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. 2008 Jun 13:9:28.
doi: 10.1186/1471-2172-9-28.

Enhanced IL-10 production in response to hepatitis C virus proteins by peripheral blood mononuclear cells from human immunodeficiency virus-monoinfected individuals

Lisa Barrett  1 , Maureen Gallant, Constance Howley, M Ian Bowmer, Geri Hirsch, Kevork Peltekian, Michael Grant
Affiliations

Enhanced IL-10 production in response to hepatitis C virus proteins by peripheral blood mononuclear cells from human immunodeficiency virus-monoinfected individuals

Lisa Barrett et al. BMC Immunol. .

Abstract

Background: Multiple immune evasion strategies by which HCV establishes chronic infection have been proposed, including manipulation of cytokine responses. Prior infection with HIV increases the likelihood of chronic HCV infection and accelerates development of HCV-related morbidity. Therefore, we investigated in vitro cytokine responses to HCV structural and non-structural proteins in peripheral blood mononuclear cells (PBMC) from uninfected, HIV-infected, HCV-infected and HIV/HCV-coinfected individuals.

Results: Intracellular flow cytometry was used to assess IL-2, IL-10, IL-12, and IFN-gamma production by freshly isolated PBMC incubated for 16 hours with recombinant HCV core, non-structural protein 3 (NS3), and NS4 proteins. Anti-HCV cellular responses were assessed in HIV/HCV-coinfected individuals by 3H-thymidine proliferation assay. Exposure to HCV antigens increased IL-10 production by PBMC, especially in uninfected and HIV-monoinfected individuals. This IL-10 response was attenuated in chronic HCV infection even with HCV/HIV-coinfection. The cells producing IL-10 in response to HCV proteins in vitro matched a PBMC subset recently shown to constitutively produce IL-10 in vivo. This subset was found at similar frequencies in uninfected, HIV-infected, HCV-infected and HIV/HCV-coinfected individuals before exposure to HCV proteins. HCV-specific T cell proliferation was detectable in only one HIV/HCV-coinfected individual who demonstrated no HCV-induced IL-10 response.

Conclusion: This pattern suggests that selective induction of IL-10 in uninfected individuals and especially in HIV-monoinfected individuals plays a role in establishing chronic HCV infection and conversely, that attenuation of this response, once chronic infection is established, favours development of hepatic immunopathology.

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Figures

Figure 1
Figure 1
Positive controls for cytokine production. (a) IFN-γ production by CD8+ and CD8- lymphoid gated PBMC after phytohemagglutinin treatment. (b) IL-2 production in total PBMC after PMA/ionomycin treatment. (c) IL-10 production in CD14+ cells after LPS stimulation. (d) IL-12 production in CD14+ cells after LPS stimulation.
Figure 2
Figure 2
Cytokine production following 16 hour exposure of PBMC to HCV proteins. Freshly isolated PBMC from HIV-infected, HCV-infected, HIV/HCV co-infected and uninfected controls were incubated for 16 hours with HCV proteins, then analyzed by intracellular flow cytometry for cytokine production as shown in representative plots. Selective IL-10 production by a small subset of CD3- PBMC with lymphoid scatter characteristics occurred in each case.
Figure 3
Figure 3
Mean percentage of PBMC from different groups expressing IL-10 ex vivo and following 16 hour incubation with HCV proteins. (a) The percentage of IL-10 producing cells (mean ± SE) was assessed by intracellular flow cytometry in freshly isolated PBMC and following 16 hour incubation with HCV proteins. Black bars represent fresh PBMC, dark grey bars represent PBMC incubated for 16 hours with recombinant HCV core, NS3 and NS4 and lighter grey bars represent the same PBMC incubated for 16 hours with β-galactosidase control protein. Statistical comparisons are made within groups using the Mann-Whitney U test and between groups using the Kruskal-Wallis test. (b) Representative flow plots of IL-10 production in PBMC from an HIV/HCV-coinfected individual after incubation of PBMC with individual HCV proteins at 2.5 μg/mL. Numbers represent the percentage of IL-10 positive PBMC.
Figure 4
Figure 4
Relationship between CD36 expression and ex vivo IL-10 production by PBMC. (a) Representative flow cytometry plot showing IL-10 production in freshly-isolated CD36+ PBMC cells with lymphoid light scatter characteristics. The percentage of CD36+ cells that are IL-10hi is indicated. (b) Mean channel fluorescence (MCF) for CD36 plotted against IL-10 MCF for six individuals. Open circles represent the CD36hi subset and dark circles represent the CD36low subset as shown in fig. 3a.
Figure 5
Figure 5
Effect of polymyxin B, chloroquine and T cell depletion on IL-10 production by PBMC exposed to recombinant HCV proteins. (a) Cells from uninfected individuals (n = 5) were pretreated with polymyxin B (grey bars) before incubation with recombinant HCV proteins to block the effects of any endotoxin contamination of the recombinant proteins. The effect of 16 hour exposure to recombinant HIV p24 on IL-10 induction was also assessed. (b) The mean percentage PBMC expressing IL-10 (± SE) from control, HIV-infected and HIV/HCV coinfected individuals was assessed after exposure to either β-galactosidase (white background bars) or HCV proteins (grey background bars). Effects of treating PBMC with chloroquine (diagonal hatch) or depleting CD3+ T cells (horizontal hatch) are also shown for comparison with the HCV protein group (grey bars). Each individual was tested on at least 2 different visits.
Figure 6
Figure 6
Anti-HCV cellular responses and IL-10 induction in HIV/HCV coinfected Individuals. (a) 3H-thymidine incorporation by PBMC from 10 HIV/HCV coinfected individuals incubated for 5 days with HCV core (black bar), NS3 (medium grey bar), NS4 (dark grey bar) or HIV p24 (light grey bar) was measured. A stimulation index of ≥3 was considered positive. Individuals 18 and 150 were anti-HCV positive but HCV RNA negative at the time of testing. (b) IL-10 production in 10 HIV/HCV coinfected individuals after 16 hour incubation with either β-galactosidase or HCV proteins. The percentage of IL-10+ PBMC within the lymphoid gate is represented on the y axis. Testing was done on the same day as the proliferation data was collected. Individuals 18 and 150 were anti-HCV positive but HCV RNA negative at the time of testing.
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