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 see more 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 selleck compound 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. 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 PIK3C2G 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.