Chronic infection caused by hepatitis C virus (HCV) is characterized by low or nil antiviral T-cell responses, whereas viral clearance is associated with strong and multispecific T-cell responses.1 Among other mechanisms, production of immunosuppressive cytokines such as interleukin 10 (IL-10)2, 3 has been postulated as responsible for this lack of efficient immunity. IL-10 is a pleiotropic cytokine traditionally considered as immunosuppressive and antiinflammatory, produced by many cell types (reviewed4), which exerts its effects by inhibiting macrophage and dendritic cell (DC) functions. In chronic HCV infection, patients have high serum levels of IL-10,5, 6 associated with incomplete responses to interferon IFN therapy.7 Interestingly, these levels decline after successful treatment.6 IL-10 is produced in these patients by antigen-stimulated CD4 and CD8 T-cells, regulatory T-cells,3, 8, 9 and DC10-12 which in turn activate IL-10-producing T-cells.13In vitro experiments have demonstrated that some HCV proteins interacting with monocytes induce the production of IL-10.14, 15 Due to its antiinflammatory properties, IL-10 has been used therapeutically in HCV patients with liver fibrosis.16 Although administration of IL-10 decreased hepatic inflammation and fibrosis, HCV RNA levels increased, and antiviral CD4 and CD8 T-cells shifted from a Th1 to a Th2 cytokine profile. All these data suggest that overexpression of IL-10 in chronic HCV infection may contribute to the lack of efficient antiviral T-cell responses. Indeed, IL-10 is a key factor in determining viral clearance versus chronic infection in the LCMV murine model, and its inhibition converted a chronic into an acute infection, which could be controlled by the immune response.17, 18 Thus, for chronic HCV infection, inhibition of IL-10 would potentially enhance the efficacy of antiviral responses and, ultimately, lead to viral clearance. With this purpose, we developed peptide inhibitors of IL-10 and tested their efficacy in the restoration of functional properties of antigen presenting cells to induce anti-HCV immunity.
The high levels of interleukin 10 (IL-10) present in chronic hepatitis C virus (HCV) infection have been suggested as responsible for the poor antiviral cellular immune responses found in these patients. To overcome the immunosuppressive effect of IL-10 on antigen-presenting cells such as dendritic cells (DCs), we developed peptide inhibitors of IL-10 to restore DC functions and concomitantly induce efficient antiviral immune responses. Two IL-10-binding peptides (p9 and p13) were selected using a phage-displayed library and their capacity to inhibit IL-10 was assessed in a bioassay and in STAT-3 (signal transducer and activator of transcription 3) phosphorylation experiments in vitro. In cultures of human leukocytes where HCV core protein induces the production of IL-10, p13 restored the ability of plasmacytoid DC to produce interferon alpha (IFN-α) after Toll-like receptor 9 (TLR9) stimulation. Similarly, when myeloid DCs were stimulated with CD40L in the presence of HCV core, p9 enhanced IL-12 production by inhibiting HCV core-induced as well as CD40L-induced IL-10. Moreover, in vitro, p13 potentiated the effect of maturation stimuli on human and murine DC, increasing their IL-12 production and stimulatory activity, which resulted in enhanced proliferation and IFN-γ production by responding T-cells. Finally, immunization with p13-treated murine DC induced stronger anti-HCV T-cell responses not only in wildtype mice but also in HCV transgenic mice and in mice transiently expressing HCV core in the liver. Conclusion: These results suggest that IL-10 inhibiting peptides may have important applications to enhance anti-HCV immune responses by restoring the immunostimulatory capabilities of DC. (HEPATOLOGY 2011.)
If you can't find a tool you're looking for, please click the link at the top of the page to "Go to old article view". Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement.
Materials and Methods
Identification of Phages Binding to IL-10.
Phages from a library expressing 15-mer random peptides near the N-terminus of phage surface protein pIII (a kind gift of GP Smith, University of Missouri-Columbia)19 were allowed to interact with biotinylated recombinant IL-10 (rIL-10) (Amersham Pharmacia Biotech, UK) as described.20 Three rounds of panning were carried out using 2.5, 0.02, and 0.002 μg/mL of rIL-10, respectively. After the third round, phages were eluted and their region coding for the 15-mer peptides was sequenced as described.20
Peptides identified using the phage library and used in initial screening assays as well as the human leukocyte antigen (HLA)-A2 restricted cytotoxic T lymphocyte (CTL) epitope from HCV NS3 1073-1081 and HCV NS3 peptide pools M2 and M4 were synthesized as described.21 Their purity was always above 80%. C-terminal amidated peptides p9 and p13, as well as control peptide p301 (amino acids 301-315 of HIV-1 gp120), were also purchased from NeoMPS (Strasbourg, France).
Bioassays Using MC/9 Cells.
MC/9 murine mast cell line (ATCC; Manassas, VA) was grown in Dulbecco's modified Eagle's medium (DMEM) containing 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol, 10% fetal bovine serum (complete medium; CM), and 5% rat T-STIM (BD-Biosciences, San Diego, CA). In IL-10-dependent proliferation assays, 2 × 104 cells were cultured in round-bottomed 96-well plates in CM containing 0.5 ng/mL of murine IL-4 (Peprotech, UK) and 1.25 ng/mL of human or murine rIL-10 (eBioscience, San Diego, CA), previously incubated for 2 hours with or without peptides. After 24 hours, 1 μCi of (methyl-3H)-thymidine (Amersham Life Science, Buckinghamshire, UK) was added per well and incubated for 12 additional hours before harvesting. Cells grown with or without IL-10 were used as positive (PC) and negative control (NC), respectively. Percentage of inhibition of IL-10 was calculated as: 100 × (cpmPC − cpmPT) / (cpmPC − cpmNC), where cpmPT corresponds to cpm obtained in the presence of IL-10 and peptides tested. Toxicity of the peptides was analyzed using similar bioassays with MC/9 cells but instead of rIL-10, murine GM-CSF (Peprotech, UK) at 0.01 ng/mL was used as stimulus.
Surface Plasmon Resonance.
Peptide binding to IL-10 was analyzed by surface plasmon resonance using a BIAcore X Biosensor (BIAcore, AB, Uppsala, Sweden). IL-10 (R&D Systems) was covalently immobilized onto the surface of flow cell 2 (FC2) of a CM5 chip (BIAcore) as described.20 Flow cell 1 (FC1) without IL-10 was used as the reference flow cell. Peptide solutions (10 μM) were injected three times in 10 mM Hepes, 150 mM NaCl, 0.005% (v/v) Tween-20, 0.1 mg/mL BSA, pH 7.4 at a flow of 30 μL/min. Curves were processed by subtracting the response in FC1 from that in FC2 and the responses at equilibrium were compared.
Western Blot Analysis.
MC/9 cells were cultured for 1 hour with rIL-10 (0.25 ng/mL) or rIL-9 (Sigma; 12.5 U/mL) previously incubated for 1 hour with or without peptides (100 μg/mL) or anti-IL-10 neutralizing antibody (eBioscience) (1 μg/mL). Then cells were harvested and phospho-STAT-3 (signal transducer and activator of transcription 3) and actin content was determined by immunoblot as described.22
Stimulation of Peripheral Blood DC to Induce Cytokine Production.
Blood was obtained from the Blood Bank of Navarra after informed consent. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki. Peripheral blood mononuclear cells (PBMCs) (2 × 105 cells/well) were stimulated in RPMI 1640 CM in 96-well plates with the Toll-like receptor 9 (TLR9) oligodeoxinucleotide ligand CpG 221623 (Sigma-Genosys) (5 μg/mL) for pDC analysis, or with a monolayer of 2 × 104 irradiated control or CD40L-expressing BHK cells24 (kindly provided by H. Engelmann; Munich, Germany) for mDC analysis, with or without recombinant HCV core protein (2.5 μg/mL) (BioDesign; Memphis, TN). IL-10-binding peptides (100 μg/mL) or anti-IL-10 antibody were added simultaneously to some wells in triplicate. Two days later, supernatants were harvested and enzyme-linked immunosorbent assay (ELISA) was used to measure content of IFN-α (Mabtech, Sweden), IL-12 and IL-10 (BD-Biosciences, San Diego, CA).
Stimulation of Human Monocyte-Derived DC and Murine DC.
Human monocyte-derived DC (MoDC) (105 cells/well), prepared as described,25 were stimulated with lipopolysaccharide (LPS) (1 μg/mL). Murine bone marrow-derived DC (BMDC) were prepared as described21 from C57BL/6 or HHD mice (transgenic for human HLA-A2.1 and beta-2 microglobulin molecules; a gift of Dr. F. Lemonnier, Institute Pasteur, Paris, France). Animals received humane care, maintained in pathogen-free conditions, and treated according to guidelines of our Institution Review Board. Murine BMDC were cultured at 106 cells/mL with LPS (0.5 μg/mL) and peptide 1073-1081 (10 μg/mL) or were infected with a recombinant adenovirus encoding HCV NS3 (AdNS3) as described.21 In all cases, cells were cultured with or without IL-10 peptide inhibitors (100 μg/mL) for 24 hours, harvested, washed, and used for in vitro stimulation experiments or immunization, in the case of murine DC.
Analysis of Cytokine Production by Flow Cytometry.
To measure IL-12 production by human mDC, PBMC were stimulated with CD40L as described above, and monensin (BD-Biosciences) was added for the last 4 hours of culture. Then cells were stained with a cocktail of FITC-labeled anti-CD3, -CD14, -CD16, -CD19, -CD20, and -CD56, PerCP-labeled anti-HLA-DR and APC-labeled anti-CD11c antibodies (all from BD-Biosciences). After fixation and permeabilization, PE-labeled anti-IL-12 antibodies (Miltenyi, Germany) were added and IL-12 production was analyzed in the mDC population, gated as Lin-HLA-DR+CD11c+. Cells were acquired in a FACSCalibur flow cytometer (BD-Biosciences) and analyzed with Flowjo software (Tree Star, Ashland, OR).
In Vitro Stimulatory Assays.
In vitro stimulatory ability of DC was assessed in mixed leukocyte reaction as described21, 25 or in assays analyzing presentation of the NS3 CTL peptide 1073-1081 to a T-cell line. In this case, peptide-pulsed DC from HHD mice (2 × 103/well) treated with LPS with or without IL-10 peptide inhibitors (100 μg/mL) during 24 hours were cocultured in anti-IFN-γ-coated ELISPOT plates with 104 1073-1081 peptide-specific T-cells. Next day, plates were developed and spot-forming cells were analyzed using an IFN-γ Elispot kit (BD-Biosciences) as described.21
Immunization and Analysis of T-Cell Responses.
HHD, C57BL/6, or FL-N transgenic mice26 expressing the full length HCV polyprotein (n = 5) were immunized subcutaneously with 2 × 105 DC pulsed with CTL peptide 1073-1081 or transfected with AdNS3. One week after immunization mice were sacrificed and splenocytes (5 × 105 cells/well) were cultured in the presence of peptide 1073-1081 or NS3 peptide pools M2 and M421 in anti-IFN-γ antibody-coated ELISPOT plates. Responses were analyzed as above.
Kruskal-Wallis and Mann-Whitney U nonparametric tests were used for comparison between groups using the SPSS v. 15.0 for Windows package. A P value <0.05 was considered significant.
Characterization of Peptide Inhibitors of IL-10.
Fifteen-mer peptides binding to IL-10 selected from the phage display library were synthesized and tested in a bioassay using the IL-10-sensitive MC/9 cell line to measure their IL-10 blocking activity. Peptides p9 (CHRCFHFRRHPVAVF) and p13 (TRH RHVPRFLPLRHV) inhibited human IL-10-induced proliferation (Fig. 1A). Inhibition of cell proliferation due to toxicity was discarded because cell stimulation by GM-CSF was not inhibited, demonstrating that inhibition was IL-10-specific (Fig. 1B). Peptide binding to IL-10 was demonstrated using surface plasmon resonance analysis. It was found that p9 and p13 bound to immobilized IL-10, as compared to a control peptide (Fig. 1C).
Finally, western blot experiments measuring IL-10-induced STAT-3 phosphorylation showed that p9 and p13 partially inhibited STAT-3 phosphorylation (Fig. 1D), but not IL-9-dependent STAT-3 phosphorylation. Moreover, in titration experiments using flow cytometry to measure phospho-STAT-3, complete inhibition was obtained with p9, and partial inhibition with p13, at the highest dose (Supporting Fig. S1).
Peptide Inhibitors of IL-10 Restore Cytokine Production by HCV Core-Treated Peripheral Blood DC.
The lack of efficient immune responses in HCV infection has been suggested to be related to a functional impairment of DC.13, 27 HCV core protein induces IL-10 production by monocytes in vitro, which inhibits functional properties of plasmacytoid DC (pDC).28 Thus, we tested whether our peptides could restore pDC functions by blocking the inhibitory effect of HCV core-induced IL-10. As described28 and shown in Fig. 2A, stimulation of pDC present in PBMC by a TLR9 ligand induced IFN-α, which was inhibited by HCV core, associated with the production of IL-10 (Fig. 2B). CpG-induced IFN-α production was restored to levels close to those induced in the absence of core when p13, but not p9 (data not shown), was included. A similar effect was observed when using only CpG (which induces low IL-10 levels) but not in the absence of core and CpG (Supporting Fig. S2), discarding an agonistic effect mediated by p13. Similar restoration was obtained when instead of HCV core, p13 was incubated with rIL-10 (0.65 ng/mL), concentration in the range of that induced in vitro by HCV core and equivalent to that found in serum of HCV patients6, 7 (Fig. 2C).
Because IL-10 not only inhibits pDC, but also myeloid DC (mDC),29 we analyzed whether core-induced IL-10 also inhibited mDC cytokine production. Stimulation of PBMC with CD40L induced IL-12 by CD11c+ mDC, as characterized by flow cytometry (Fig. 3A), which was inhibited by HCV core. Peptide p9, but not p13, partially restored the percentage of IL-12-producing mDC inhibited by HCV core. Restoration of IL-12 production by p9 was more clearly observed when measuring IL-12 secreted to the supernatants after CD40L stimulation in the presence of HCV core (Fig. 3B) or rIL-10 (Fig. 3C). Because IL-10 was also induced by CD40L (Fig. 3D), we tested the effect of p9 in the absence of exogenous IL-10 or HCV core. Stimulation with CD40L in the presence of p9 increased IL-12 production (Fig. 3E). In this case, IL-10, but not IL-12, was mainly produced by monocytes (Supporting Fig. S3). No effect was observed when p9 was added in the absence of CD40L (Supporting Fig. S4). Enhancement of IL-12 production by p9 did not reach statistical significance when added after blocking IL-10R (Fig. 3E). Moreover, p9 did not enhance IL-12 production after stimulation with CD40L of PBMC depleted of IL-10-producing CD14+ cells (Supporting Fig. S3). This suggests that p9 acts by inhibiting exogenous, HCV core-induced and endogenous maturation-induced IL-10.
Peptide Inhibitors of IL-10 Enhance the T-Cell Stimulatory Properties of DC.
Enhanced production of IL-12 after inhibition of endogenous IL-10 during DC activation suggested that peptide inhibitors of IL-10 could be useful not only in the presence of HCV proteins inducing IL-10, but also when using maturation stimuli inducing IL-10. To test this hypothesis in vitro and in vivo, human and murine DC were used. Human MoDC stimulated with LPS induced high levels of IL-10 (Fig. 4A). Treatment of MoDC with LPS in the presence of p13 did not modify their phenotype (data not shown). However, it induced higher IL-12 production (Fig. 4B), but only in the presence of LPS (Supporting Fig. S5). Moreover, p13 enhanced T-cell stimulatory ability of MoDC, measured as lymphocyte proliferation (Fig. 4C) and IFN-γ production (Fig. 4D). No effect was seen for p9 (data not shown).
Before testing our peptides in vivo we characterized them in vitro in a murine model. p13 and p9 bound to murine IL-10 and inhibited its activity in the MC9 bioassay (Supporting Fig. S6). Murine DC stimulated with LPS induced IL-10 (Fig. 5A), and in the presence of p13, higher IL-12 levels were induced (Fig. 5B), which did not occur with p9 (data not shown). When these DC were used as stimulators in vitro in MLR, enhanced proliferation (Fig. 5C) and IFN-γ production (Fig. 5D) were observed in those lymphocytes stimulated with p13-treated DC. Moreover, treatment with p13 of LPS-activated DC from HHD mice, which express human HLA-A2 molecules, enhanced their ability to present in vitro the HLA-A2-restricted NS3 CTL epitope 1073-1081 to 1073-1081-specific CD8 T-cells (Fig. 5E).
The in vivo stimulatory ability of p13-treated DC was then tested in HHD mice immunized with LPS-stimulated DC pulsed with CTL epitope 1073-1081 and in C57BL/6 mice immunized with DC transduced with a recombinant adenovirus expressing HCV NS3, known to induce IL-10.21 In both cases, treatment of DC with p13 clearly increased their in vivo immunogenicity, as measured by their ability to induce anti-NS3 T-cell responses (Fig. 6A,B). HCV core protein induces IL-10 by murine splenocytes (Supporting Fig. S7). Thus, in order to mimic the effect of HCV core in infected patients, we immunized HHD mice transiently expressing in the liver a secretable version of core protein (Supporting Fig. S7). In this model, as in previous experiments, higher responses against peptide 1073-1081 were induced by p13-treated DC (Fig. 6C). Finally, in transgenic mice expressing HCV full-length polyprotein in the liver, immunization with p13-treated DC also induced stronger anti-NS3 T-cell responses (Fig. 6D).
HCV chronic infection is characterized by poor cellular immune responses, which might be in part due to the production of immunosuppressive cytokines like IL-10.8 Due to the role that IL-10 plays in the development of a chronic viral infection17, 18 and in the efficacy of antiviral immunotherapy,30 we tested the effect of peptide inhibitors of IL-10 on the functional properties of DC, a cell population responsible for the activation of cellular immunity, which can be suppressed by IL-10. We identified two IL-10 inhibiting peptides capable of blocking the ability of IL-10 to activate STAT-3 as well as the biological activity of the cytokine in specific bioassays. Importantly, these peptides rescued the functional properties of DC activated in the presence of HCV core, a known inducer of IL-10 and a repressor of DC immunostimulatory functions.28 Indeed, p13 restored IFN-α production by pDC after TLR9 stimulation in a well-characterized model of IL-10-dependent DC inhibition,28 whereas p9 restored IL-12 production by mDC after stimulation with CD40L. Production of IFN-α and IL-12 by activated pDC and mDC, respectively, are important functional features of these DC populations. IFN-α has important direct antiviral properties and immunostimulatory effects. At the same time, IL-12 facilitates the induction of Th1 responses, known to help viral clearance. In HCV infection, several sources of IL-10 have been described,11-13 viral proteins induce IL-10,14, 15 and high IL-10 serum levels have been correlated with poor treatment response.7 Thus, inhibition of IL-10 in HCV infection by using peptide inhibitors described in the present work might contribute to restore the ability of DC to activate T-cell responses. In this line, in vitro neutralization of IL-10 in PBMC from HCV-infected patients recovered the activity of low-responsive T-cells.3, 31 Although the mechanism responsible for this recovery is not characterized, restoration of functional properties of DC and concomitantly of T-cells might explain these results. Thus, IL-10 inhibition in HCV infection might enhance T-cell immunity facilitating viral clearance.
An important finding obtained using peptide inhibitors of IL-10 is that they not only inhibit IL-10 released in response to HCV proteins, but also IL-10 induced by maturation stimuli. Indeed, activation of mDC with CD40L in the presence of p9 enhanced IL-12 production. Thus, we hypothesized that inhibiting an endogenous brake, like IL-10, synthesized upon CD40L stimulation, may be useful to improve the functional properties of DC. This strategy may increase the immunogenicity of DC, leading to higher efficacy of DC-based vaccination procedures. Using human MoDC (DC population commonly used in vaccination), we found that inhibition of endogenous IL-10 with p13 enhanced their immunogenicity in vitro, increasing lymphocyte proliferation and IFN-γ production, the prototypical Th1 cytokine. Similar results were obtained with murine DC, in agreement with the ability of these peptides to bind and inhibit murine IL-10, which has more than 70% homology with hIL-10. More important, immunization with p13-treated DC in different antigenic models, including mice expressing a secretable version of HCV core as well as transgenic mice expressing the full HCV polyprotein, led to the induction of stronger anti-NS3 T-cell responses, measured as IFN-γ production. Thus, these peptides may have important applications in HCV infection not only in vivo, to inhibit IL-10, thus modulating immune responses, but also ex vivo, in clinical protocols based on the use of DC loaded with HCV antigens for further administration in therapeutic vaccination.
An interesting result regarding the activity of p13 and p9 is their selective effect on their ability to restore cytokine production by different DC populations. We do not have a clear explanation, but it might be speculated that these DC populations and their functions have a different sensitivity to be inhibited by IL-10. This might be related to differences in the site of binding to IL-10 by p9 and p13, resulting in specific effects on both types of DC populations. Similarly, binding of the anti-IL-10 antibody to another site on the cytokine may also explain differences in its effect.
Finally, because IL-10 plays an immunosuppressive role32 in other diseases (infections by HBV, human immunodeficiency virus [HIV], Epstein-Barr virus [EBV], or cancer), we believe that these peptides might be also useful in these settings. Neutralizing antibodies have been used to block IL-10; however, peptides like those described here can also be considered therapeutic tools, because they have advantages such as lower manufacturing cost, higher activity per unit mass, greater stability for storage, better organ penetration, and the possibility of sequence modification to improve activity, half-life, and specificity.33
In summary, we have developed two peptide inhibitors of IL-10 that can restore and enhance functional properties of DC. Due to the important role that IL-10 plays in chronic HCV infection as well as on the development of other diseases, we believe that these compounds might have important immunotherapeutic applications.
The authors thank Drs. G.P. Smith and H. Engelmann for their kind gift of different reagents.