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Noninvasive diagnostic criteria for nonalcoholic steatohepatitis based on gene expression levels in peripheral blood mononuclear cells

Journal of Gastroenterology volume 54, pages 730–741 (2019)Cite this article

Abstract

Background

Nonalcoholic fatty liver disease (NAFLD) consists of nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH); the latter progresses to liver cirrhosis and hepatocellular carcinoma. Discriminating NASH from NAFL typically involves liver biopsy. The mechanism of NASH progression is unclear but may involve immunological pathways. In this study, we examined expression levels of cytokine- and chemokine-encoding genes in peripheral blood mononuclear cells (PBMCs) from NAFLD patients and established immunological criteria for discriminating NASH from NAFL.

Methods

PBMCs were obtained from 54 patients diagnosed histologically with NAFLD (NAFL, 18; NASH, 36). mRNA was extracted from PBMCs, and expression levels of cytokine- and chemokine-encoding genes were determined by quantitative real-time PCR. Statistical analysis was performed by nonparametric test.

Results

Expression levels of interferon (IFN)γ, interleukin (IL)2, IL15, C–C-motif chemokine ligand (CCL)2, IL10, and C-X-C-motif chemokine ligand (CXCL)11 were significantly upregulated in NASH patients compared with NAFL patients. Moreover, their expression levels were positively correlated with the degree of ballooning of hepatocytes but not of steatosis or lobular inflammation. We focused on those encoding IL10, IFNγ, and CCL2, and developed a scoring system to discriminate NASH from NAFL. The discriminatory power of the criteria was validated in an independent cohort.

Conclusions

Expression levels of the cytokine- and chemokine-encoding genes in PBMCs were positively correlated with ballooning, suggesting their utility for the diagnosis of NASH. The data indicate that peripheral as well as intrahepatic immunity is involved in the progression of NASH. Our findings afford new insight into immunological mechanisms of NASH and will facilitate its noninvasive diagnosis.

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Fig. 1
Fig. 2

Abbreviations

NAFLD:

Nonalcoholic fatty liver disease

NAFL:

Nonalcoholic fatty liver

NASH:

Nonalcoholic steatohepatitis

PBMC:

Peripheral blood mononuclear cells

IFN:

Interferon

IL:

Interleukin

CCL:

C–C motif chemokine ligand

CXCL:

C-X-C motif chemokine ligand

CXCR:

C-X-C chemokine receptor

NAS:

NAFLD activity score

LPS:

Lipopolysaccharide

MCP:

Monocyte chemoattractant protein

TLR:

Toll-like receptor

BMI:

Body mass index

AST:

Aspartate aminotransferase

ALT:

Alanine aminotransferase

GGT:

Gamma-glutamyl transpeptidase

TG:

Triglyceride

HDL:

High-density lipoprotein

LDL:

Low-density lipoprotein

CRP:

C-reactive protein

PT:

Prothrombin time

HbA1c:

Hemoglobin A1c

HOMA-IR:

Homeostasis model assessment insulin resistance

cDNA:

Complementary DNA

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

SD:

Standard deviation

LI:

Lobular inflammation

Treg:

Regulatory T cells

ROC:

Receiver operating characteristic

AUC:

Area under curve

DC:

Dendritic cells

NK:

Natural killer

NKT:

Natural killer T

CTL:

Cytotoxic T lymphocytes

HCC:

Hepatocellular carcinoma

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Acknowledgements

We thank Ms. Seiko Shinzawa (Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo) for providing technical assistance. We thank the staff of the Department of Pathology, Graduate School of Medicine, The University of Tokyo for processing and pathological examination of specimens. This research was supported by Health Sciences Research Grant from the Ministry of Health, Labor, and Welfare of Japan (Research on Hepatitis), and Grant from the Japan Agency for Medical Research and Development (AMED) under Grant Number JP17fk0210304 and JP18fk0210040. No additional external funding was received. The funders played no role in the study design, data collection, or analysis, decision to publish, or preparation of the manuscript.

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Affiliations

  1. Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan

    Akira Kado, Kenichiro Enooku, Hidetaka Fujinaga, Kazuya Okushin & Kazuhiko Koike

  2. Division of Infectious Diseases, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan

    Takeya Tsutsumi & Hiroshi Yotsuyanagi

  3. Department of Infectious Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Kazuhiko Ikeuchi & Kyoji Moriya

  4. Department of Infection Control and Prevention, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Kazuya Okushin & Kyoji Moriya

Authors
  1. Akira Kado
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  2. Takeya Tsutsumi
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  3. Kenichiro Enooku
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  4. Hidetaka Fujinaga
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  5. Kazuhiko Ikeuchi
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  6. Kazuya Okushin
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  7. Kyoji Moriya
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  8. Hiroshi Yotsuyanagi
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  9. Kazuhiko Koike
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Contributions

AK, TT, HF, and KE contributed to the study design, acquisition of data, analysis and interpretation of data, and drafting of the manuscript. KI, KO, KM, and HY participated in critical revision of the manuscript. KK participated in the study design, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript, and supervision. We confirm that all authors have reviewed and approved the final version of the manuscript.

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Correspondence to Kazuhiko Koike.

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Kado, A., Tsutsumi, T., Enooku, K. et al. Noninvasive diagnostic criteria for nonalcoholic steatohepatitis based on gene expression levels in peripheral blood mononuclear cells. J Gastroenterol 54, 730–741 (2019). https://doi.org/10.1007/s00535-019-01565-x

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Keywords

  • Nonalcoholic steatohepatitis
  • Peripheral blood mononuclear cells
  • Cytokine
  • Chemokine