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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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CTB-INS vaccination of monocytes does not interfere with dendritic cell differentiation.Flow cytometric analysis of vaccinated monocyte differentiation into dendritic cells. (A) The CD14 monocyte marker becomes progressively less positive (number inside the box (gate), as DC differentiation proceeds in the presence of the CTB-INS fusion protein. (B) The HLA-DR marker indicative of antigen presenting cell DC morphogenesis increases as dendritic cell differentiation progresses in the presence of CTB-INS. (C) The co-stimulatory factor marker for DC maturation CD86 is observed to decrease with time in vaccinated DCs in comparison with background levels. (D) With time after inoculation with CTB-INS, the dendritic cell marker CD83 indicative of DC maturation decreases in comparison with unvaccinated DC background levels.
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pone.0118562.g006: CTB-INS vaccination of monocytes does not interfere with dendritic cell differentiation.Flow cytometric analysis of vaccinated monocyte differentiation into dendritic cells. (A) The CD14 monocyte marker becomes progressively less positive (number inside the box (gate), as DC differentiation proceeds in the presence of the CTB-INS fusion protein. (B) The HLA-DR marker indicative of antigen presenting cell DC morphogenesis increases as dendritic cell differentiation progresses in the presence of CTB-INS. (C) The co-stimulatory factor marker for DC maturation CD86 is observed to decrease with time in vaccinated DCs in comparison with background levels. (D) With time after inoculation with CTB-INS, the dendritic cell marker CD83 indicative of DC maturation decreases in comparison with unvaccinated DC background levels.

Mentions: Despite phenotypic and functional differences based on their origin, maturation stage, and culture conditions, DCs display common features among their subsets. These features include similar morphology, high density of membrane HLA class II co-stimulatory molecules, low phagocytic activity, and a strong capacity for antigen uptake and presentation to T lymphocytes [45,46]. Under normal conditions of immunological homeostasis, monocytes are more numerous than DCs and can differentiate into dendritic cells [47]. To assess vaccine safety for clinical applications, we examined potential detrimental effects of vaccine inoculation on the differentiation of monocytes into dendritic cells (Fig. 6). Peripheral blood monocytes were isolated and inoculated with CTB-INS plus GM-CSF and IL-4 in one experimental group and another group of monocytes were inoculated with only GM-CSF and IL-4 only. Both experimental groups were cultured for 2, 4 and 6 days post inoculation. The disappearance of the CD14 monocyte markers and appearance of the dendritic cell MHC Class II HLA-DR markers were monitored during DC differentiation. The morphogenesis of monocytes into dendritic cells was not impeded by inoculation with CTB-INS and the levels of DC maturation markers CD86 and CD83 measured by flow cytometry were lower than the controls in monocyte cultures inoculated with CTB-INS (Fig. 6C-D). The time required for differentiation from monocytes to DCs was not altered by inoculation with CTB-INS. In addition, DCs treated with the vaccine showed a modulation of proteins involved in the suppression of DC maturation and activation (Table 1) along with down regulation of many proteins involved in the onset of type 1 diabetes (Table 2).


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)

CTB-INS vaccination of monocytes does not interfere with dendritic cell differentiation.Flow cytometric analysis of vaccinated monocyte differentiation into dendritic cells. (A) The CD14 monocyte marker becomes progressively less positive (number inside the box (gate), as DC differentiation proceeds in the presence of the CTB-INS fusion protein. (B) The HLA-DR marker indicative of antigen presenting cell DC morphogenesis increases as dendritic cell differentiation progresses in the presence of CTB-INS. (C) The co-stimulatory factor marker for DC maturation CD86 is observed to decrease with time in vaccinated DCs in comparison with background levels. (D) With time after inoculation with CTB-INS, the dendritic cell marker CD83 indicative of DC maturation decreases in comparison with unvaccinated DC background levels.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118562.g006: CTB-INS vaccination of monocytes does not interfere with dendritic cell differentiation.Flow cytometric analysis of vaccinated monocyte differentiation into dendritic cells. (A) The CD14 monocyte marker becomes progressively less positive (number inside the box (gate), as DC differentiation proceeds in the presence of the CTB-INS fusion protein. (B) The HLA-DR marker indicative of antigen presenting cell DC morphogenesis increases as dendritic cell differentiation progresses in the presence of CTB-INS. (C) The co-stimulatory factor marker for DC maturation CD86 is observed to decrease with time in vaccinated DCs in comparison with background levels. (D) With time after inoculation with CTB-INS, the dendritic cell marker CD83 indicative of DC maturation decreases in comparison with unvaccinated DC background levels.
Mentions: Despite phenotypic and functional differences based on their origin, maturation stage, and culture conditions, DCs display common features among their subsets. These features include similar morphology, high density of membrane HLA class II co-stimulatory molecules, low phagocytic activity, and a strong capacity for antigen uptake and presentation to T lymphocytes [45,46]. Under normal conditions of immunological homeostasis, monocytes are more numerous than DCs and can differentiate into dendritic cells [47]. To assess vaccine safety for clinical applications, we examined potential detrimental effects of vaccine inoculation on the differentiation of monocytes into dendritic cells (Fig. 6). Peripheral blood monocytes were isolated and inoculated with CTB-INS plus GM-CSF and IL-4 in one experimental group and another group of monocytes were inoculated with only GM-CSF and IL-4 only. Both experimental groups were cultured for 2, 4 and 6 days post inoculation. The disappearance of the CD14 monocyte markers and appearance of the dendritic cell MHC Class II HLA-DR markers were monitored during DC differentiation. The morphogenesis of monocytes into dendritic cells was not impeded by inoculation with CTB-INS and the levels of DC maturation markers CD86 and CD83 measured by flow cytometry were lower than the controls in monocyte cultures inoculated with CTB-INS (Fig. 6C-D). The time required for differentiation from monocytes to DCs was not altered by inoculation with CTB-INS. In addition, DCs treated with the vaccine showed a modulation of proteins involved in the suppression of DC maturation and activation (Table 1) along with down regulation of many proteins involved in the onset of type 1 diabetes (Table 2).

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