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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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Mass spectrometric identification of the CTB-INS vaccinated DC proteome.(A) Venn diagram of significantly up or down regulated proteins in human monocyte derived dendritic cells inoculated with CTB-INS in comparison with monocytes as the baseline. A total of 845 proteins were identified in untreated DCs and 654 up and down-regulated proteins were unique in CTB-INS treated DCs, while 131 proteins were common to both treated and untreated DCs. (B) Canonical, metabolic and signaling pathways that correlate with changes in protein expression in CTB-INS vaccinated dendritic cells identified by IPA program analysis. The threshold line represents the value at which statistical significance is reported. The grey line connects the ratios between the number of up and down-regulated proteins over the total number of proteins identified in each of the pathways shown.
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pone.0118562.g001: Mass spectrometric identification of the CTB-INS vaccinated DC proteome.(A) Venn diagram of significantly up or down regulated proteins in human monocyte derived dendritic cells inoculated with CTB-INS in comparison with monocytes as the baseline. A total of 845 proteins were identified in untreated DCs and 654 up and down-regulated proteins were unique in CTB-INS treated DCs, while 131 proteins were common to both treated and untreated DCs. (B) Canonical, metabolic and signaling pathways that correlate with changes in protein expression in CTB-INS vaccinated dendritic cells identified by IPA program analysis. The threshold line represents the value at which statistical significance is reported. The grey line connects the ratios between the number of up and down-regulated proteins over the total number of proteins identified in each of the pathways shown.

Mentions: Dendritic cells were both treated with 10μg/ml of CTB-INS or PBS for 24 hours and lysed. To determine the effects of CTB-INS vaccination on the DC proteome, we employed Orbitrap mass spectrometry to identify and quantify vaccine regulated proteins and Ingenuity Pathway Analysis (IPA) algorithms, to assess their role in signaling or metabolic functions in vaccinated DCs. A total of 845 proteins were identified in unvaccinated DCs and 654 proteins were identified as unique and significantly up or down regulated in CTB-INS vaccinated DCs. About 131 proteins were detected as common in both vaccinated and unvaccinated DCs (Fig. 1A). Signaling pathways identified by IPA that correlated with changes in protein expression in vaccinated DCs were arranged by decreasing—log (p-values) (Fig. 1B). Identified among these pathways were 2 types of tryptophan degradation pathways, namely the tryptophan degradation pathways III and X [34]. Ingenuity pathway analysis of the ratios of up and down-regulated proteins over the total number of proteins in each DC metabolic or signaling pathway showed that the tryptophan degradation pathway, the aryl hydrocarbon receptor signaling pathway and the dopamine degradation pathway were significantly up-regulated in the presence of the vaccine (Fig. 1B).


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)

Mass spectrometric identification of the CTB-INS vaccinated DC proteome.(A) Venn diagram of significantly up or down regulated proteins in human monocyte derived dendritic cells inoculated with CTB-INS in comparison with monocytes as the baseline. A total of 845 proteins were identified in untreated DCs and 654 up and down-regulated proteins were unique in CTB-INS treated DCs, while 131 proteins were common to both treated and untreated DCs. (B) Canonical, metabolic and signaling pathways that correlate with changes in protein expression in CTB-INS vaccinated dendritic cells identified by IPA program analysis. The threshold line represents the value at which statistical significance is reported. The grey line connects the ratios between the number of up and down-regulated proteins over the total number of proteins identified in each of the pathways shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118562.g001: Mass spectrometric identification of the CTB-INS vaccinated DC proteome.(A) Venn diagram of significantly up or down regulated proteins in human monocyte derived dendritic cells inoculated with CTB-INS in comparison with monocytes as the baseline. A total of 845 proteins were identified in untreated DCs and 654 up and down-regulated proteins were unique in CTB-INS treated DCs, while 131 proteins were common to both treated and untreated DCs. (B) Canonical, metabolic and signaling pathways that correlate with changes in protein expression in CTB-INS vaccinated dendritic cells identified by IPA program analysis. The threshold line represents the value at which statistical significance is reported. The grey line connects the ratios between the number of up and down-regulated proteins over the total number of proteins identified in each of the pathways shown.
Mentions: Dendritic cells were both treated with 10μg/ml of CTB-INS or PBS for 24 hours and lysed. To determine the effects of CTB-INS vaccination on the DC proteome, we employed Orbitrap mass spectrometry to identify and quantify vaccine regulated proteins and Ingenuity Pathway Analysis (IPA) algorithms, to assess their role in signaling or metabolic functions in vaccinated DCs. A total of 845 proteins were identified in unvaccinated DCs and 654 proteins were identified as unique and significantly up or down regulated in CTB-INS vaccinated DCs. About 131 proteins were detected as common in both vaccinated and unvaccinated DCs (Fig. 1A). Signaling pathways identified by IPA that correlated with changes in protein expression in vaccinated DCs were arranged by decreasing—log (p-values) (Fig. 1B). Identified among these pathways were 2 types of tryptophan degradation pathways, namely the tryptophan degradation pathways III and X [34]. Ingenuity pathway analysis of the ratios of up and down-regulated proteins over the total number of proteins in each DC metabolic or signaling pathway showed that the tryptophan degradation pathway, the aryl hydrocarbon receptor signaling pathway and the dopamine degradation pathway were significantly up-regulated in the presence of the vaccine (Fig. 1B).

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