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Expression of Cytokine Signaling Genes in the Adipose Tissue of Patients with Non-alcoholic Steatohepatitis and Fibrosis
Michael Estep, Ancha Baranova, Noreen Hossain, Hazem Elariny, Maria Stepanova, Yun Fang, Z. Goodman, Vikas Chandhoke, Zobair M. Younossi

This is a collaborative project between

Molecular and Microbiology Department, College of Science,George Mason University, Fairfax, VA

Translational Reseach Institute, Inova Hospital, VA

This study was published in: Obesity Surgery 2009 Mar 12.

Background and Aims: The role of visceral obesity in the development of non-alcoholic fatty liver disease (NAFLD), and its progressive type, non-alcoholic steatohepatitis (NASH), is partially due to cytokines and adipokines produced by the white adipose tissue. Our aim was to assess the association of hepatic fibrosis and NASH by evaluating the intrinsic differences in the inflammatory cytokine signaling in the visceral adipose tissue.

Methods: We used targeted microarrays representing human genes involved in the inflammatory and fibrogenic reactions to profile visceral adipose samples of 15 well-matched NASH patients with and without fibrosis. Additionally, visceral adipose samples were subjected to Real Time PCR profiling of 84 inflammations related genes.

Results: Eight genes (CCL2, CCL4, CCL18, CCR1, IL10RB, IL15RA and LTB) were differentially expressed in NASH with fibrosis. Additionally, an overlapping but distinct list of four differentially expressed genes were found in NASH with type-II diabetes (DM) (IL8, BLR1, IL2RA, CD40LG, IL1RN, IL15RA and CCL4) as compared to NASH without DM.

An expression of CCL2 encoding MCP1 in adipose has been associated with liver fibrosis. The higher levels of CCL2 production by macrophages embedded in adipose tissue as compared to pro-inflammatory M1 macrophages are generally thought to play a role in the development of insulin resistance. However, in this study the comparison of CCL2 mRNA levels were shown to be related to fibrosis rather than to diabetes. Interestingly, in NASH patients with fibrosis, the levels of RNAs were increased both for CCL2 as well as its receptor which could cause a local increase in pro-inflammatory signaling. Additionally, excessive CLL2 produced by adipose may be delivered to the liver by portal vein and exert a direct fibrogenic effect.

We also registered exclusive over-expression of mRNA for CD40 ligand (CD40LG) in the in the diabetic patients with NASH and fibrosis. In fact, the expression level of CD40 ligand mRNA was below the detection threshold in all other groups (Supplementary Table 1), including NASH patients who had diabetes but not fibrosis. CD40LG has been previously implicated in the pathogenesis of metabolic syndrome in relation to heart disease and diabetes and also shown to induce apoptosis in hepatocytes and biliary epithelial cells as well as stimulating NF- k B signaling. Our findings support further investigation into the possibility of the using soluble CD40LG as a component for the predictive diagnostic of the fibrosis patients with NASH.

Conclusions: Inflammatory cytokines are differentially expressed in the adipose tissue of NASH with fibrosis, as well in NASH with DM. These findings points at the interaction of adipose inflammatory cytokines, DM and hepatic fibrosis in NASH.

Supplementary Table 1 . List of human genes profiled in adipose samples of NASH patients by microarray. Genes that were present only in microarray experiment, but not in RT-PCR experiment are in italic.

Supplementary Table 2 . List of human genes profiled in adipose samples of NASH patients by RT-PCR. Genes that were present only in RT PCR experiment, but not in microarray experiment are in italic. B

Supplementary Table 3 . Cytokine signaling related (CSR) genes expressed in the adipose tissue in each of the patients' cohorts.

Table 4 . Genes differentially expressed between F+ and F- NASH patients.

Table 5 . Genes differentially expressed in D+ and D- NASH patients

Figure 1 . Patient sample clustering by gene expression pattern evaluated on microarrays: A: Hierarchical trees of patient samples were constructed using Pearson correlation and average linkage. Presence of the hepatic fibrosis marked by letter “F+”, presence of diabetes by “D+”. C. Patient sample clustering by subset of genes significantly different by their expression in the adipose samples of the patients with NASH-related fibrosis when compared to NASH. D. Patient clustering by subset of genes significantly different by their expression in the adipose samples of the insulin resistant patients as compared to the patients without prior diagnosis of diabetes type II. On C. and D., only genes yielding Mann-Whitney significance value of <0.05 have been used for clustering.

Figure 2 . Patient clustering by subset of genes significantly different in D+ F+ NASH patients and D- F- NASH patients: Genes with Mann-Whitney significance values of <0.05 and a GE ratio with an absolute value >1.4 in a comparison between D+ F+ NASH patients vs. D- F- NASH were used to generate hierarchical clustering.