Beta-defensin 1, aryl hydrocarbon receptor and plasma kynurenine in major depressive disorder: metabolomics-informed genomics

Abstract

Major depressive disorder (MDD) is a heterogeneous disease. Efforts to identify biomarkers for sub-classifying MDD and antidepressant therapy by genome-wide association studies (GWAS) alone have generally yielded disappointing results. We applied a metabolomics-informed genomic research strategy to study the contribution of genetic variation to MDD pathophysiology by assaying 31 metabolites, including compounds from the tryptophan, tyrosine, and purine pathways, in plasma samples from 290 MDD patients. Associations of metabolite concentrations with depressive symptoms were determined, followed by GWAS for selected metabolites and functional validation studies of the genes identified. Kynurenine (KYN), the baseline plasma metabolite that was most highly associated with depressive symptoms, was negatively correlated with severity of those symptoms. GWAS for baseline plasma KYN concentrations identified SNPs across the beta-defensin 1 (DEFB1) and aryl hydrocarbon receptor (AHR) genes that were cis-expression quantitative trait loci (eQTLs) for DEFB1 and AHR mRNA expression, respectively. Furthermore, the DEFB1 locus was associated with severity of MDD symptoms in a larger cohort of 803 MDD patients. Functional studies demonstrated that DEFB1 could neutralize lipopolysaccharide-stimulated expression of KYN-biosynthesizing enzymes in monocytic cells, resulting in altered KYN concentrations in the culture media. In addition, we demonstrated that AHR was involved in regulating the expression of enzymes in the KYN pathway and altered KYN biosynthesis in cell lines of hepatocyte and astrocyte origin. In conclusion, these studies identified SNPs that were cis-eQTLs for DEFB1 and AHR and, which were associated with variation in plasma KYN concentrations that were related to severity of MDD symptoms.

Trial registration: ClinicalTrials.gov NCT00613470.

Conflict of interest statement

Dr. Balmiki Ray is a salaried employee of Assurex Health Inc. Dr. Wayne Matxon and Dr. Swati Bhasin are salaried employees of Ixcela. Dr. Mark Frye receives grant support from AssureRx, Janssen Research & Development, Myriad, and Pfizer. He is also a consultant for Janssen Research & Development, LLC, Mitsubishi Tanabe Pharma Corporation, Myriad Genetics, Sunovion, Supernus Pharmaceuticals, Teva Pharmaceuticals, and Neuralstem, Inc. and he receives CME/Travel Support from the American Physician Institute and CME Outfitters. Drs. Wang and Weinshilboum are founders and stockholders in OneOme LLC.

Figures

Fig. 1. Tryptophan metabolism, kynurenine pathway and major depressive disorder

Tryptophan (TRP) is metabolized by two major pathways: the “kynurenine (KYN) pathway” and the “serotonin (5-HT)” pathways. 5-HT cannot pass the blood–brain barrier (BBB). The majority of TRP is metabolized to form KYN in the liver and is released into peripheral blood. The initial and rate-limiting step in the KYN pathway is catalyzed by indoleamine 2,3-dioxygenase 1 (IDO1), IDO2 and/or tryptophan 2,3-dioxygenase (TDO2)—depending on the tissue involved—to form N-formyl-kynurenine. N-formyl-kynurenine is metabolized by formamidase to produce KYN, which can cross the BBB. Approximately 60% of KYN in the CNS originates from the liver. In the periphery and in the CNS, KYN can be further metabolized by either kynurenine aminotransferases (KATs) or by kynurenine 3-monooxygenase (KMO) and kynureninase (KYNU), leading to the generation of the neuroactive metabolites kynurenic acid (KYNA) or quinolinic acid (QUIN), respectively. KYNA, which is neuroprotective, and QUIN, which is neuroactive in the CNS, have opposite effects on the NMDA receptor. Four KATs, encoded by AADAT, CCBL1, CCBL2, and GOT2, have been shown to catalyze the conversion of KYN to KYNA. The other branches of the KYN pathway involve KMO and KYNU, which catalyze the metabolism of kynurenine to form 3-hydroxykynurenine and 3-hydroxyanthranilic acid, respectively. KYNU can also metabolize KYN to form anthranilic acid, which can then be coverted to 3-hydroxyanthranilic acid by nonspecific hydroxylation. 3-Hydroxyanthranilic acid is metabolized further by 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) to form either QUIN or, after a series of reactions, picolinic acid

Fig. 2. Plasma KYN concentrations GWAS

a Manhattan plot for baseline plasma KYN concentrations. SNPs across the DEFB1 and AHR genes have been highlighted, with rs5743467 as the “top” DEFB1 SNP (P-value = 8.18E−07) and, rs17137566 as the “top” AHR SNP (P-value = 6.22E−06). Regional association plots (Locus Zooms) for the DEFB1 gene B and the AHR gene C are also shown. Circles and diamonds represent observed and imputed SNPs, respectively. The color of each SNP represents its’ linkage disequilibrium (LD) with the “top SNP”, which is colored purple

Fig. 3

eQTL analysis for the top DEFB1A and AHRB SNPs based on the GTEx dataset. ADEFB1 mRNA expression was significantly decreased in human cerebellum (left), transverse colon (middle) and esophageal mucosa (right) from individuals with rs5743467 variant genotypes (G) when compared with WT genotype (C) (P < 0.05). BAHR mRNA expression was significantly decreased in human cerebellum (left), sigmoid colon (middle) and esophageal mucosa (right) from subjects with rs17137566 variant genotypes (C) when compared with those for subjects homozygous for the WT genotype (T) (P < 0.05). mRNA levels were determined by RNA sequencing data available in GTEx

Fig. 4. DEFB1 functional studies in THP-1 cells

A mRNA expression was determined by qRT-PCR after THP-1 cells were exposed to 10 and 100 ng/ml of LPS at different time points. Compared to vehicle treated cells, IDO1 mRNA levels were significantly increased after LPS treatment. mRNA levels for TNF, a pro-inflammatory cytokine used as a positive control for LPS effect, were also significantly increased after LPS treatment. B IDO1 protein expression was increased as analyzed by Western blot after LPS treatment. C KYN concentrations (left) in cell culture media were undetectable after 24 and 48 h of vehicle treatments, but were significantly increased after 10 ng/ml LPS treatment. At the same time, TRP concentrations (middle) were significantly decreased in cell culture media after LPS treatment and the K/T ratio was increased (right). After 3, 6, or 12 h of LPS treatment, KYN concentrations were undetectable. D mRNA levels for IDO1 and TNF were significantly increased after 10 ng/ml LPS treatment, but recombinant human DEFB1 co-incubation with LPS significantly decreased mRNA levels for IDO1 and TNF when compared with LPS treatment alone. E When DEFB1 was co-incubated with LPS as compared to LPS alone, KYN concentrations (left) in cell culture media were significantly decreased, TRP concentrations were increased (middle) and K/T ratios (right) were decreased after DEFB1 was pre-incubated with LPS when compared with results for cells treated with LPS alone. N ≥ 3 for all the experiments. Data = mean ± SEM, with statistical significance determined by two-tailed t-test denoted as *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 5. AHR functional studies in HepaRG and U-87 MG cells

A mRNA expression determined by qRT-PCR after AHR KD in HepaRG cells. TDO2, KMO, and KYNU expression was significantly increased and AHRR and CYP1A1 expression was significantly decreased following AHR KD. B Protein expression analysis for AHR, TDO2, KMO, and KYNU by Western blot analysis after AHR KD in HepaRG cells. C mRNA expression in HepaRG cells determined by qRT-PCR after 24-h treatment with 1 µM 3-MC, an AHR agonist. TDO2, KMO, and KYNU expression was significantly decreased. AHRR, CYP1A1 (in HepaRG), was  induced by 3-MC, indicating that AHR was activated by the treatment. D KYN concentrations in HepaRG cell culture media after AHR and AHR plus KMO or KYNU KD. ETDO2, KMO, and KYNU expression was significantly increased while AHRR and CYP1B1 expression was significantly decreased following AHR KD. F AHR, TDO2, KMO, and KYNU protein concentrations were significantly altered in U-87 MG cells after AHR KD. G mRNA expression in U-87 MG cells after 1 µM 3-MC treatment showed significant decreases in TDO2, KMO, and KYNU expression and significant increases in AHRR and CYP1B1 expression. H KYN concentrations in U-87 MG cell culture media were significantly decreased after AHR KD but were less decreased after AHR KD together with KMO or KYNU KD. N ≥ 3 for all experiments. Data = mean ± SEM, with statistical significance determined by two-tailed t-test denoted as *P < 0.05, **P < 0.01 and ***P < 0.001 when compared with control. ns = not significant