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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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Induction of Th17 response by DC loaded with apoptotic cells.A) Representative dot plot analysis (one out of three) of CD154 induction in CD4 T cells after 6 days of culture with autologous IFN-DC or IL4-DC fed with apoptotic cells. The upper dot plots show CD154 expression in purified naïve CD4 T cells prior to use. CTR dot plots show the percentage of CD154 expression by CD4 T cells cultivated in the presence of autologous DC which had not phagocytosed apoptotic tumor cells. Analysis was performed on electronically gated CD4+ T cells. The percentage of early and late apoptotic tumor cells was evaluated by Annexin-V/propidium iodide staining. B) ELISA detection of IL-1β, IL-6 and IL-23 release by DC upon phagocytosis of apoptotic tumor cell lines. Analysis was performed after 18 hours from tumor cell uptake. The values represent the mean +/− SD of three independent experiments. C) ELISA detection of IL-1β, IL-6 and IL-23 in supernatants from co-culture of CD4 T cells with autologous DC which had phagocytosed apoptotic cells. After three days of co-culture, CD4 T cells were restimulated overnight with anti-CD3 coated beads. At day four the supernatant was collected for cytokine detection. All data are expressed as the mean +/− SD of three independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05. D) Dot plot analysis of CCR6/CCR4 expression by CD4 T cells after six days of stimulation in the presence of DC fed with apoptotic tumor cells or autologous PBL. The results of one representative experiment out of three are shown. In all case, the analysis was performed on electronically gated CD4+ T cells. E) Evaluation of IL-22, IL-17 and IFN-γ production at day 4, after overnight restimulation of CD4 T cells with anti-CD3 coated beads. All data are representative of one out of three independent experiments performed with cells derived from different donors. The 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-g007: Induction of Th17 response by DC loaded with apoptotic cells.A) Representative dot plot analysis (one out of three) of CD154 induction in CD4 T cells after 6 days of culture with autologous IFN-DC or IL4-DC fed with apoptotic cells. The upper dot plots show CD154 expression in purified naïve CD4 T cells prior to use. CTR dot plots show the percentage of CD154 expression by CD4 T cells cultivated in the presence of autologous DC which had not phagocytosed apoptotic tumor cells. Analysis was performed on electronically gated CD4+ T cells. The percentage of early and late apoptotic tumor cells was evaluated by Annexin-V/propidium iodide staining. B) ELISA detection of IL-1β, IL-6 and IL-23 release by DC upon phagocytosis of apoptotic tumor cell lines. Analysis was performed after 18 hours from tumor cell uptake. The values represent the mean +/− SD of three independent experiments. C) ELISA detection of IL-1β, IL-6 and IL-23 in supernatants from co-culture of CD4 T cells with autologous DC which had phagocytosed apoptotic cells. After three days of co-culture, CD4 T cells were restimulated overnight with anti-CD3 coated beads. At day four the supernatant was collected for cytokine detection. All data are expressed as the mean +/− SD of three independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05. D) Dot plot analysis of CCR6/CCR4 expression by CD4 T cells after six days of stimulation in the presence of DC fed with apoptotic tumor cells or autologous PBL. The results of one representative experiment out of three are shown. In all case, the analysis was performed on electronically gated CD4+ T cells. E) Evaluation of IL-22, IL-17 and IFN-γ production at day 4, after overnight restimulation of CD4 T cells with anti-CD3 coated beads. All data are representative of one out of three independent experiments performed with cells derived from different donors. The values represent the mean +/− SD of three independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05.

Mentions: To investigate whether apoptotic cell-loaded IFN-DC could promote the expansion of IL-17-secreting cells, we cultivated naïve CD4 T cells with autologous DC which had phagocytosed apoptotic melanoma (Me501) or cervical cancer (CaSki) cells. Interestingly, a notable increase in the percentage of CD154+ cells was induced by the co-cultivation of naïve CD4 T cells with apoptotic cell-loaded IFN-DC (Fig. 7A). As shown in Fig. 7B, the uptake of apoptotic tumor cells induced a massive production of IL-6 in both IFN-DC and IL-4-DC, while the production of IL-23 was selectively induced in IFN-DC. On the contrary, only irrelevant levels of IL-1β could be detected in all the conditions tested. Upon culture of naïve CD4 T cells with autologous DC loaded with apoptotic cells, high levels of IL-6 were found in the supernatants of all the different DC-CD4 T cells co-cultures, whereas IL-1β and IL-23 were produced mainly in the co-cultures of CD4 T cells with apoptotic cell-loaded IFN-DC (Fig. 7C). High levels of TNF-α were present in all culture conditions, while TGF-β was in all cases undetectable (data not shown).


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)

Induction of Th17 response by DC loaded with apoptotic cells.A) Representative dot plot analysis (one out of three) of CD154 induction in CD4 T cells after 6 days of culture with autologous IFN-DC or IL4-DC fed with apoptotic cells. The upper dot plots show CD154 expression in purified naïve CD4 T cells prior to use. CTR dot plots show the percentage of CD154 expression by CD4 T cells cultivated in the presence of autologous DC which had not phagocytosed apoptotic tumor cells. Analysis was performed on electronically gated CD4+ T cells. The percentage of early and late apoptotic tumor cells was evaluated by Annexin-V/propidium iodide staining. B) ELISA detection of IL-1β, IL-6 and IL-23 release by DC upon phagocytosis of apoptotic tumor cell lines. Analysis was performed after 18 hours from tumor cell uptake. The values represent the mean +/− SD of three independent experiments. C) ELISA detection of IL-1β, IL-6 and IL-23 in supernatants from co-culture of CD4 T cells with autologous DC which had phagocytosed apoptotic cells. After three days of co-culture, CD4 T cells were restimulated overnight with anti-CD3 coated beads. At day four the supernatant was collected for cytokine detection. All data are expressed as the mean +/− SD of three independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05. D) Dot plot analysis of CCR6/CCR4 expression by CD4 T cells after six days of stimulation in the presence of DC fed with apoptotic tumor cells or autologous PBL. The results of one representative experiment out of three are shown. In all case, the analysis was performed on electronically gated CD4+ T cells. E) Evaluation of IL-22, IL-17 and IFN-γ production at day 4, after overnight restimulation of CD4 T cells with anti-CD3 coated beads. All data are representative of one out of three independent experiments performed with cells derived from different donors. The values represent the mean +/− SD of three independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017364-g007: Induction of Th17 response by DC loaded with apoptotic cells.A) Representative dot plot analysis (one out of three) of CD154 induction in CD4 T cells after 6 days of culture with autologous IFN-DC or IL4-DC fed with apoptotic cells. The upper dot plots show CD154 expression in purified naïve CD4 T cells prior to use. CTR dot plots show the percentage of CD154 expression by CD4 T cells cultivated in the presence of autologous DC which had not phagocytosed apoptotic tumor cells. Analysis was performed on electronically gated CD4+ T cells. The percentage of early and late apoptotic tumor cells was evaluated by Annexin-V/propidium iodide staining. B) ELISA detection of IL-1β, IL-6 and IL-23 release by DC upon phagocytosis of apoptotic tumor cell lines. Analysis was performed after 18 hours from tumor cell uptake. The values represent the mean +/− SD of three independent experiments. C) ELISA detection of IL-1β, IL-6 and IL-23 in supernatants from co-culture of CD4 T cells with autologous DC which had phagocytosed apoptotic cells. After three days of co-culture, CD4 T cells were restimulated overnight with anti-CD3 coated beads. At day four the supernatant was collected for cytokine detection. All data are expressed as the mean +/− SD of three independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05. D) Dot plot analysis of CCR6/CCR4 expression by CD4 T cells after six days of stimulation in the presence of DC fed with apoptotic tumor cells or autologous PBL. The results of one representative experiment out of three are shown. In all case, the analysis was performed on electronically gated CD4+ T cells. E) Evaluation of IL-22, IL-17 and IFN-γ production at day 4, after overnight restimulation of CD4 T cells with anti-CD3 coated beads. All data are representative of one out of three independent experiments performed with cells derived from different donors. The values represent the mean +/− SD of three independent experiments. Statistical analysis was performed by Mann-Whitney test. * p<0.05.
Mentions: To investigate whether apoptotic cell-loaded IFN-DC could promote the expansion of IL-17-secreting cells, we cultivated naïve CD4 T cells with autologous DC which had phagocytosed apoptotic melanoma (Me501) or cervical cancer (CaSki) cells. Interestingly, a notable increase in the percentage of CD154+ cells was induced by the co-cultivation of naïve CD4 T cells with apoptotic cell-loaded IFN-DC (Fig. 7A). As shown in Fig. 7B, the uptake of apoptotic tumor cells induced a massive production of IL-6 in both IFN-DC and IL-4-DC, while the production of IL-23 was selectively induced in IFN-DC. On the contrary, only irrelevant levels of IL-1β could be detected in all the conditions tested. Upon culture of naïve CD4 T cells with autologous DC loaded with apoptotic cells, high levels of IL-6 were found in the supernatants of all the different DC-CD4 T cells co-cultures, whereas IL-1β and IL-23 were produced mainly in the co-cultures of CD4 T cells with apoptotic cell-loaded IFN-DC (Fig. 7C). High levels of TNF-α were present in all culture conditions, while TGF-β was in all cases undetectable (data not shown).

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