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Intranasal delivery of cholera toxin induces th17-dominated T-cell response to bystander antigens.

Lee JB, Jang JE, Song MK, Chang J - PLoS ONE (2009)

Bottom Line: It has been suggested that CT promotes T helper type 2 (Th2) response and suppresses Th1 response.This dramatic Th17-driving effect of CT, which was dependent on the B subunit, was observed even in Th1 or Th2-favored conditions of respiratory virus infection.These data demonstrate a novel Th17-driving activity of CT, and help understand the mechanisms of CT adjuvanticity to demarcate T helper responses.

View Article: PubMed Central - PubMed

Affiliation: Division of Life and Pharmaceutical Sciences, and Center for Cell Signaling & Drug Discovery Research, Ewha Womans University, Seoul, Republic of Korea.

ABSTRACT
Cholera toxin (CT) is a potent vaccine adjuvant, which promotes mucosal immunity to protein antigen given by nasal route. It has been suggested that CT promotes T helper type 2 (Th2) response and suppresses Th1 response. We here report the induction of Th17-dominated responses in mice by intranasal delivery of CT. This dramatic Th17-driving effect of CT, which was dependent on the B subunit, was observed even in Th1 or Th2-favored conditions of respiratory virus infection. These dominating Th17 responses resulted in the significant neutrophil accumulation in the lungs of mice given CT. Both in vitro and in vivo treatment of CT induced strongly augmented IL-6 production, and Th17-driving ability of CT was completely abolished in IL-6 knockout mice, indicating a role of this cytokine in the Th17-dominated T-cell responses by CT. These data demonstrate a novel Th17-driving activity of CT, and help understand the mechanisms of CT adjuvanticity to demarcate T helper responses.

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CT induces IL-6 production in vitro and in vivo, which is required for Th17-driving activity.(A) Dendritic cells were generated from bone marrow of BALB/c mice by culturing with GM-CSF and IL-4. After 6 days, DCs were purified and cultured in the presence or absence of CT. At indicated time points, the levels of IL-6 cytokine in the supernatant were determined by ELISA. (B) B6 mice were intranasally injected with 2 µg of CT and BAL samples were prepared at indicated time points. The levels of IL-6 in the BAL fluids were measured by ELISA. (C, D) IL-6KO mice with B6 background were intranasally injected with CT, and lung mononuclear cells were prepared at day 7 and IL-17- and IFN-γ-producing CD4 T cells were measured by ICS upon stimulation. (E) For blocking experiment, CT was pre-incubated with 5-fold molar excess of ganglioside GM1 and then added to the DC culture. Data are average ± SEM, and representative of two experiments. (F) CT was heat-inactivated by boiling for 30 min and intranasally administered into normal B6 mice. At day 7, lung tissues were isolated, stimulated with PMA/ionomycin, and stained for CD4, IFN-γ, and IL-17. *, P<0.05; **, P<0.01; ***, P<0.001; n.s., not significant.
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pone-0005190-g005: CT induces IL-6 production in vitro and in vivo, which is required for Th17-driving activity.(A) Dendritic cells were generated from bone marrow of BALB/c mice by culturing with GM-CSF and IL-4. After 6 days, DCs were purified and cultured in the presence or absence of CT. At indicated time points, the levels of IL-6 cytokine in the supernatant were determined by ELISA. (B) B6 mice were intranasally injected with 2 µg of CT and BAL samples were prepared at indicated time points. The levels of IL-6 in the BAL fluids were measured by ELISA. (C, D) IL-6KO mice with B6 background were intranasally injected with CT, and lung mononuclear cells were prepared at day 7 and IL-17- and IFN-γ-producing CD4 T cells were measured by ICS upon stimulation. (E) For blocking experiment, CT was pre-incubated with 5-fold molar excess of ganglioside GM1 and then added to the DC culture. Data are average ± SEM, and representative of two experiments. (F) CT was heat-inactivated by boiling for 30 min and intranasally administered into normal B6 mice. At day 7, lung tissues were isolated, stimulated with PMA/ionomycin, and stained for CD4, IFN-γ, and IL-17. *, P<0.05; **, P<0.01; ***, P<0.001; n.s., not significant.

Mentions: The cytokines thought to be critical for promoting murine Th17 differentiation are IL-6 and TGF-β [22]–[24]. The findings that CT actively induces Th17 differentiation and airway neutrophilia suggest that IL-6 and/or TGF-β could be involved in the skewed Th17 differentiation by CT. We thus examined whether the expression of IL-6 and/or TGF-β could be induced by CT using bone marrow-derived dendritic cells in vitro, since CT has been found to directly affect cytokine profiles of APC [7], [25]. Significantly increased IL-6 production was detected in the DC cultures upon 24 h of CT treatment, and there was more than 30-fold increase of IL-6 production during 72 h treatment (Fig. 5A). However, we failed to detect any measurable TGF-β by ELISA in the same culture supernatant (data not shown). In an in vivo setting, significantly increased IL-6 production was also observed in the lungs when bronchoalveolar lavage samples from CT-treated B6 mice were examined (Fig. 5B). We also checked the levels of TGF-β in the lungs, and intriguingly, constitutively high levels of TGF-β (more than 500 pg/ml in the lung homogenates) were detected by ELISA in mice during the experimental periods (data not shown). These results strongly suggest that induction of IL-6 mediates the ability of CT inducing Th17-dominated responses. To determine whether endogenous IL-6 production is necessary for the Th17-driving ability of CT, IL-6 knockout (IL-6KO) mice were used for the experiment. Compared to CT-treated wild type mice, IL-6KO mice are almost completely resistant to the Th-17-driving effect of CT (Fig. 5C, D), indicating that IL-6 induction is required for the effect.


Intranasal delivery of cholera toxin induces th17-dominated T-cell response to bystander antigens.

Lee JB, Jang JE, Song MK, Chang J - PLoS ONE (2009)

CT induces IL-6 production in vitro and in vivo, which is required for Th17-driving activity.(A) Dendritic cells were generated from bone marrow of BALB/c mice by culturing with GM-CSF and IL-4. After 6 days, DCs were purified and cultured in the presence or absence of CT. At indicated time points, the levels of IL-6 cytokine in the supernatant were determined by ELISA. (B) B6 mice were intranasally injected with 2 µg of CT and BAL samples were prepared at indicated time points. The levels of IL-6 in the BAL fluids were measured by ELISA. (C, D) IL-6KO mice with B6 background were intranasally injected with CT, and lung mononuclear cells were prepared at day 7 and IL-17- and IFN-γ-producing CD4 T cells were measured by ICS upon stimulation. (E) For blocking experiment, CT was pre-incubated with 5-fold molar excess of ganglioside GM1 and then added to the DC culture. Data are average ± SEM, and representative of two experiments. (F) CT was heat-inactivated by boiling for 30 min and intranasally administered into normal B6 mice. At day 7, lung tissues were isolated, stimulated with PMA/ionomycin, and stained for CD4, IFN-γ, and IL-17. *, P<0.05; **, P<0.01; ***, P<0.001; n.s., not significant.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2663811&req=5

pone-0005190-g005: CT induces IL-6 production in vitro and in vivo, which is required for Th17-driving activity.(A) Dendritic cells were generated from bone marrow of BALB/c mice by culturing with GM-CSF and IL-4. After 6 days, DCs were purified and cultured in the presence or absence of CT. At indicated time points, the levels of IL-6 cytokine in the supernatant were determined by ELISA. (B) B6 mice were intranasally injected with 2 µg of CT and BAL samples were prepared at indicated time points. The levels of IL-6 in the BAL fluids were measured by ELISA. (C, D) IL-6KO mice with B6 background were intranasally injected with CT, and lung mononuclear cells were prepared at day 7 and IL-17- and IFN-γ-producing CD4 T cells were measured by ICS upon stimulation. (E) For blocking experiment, CT was pre-incubated with 5-fold molar excess of ganglioside GM1 and then added to the DC culture. Data are average ± SEM, and representative of two experiments. (F) CT was heat-inactivated by boiling for 30 min and intranasally administered into normal B6 mice. At day 7, lung tissues were isolated, stimulated with PMA/ionomycin, and stained for CD4, IFN-γ, and IL-17. *, P<0.05; **, P<0.01; ***, P<0.001; n.s., not significant.
Mentions: The cytokines thought to be critical for promoting murine Th17 differentiation are IL-6 and TGF-β [22]–[24]. The findings that CT actively induces Th17 differentiation and airway neutrophilia suggest that IL-6 and/or TGF-β could be involved in the skewed Th17 differentiation by CT. We thus examined whether the expression of IL-6 and/or TGF-β could be induced by CT using bone marrow-derived dendritic cells in vitro, since CT has been found to directly affect cytokine profiles of APC [7], [25]. Significantly increased IL-6 production was detected in the DC cultures upon 24 h of CT treatment, and there was more than 30-fold increase of IL-6 production during 72 h treatment (Fig. 5A). However, we failed to detect any measurable TGF-β by ELISA in the same culture supernatant (data not shown). In an in vivo setting, significantly increased IL-6 production was also observed in the lungs when bronchoalveolar lavage samples from CT-treated B6 mice were examined (Fig. 5B). We also checked the levels of TGF-β in the lungs, and intriguingly, constitutively high levels of TGF-β (more than 500 pg/ml in the lung homogenates) were detected by ELISA in mice during the experimental periods (data not shown). These results strongly suggest that induction of IL-6 mediates the ability of CT inducing Th17-dominated responses. To determine whether endogenous IL-6 production is necessary for the Th17-driving ability of CT, IL-6 knockout (IL-6KO) mice were used for the experiment. Compared to CT-treated wild type mice, IL-6KO mice are almost completely resistant to the Th-17-driving effect of CT (Fig. 5C, D), indicating that IL-6 induction is required for the effect.

Bottom Line: It has been suggested that CT promotes T helper type 2 (Th2) response and suppresses Th1 response.This dramatic Th17-driving effect of CT, which was dependent on the B subunit, was observed even in Th1 or Th2-favored conditions of respiratory virus infection.These data demonstrate a novel Th17-driving activity of CT, and help understand the mechanisms of CT adjuvanticity to demarcate T helper responses.

View Article: PubMed Central - PubMed

Affiliation: Division of Life and Pharmaceutical Sciences, and Center for Cell Signaling & Drug Discovery Research, Ewha Womans University, Seoul, Republic of Korea.

ABSTRACT
Cholera toxin (CT) is a potent vaccine adjuvant, which promotes mucosal immunity to protein antigen given by nasal route. It has been suggested that CT promotes T helper type 2 (Th2) response and suppresses Th1 response. We here report the induction of Th17-dominated responses in mice by intranasal delivery of CT. This dramatic Th17-driving effect of CT, which was dependent on the B subunit, was observed even in Th1 or Th2-favored conditions of respiratory virus infection. These dominating Th17 responses resulted in the significant neutrophil accumulation in the lungs of mice given CT. Both in vitro and in vivo treatment of CT induced strongly augmented IL-6 production, and Th17-driving ability of CT was completely abolished in IL-6 knockout mice, indicating a role of this cytokine in the Th17-dominated T-cell responses by CT. These data demonstrate a novel Th17-driving activity of CT, and help understand the mechanisms of CT adjuvanticity to demarcate T helper responses.

Show MeSH
Related in: MedlinePlus