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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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Expression of T-bet and RoR-γt transcription factors. IL-17 and IL-22 production by CD4 T cells stimulated in the presence of IFN-DC.A) CLSM analysis (central optical sections) was performed on CD4 T cells activated by anti-CD3-coated beads in the presence of autologous DC for six days to evaluate the expression of the transcription factors T-bet and RoR-γt (pseudo-color grey). Cell nuclei were stained with DAPI (blue). Insets represent background labelling of isotype control antibodies. One representative experiment out of three independently performed is reported. B) Co-expression of the transcription factors T-bet (red) and RoR-γt (green) in CD4 T cells activated with anti-CD3-coated beads in the presence of autologous DC for six days, as observed in a representative experiment out of three. Cell nuclei were stained with DAPI (blue). One representative experiment out of three independently performed is reported. C) Production of IL-17 and IL-22 in the culture supernatants of CD4 T cells stimulated in the presence of autologous DC. Naïve CD4 T cells were stimulated with SEA or anti-CD3 in the presence of autologous DC for six days. The values represent the mean +/− SD of four independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05. D) Production of IL-17 by a fraction of CD4 T cells, as demonstrated by intracellular cytokine staining. The dot plot analysis, representative of one out of five experiments, shows the intracellular IFN-γ and L-17 expression by CD4 T cells primed with SEA or anti-CD3 in the presence of autologous DC for six days. Upon activation with PMA/ionomycin in the presence of brefeldin-A for 5 hours, as described in Materials and Methods, cells were stained for the CD4 antigen, permeabilized and stained for intracellular IFN-γ and IL-17. Analysis was performed on electronically gated CD4+ T cells.
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pone-0017364-g005: Expression of T-bet and RoR-γt transcription factors. IL-17 and IL-22 production by CD4 T cells stimulated in the presence of IFN-DC.A) CLSM analysis (central optical sections) was performed on CD4 T cells activated by anti-CD3-coated beads in the presence of autologous DC for six days to evaluate the expression of the transcription factors T-bet and RoR-γt (pseudo-color grey). Cell nuclei were stained with DAPI (blue). Insets represent background labelling of isotype control antibodies. One representative experiment out of three independently performed is reported. B) Co-expression of the transcription factors T-bet (red) and RoR-γt (green) in CD4 T cells activated with anti-CD3-coated beads in the presence of autologous DC for six days, as observed in a representative experiment out of three. Cell nuclei were stained with DAPI (blue). One representative experiment out of three independently performed is reported. C) Production of IL-17 and IL-22 in the culture supernatants of CD4 T cells stimulated in the presence of autologous DC. Naïve CD4 T cells were stimulated with SEA or anti-CD3 in the presence of autologous DC for six days. The values represent the mean +/− SD of four independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05. D) Production of IL-17 by a fraction of CD4 T cells, as demonstrated by intracellular cytokine staining. The dot plot analysis, representative of one out of five experiments, shows the intracellular IFN-γ and L-17 expression by CD4 T cells primed with SEA or anti-CD3 in the presence of autologous DC for six days. Upon activation with PMA/ionomycin in the presence of brefeldin-A for 5 hours, as described in Materials and Methods, cells were stained for the CD4 antigen, permeabilized and stained for intracellular IFN-γ and IL-17. Analysis was performed on electronically gated CD4+ T cells.

Mentions: We have then assessed by CLSM analysis the expression of the transcriptional factors T-bet and RoR-γt, specific for the Th1 and Th17 subsets, respectively. The majority of CD4 T cells activated in the presence of autologous DC selectively expressed T-bet, while RoR-γt was detected in only a minority of the cells (Fig. 5A, B). Consistently with the results indicating the IFN-DC-induced expansion of Th17 cells, the percentage of CD4 T cells expressing RoR-γt was higher in the cultures stimulated with anti-CD3-coated beads in the presence of autologous IFN-DC (mean 9.7±SD 1.8%) than in the co-cultures with IL-4-DC (mean 2.5±SD 2.4%) (Fig. 5A, B). The co-expression of T-bet and RoR-γt was detected in about 1% of CD4 T cells expanded by anti-CD3-coated beads and IFN-DC, whereas it was virtually absent in the CD4 T cell-IL-4-DC co-cultures (Fig. 5B).


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)

Expression of T-bet and RoR-γt transcription factors. IL-17 and IL-22 production by CD4 T cells stimulated in the presence of IFN-DC.A) CLSM analysis (central optical sections) was performed on CD4 T cells activated by anti-CD3-coated beads in the presence of autologous DC for six days to evaluate the expression of the transcription factors T-bet and RoR-γt (pseudo-color grey). Cell nuclei were stained with DAPI (blue). Insets represent background labelling of isotype control antibodies. One representative experiment out of three independently performed is reported. B) Co-expression of the transcription factors T-bet (red) and RoR-γt (green) in CD4 T cells activated with anti-CD3-coated beads in the presence of autologous DC for six days, as observed in a representative experiment out of three. Cell nuclei were stained with DAPI (blue). One representative experiment out of three independently performed is reported. C) Production of IL-17 and IL-22 in the culture supernatants of CD4 T cells stimulated in the presence of autologous DC. Naïve CD4 T cells were stimulated with SEA or anti-CD3 in the presence of autologous DC for six days. The values represent the mean +/− SD of four independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05. D) Production of IL-17 by a fraction of CD4 T cells, as demonstrated by intracellular cytokine staining. The dot plot analysis, representative of one out of five experiments, shows the intracellular IFN-γ and L-17 expression by CD4 T cells primed with SEA or anti-CD3 in the presence of autologous DC for six days. Upon activation with PMA/ionomycin in the presence of brefeldin-A for 5 hours, as described in Materials and Methods, cells were stained for the CD4 antigen, permeabilized and stained for intracellular IFN-γ and IL-17. Analysis was performed on electronically gated CD4+ T cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046151&req=5

pone-0017364-g005: Expression of T-bet and RoR-γt transcription factors. IL-17 and IL-22 production by CD4 T cells stimulated in the presence of IFN-DC.A) CLSM analysis (central optical sections) was performed on CD4 T cells activated by anti-CD3-coated beads in the presence of autologous DC for six days to evaluate the expression of the transcription factors T-bet and RoR-γt (pseudo-color grey). Cell nuclei were stained with DAPI (blue). Insets represent background labelling of isotype control antibodies. One representative experiment out of three independently performed is reported. B) Co-expression of the transcription factors T-bet (red) and RoR-γt (green) in CD4 T cells activated with anti-CD3-coated beads in the presence of autologous DC for six days, as observed in a representative experiment out of three. Cell nuclei were stained with DAPI (blue). One representative experiment out of three independently performed is reported. C) Production of IL-17 and IL-22 in the culture supernatants of CD4 T cells stimulated in the presence of autologous DC. Naïve CD4 T cells were stimulated with SEA or anti-CD3 in the presence of autologous DC for six days. The values represent the mean +/− SD of four independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05. D) Production of IL-17 by a fraction of CD4 T cells, as demonstrated by intracellular cytokine staining. The dot plot analysis, representative of one out of five experiments, shows the intracellular IFN-γ and L-17 expression by CD4 T cells primed with SEA or anti-CD3 in the presence of autologous DC for six days. Upon activation with PMA/ionomycin in the presence of brefeldin-A for 5 hours, as described in Materials and Methods, cells were stained for the CD4 antigen, permeabilized and stained for intracellular IFN-γ and IL-17. Analysis was performed on electronically gated CD4+ T cells.
Mentions: We have then assessed by CLSM analysis the expression of the transcriptional factors T-bet and RoR-γt, specific for the Th1 and Th17 subsets, respectively. The majority of CD4 T cells activated in the presence of autologous DC selectively expressed T-bet, while RoR-γt was detected in only a minority of the cells (Fig. 5A, B). Consistently with the results indicating the IFN-DC-induced expansion of Th17 cells, the percentage of CD4 T cells expressing RoR-γt was higher in the cultures stimulated with anti-CD3-coated beads in the presence of autologous IFN-DC (mean 9.7±SD 1.8%) than in the co-cultures with IL-4-DC (mean 2.5±SD 2.4%) (Fig. 5A, B). The co-expression of T-bet and RoR-γt was detected in about 1% of CD4 T cells expanded by anti-CD3-coated beads and IFN-DC, whereas it was virtually absent in the CD4 T cell-IL-4-DC co-cultures (Fig. 5B).

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