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Interferon-α-conditioned human monocytes combine a Th1-orienting attitude with the induction of autologous Th17 responses: role of IL-23 and IL-12.

Santini SM, Lapenta C, Donati S, Spadaro F, Belardelli F, Ferrantini M - PLoS ONE (2011)

Bottom Line: Priming of naïve CD4 T cells with autologous IFN-DC in the presence of either SEA or anti-CD3, resulted, in addition to a prominent expansion of CXCR3+ IFN-γ-producing CD4 Th1 cells, in the emergence of two distinct subsets of IL-17-producing CD4 T cells: i) a predominant Th17 population selectively producing IL-17 and expressing CCR6; ii) a minor Th1/Th17 population, producing both IL-17 and IFN-γ.After phagocytosis of apoptotic cells, IFN-DC induced Th17 cell expansion and IL-17 release.Notably, the use of neutralizing antibodies revealed that IL-23 was an essential cytokine in mediating Th17 cell development by IFN-DC.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy.

ABSTRACT
IFN-α exerts multiple effects leading to immune protection against pathogens and cancer as well to autoimmune reactions by acting on monocytes and dendritic cells. We analyzed the versatility of human monocytes conditioned by IFN-α towards dendritic cell differentiation (IFN-DC) in shaping the autologous T-helper response. Priming of naïve CD4 T cells with autologous IFN-DC in the presence of either SEA or anti-CD3, resulted, in addition to a prominent expansion of CXCR3+ IFN-γ-producing CD4 Th1 cells, in the emergence of two distinct subsets of IL-17-producing CD4 T cells: i) a predominant Th17 population selectively producing IL-17 and expressing CCR6; ii) a minor Th1/Th17 population, producing both IL-17 and IFN-γ. After phagocytosis of apoptotic cells, IFN-DC induced Th17 cell expansion and IL-17 release. Notably, the use of neutralizing antibodies revealed that IL-23 was an essential cytokine in mediating Th17 cell development by IFN-DC. The demonstration of the IFN-DC-induced expansion of both Th1 and Th17 cell populations reveals the intrinsic plasticity of these DC in orienting the immune response and provides a mechanistic link between IFN-α and the onset of autoimmune phenomena, which have been correlated with both IL-17 production and exposure to IFN-α.

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Inhibition of IL-17 production by selective cytokine neutralization.A) Inhibition of IL-17 production by selected couples or cocktail of cytokine blocking antibodies. Naïve CD4+ T cells were activated with anti-CD3 coated beads and autologous DC at a cell ratio of 1∶4 in the presence of the indicated couples of cytokine-neutralizing antibodies, or of the respective control isotypes. The presence of IL-17 was analyzed in the supernatants of cell cultures after 6 days by ELISA. The effect of the control isotypes couples on IL-17 production was not significantly different from that of the control isotypes cocktail. The values represent the mean +/− SD of three independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05. B) Inhibition of IFN-γ and IL-23 production by selective IL-23/IL-12 neutralization as compared to the inhibition obtained with the complete neutralizing cocktail, including antibodies to the p40 subunit of IL-12 and IL-23, p19 (IL-23), IL-1β, TNF-α and IL-6. Th values represent the mean +/− SD of three independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05.
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pone-0017364-g006: Inhibition of IL-17 production by selective cytokine neutralization.A) Inhibition of IL-17 production by selected couples or cocktail of cytokine blocking antibodies. Naïve CD4+ T cells were activated with anti-CD3 coated beads and autologous DC at a cell ratio of 1∶4 in the presence of the indicated couples of cytokine-neutralizing antibodies, or of the respective control isotypes. The presence of IL-17 was analyzed in the supernatants of cell cultures after 6 days by ELISA. The effect of the control isotypes couples on IL-17 production was not significantly different from that of the control isotypes cocktail. The values represent the mean +/− SD of three independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05. B) Inhibition of IFN-γ and IL-23 production by selective IL-23/IL-12 neutralization as compared to the inhibition obtained with the complete neutralizing cocktail, including antibodies to the p40 subunit of IL-12 and IL-23, p19 (IL-23), IL-1β, TNF-α and IL-6. Th values represent the mean +/− SD of three independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05.

Mentions: We then asked which IFN-DC cytokines were required for IL-17 induction in naïve CD4 T cells. As expected, the antibody blockade of an ensemble of cytokines (i.e. IL-1β, IL-6, TNF-α, and IL-12/IL-23) markedly inhibited IL-17 production (Fig. 6A), whereas neutralization of single cytokines did not significantly prevent IL-17 production (data not shown). Interestingly, neutralizing antibodies against the IL-23p19 and the IL-12/IL-23p40 consistently and markedly inhibited IL-17 production in the co-cultures of naïve CD4 T cells with IFN-DC (Fig. 6A). The combination of anti-p19 and anti-p40 antibodies was used to obtain the highest possible neutralization of IL-23 as, in our experimental setting, the addition of the anti-p19 antibody alone reproducibly resulted in only a limited neutralization of IL-23 and a slight inhibition of IL-17 production (data not shown). In contrast, the addition of both anti-p19 and anti-p40 antibodies resulted not only in a virtually complete disappearance of IL-23 (Fig. 6B, upper graph), but also in a sharp reduction of IFN-γ production in the co-cultures of naïve CD4 T cells with autologous IFN-DC (Fig. 6B, lower graph). No significant increase in the inhibition of IL-17 production was observed when anti-IL-1β, anti-IL-6 or anti-TNF-α neutralizing antibodies were added together with the anti-p40 antibodies to the anti-CD3-activated naïve CD4 T cell-IFN-DC co-cultures (Fig. 6A). Overall, these results together with the observation that much higher levels of IL-23 as compared to IL-12 were reproducibly detected in those co-cultures (data not shown), strongly indicate IL-23 as an essential cytokine in mediating human Th17 cell development by IFN-DC.


Interferon-α-conditioned human monocytes combine a Th1-orienting attitude with the induction of autologous Th17 responses: role of IL-23 and IL-12.

Santini SM, Lapenta C, Donati S, Spadaro F, Belardelli F, Ferrantini M - PLoS ONE (2011)

Inhibition of IL-17 production by selective cytokine neutralization.A) Inhibition of IL-17 production by selected couples or cocktail of cytokine blocking antibodies. Naïve CD4+ T cells were activated with anti-CD3 coated beads and autologous DC at a cell ratio of 1∶4 in the presence of the indicated couples of cytokine-neutralizing antibodies, or of the respective control isotypes. The presence of IL-17 was analyzed in the supernatants of cell cultures after 6 days by ELISA. The effect of the control isotypes couples on IL-17 production was not significantly different from that of the control isotypes cocktail. The values represent the mean +/− SD of three independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05. B) Inhibition of IFN-γ and IL-23 production by selective IL-23/IL-12 neutralization as compared to the inhibition obtained with the complete neutralizing cocktail, including antibodies to the p40 subunit of IL-12 and IL-23, p19 (IL-23), IL-1β, TNF-α and IL-6. Th values represent the mean +/− SD of three independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046151&req=5

pone-0017364-g006: Inhibition of IL-17 production by selective cytokine neutralization.A) Inhibition of IL-17 production by selected couples or cocktail of cytokine blocking antibodies. Naïve CD4+ T cells were activated with anti-CD3 coated beads and autologous DC at a cell ratio of 1∶4 in the presence of the indicated couples of cytokine-neutralizing antibodies, or of the respective control isotypes. The presence of IL-17 was analyzed in the supernatants of cell cultures after 6 days by ELISA. The effect of the control isotypes couples on IL-17 production was not significantly different from that of the control isotypes cocktail. The values represent the mean +/− SD of three independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05. B) Inhibition of IFN-γ and IL-23 production by selective IL-23/IL-12 neutralization as compared to the inhibition obtained with the complete neutralizing cocktail, including antibodies to the p40 subunit of IL-12 and IL-23, p19 (IL-23), IL-1β, TNF-α and IL-6. Th values represent the mean +/− SD of three independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05.
Mentions: We then asked which IFN-DC cytokines were required for IL-17 induction in naïve CD4 T cells. As expected, the antibody blockade of an ensemble of cytokines (i.e. IL-1β, IL-6, TNF-α, and IL-12/IL-23) markedly inhibited IL-17 production (Fig. 6A), whereas neutralization of single cytokines did not significantly prevent IL-17 production (data not shown). Interestingly, neutralizing antibodies against the IL-23p19 and the IL-12/IL-23p40 consistently and markedly inhibited IL-17 production in the co-cultures of naïve CD4 T cells with IFN-DC (Fig. 6A). The combination of anti-p19 and anti-p40 antibodies was used to obtain the highest possible neutralization of IL-23 as, in our experimental setting, the addition of the anti-p19 antibody alone reproducibly resulted in only a limited neutralization of IL-23 and a slight inhibition of IL-17 production (data not shown). In contrast, the addition of both anti-p19 and anti-p40 antibodies resulted not only in a virtually complete disappearance of IL-23 (Fig. 6B, upper graph), but also in a sharp reduction of IFN-γ production in the co-cultures of naïve CD4 T cells with autologous IFN-DC (Fig. 6B, lower graph). No significant increase in the inhibition of IL-17 production was observed when anti-IL-1β, anti-IL-6 or anti-TNF-α neutralizing antibodies were added together with the anti-p40 antibodies to the anti-CD3-activated naïve CD4 T cell-IFN-DC co-cultures (Fig. 6A). Overall, these results together with the observation that much higher levels of IL-23 as compared to IL-12 were reproducibly detected in those co-cultures (data not shown), strongly indicate IL-23 as an essential cytokine in mediating human Th17 cell development by IFN-DC.

Bottom Line: Priming of naïve CD4 T cells with autologous IFN-DC in the presence of either SEA or anti-CD3, resulted, in addition to a prominent expansion of CXCR3+ IFN-γ-producing CD4 Th1 cells, in the emergence of two distinct subsets of IL-17-producing CD4 T cells: i) a predominant Th17 population selectively producing IL-17 and expressing CCR6; ii) a minor Th1/Th17 population, producing both IL-17 and IFN-γ.After phagocytosis of apoptotic cells, IFN-DC induced Th17 cell expansion and IL-17 release.Notably, the use of neutralizing antibodies revealed that IL-23 was an essential cytokine in mediating Th17 cell development by IFN-DC.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy.

ABSTRACT
IFN-α exerts multiple effects leading to immune protection against pathogens and cancer as well to autoimmune reactions by acting on monocytes and dendritic cells. We analyzed the versatility of human monocytes conditioned by IFN-α towards dendritic cell differentiation (IFN-DC) in shaping the autologous T-helper response. Priming of naïve CD4 T cells with autologous IFN-DC in the presence of either SEA or anti-CD3, resulted, in addition to a prominent expansion of CXCR3+ IFN-γ-producing CD4 Th1 cells, in the emergence of two distinct subsets of IL-17-producing CD4 T cells: i) a predominant Th17 population selectively producing IL-17 and expressing CCR6; ii) a minor Th1/Th17 population, producing both IL-17 and IFN-γ. After phagocytosis of apoptotic cells, IFN-DC induced Th17 cell expansion and IL-17 release. Notably, the use of neutralizing antibodies revealed that IL-23 was an essential cytokine in mediating Th17 cell development by IFN-DC. The demonstration of the IFN-DC-induced expansion of both Th1 and Th17 cell populations reveals the intrinsic plasticity of these DC in orienting the immune response and provides a mechanistic link between IFN-α and the onset of autoimmune phenomena, which have been correlated with both IL-17 production and exposure to IFN-α.

Show MeSH
Related in: MedlinePlus