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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 vaccine induced changes in DC cellular functions.(A) Heat map representing cellular functions (z-scores*) identified in vaccinated dendritic cells. Red bands indicate up-regulated cellular functions, yellow bands indicate no change in function and green bands indicate down-regulated cellular functions. (B) Principal component (PC) analysis of dendritic cell functions, z-scores. The Bi-plot correlates dendritic cell functions (+/-) vaccine treatments as indicated by the proximity of individual DC functions (numbers) to either vector arrowhead [a = CTB-INS (-), b = CTB-INS (+)]. Numbers further from the mean in the direction of the vector exhibit an increased correlation of the DC function with a or b. Example: Function #12 is highly correlated with unvaccinated DCs, [CTB-INS (-)] and Function #2 is poorly correlated with vaccinated DCs, [CTB-INS (+)]. Zero (0) is the weighted mean of the multivariate dataset. The values on the (x; y) axis represent (+/-) deviation from the mean of the dataset (0). (*z-score = statistical measurement of a score’s relationship to the mean in a group of scores. A z-score of 0 means the score is the same as the mean. A z-score can also be positive or negative, indicating whether it is above or below the mean and by how many standard deviations.)
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pone.0118562.g002: CTB-INS vaccine induced changes in DC cellular functions.(A) Heat map representing cellular functions (z-scores*) identified in vaccinated dendritic cells. Red bands indicate up-regulated cellular functions, yellow bands indicate no change in function and green bands indicate down-regulated cellular functions. (B) Principal component (PC) analysis of dendritic cell functions, z-scores. The Bi-plot correlates dendritic cell functions (+/-) vaccine treatments as indicated by the proximity of individual DC functions (numbers) to either vector arrowhead [a = CTB-INS (-), b = CTB-INS (+)]. Numbers further from the mean in the direction of the vector exhibit an increased correlation of the DC function with a or b. Example: Function #12 is highly correlated with unvaccinated DCs, [CTB-INS (-)] and Function #2 is poorly correlated with vaccinated DCs, [CTB-INS (+)]. Zero (0) is the weighted mean of the multivariate dataset. The values on the (x; y) axis represent (+/-) deviation from the mean of the dataset (0). (*z-score = statistical measurement of a score’s relationship to the mean in a group of scores. A z-score of 0 means the score is the same as the mean. A z-score can also be positive or negative, indicating whether it is above or below the mean and by how many standard deviations.)

Mentions: The effect of CTB-INS vaccination on changes in DC cellular functions including protein synthesis, maturation, and the immune response are presented graphically in the heat map (Fig. 2A). Dendritic cell functions (shown as z-scores), are displayed as up-regulated (red bands), down-regulated (green bands) or insignificantly changed (yellow bands), following DC inoculation with the vaccine. Dendritic cell functions identified by IPA that correlate with bands in the heat map are labeled as numbers to the right of the map. Functions associated with mechanisms leading to increased dendritic cell maturation, such as the immune responses of dendritic cells, cell movement, and cell migration, were found to be significantly down-regulated in CTB-INS treated dendritic cells [35–37]. Integrin associated protein (CD47), involved in a wide range of inflammatory cellular processes including activation, proliferation, migration and immune responses [38–40], was also found to be markedly down-regulated (Table 1). The dataset used to generate the heat map was analyzed by principal component (PC) analysis in order to identify the extent of cellular function correlation with CTB-INS treatment (Fig. 2B). As predicted from the heat map, CTB-INS vaccination induced distinct cellular functions in DCs that correlated with a change toward immune suppression which were absent from unvaccinated DCs. Cellular functions up-regulated in CTB-INS (+) treated DCs include RNA splicing and processing of mRNA which appear on the plot near the arrowhead of the CTB-INS (+) vector, indicating a high level of correlation with DC vaccination. Alternatively, pro-inflammatory cellular functions such as infection of cells, are strongly down-regulated in CTB-INS (+) DCs, and are located near the arrowhead of the CTB-INS (-) vector.


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 vaccine induced changes in DC cellular functions.(A) Heat map representing cellular functions (z-scores*) identified in vaccinated dendritic cells. Red bands indicate up-regulated cellular functions, yellow bands indicate no change in function and green bands indicate down-regulated cellular functions. (B) Principal component (PC) analysis of dendritic cell functions, z-scores. The Bi-plot correlates dendritic cell functions (+/-) vaccine treatments as indicated by the proximity of individual DC functions (numbers) to either vector arrowhead [a = CTB-INS (-), b = CTB-INS (+)]. Numbers further from the mean in the direction of the vector exhibit an increased correlation of the DC function with a or b. Example: Function #12 is highly correlated with unvaccinated DCs, [CTB-INS (-)] and Function #2 is poorly correlated with vaccinated DCs, [CTB-INS (+)]. Zero (0) is the weighted mean of the multivariate dataset. The values on the (x; y) axis represent (+/-) deviation from the mean of the dataset (0). (*z-score = statistical measurement of a score’s relationship to the mean in a group of scores. A z-score of 0 means the score is the same as the mean. A z-score can also be positive or negative, indicating whether it is above or below the mean and by how many standard deviations.)
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118562.g002: CTB-INS vaccine induced changes in DC cellular functions.(A) Heat map representing cellular functions (z-scores*) identified in vaccinated dendritic cells. Red bands indicate up-regulated cellular functions, yellow bands indicate no change in function and green bands indicate down-regulated cellular functions. (B) Principal component (PC) analysis of dendritic cell functions, z-scores. The Bi-plot correlates dendritic cell functions (+/-) vaccine treatments as indicated by the proximity of individual DC functions (numbers) to either vector arrowhead [a = CTB-INS (-), b = CTB-INS (+)]. Numbers further from the mean in the direction of the vector exhibit an increased correlation of the DC function with a or b. Example: Function #12 is highly correlated with unvaccinated DCs, [CTB-INS (-)] and Function #2 is poorly correlated with vaccinated DCs, [CTB-INS (+)]. Zero (0) is the weighted mean of the multivariate dataset. The values on the (x; y) axis represent (+/-) deviation from the mean of the dataset (0). (*z-score = statistical measurement of a score’s relationship to the mean in a group of scores. A z-score of 0 means the score is the same as the mean. A z-score can also be positive or negative, indicating whether it is above or below the mean and by how many standard deviations.)
Mentions: The effect of CTB-INS vaccination on changes in DC cellular functions including protein synthesis, maturation, and the immune response are presented graphically in the heat map (Fig. 2A). Dendritic cell functions (shown as z-scores), are displayed as up-regulated (red bands), down-regulated (green bands) or insignificantly changed (yellow bands), following DC inoculation with the vaccine. Dendritic cell functions identified by IPA that correlate with bands in the heat map are labeled as numbers to the right of the map. Functions associated with mechanisms leading to increased dendritic cell maturation, such as the immune responses of dendritic cells, cell movement, and cell migration, were found to be significantly down-regulated in CTB-INS treated dendritic cells [35–37]. Integrin associated protein (CD47), involved in a wide range of inflammatory cellular processes including activation, proliferation, migration and immune responses [38–40], was also found to be markedly down-regulated (Table 1). The dataset used to generate the heat map was analyzed by principal component (PC) analysis in order to identify the extent of cellular function correlation with CTB-INS treatment (Fig. 2B). As predicted from the heat map, CTB-INS vaccination induced distinct cellular functions in DCs that correlated with a change toward immune suppression which were absent from unvaccinated DCs. Cellular functions up-regulated in CTB-INS (+) treated DCs include RNA splicing and processing of mRNA which appear on the plot near the arrowhead of the CTB-INS (+) vector, indicating a high level of correlation with DC vaccination. Alternatively, pro-inflammatory cellular functions such as infection of cells, are strongly down-regulated in CTB-INS (+) DCs, and are located near the arrowhead of the CTB-INS (-) vector.

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