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Induction of indoleamine 2, 3-dioxygenase in human dendritic cells by a cholera toxin B subunit-proinsulin vaccine.

Mbongue JC, Nicholas DA, Zhang K, Kim NS, Hamilton BN, Larios M, Zhang G, Umezawa K, Firek AF, Langridge WH - PLoS ONE (2015)

Bottom Line: Vaccination did not interfere with monocytes differentiation into DC, suggesting the vaccine can function safely in the human immune system.Together, our experimental data indicate that CTB-INS vaccine induction of IDO1 biosynthesis in human DCs may result in the inhibition of DC maturation generating a durable state of immunological tolerance.Understanding how CTB-INS modulates IDO1 activity in human DCs will facilitate vaccine efficacy and safety, moving this immunosuppressive strategy closer to clinical applications for prevention of type 1 diabetes autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Center for Health Disparities and Molecular Medicine, Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA, United States of America; Loma Linda University School of Medicine, Department of Basic Sciences, Division of Physiology, Loma Linda, CA, United States of America.

ABSTRACT
Dendritic cells (DC) interact with naïve T cells to regulate the delicate balance between immunity and tolerance required to maintain immunological homeostasis. In this study, immature human dendritic cells (iDC) were inoculated with a chimeric fusion protein vaccine containing the pancreatic β-cell auto-antigen proinsulin linked to a mucosal adjuvant the cholera toxin B subunit (CTB-INS). Proteomic analysis of vaccine inoculated DCs revealed strong up-regulation of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1). Increased biosynthesis of the immunosuppressive enzyme was detected in DCs inoculated with the CTB-INS fusion protein but not in DCs inoculated with proinsulin, CTB, or an unlinked combination of the two proteins. Immunoblot and PCR analyses of vaccine treated DCs detected IDO1mRNA by 3 hours and IDO1 protein synthesis by 6 hours after vaccine inoculation. Determination of IDO1 activity in vaccinated DCs by measurement of tryptophan degradation products (kynurenines) showed increased tryptophan cleavage into N-formyl kynurenine. Vaccination did not interfere with monocytes differentiation into DC, suggesting the vaccine can function safely in the human immune system. Treatment of vaccinated DCs with pharmacological NF-κB inhibitors ACHP or DHMEQ significantly inhibited IDO1 biosynthesis, suggesting a role for NF-κB signaling in vaccine up-regulation of dendritic cell IDO1. Heat map analysis of the proteomic data revealed an overall down-regulation of vaccinated DC functions, suggesting vaccine suppression of DC maturation. Together, our experimental data indicate that CTB-INS vaccine induction of IDO1 biosynthesis in human DCs may result in the inhibition of DC maturation generating a durable state of immunological tolerance. Understanding how CTB-INS modulates IDO1 activity in human DCs will facilitate vaccine efficacy and safety, moving this immunosuppressive strategy closer to clinical applications for prevention of type 1 diabetes autoimmunity.

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ACHP and DHMEQ inhibitors of NF-κB block CTB-INS induced IDO1 biosynthesis.(A) Dendritic cell viability was measured by determination of the percentage of DCs negative for annexin V and propidium iodide following treatment with ACHP (500 nM) and DHMEQ (3μg/ml). The measurement of DC viability shows vaccinated DCs are unaffected by treatment with the inhibitors. (B, C) Agarose gel electrophoresis and graphic representation showing changes in IDO1 mRNA levels in DCs from three representative subjects determined by qRT-PCR after 3 hours incubation of subject monocyte derived DCs with CTB-INS (10 μg/mL) or CTB-INS and ACHP. Statistical significance (p<0.05) was achieved by analysis of a total of 5 subjects. (The vehicle used is the DMSO solvent in which ACHP and DHMEQ are dissolved). (D, E) Immunoblot and graphic representation of the fold change in IDO protein synthesized 24hrs after vaccine inoculation of subjects DCs inoculated with CTB-INS +/- ACHP. The experimental data was collected from five normal subjects, although only one representative subject is shown. Statistical significance between vaccine treated and vaccine + ACHP treated groups was measured by paired Student’s t test, (p = 0.0273). (F, G) Immunoblot and graphic representation of the fold change in IDO1 protein synthesized in subject DCs following inoculation with CTB-INS with and without DHMEQ. While experimental data was collected from five normal subjects, only two are shown to highlight the variability in IDO1 levels observed among subjects. Statistical significance was measured by paired Student’s t test (p = 0.017). (H) Graphic representation showing the relative efficacy of ACHP and DHMEQ inhibitors for blocking NF-κB induction of IDO1 biosynthesis.
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pone.0118562.g004: ACHP and DHMEQ inhibitors of NF-κB block CTB-INS induced IDO1 biosynthesis.(A) Dendritic cell viability was measured by determination of the percentage of DCs negative for annexin V and propidium iodide following treatment with ACHP (500 nM) and DHMEQ (3μg/ml). The measurement of DC viability shows vaccinated DCs are unaffected by treatment with the inhibitors. (B, C) Agarose gel electrophoresis and graphic representation showing changes in IDO1 mRNA levels in DCs from three representative subjects determined by qRT-PCR after 3 hours incubation of subject monocyte derived DCs with CTB-INS (10 μg/mL) or CTB-INS and ACHP. Statistical significance (p<0.05) was achieved by analysis of a total of 5 subjects. (The vehicle used is the DMSO solvent in which ACHP and DHMEQ are dissolved). (D, E) Immunoblot and graphic representation of the fold change in IDO protein synthesized 24hrs after vaccine inoculation of subjects DCs inoculated with CTB-INS +/- ACHP. The experimental data was collected from five normal subjects, although only one representative subject is shown. Statistical significance between vaccine treated and vaccine + ACHP treated groups was measured by paired Student’s t test, (p = 0.0273). (F, G) Immunoblot and graphic representation of the fold change in IDO1 protein synthesized in subject DCs following inoculation with CTB-INS with and without DHMEQ. While experimental data was collected from five normal subjects, only two are shown to highlight the variability in IDO1 levels observed among subjects. Statistical significance was measured by paired Student’s t test (p = 0.017). (H) Graphic representation showing the relative efficacy of ACHP and DHMEQ inhibitors for blocking NF-κB induction of IDO1 biosynthesis.

Mentions: Based on the observation that CD40 ligand (CD40L) induces IDO1 biosynthesis through NF-κB signaling in human DCs [41], we investigated the role of NF-κB signaling pathway involvement in CTB-INS induced IDO1 biosynthesis. To assess the requirement for NF-κB in vaccine up-regulation of IDO1 we attempted to block vaccine stimulated IDO1 biosynthesis in DCs with two different NF-κB pharmacological inhibitors, ACHP and DHMEQ. Assessment of DC viability following ACHP and DHMEQ treatment indicated both inhibitors were not toxic to DCs (Fig. 4A). Immature dendritic cells were incubated with CTB-INS +/- the NF-κB inhibitor ACHP. The levels of IDO1 mRNA were found to be significantly increased in vaccinated DCs while addition of ACHP to vaccinated DCs completely inhibited IDO1 transcription (Fig. 4B-C). Correspondingly, IDO1 protein levels were significantly higher in cells inoculated with CTB-INS than in DCs incubated for 24 hours with CTB-INS + either ACHP or DHMEQ (Fig. 4D-G). Both DHMEQ and ACHP were shown to be equally effective in blocking DC biosynthesis of IDO1 (Fig. 4H).


Induction of indoleamine 2, 3-dioxygenase in human dendritic cells by a cholera toxin B subunit-proinsulin vaccine.

Mbongue JC, Nicholas DA, Zhang K, Kim NS, Hamilton BN, Larios M, Zhang G, Umezawa K, Firek AF, Langridge WH - PLoS ONE (2015)

ACHP and DHMEQ inhibitors of NF-κB block CTB-INS induced IDO1 biosynthesis.(A) Dendritic cell viability was measured by determination of the percentage of DCs negative for annexin V and propidium iodide following treatment with ACHP (500 nM) and DHMEQ (3μg/ml). The measurement of DC viability shows vaccinated DCs are unaffected by treatment with the inhibitors. (B, C) Agarose gel electrophoresis and graphic representation showing changes in IDO1 mRNA levels in DCs from three representative subjects determined by qRT-PCR after 3 hours incubation of subject monocyte derived DCs with CTB-INS (10 μg/mL) or CTB-INS and ACHP. Statistical significance (p<0.05) was achieved by analysis of a total of 5 subjects. (The vehicle used is the DMSO solvent in which ACHP and DHMEQ are dissolved). (D, E) Immunoblot and graphic representation of the fold change in IDO protein synthesized 24hrs after vaccine inoculation of subjects DCs inoculated with CTB-INS +/- ACHP. The experimental data was collected from five normal subjects, although only one representative subject is shown. Statistical significance between vaccine treated and vaccine + ACHP treated groups was measured by paired Student’s t test, (p = 0.0273). (F, G) Immunoblot and graphic representation of the fold change in IDO1 protein synthesized in subject DCs following inoculation with CTB-INS with and without DHMEQ. While experimental data was collected from five normal subjects, only two are shown to highlight the variability in IDO1 levels observed among subjects. Statistical significance was measured by paired Student’s t test (p = 0.017). (H) Graphic representation showing the relative efficacy of ACHP and DHMEQ inhibitors for blocking NF-κB induction of IDO1 biosynthesis.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4340906&req=5

pone.0118562.g004: ACHP and DHMEQ inhibitors of NF-κB block CTB-INS induced IDO1 biosynthesis.(A) Dendritic cell viability was measured by determination of the percentage of DCs negative for annexin V and propidium iodide following treatment with ACHP (500 nM) and DHMEQ (3μg/ml). The measurement of DC viability shows vaccinated DCs are unaffected by treatment with the inhibitors. (B, C) Agarose gel electrophoresis and graphic representation showing changes in IDO1 mRNA levels in DCs from three representative subjects determined by qRT-PCR after 3 hours incubation of subject monocyte derived DCs with CTB-INS (10 μg/mL) or CTB-INS and ACHP. Statistical significance (p<0.05) was achieved by analysis of a total of 5 subjects. (The vehicle used is the DMSO solvent in which ACHP and DHMEQ are dissolved). (D, E) Immunoblot and graphic representation of the fold change in IDO protein synthesized 24hrs after vaccine inoculation of subjects DCs inoculated with CTB-INS +/- ACHP. The experimental data was collected from five normal subjects, although only one representative subject is shown. Statistical significance between vaccine treated and vaccine + ACHP treated groups was measured by paired Student’s t test, (p = 0.0273). (F, G) Immunoblot and graphic representation of the fold change in IDO1 protein synthesized in subject DCs following inoculation with CTB-INS with and without DHMEQ. While experimental data was collected from five normal subjects, only two are shown to highlight the variability in IDO1 levels observed among subjects. Statistical significance was measured by paired Student’s t test (p = 0.017). (H) Graphic representation showing the relative efficacy of ACHP and DHMEQ inhibitors for blocking NF-κB induction of IDO1 biosynthesis.
Mentions: Based on the observation that CD40 ligand (CD40L) induces IDO1 biosynthesis through NF-κB signaling in human DCs [41], we investigated the role of NF-κB signaling pathway involvement in CTB-INS induced IDO1 biosynthesis. To assess the requirement for NF-κB in vaccine up-regulation of IDO1 we attempted to block vaccine stimulated IDO1 biosynthesis in DCs with two different NF-κB pharmacological inhibitors, ACHP and DHMEQ. Assessment of DC viability following ACHP and DHMEQ treatment indicated both inhibitors were not toxic to DCs (Fig. 4A). Immature dendritic cells were incubated with CTB-INS +/- the NF-κB inhibitor ACHP. The levels of IDO1 mRNA were found to be significantly increased in vaccinated DCs while addition of ACHP to vaccinated DCs completely inhibited IDO1 transcription (Fig. 4B-C). Correspondingly, IDO1 protein levels were significantly higher in cells inoculated with CTB-INS than in DCs incubated for 24 hours with CTB-INS + either ACHP or DHMEQ (Fig. 4D-G). Both DHMEQ and ACHP were shown to be equally effective in blocking DC biosynthesis of IDO1 (Fig. 4H).

Bottom Line: Vaccination did not interfere with monocytes differentiation into DC, suggesting the vaccine can function safely in the human immune system.Together, our experimental data indicate that CTB-INS vaccine induction of IDO1 biosynthesis in human DCs may result in the inhibition of DC maturation generating a durable state of immunological tolerance.Understanding how CTB-INS modulates IDO1 activity in human DCs will facilitate vaccine efficacy and safety, moving this immunosuppressive strategy closer to clinical applications for prevention of type 1 diabetes autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Center for Health Disparities and Molecular Medicine, Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA, United States of America; Loma Linda University School of Medicine, Department of Basic Sciences, Division of Physiology, Loma Linda, CA, United States of America.

ABSTRACT
Dendritic cells (DC) interact with naïve T cells to regulate the delicate balance between immunity and tolerance required to maintain immunological homeostasis. In this study, immature human dendritic cells (iDC) were inoculated with a chimeric fusion protein vaccine containing the pancreatic β-cell auto-antigen proinsulin linked to a mucosal adjuvant the cholera toxin B subunit (CTB-INS). Proteomic analysis of vaccine inoculated DCs revealed strong up-regulation of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1). Increased biosynthesis of the immunosuppressive enzyme was detected in DCs inoculated with the CTB-INS fusion protein but not in DCs inoculated with proinsulin, CTB, or an unlinked combination of the two proteins. Immunoblot and PCR analyses of vaccine treated DCs detected IDO1mRNA by 3 hours and IDO1 protein synthesis by 6 hours after vaccine inoculation. Determination of IDO1 activity in vaccinated DCs by measurement of tryptophan degradation products (kynurenines) showed increased tryptophan cleavage into N-formyl kynurenine. Vaccination did not interfere with monocytes differentiation into DC, suggesting the vaccine can function safely in the human immune system. Treatment of vaccinated DCs with pharmacological NF-κB inhibitors ACHP or DHMEQ significantly inhibited IDO1 biosynthesis, suggesting a role for NF-κB signaling in vaccine up-regulation of dendritic cell IDO1. Heat map analysis of the proteomic data revealed an overall down-regulation of vaccinated DC functions, suggesting vaccine suppression of DC maturation. Together, our experimental data indicate that CTB-INS vaccine induction of IDO1 biosynthesis in human DCs may result in the inhibition of DC maturation generating a durable state of immunological tolerance. Understanding how CTB-INS modulates IDO1 activity in human DCs will facilitate vaccine efficacy and safety, moving this immunosuppressive strategy closer to clinical applications for prevention of type 1 diabetes autoimmunity.

Show MeSH
Related in: MedlinePlus