Early IL-10 predominant responses are associated with progression to chronic hepatitis C virus infection in injecting drug users - PubMed (original) (raw)

Multicenter Study

Early IL-10 predominant responses are associated with progression to chronic hepatitis C virus infection in injecting drug users

J K Flynn et al. J Viral Hepat. 2011 Aug.

Abstract

The critical events in clearance or persistence of hepatitis C virus (HCV) infection are unknown but likely to be determined early in acute infection. Type 1 and type 2 cytokine production was assessed by HCV peptide ELISpot and multiplex in vitro cytokine production assays in longitudinally collected samples from 20 untreated participants enrolled in the Australian Trial in Acute Hepatitis C (ATAHC); a prospective cohort of acute HCV infection (77% injecting drug users, IDU). Significantly higher interleukin-10 (IL-10) production (P = 0.048), in the relative absence of interferon-gamma (IFN-γ) and IL-2 production, was present early in HCV infection in those who progressed to chronic infection. In contrast, viral clearance was associated with a greater magnitude and broader specificity of IFN-γ (magnitude P < 0.001, breadth P = 0.004) and IL-2 responses, in the relative absence of IL-10. Early IL-10 production was correlated with higher HCV RNA level at baseline (P = 0.046) and week 12 (P = 0.018), while IFN-γ and IL-2 production was inversely correlated with HCV RNA level at baseline (IFN-γ P = 0.020, IL-2 P = 0.050) and week 48 (IFN-γ P = 0.045, IL-2 P = 0.026). Intracellular staining (ICS) indicated the HCV-specific IFN-γ response was primarily from CD8(+) T cells and NK cells, whereas IL-10 production was predominantly from monocytes, with a subset of IL-10 producing CD8(+) T cells present only in those who progressed to chronic infection. IL-10, an immunoregulatory cytokine, appears to play a key role in progression to chronic HCV infection.

© 2010 Blackwell Publishing Ltd.

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Conflict of interest statement

Author Statement:

No author has a commercial or other association that might pose a conflict of interest.

Figures

Figure 1

Figure 1

Longitudinal analysis of the magnitude (A-B) and breadth (C-D) of HCV-specific IFN-γ and IL-2 responses in clearers (n=12, white bars) and persisters (n=8, hatched bars) from baseline to week 48. A) A significantly higher magnitude of IFN-γ production was detected in clearers compared to persisters at baseline (p<0.001) and week 12 (p=0.019) by Mann-Whitney. B) Clearers maintained their HCV-specific IL-2 response with higher magnitude of IL-2 production compared to persisters screening to week 48. C) Clearers of HCV infection had significantly broader HCV-specific IFN-γ responses compared to persisters at baseline (p=0.004, Mann-Whitney) and week 12 (p=0.007, Mann-Whitney), and maintained a boarder response to week 48. D) Clearers demonstrated a trend of a broader IL-2 response baseline to week 48 compared to persisters. The horizontal solid lines in the box and whisker plots represent the median response. Four asterisks represent p<0.001, two asterisks represent p<0.01 and one asterisk represents p<0.05.

Figure 1

Figure 1

Longitudinal analysis of the magnitude (A-B) and breadth (C-D) of HCV-specific IFN-γ and IL-2 responses in clearers (n=12, white bars) and persisters (n=8, hatched bars) from baseline to week 48. A) A significantly higher magnitude of IFN-γ production was detected in clearers compared to persisters at baseline (p<0.001) and week 12 (p=0.019) by Mann-Whitney. B) Clearers maintained their HCV-specific IL-2 response with higher magnitude of IL-2 production compared to persisters screening to week 48. C) Clearers of HCV infection had significantly broader HCV-specific IFN-γ responses compared to persisters at baseline (p=0.004, Mann-Whitney) and week 12 (p=0.007, Mann-Whitney), and maintained a boarder response to week 48. D) Clearers demonstrated a trend of a broader IL-2 response baseline to week 48 compared to persisters. The horizontal solid lines in the box and whisker plots represent the median response. Four asterisks represent p<0.001, two asterisks represent p<0.01 and one asterisk represents p<0.05.

Figure 2

Figure 2

Multiplex cytokine production at baseline in a representative example of A) a clearer (duration of infection 25 weeks) and B) a persister (duration of infection 28 weeks) after stimulation with the three HCV peptide pools (Core-p7, NS2-3, NS4-5), control CEF peptides or PHA. Strong Type 1 cytokine responses (IFN-γ/IL-2) to HCV antigens are present in the clearer and absent in the persister. The dotted black line at 10 pg/mL represents the background cytokine production (media alone) in the assay.

Figure 3

Figure 3

Longitudinal analysis of HCV-specific A) IL-10 and B) IFN-γ production in clearers (baseline n=12, white bars) and persisters (baseline n=8, hatched bars) by multiplex cytokine assay. A) A significantly higher magnitude of IL-10 production was detected in persisters at baseline (p=0.048) compared to clearers. IL-10 production was maintained in persisters longitudinally from baseline to week 48. B) In contrast, a significantly higher magnitude of IFN-γ production was detected in clearers at baseline (p=0.04), which was maintained in clearers longitudinally from baseline to week 48. C) The ratio of IL-10 production to IFN-γ production at baseline was significantly higher in persisters (p=0.002). In graphs A to C the horizontal line indicates the median and in graph C, each dot represents a subject in the study. Three asterisks represent p<0.005 and one asterisk represents p<0.05.

Figure 4

Figure 4

Characterization of HCV-specific IFN-γ and IL-10 producing cells by ICS. PBMCs from 4 persisters and 4 clearers were stimulated with media alone or HCV antigens (Core-p7 or NS4/5) for 3 days prior to ICS. A) IL-10 producing cells from subject P7 are presented where cells were stimulated with media alone (top row) or HCV antigen C-P7 (bottom row). Dot plot analysis of IL-10 producing cells revealed a low background in the media control, whereas C-P7 stimulated CD3+CD4+, CD3+CD8+ and CD14+ IL-10 producing cells were 3.1, 2.7 and 2.3 fold higher in number respectively. B) IL-10 production was more frequently detected following stimulation with Core-P7 antigens and from persisters. HCV-specific IL-10 production was from CD14+ cells in both persisters and clearers, while only persisters had a subset of IL-10 producing CD8+ and CD4+ T cells. C) IFN-γ producing cells were detected following stimulation with NS4/5 antigens. HCV-specific IFN-γ production was mainly from CD8+ T cells and CD56+ cells. The white bars represent CD4+ T cells, the speckled bars represent CD8+ T cells, CD56+ cells are represented by horizontally striped bars and the CD14+ cells by speckled bars. To determine the percentage of cytokine producing cells, viable cells were first gated on either CD14+ cells, CD56+ cells or CD3+ cells. The CD3+ cells were then gated on CD3+CD4+ and CD3+CD8+ T cells.

Figure 5

Figure 5

Correlation of HCV-specific cytokine production with HCV RNA levels from A) IFN-γ ELISpot assay B) IL-2 ELISpot assay and C) IL-10 production from the multiplex in vitro cytokine assay at baseline. A) High IFN-γ production was inversely correlated with HCV RNA (Spearman correlation rho=-0.51 p=0.02) B) The magnitude of IL-2 was also inversely correlated with HCV RNA (Spearman correlation rho=-0.44 p=0.05) C) In the multiarray assay, high IL-10 production was positively correlated with high HCV RNA levels (Spearman correlation rho=0.45, p=0.046).

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