Association of interleukin-15-induced peripheral immune activation with hepatic stellate cell activation in persons coinfected with hepatitis C virus and HIV

Abstract

Hepatic stellate cells (HSCs) mediate hepatitis C virus (HCV)-related liver fibrosis, and increased HSC activation in human immunodeficiency virus (HIV)/HCV coinfection may be associated with accelerated fibrosis. We examined the level of HSC activation in HIV/HCV-coinfected and HCV-monoinfected subjects and its relationship to the level of activation and gene expression of peripheral immune cells in coinfected subjects. HSC activation levels positively correlated with peripheral CD4+ and CD8+ T cell immune activation and were associated with enhanced interleukin-15 (IL-15) gene expression, suggesting a pathogenic role for IL-15-driven immunomediated hepatic fibrosis. Future strategies that reduce immune activation and HSC activation may delay progression of liver fibrosis.

Figures

Figure 1

At baseline, these genes were significantly associated with an increase (group C) or decrease (group B) in α-smooth muscle actin (ASMA), a marker of hepatic stellate cell (HSC) activation (P < .05; absolute mean fold difference, >1.3). The gene encoding interleukin-15 (IL-15) was the only gene that presented consistently significant values in the pair-wise comparison between groups C and B, before treatment (n = 29 signature genes) and after treatment (n = 71 signature genes; data not shown). Group A consisted of subjects who experienced no changes in the ASMA score after therapy. ASMA was not associated with treatment response (P = .702).

Figure 2

Increased levels of expression of the gene encoding interleukin-15 (IL-15) were significantly associated with increased α-smooth muscle actin (ASMA) levels before and after interferon-α/ribavirin therapy in microarray analysis (group C; P < .01). These results were validated by real-time polymerase chain reaction (PCR), reflecting significant differences in the IL-15 gene expression in the pair-wise comparisons between group B and C and between groups A and C before and after interferon-α/ribavirin therapy (P < .01). Although real-time PCR revealed that IL-15 expression appeared to increase for all groups (A, B, and C) after therapy, these changes were not significant. Results are for 22 liver biopsy specimens obtained before and 22 obtained after treatment that were included in microarray analysis. The mean fold difference (MFD) was calculated for each gene at 2 different time points (i.e., before and after therapy) as follows: for microarray analysis and real-time PCR, (log2) MFD = [mean expression (log2) in group C] – [mean expression (log2) in group B]; for real-time PCR, (log2) MFD = [mean expression (log2) in group C] – [mean expression (log2) in group A. *Cutoff for statistical significance: P < .05 and (log2) MFD >0.38 or <−0.38 (which is equal to an absolute MFD of >1.3 or <−1.3). The mean fold change (MFC) was calculated for each gene within each group (A, B, and C) at 2 different time points (i.e., before and after therapy) as follows: for microarray analysis and real-time PCR, (log2) MFC = [mean expression (log2) after therapy] – [mean expression (log2) before therapy]. **Cutoff for statistical significance: P < .05 and (log2) MFC >0.38 or <−0.38 (which is equal to an absolute MFC of >1.3 or <−1.3).

Figure 3

Total RNA was isolated from peripheral blood mononuclear cells (PBMCs), and 150 ng was used for reverse transcription (RT) analysis, using the manufacturer’s recommend protocol (ABI reverse transcription kit). One-fifteenth of the RT reaction mix was used for real-time polymerase chain reaction (PCR; in triplicate), using ABI′ Taqman gene expression master mix and the ABI 7900HT fast real-time PCR system. IL-15 and GAPDH primers and probes were ordered through ABI’s premade Taqman gene expression assays system: IL-15 probe set A (ABI assay ID: HS9999039_m1) specific for an IL-15 coding exon; IL-15 probe set B (ABI assay ID: Hs01003716_m1) specific for the IL-15 3′ UTR target by Affymetrix’s U133A IL-15 probe set 205992_s_at; and GAPDH (ABI assay ID: Hs01003716_m1). IL-15 expression data were normalized to GAPDH expression levels and expressed as relative log2 values.