrointest Liver Physiol. 2020;318:G211–24.
    Article CAS PubMed Google Scholar
  42. Bhattacharjee J, Kirby M, Softic S, et al. Hepatic natural killer T-cell and CD8+ T-cell signatures in mice with nonalcoholic steatohepatitis. Hepatol Commun. 2017;1:299–310.
    Article CAS PubMed PubMed Central Google Scholar
  43. Wolf MJ, Adili A, Piotrowitz K, 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. 2014;26:549–64.
    Article CAS PubMed Google Scholar
  44. Eickmeier I, Seidel D, Grün JR, et al. Influence of CD8 T cell priming in liver and gut on the enterohepatic circulation. J Hepatol. 2014;60:1143–50.
    Article CAS PubMed Google Scholar
  45. Dudek M, Pfister D, Donakonda S, et al. Auto-aggressive CXCR6(+) CD8 T cells cause liver immune pathology in NASH. Nature. 2021;592:444–9.
    Article CAS PubMed Google Scholar
  46. Burtis AEC, DeNicola DMC, Ferguson ME, et al. Ag-driven CD8 + T cell clonal expansion is a prominent feature of MASH in humans and mice. Hepatology. 2025;81:591–608.
    Article PubMed Google Scholar
  47. Sekine T, Perez-Potti A, Nguyen S, et al. TOX is expressed by exhausted and polyfunctional human effector memory CD8(+) T cells. Sci Immunol. 2020;5(49):eaba7918.
    Article CAS PubMed Google Scholar

Download references

Acknowledgements

Assistance with the study: none.

Funding

This work was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (RS-2024-00438542 to S.H.B). Additional support was provided in part by the NRF grant funded by the Korean government (MSIT) (RS-2023-00208767 to S.H.B) and (RS-2024-00337298 to P.S.S).

Author information

Author notes

  1. Jaejun Lee and Jung Hoon Cha have contributed equally to this work.

Authors and Affiliations

  1. Division of Hepatology, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
    Jaejun Lee, Hee Sun Cho, Keungmo Yang, Hyun Yang, Heechul Nam, Pil Soo Sung & Si Hyun Bae
  2. The Catholic University Liver Research Center, Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
    Jaejun Lee, Jung Hoon Cha, Hee Sun Cho, Keungmo Yang, Hyun Yang, Heechul Nam, Pil Soo Sung & Si Hyun Bae
  3. Xenohelix Research Institute, Incheon, Republic of Korea
    Mi Young Byun & Seok Keun Cho
  4. Department of Biomechatronic Engineering, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Republic of Korea
    Jinsung Park
  5. Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea
    Hyuk Wan Ko
  6. Department of Systems Biology, Institute of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea
    Seong Wook Yang
  7. Division of Gastroenterology and Hepatology, Department of Internal Medicine, College of Medicine, Seoul St. Mary’s Hospital, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul, 06591, Republic of Korea
    Pil Soo Sung
  8. Division of Gastroenterology and Hepatology, Department of Internal Medicine, College of Medicine, Eunpyeong St. Mary’s Hospital, The Catholic University of Korea, 1021 Tongil-ro, Eunpyeong-gu, Seoul, 03312, Republic of Korea
    Si Hyun Bae

Authors

  1. Jaejun Lee
  2. Jung Hoon Cha
  3. Hee Sun Cho
  4. Keungmo Yang
  5. Hyun Yang
  6. Heechul Nam
  7. Mi Young Byun
  8. Seok Keun Cho
  9. Jinsung Park
  10. Hyuk Wan Ko
  11. Seong Wook Yang
  12. Pil Soo Sung
  13. Si Hyun Bae

Contributions

Study concept and design: Pil Soo Sung and Si Hyun Bae. Data collection: Jaejun Lee, Jung Hoon Cha, Hyun Yang, Pil Soo Sung, and Si Hyun Bae. Data analysis and interpretation: Jaejun Lee, Jung Hoon Cha, Mi Young Byun, Seok keun Cho, Jin Sung Park, Hyuk Wan Ko, Seong Wook Yang, Pil Soo Sung, and Si Hyun Bae. Manuscript writing: Jaejun Lee, Jung Hoon Cha, and Pil Soo Sung. Conceptualization, methodology, and supervision: Pil Soo Sung and Si Hyun Bae. Final approval of the version to be published: all authors.

Corresponding authors

Correspondence toPil Soo Sung or Si Hyun Bae.

Ethics declarations

Conflict of interests

The authors declare no conflicts of interest.

Informed consent was obtained from all participants prior to the study enrolment.

Institutional Review Board statement

This study was approved by the Institutional Review Board of the Catholic University of Korea (approval number: XC24TIDI0025). Informed consent was obtained from all participants prior to the study enrolment.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Cite this article

Lee, J., Cha, J.H., Cho, H.S. et al. The PNPLA3 I148M variant is associated with immune cell infiltration and advanced fibrosis in MASLD: a prospective genotype–phenotype study.J Gastroenterol 60, 1284–1295 (2025). https://doi.org/10.1007/s00535-025-02285-1

Download citation

Keywords