The Cytochrome P450 2C8*3 Variant (rs11572080) Is Associated with Improved Asthma Symptom Control in Children and Altered Lipid Mediator Production and Inflammatory Response in Human Bronchial Epithelial Cells

Abstract

This study investigated an association between the cytochrome P450 (CYP) 2C8*3 polymorphism with asthma symptom control in children and changes in lipid metabolism and pro-inflammatory signaling by human bronchial epithelial cells (HBECs) treated with cigarette smoke condensate (CSC). CYP genes are inherently variable in sequence, and while such variations are known to produce clinically relevant effects on drug pharmacokinetics and pharmacodynamics, the effects on endogenous substrate metabolism and associated physiologic processes are less understood. In this study, CYP2C8*3 was associated with improved asthma symptom control among children: Mean asthma control scores were 3.68 (n = 207) for patients with one or more copies of the CYP2C8*3 allele versus 4.42 (n = 965) for CYP2C8*1/*1 (P = 0.0133). In vitro, CYP2C8*3 was associated with an increase in montelukast 36-hydroxylation and a decrease in linoleic acid metabolism despite lower mRNA and protein expression. Additionally, CYP2C8*3 was associated with reduced mRNA expression of interleukin-6 (IL-6) and C-X-C motif chemokine ligand 8 (CXCL-8) by HBECs in response to CSC, which was replicated using the soluble epoxide hydrolase inhibitor, 12-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]-dodecanoic acid. Interestingly, 9(10)- and 12(13)- dihydroxyoctadecenoic acid, the hydrolyzed metabolites of 9(10)- and 12(13)- epoxyoctadecenoic acid, increased the expression of IL-6 and CXCL-8 mRNA by HBECs. This study reveals previously undocumented effects of the CYP2C8*3 variant on the response of HBECs to exogenous stimuli. SIGNIFICANCE STATEMENT: These findings suggest a role for CYP2C8 in regulating the epoxyoctadecenoic acid:dihydroxyoctadecenoic acid ratio leading to a change in cellular inflammatory responses elicited by environmental stimuli that exacerbate asthma.

Graphical abstract

Fig. 1.

Comparison of asthma control scores as a function of rs11572080 genotype in a population of children with asthma. Genotype denotes an individual’s combination of alleles in the CYP2C8 G-7225 > A (rs11572080) gene polymorphism, where G is the reference allele, A is the variant allele, and X is representative of the presence of either allele. Asthma control scores are scaled from 0 (optimal control) to 15 (poor control). Data are represented as the median and interquartile range (IQR), where the horizontal line indicates the median asthma control score (50th percentile) and the top and bottom of the box is the third quartile (75th percentile) and first quartile (25th percentile), respectively. The whiskers mark the 2.5th and 97.5th percentiles. Data were analyzed for significant differences using the Mann–Whitney U test. **P < 0.01.

Fig. 2.

Comparison of asthma control scores as a function of rs11572080 genotype and treatment with the glucocorticoids (GC) or montelukast (MKT) in a population of children with a confirmed asthma diagnosis. Genotype denotes an individual’s combination of alleles in the CYP2C8 G-7225 > A (rs11572080) gene polymorphism, where G is the reference allele, A is the variant allele, and X is representative of the presence of either allele. Asthma control scores are scaled from 0 (optimal control) to 15 (poor control). Data are represented as the median and interquartile range (IQR), where the horizontal line indicates the median asthma control score (50th percentile) and the top and bottom of the box is the third quartile (75th percentile) and first quartile (25th percentile), respectively. The whiskers mark the 2.5th and 97.5th percentiles. Data were analyzed for significant differences using the Kruskal–Wallis H test followed by Dunn’s post hoc testing to correct for multiple comparisons. *P < 0.05; ***P < 0.0005; ****P < 0.0001.

Fig. 3.

Relative (A) mRNA and (B) protein expression of CYP2C8 in cells engineered to overexpress CYP2C8*1 and CYP2C8*3. Data were normalized to glyceraldehyde 3-phosphate dehydrogenase expression and are represented as fold change relative to the Flp-In BEAS-2B cell line transfected with the pcDNA5 FRT empty vector. Results are mean ± S.D. (error bars) from n = 3 replicates. Data were analyzed for significant differences by one-way ANOVA followed by Tukey post hoc testing to correct for multiple comparisons. *P < 0.05; **P < 0.01; ****P < 0.0001.

Fig. 4.

Effect of the CYP2C8*3 variant on the response of BEAS-2B to CSC (89 μg/cm²). Relative mRNA expression of (A) IL-6 and (B) CXCL-8 4 hours following exposure to CSC. Data were normalized to glyceraldehyde 3-phosphate dehydrogenase expression and are represented as fold change relative to the pcDNA5 vehicle control. Results are mean ± S.D. from n = 3 replicates. Data were analyzed for significant differences by two-way ANOVA followed by Tukey post hoc testing to correct for multiple comparisons. *P < 0.05; **P < 0.01; ****P < 0.0001.

Fig. 5.

Metabolism of LA by cells engineered to overexpress CYP2C8*1 and CYP2C8*3. Data are represented as the mean ± S.D. (error bars) from n = 3 replicates. Data were analyzed for significant differences by one-way ANOVA followed by Tukey post hoc testing to correct for multiple comparisons.

Fig. 6.

Formation of the LA-derived epoxides and corresponding diols by cells engineered to overexpress CYP2C8*1 and CYP2C8*3. Data are represented as the mean ± S.D. (error bars) from n = 3 replicates. Data were analyzed for significant differences by one-way ANOVA followed by Tukey post hoc testing to correct for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 7.

Effect of sEH inhibition on the response of BEAS-2B to CSC (89 μg/cm²). Relative mRNA expression of (A) IL-6 and (B) CXCL-8 following a 4-hour exposure to cigarette smoke condensate in the presence or absence of AUDA (10 μM). Data were normalized to glyceraldehyde 3-phosphate dehydrogenase expression and are represented as fold change relative to the vehicle control. Results are mean ± S.D. from n = 3 replicates. Data were analyzed for significant differences by one-way ANOVA followed by Tukey post hoc testing to correct for multiple comparisons. *P < 0.05; ****P < 0.0001.

Fig. 8.

Effect of 9(10)-EpOME, 9(10)-DiHOME, 12(13)-EpOME, and 12(13)-DiHOME (1, 2, or 10 μM) on cytokine expression by normal HBEC-3KT. Expression of (A and C) IL-6 and (B and D) CXCL-8 mRNA following treatment with 9(10)-EpOME, 9(10)-DiHOME, 12(13)-EpOME, or 12(13)-DiHOME for 6 hours. Data were normalized to β2-microglobulin expression and are represented as fold change relative to the vehicle control. Results are mean ± S.D. from n = 3 replicates. Data were analyzed for significant differences by one-way ANOVA followed by Tukey post hoc testing to correct for multiple comparisons. ****P < 0.0001.