Anthony Firek

The Role of Indoleamine 2, 3-Dioxygenase in Immune Suppression and Autoimmunity

Indoleamine 2, 3-dioxygenase (IDO) is the first and rate limiting catabolic enzyme in the degradation pathway of the essential amino acid tryptophan. By cleaving the aromatic indole ring of tryptophan, IDO initiates the production of a variety of tryptophan degradation products called “kynurenines” that are known to exert important immuno-regulatory functions. Because tryptophan must be supplied in the diet, regulation of tryptophan catabolism may exert profound effects by activating or inhibiting metabolism and immune responses. Important for survival, the regulation of IDO biosynthesis and its activity in cells of the immune system can critically alter their responses to immunological insults, such as infection, autoimmunity and cancer. In this review, we assess how IDO-mediated catabolism of tryptophan can modulate the immune system to arrest inflammation, suppress immunity to cancer and inhibit allergy, autoimmunity and the rejection of transplanted tissues. Finally, we examine how vaccines may enhance immune suppression of autoimmunity through the upregulation of IDO biosynthesis in human dendritic cells.

The Role of Dendritic Cells in Tissue-Specific Autoimmunity

In this review, we explore the role of dendritic cell subsets in the development of tissue-specific autoimmune diseases. From the increasing list of dendritic cell subclasses, it is becoming clear that we are only at the beginning of understanding the role of these antigen presenting cells in mediating autoimmunity. Emerging research areas for the study of dendritic cell involvement in the onset and inhibition of tissue-specific autoimmunity are presented. Further, we compare tissue specific to systemic autoimmunity to demonstrate how development of dendritic cell-based therapies may be broadly applicable to both classes of autoimmunity. Continued development of these research areas will lead us closer to clinical assessment of novel immunosuppressive therapy for the reversal and prevention of tissue-specific autoimmunity. Through description of dendritic cell functions in the modulation of tissue-specific autoimmunity, we hope to stimulate a greater appreciation and understanding of the role dendritic cells play in the development and treatment of autoimmunity.

En Balance Participants Decrease Dietary Fat and Cholesterol Intake as Part of a Culturally Sensitive Hispanic Diabetes Education Program

Purpose The purpose of this study was to assess dietary intake habits of Mexican American Hispanic adults participating in the En Balance diabetes education program. Methods En Balance is a 3-month culturally sensitive diabetes education intervention for Spanish-speaking Hispanics. Of the 46 participants enrolled, 39 mainly Mexican American Hispanic adults with type 2 diabetes completed the En Balance program. Participants lived in the Riverside and San Bernardino counties of California, and all participants completed the program by June 2008. Dietary intake was assessed at baseline and at 3 months using the validated Southwest Food Frequency Questionnaire. Results Clinically important decreases in glycemic control and serum lipid levels were observed at the end of the 3-month program. The baseline diet was characterized by a high intake of energy (2478 ± 1140 kcal), total fat (87 ± 44 g/day), saturated fat (28 ± 15 g/day), dietary cholesterol (338 ± 217 mg/day), and sodium (4236 ± 2055 mg/ day). At 3 months, the En Balance group mean intake of dietary fat (P = .045) and dietary cholesterol ( P = .033) decreased significantly. Low dietary intakes of docosahexaenoic acid, eicosapentaenoic acid, and vitamin E were also observed in these adults with type 2 diabetes. Conclusions The En Balance program improved glycemic control and lipid profiles in a group of Hispanic diabetic participants. En Balance also promoted decreases in dietary fat and dietary cholesterol intake.

Long-term, progressive, aerobic training increases adiponectin in middle-aged, overweight, untrained males and females

Abstract Adipose tissue secretes the adipokine, adiponectin (ADPN), which increases insulin sensitivity. Because some of the metabolic effects of exercise and ADPN are similar, exercise has been proposed to increase ADPN. However, most short-term (≤3 mos) and constant-effort exercise protocols have not produced increases in ADPN. Furthermore, no direct comparisons of male and female subjects on the effect of exercise on ADPN levels have been reported. We hypothesized that long-term (6 mos), progressive training would increase ADPN levels in both males and females. We recruited middle-aged, untrained males and females to participate in an interventional study employing a marathon training regimen progressing from 9.7 to 88.5 km (6 to 55 miles) per week over 6 mos. At baseline, we matched the mean ages of the male and female groups. We collected and stored fasting plasma samples and recorded body measurements at 0 (baseline) and 6 mos. Stored samples were analysed for insulin, glucose, and ADPN. ADPN increased significantly among both males (from 5.89 ± 2.46 (mean ± SD) to 7.65 ± 3.18 μg/ml; p < 0.05) and females (from 8.48 ± 3.22 to 10.56 ± 4.05 μg/ml; p < 0.05). The extent of the increase in ADPN was similar in the male (40.7 ± 50%; median, 12.1%) and female (27.0 ± 31.1%; median, 22.3%) groups. However, there was no significant reduction in insulin resistance as measured by the HOMA-IR scores in either group. We conclude that long-term, progressive aerobic training increases circulating ADPN levels in middle-aged, untrained males and females.

Induction of Indoleamine 2, 3-Dioxygenase in Human Dendritic Cells by a Cholera Toxin B Subunit—Proinsulin Vaccine

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.

En Balance: the effects of Spanish diabetes education on physical activity changes and diabetes control.

Purpose This study was designed to assess the feasibility of culturally and language-sensitive diabetes education as a way to increase physical activity and to improve health/diabetes management in a group of Spanish-speaking Hispanics in the Inland Empire region of Southern California. Methods En Balance is a culturally sensitive diabetes education program designed for Spanish-speaking Hispanic adults. The 3-month educational intervention assessed 16 males and 23 females living in Riverside and San Bernardino counties of Southern California. Baseline and 3-month evaluations of physical activity were assessed using the validated Arizona Activity Frequency Questionnaire. Results After 3 months on the En Balance program, there was a significant increase in moderate intensity physical activity energy expenditure (M = 368 ± 894 kcal/day, P < 0.01) and high intensity physical activity energy expenditure (M = 405 ± 2569 kcal/day, P = 0.05) compared to baseline and significant reductions in A1C (−0.90%, P = 0.01), total cholesterol (−13.44 mg/dl, P = 0.01), LDL cholesterol (−10.28 mg/dl, P = 0.03), and waist circumference (−1.52 cm, P = 0.04). Conclusion En Balance program resulted in significant mean increases in both moderate and high intensity physical activity energy expenditure among this group of Hispanic diabetic participants, indicating that despite a general pattern of low physical activity in this group, an intervention that stresses both nutrition and exercise in culturally sensitive ways can positively impact participant’s physical activity levels as well as impact nutritional changes.

Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway.

A chimeric protein vaccine composed of the cholera toxin B subunit fused to proinsulin (CTB-INS) was shown to suppress type 1 diabetes onset in NOD mice and upregulate biosynthesis of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) in human dendritic cells (DCs). Here we demonstrate siRNA inhibition of the NF-κB-inducing kinase (NIK) suppresses vaccine-induced IDO1 biosynthesis as well as IKKα phosphorylation. Chromatin immunoprecipitation (ChIP) analysis of CTB-INS inoculated DCs showed that RelB bound to NF-κB consensus sequences in the IDO1 promoter, suggesting vaccine stimulation of the non-canonical NF-κB pathway activates IDO1 expression in vivo. The addition of Tumor Necrosis Factor Associated Factors (TRAF) TRAF 2, 3 and TRAF6 blocking peptides to vaccine inoculated DCs was shown to inhibit IDO1 biosynthesis. This experimental outcome suggests vaccine activation of the TNFR super-family receptor pathway leads to upregulation of IDO1 biosynthesis in CTB-INS inoculated dendritic cells. Together, our experimental data suggest the CTB-INS vaccine uses a TNFR-dependent signaling pathway of the non-canonical NF-κB signaling pathway resulting in suppression of dendritic cell mediated type 1 diabetes autoimmunity.

The Ala54Thr Polymorphism of the Fatty Acid Binding Protein 2 Gene Modulates HDL Cholesterol in Mexican-Americans with Type 2 Diabetes

The alanine to threonine amino acid substitution at codon 54 (Ala54Thr) of the intestinal fatty acid binding protein (FABP2) has been associated with elevated levels of insulin and blood glucose as well as with dyslipidemia. The aim of this study was to characterize the effect of this FABP2 polymorphism in Mexican-Americans with type 2 diabetes (T2D) in the context of a three-month intervention to determine if the polymorphism differentially modulates selected clinical outcomes. For this study, we genotyped 43 participant samples and performed post-hoc outcome analysis of the profile changes in fasting blood glucose, HbA1c, insulin, lipid panel and body composition, stratified by the Ala54Thr polymorphism. Our results show that the Thr54 allele carriers (those who were heterozygous or homozygous for the threonine-encoding allele) had lower HDL cholesterol and higher triglyceride levels at baseline compared to the Ala54 homozygotes (those who were homozygous for the alanine-encoding allele). Both groups made clinically important improvements in lipid profiles and glycemic control as a response to the intervention. Whereas the Ala54 homozygotes decreased HDL cholesterol in the context of an overall total cholesterol decrease, Thr54 allele carriers increased HDL cholesterol as part of an overall total cholesterol decrease. We conclude that the Ala54Thr polymorphism of FABP2 modulates HDL cholesterol in Mexican-Americans with T2D and that Thr54 allele carriers may be responsive in interventions that include dietary changes.

Identification of Anti-Long Chain Saturated Fatty Acid IgG Antibodies in Serum of Patients with Type 2 Diabetes

High levels of serum long chain saturated fatty acids (LCSFAs) have been associated with inflammation in type 2 diabetes. Dietary SFAs can promote inflammation, the secretion of IgG antibodies, and secretion of the proinflammatory cytokine IL-1β. This study characterizes anti-LCSFA IgG antibodies from patients with type 2 diabetes. Serum samples from several cohorts with type 2 diabetes were analyzed for the presence of anti-LCSFA IgG, the cytokine IL-1β, and nonesterified fatty acids. Anti-LCSFA IgG was isolated from patient samples and used for in vitro characterization of avidity and specificity. A cohort participating in En Balance, a diabetes health education program that improved diabetes management, tested positive for anti-LCSFA IgG. Following the 3-month program, the cohort showed a significant reduction in anti-LCSFA IgG levels. Anti-LCSFA antibodies isolated from these patients demonstrated high avidity, were specific for long chain SFAs, and correlated with serum fatty acids in patients with managed type 2 diabetes. Interestingly, anti-LCSFA IgG neutralized PA-induced IL-1β secretion by dendritic cells. Our data shows that nonesterified SFAs are recognized by IgG antibodies present in human blood. The identification of anti-LCSFA IgG antibodies in human sera establishes a basis for further exploration of lipid induced immune responses in diabetic patients.