Liver transcriptomics highlights interleukin-32 as novel NAFLD-related cytokine and candidate biomarker

Abstract

Objective: Efforts to manage non-alcoholic fatty liver disease (NAFLD) are limited by the incomplete understanding of the pathogenic mechanisms and the absence of accurate non-invasive biomarkers. The aim of this study was to identify novel NAFLD therapeutic targets andbiomarkers by conducting liver transcriptomic analysis in patients stratified by the presence of the PNPLA3 I148M genetic risk variant.

Design: We sequenced the hepatic transcriptome of 125 obese individuals. 'Severe NAFLD' was defined as the presence of steatohepatitis, NAFLD activity score ≥4 or fibrosis stage ≥2. The circulating levels of the most upregulated transcript, interleukin-32 (IL32), were measured by ELISA.

Results: Carriage of the PNPLA3 I148M variant correlated with the two major components of hepatic transcriptome variability and broadly influenced gene expression. In patients with severe NAFLD, there was an upregulation of inflammatory and lipid metabolism pathways. IL32 was the most robustly upregulated gene in the severe NAFLD group (adjusted p=1×10^-6), and its expression correlated with steatosis severity, both in I148M variant carriers and non-carriers. In 77 severely obese, and in a replication cohort of 160 individuals evaluated at the hepatology service, circulating IL32 levels were associated with both NAFLD and severe NAFLD independently of aminotransferases (p<0.01 for both). A linear combination of IL32-ALT-AST showed a better performance than ALT-AST alone in NAFLD diagnosis (area under the curve=0.92 vs 0.81, p=5×10^-5).

Conclusion: Hepatic IL32 is overexpressed in NAFLD, correlates with hepatic fat and liver damage, and is detectable in the circulation, where it is independently associated with the presence and severity of NAFLD.

Keywords: cytokines; genetics; nonalcoholic steatohepatitis; rna expression.

Figure 1

Principal components correlation and differential pathway expression. (A) Correlation matrix of the main five principal components (PCs) of hepatic transcriptome variability with clinical features as assessed by multivariate regression. (B, C, D, E) Pathways enriched in genes differentially (multiplicity adjusted p<0.1, negative binomial generalised linear model) expressed in: PNPLA3 I148M carriers versus non-carriers (B), severe NAFLD versus NAFLD and healthy individuals (C), severe versus non-severe in PNPLA3 I148M non-carriers (D), severe versus non-severe NAFLD among PNPLA3 I148M carriers (E). BMI, body max index; EMT, epithelial-to-mesenchymal transition; IFN, interferon; NAFLD, non-alcoholic fatty liver disease; NES, normalised enrichment score; PC, principal component; PNPLA3, patatin-like phospholipase domain-containing protein 3; TM6SF2, transmembrane 6 superfamily 2 human gene.

Figure 2

IL32 isoform expression. (A) Percentage of the IL32 gene products. ***p<0.001 versus all the other isoforms, post hoc Tukey’s test. (B) Isoform absolute expression value. ***p<0.001 versus NAFLD and healthy patients, post hoc Tukey’s test. ANOVA, analysis of variance; IL32, interleukin-32; NAFLD, non-alcoholic fatty liver disease; PNPLA3, patatin-like phospholipase domain-containing protein 3; TPM, transcripts per million.

Figure 3

IL32 expression correlations. (A) Pathway enriched in IL32 correlated genes (adjusted p<0.1). (B) IL32 expression stratified by steatosis grade; statistical analysis was performed by ordinal logistic regression. (C, D, E) IL32 expression stratified by the presence of lobular inflammation (C), ballooning (D), fibrosis (E). **p<0.01; ***p<0.001, Student’s t-test. EMT, epithelial-to-mesenchymal transition; IFN: interferon; IL32, interleukin-32; NAFLD, non-alcoholic fatty liver disease; NES, normalised enrichment score; PNPLA3, patatin-like phospholipase domain-containing protein 3; ROS, reactive oxygen species.

Figure 4

IL32 expression by cell type and IL32 plasma levels. (A) IL32 expression in different cell types assessed by RT-qPCR. ***p<0.001 versus human hepatocytes (HH), post hoc Tukey’s test. (B) Spearman correlation between IL32 liver expression and plasma levels in 16 patients, who belong to both transcriptomic and Bariatric surgery cohorts. (C, D, E, F, G, H) IL32 plasma levels in patients stratified either by the presence of NAFLD or severe NAFLD as assessed in the Bariatric surgery cohort (C and D), Hepatology service cohort (E and F) and the overall cohort (G and H). *p<0.05; **p<0.01; ***p<0.001, Wilcoxon rank-sum test. ANOVA, analysis of variance; IL32, interleukin-32; HSCs, hepatic stellate cells; HUVEC, human umbilical vein endothelial cell; M1, M1-like human macrophages; M2, M2-like human macrophages; NAFLD, non-alcoholic fatty liver disease.

Figure 5

IL32 diagnostic accuracy. (A, B, C) NAFLD diagnostic accuracy in the Bariatric surgery cohort (A), the Hepatology service cohort (B) and the overall cohort (C). (D, E, F) Severe NAFLD diagnostic accuracy in the Bariatric surgery cohort (D), the Hepatology service cohort (E) and the overall cohort (F). IL32-ALT-AST score (red curve) was compared with ALT-AST score (blue). Comparison was performed by Delong test. AUC, area under the curve; NAFLD, non-alcoholic fatty liver disease.