Immune activation in HIV/HCV-infected patients is associated with low-level expression of liver expressed antimicrobial peptide-2 (LEAP-2)

Abstract

Background: Immune activation is one of the main features of HIV/Hepatitis C virus (HCV) infections and has been linked to the disturbance of the gut-associated lymphoid tissue (GALT). In chronic HIV infection, loss of GALT integrity results in translocation of microbial products and chronic immune activation. We explored the relationship between bacterial translocation and specific colonic proteins, including liver expressed antimicrobial peptide (LEAP 2) which may play a role in modulating the bacterial translocation process.

Methods: A total of 40 subjects (10 HIV/HCV, 10 HIV, 10 HCV-infected patients and 10 controls) were enrolled and underwent serum and colonic tissue sampling. The levels of immune activation were evaluated by measuring plasma sCD27, and the levels of selected proinflammatory, Th2 and regulatory cytokines in both the plasma and supernatant of CD3-stimulated intraepithelial lymphocytes. We also evaluated LEAP-2 expression in the colon biopsies using Affymetrix Human Gene 1.0 ST (HuGene) and fluorescent immunohistochemistry.

Results: Increased levels of sCD27 were observed in HIV/HCV coinfected (p=0.03) and HIV monoinfected (p=0.04) patients compared with controls consistent with the presence of immune activation. The chip array identified LEAP-2 expression as a key marker associated with immune activation. LEAP-2 expression in HIV, HCV and HIV/HCV-infected patients was significantly lower compared with controls, and was significantly negatively correlated (p=0.03, r=-0.44) with sCD27.

Conclusions: Our data suggests that HCV and HIV infections are associated with decreased expression of LEAP-2 in colonic tissue. This may represent a key mechanism for enhanced microbial translocation and immune activation in HIV/HCV-infected patients.

Keywords: HIV; Hepatitis; Immunopathology; Liver; Microbial Pathogenic.

Figures

Figure 1

Heat map of gene array data of colon biopsies from control, HIV, HCV and HIV/HCV-coinfected patients. Log-fold changes of gene expressions (Y axis) for the colon tissues from control, HIV, HCV and HIV/HCV-coinfected patients are plotted in a heat map graph. Red represents up-regulated gene expression, while green represents down-regulated gene expression. Degree of colour reflects the levels of changes.

Figure 2

Liver expressed antimicrobial peptide-2 (LEAP-2) gene expression in colon tissue. Relative expression of LEAP-2 mRNA was measured using gene array as described in the methods. The levels of LEAP-2 expressions were compared between the enrolled groups using the Student t test. Data was presented using vertical box and whiskers graph. The horizontal lines represent the median values. The vertical lines represent the SDs. The box represents the 75% percentile of the data. LEAP-2 expression is significantly higher (p

Figure 3

Expression of liver expressed antimicrobial peptide-2 (LEAP-2) protein in the colon tissues. Expression of LEAP-2 protein in the colonic tissue was evaluated using fluorescent immunohistochemistry. (A) Represents the expression of LEAP-2 protein in colonic tissue from normal healthy control subject using anti-LEAP-2 antibody (A2), positive control colon biopsy samples from a patient with Crohn’s disease (A3) or patient with ulcerative colitis (A4) or isotype control (A1) and the secondary antibody Alexa Fluor 594 donkey antirabbit IgG. LEAP-2 appears as red particles in intraepithelial and interstitial tissue of the colon. (B) Shows representatives data of the comparative evaluation of LEAP-2 expression in colonic tissues from a healthy control subject (#105), HIV-monoinfected (#205), HCV-monoinfected (#303) and HIV/HCV-coinfected (#404) patients. The relative expression levels of LEAP-2 for control (#105), HIV patient (#205), HCV-monoinfected patient (#303), and HIV/HCV-coinfected patient (#404) were 7.64, 5.35, 5.14 and 5.61, respectively. The corresponding haematoxylin and eosin (H&E) staining for colonic tissues of the subjects demonstrated an increase in infiltrating inflammatory cells (markers of immune activation) in colonic tissues of patients compared with control.

Figure 4

Levels of sCD27 in the plasma of enrolled subjects. (A) Plasma levels of sCD27 were measured by ELISA as described in methods. Data are presented using vertical scatterplot graph. The horizontal lines represent the mean values with each symbol represents one subject; controls (open circle), HIV-monoinfected (filled circle), HCV-monoinfected (filled triangle), and HIV/HCV-coinfected (filled rectangle) patients. The levels of sCD27 in the control were significantly lower than the levels in HIV-monoinfected (p=0.04), and HIV/HCV-coinfected patients (p=0.03) (mean±SD: 0.5±0.2, 11.9±4.7 and 63.4±23.5 U/mL, respectively), but not significantly different from the level of sCD27 in the HCV-monoinfected patients (p=0.19, mean±SD: 12.9±8.8 U/mL). (B) A significant negative correlation (p=0.03 r= −0.44) between plasma sCD27 levels of the patients with their relative expression of liver expressed antimicrobial peptide-2 in colon tissue compared with controls. The data was evaluated using Spearman’s correlation coefficient.