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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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Vaccine induction of IDO1 biosynthesis in dendritic cells.(A) Immunoblot detection of IDO1 protein synthesis in human DCs in the presence of CTB-INS. (B) Immunoblot analysis of IDO1 up-regulation in DCs inoculated with all components of the CTB-INS fusion protein including insulin C-peptide. The images are representative of five subjects. (C) Reverse transcriptase polymerase chain reaction (RT-PCR) for assessment of IDO1 mRNA expression in a time dependent manner from DCs incubated with and without CTB-INS. RNA (2 μg). The cDNA samples were used as templates for PCR amplification. The DNA amplification products were visualized on X ray film after electrophoresis in 2.0% agarose gels. Lane M: 100 bps DNA ladder marker (Gibco); lane NC: negative control RNA after treatment with DNase I from DCs as template; lane 0: cDNA from DCs without CTB-INS inoculation (-); lanes 1, 3, 6, 9, 12 and 24: cDNA from time-dependent DCs incubated with CTB-INS (+). A β-actin gene was used as an internal standard in RT-PCR.The image is representative of 3 different subjects. (D, E) Immunoblot of a time course of IDO1 expression in DCs after inoculation with CTB-INS (10μg/mL). Cell samples were harvested at 0, 3, 6, 8, 12 and 24 hours after vaccine inoculation. The data is representative of 6 different subject samples. (Shapes on Panel E represent different subjects). (F, G) Assessment of vaccine induced IDO1 activity and L-1-methyl-L-tryptophan (L-1-MT) inhibition of IDO1 activity in vaccinated DCs identified by ELISA. Top wells in the plate (F), show the production of tryptophan degradation products (kynurenines) in vaccinated DCs. Bottom wells show the inhibition of IDO1 kynurenine biosynthesis (absence of yellow color), in vaccinated DCs treated with L-1-MT (bottom wells). The data was compared for significance using a paired t-test, p = 0.018.
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pone.0118562.g003: Vaccine induction of IDO1 biosynthesis in dendritic cells.(A) Immunoblot detection of IDO1 protein synthesis in human DCs in the presence of CTB-INS. (B) Immunoblot analysis of IDO1 up-regulation in DCs inoculated with all components of the CTB-INS fusion protein including insulin C-peptide. The images are representative of five subjects. (C) Reverse transcriptase polymerase chain reaction (RT-PCR) for assessment of IDO1 mRNA expression in a time dependent manner from DCs incubated with and without CTB-INS. RNA (2 μg). The cDNA samples were used as templates for PCR amplification. The DNA amplification products were visualized on X ray film after electrophoresis in 2.0% agarose gels. Lane M: 100 bps DNA ladder marker (Gibco); lane NC: negative control RNA after treatment with DNase I from DCs as template; lane 0: cDNA from DCs without CTB-INS inoculation (-); lanes 1, 3, 6, 9, 12 and 24: cDNA from time-dependent DCs incubated with CTB-INS (+). A β-actin gene was used as an internal standard in RT-PCR.The image is representative of 3 different subjects. (D, E) Immunoblot of a time course of IDO1 expression in DCs after inoculation with CTB-INS (10μg/mL). Cell samples were harvested at 0, 3, 6, 8, 12 and 24 hours after vaccine inoculation. The data is representative of 6 different subject samples. (Shapes on Panel E represent different subjects). (F, G) Assessment of vaccine induced IDO1 activity and L-1-methyl-L-tryptophan (L-1-MT) inhibition of IDO1 activity in vaccinated DCs identified by ELISA. Top wells in the plate (F), show the production of tryptophan degradation products (kynurenines) in vaccinated DCs. Bottom wells show the inhibition of IDO1 kynurenine biosynthesis (absence of yellow color), in vaccinated DCs treated with L-1-MT (bottom wells). The data was compared for significance using a paired t-test, p = 0.018.

Mentions: To confirm the mass spectrometry data, we assessed CTB-INS induced up-regulation of IDO1 in DCs by immunoblotting and found that DCs treated for 24 hours with CTB-INS (10μg/ml) showed a significant increase in IDO1 biosynthesis (Fig. 3A). To determine the specificity of CTB-INS fusion protein for induction of IDO1, dendritic cells were incubated with CTB, insulin, proinsulin, c-peptide, a combination of both CTB and insulin, and CTB-INS fusion protein for 24 hours followed by immunoblot analysis. Unlike CTB-INS, none of the vaccine components delivered alone or together induced IDO1 biosynthesis (Fig. 3B). The CTB-INS fusion protein induced detectable levels of IDO1 in DCs as early as 3–6 hours after treatment and IDO1 RNA levels as early as 3 hours. (Fig. 3C-E). These results clearly demonstrated CTB-INS-induced IDO1 is enzymatically active in conversion of L-tryptophan into L-kynurenine. Kynurenine biosynthesis was arrested by 100 μM of the IDO1 inhibitor L-1-methyl-tryptophan (1-MT) as shown in Fig. 3F-G.


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)

Vaccine induction of IDO1 biosynthesis in dendritic cells.(A) Immunoblot detection of IDO1 protein synthesis in human DCs in the presence of CTB-INS. (B) Immunoblot analysis of IDO1 up-regulation in DCs inoculated with all components of the CTB-INS fusion protein including insulin C-peptide. The images are representative of five subjects. (C) Reverse transcriptase polymerase chain reaction (RT-PCR) for assessment of IDO1 mRNA expression in a time dependent manner from DCs incubated with and without CTB-INS. RNA (2 μg). The cDNA samples were used as templates for PCR amplification. The DNA amplification products were visualized on X ray film after electrophoresis in 2.0% agarose gels. Lane M: 100 bps DNA ladder marker (Gibco); lane NC: negative control RNA after treatment with DNase I from DCs as template; lane 0: cDNA from DCs without CTB-INS inoculation (-); lanes 1, 3, 6, 9, 12 and 24: cDNA from time-dependent DCs incubated with CTB-INS (+). A β-actin gene was used as an internal standard in RT-PCR.The image is representative of 3 different subjects. (D, E) Immunoblot of a time course of IDO1 expression in DCs after inoculation with CTB-INS (10μg/mL). Cell samples were harvested at 0, 3, 6, 8, 12 and 24 hours after vaccine inoculation. The data is representative of 6 different subject samples. (Shapes on Panel E represent different subjects). (F, G) Assessment of vaccine induced IDO1 activity and L-1-methyl-L-tryptophan (L-1-MT) inhibition of IDO1 activity in vaccinated DCs identified by ELISA. Top wells in the plate (F), show the production of tryptophan degradation products (kynurenines) in vaccinated DCs. Bottom wells show the inhibition of IDO1 kynurenine biosynthesis (absence of yellow color), in vaccinated DCs treated with L-1-MT (bottom wells). The data was compared for significance using a paired t-test, p = 0.018.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118562.g003: Vaccine induction of IDO1 biosynthesis in dendritic cells.(A) Immunoblot detection of IDO1 protein synthesis in human DCs in the presence of CTB-INS. (B) Immunoblot analysis of IDO1 up-regulation in DCs inoculated with all components of the CTB-INS fusion protein including insulin C-peptide. The images are representative of five subjects. (C) Reverse transcriptase polymerase chain reaction (RT-PCR) for assessment of IDO1 mRNA expression in a time dependent manner from DCs incubated with and without CTB-INS. RNA (2 μg). The cDNA samples were used as templates for PCR amplification. The DNA amplification products were visualized on X ray film after electrophoresis in 2.0% agarose gels. Lane M: 100 bps DNA ladder marker (Gibco); lane NC: negative control RNA after treatment with DNase I from DCs as template; lane 0: cDNA from DCs without CTB-INS inoculation (-); lanes 1, 3, 6, 9, 12 and 24: cDNA from time-dependent DCs incubated with CTB-INS (+). A β-actin gene was used as an internal standard in RT-PCR.The image is representative of 3 different subjects. (D, E) Immunoblot of a time course of IDO1 expression in DCs after inoculation with CTB-INS (10μg/mL). Cell samples were harvested at 0, 3, 6, 8, 12 and 24 hours after vaccine inoculation. The data is representative of 6 different subject samples. (Shapes on Panel E represent different subjects). (F, G) Assessment of vaccine induced IDO1 activity and L-1-methyl-L-tryptophan (L-1-MT) inhibition of IDO1 activity in vaccinated DCs identified by ELISA. Top wells in the plate (F), show the production of tryptophan degradation products (kynurenines) in vaccinated DCs. Bottom wells show the inhibition of IDO1 kynurenine biosynthesis (absence of yellow color), in vaccinated DCs treated with L-1-MT (bottom wells). The data was compared for significance using a paired t-test, p = 0.018.
Mentions: To confirm the mass spectrometry data, we assessed CTB-INS induced up-regulation of IDO1 in DCs by immunoblotting and found that DCs treated for 24 hours with CTB-INS (10μg/ml) showed a significant increase in IDO1 biosynthesis (Fig. 3A). To determine the specificity of CTB-INS fusion protein for induction of IDO1, dendritic cells were incubated with CTB, insulin, proinsulin, c-peptide, a combination of both CTB and insulin, and CTB-INS fusion protein for 24 hours followed by immunoblot analysis. Unlike CTB-INS, none of the vaccine components delivered alone or together induced IDO1 biosynthesis (Fig. 3B). The CTB-INS fusion protein induced detectable levels of IDO1 in DCs as early as 3–6 hours after treatment and IDO1 RNA levels as early as 3 hours. (Fig. 3C-E). These results clearly demonstrated CTB-INS-induced IDO1 is enzymatically active in conversion of L-tryptophan into L-kynurenine. Kynurenine biosynthesis was arrested by 100 μM of the IDO1 inhibitor L-1-methyl-tryptophan (1-MT) as shown in Fig. 3F-G.

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