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ONLINE ARTICLE: Original Articles

Genetic Variations at rs3129891 and rs77005575 are Associated With Reduced Expression of Enteric α-defensins in IBD Patients

Deng, Qiliang MD; Tan, Gao MD; Deng, Feihong MD; Zhi, Fachao MD

Author Information
Journal of Clinical Gastroenterology: July 2020 - Volume 54 - Issue 6 - p e50-e55
doi: 10.1097/MCG.0000000000001146

Abstract

Inflammatory bowel disease (IBD) is a chronic idiopathic inflammation of the intestine, and its 2 major forms include ulcerative colitis (UC) and Crohn’s disease (CD), based on clinical features and histopathology.1,2 Although its etiology is still unclear, a strongly advocated view is that ineffective bacterial clearance resulted from the diminished expression of enteric antimicrobial peptides triggers and sustains the abnormal immune responses observed in IBD patients.3–6

Human enteric antimicrobial peptides are composed predominantly of human enteric α-defensins (HD5 and HD6) as well as lysozyme and secretory phospholipase A2 (SPLA2), to a lesser extent.7 In humans, there are only 2 enteric α-defensins, namely HD5 and HD6.8,9 They are the main antimicrobial peptides in the intestine. Matrilysin-knockout mice failed to produce mature enteric α-defensins have increased susceptibility toward infection with orally administered enteric pathogen salmonella.10,11 However, HD5 transgenic mice have a protection against salmonella infection and have observably loss of segmented filamentous bacteria.10,12 In addition, HD6 can kill specific microbes depending on the local environmental conditions13 and HD6 transgenic mice have a protection against invasion by diverse enteric pathogens.14 Their antimicrobial activities contribute to their roles in maintaining intestinal homeostasis.

IBD has been at the vanguard of progress in understanding the genetic framework of complex diseases, with 200 susceptibility genetic loci identified so far.15 Genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) across these loci as being associated with an increased risk of developing IBD.15 Genotype-phenotype correlation studies have revealed that NOD2 mutations (SNP8, rs2066844; SNP12, rs2066845; SNP13, rs2066847) are associated with ileal CD. Expression of enteric α-defensins has been shown to be diminished in terminal ileal biopsies from CD patients harboring these mutations, with diminished levels being more pronounced in the presence of an SNP13 mutation.16,17 However, it is still unclear which genetic variations are associated with mucosal expression of antimicrobial peptides in colonic CD and UC patients.

The purpose of this study was to determine which genetic variations can influence mucosal expression of antimicrobial peptides in colonic CD and UC. To address this issue, we compared mucosal levels of human enteric antimicrobial peptides in colonic CD and UC patients with respect to genotypes at 22 IBD-associated SNPs. These SNPs are associated with IBD subphenotypes and disease location, including rs2066844, rs2066845, rs2066847, rs35261698, rs2172252, rs3197999, rs4151651, rs6930777, rs77005575, rs9268832, rs3129891, rs3115674, rs2066842, rs10885394, rs10885395, rs3814570, rs11209026, rs11805303, rs12212067, rs2241880, rs2542151, and rs7234029.18–21

MATERIALS AND METHODS

Patients

In total, 16 patients undergoing a routine colonoscopy without significant pathology were randomly selected as controls. A cohort of 42 UC and 60 CD patients from Department of Gastroenterology at Nanfang Hospital were screened for mutant genotypes of 22 IBD-associated SNPs. Colonic biopsies were available from the 118 patients. In the case of UC and CD, samples were taken from macroscopically inflamed and noninflamed mucosa. Samples were immediately snap frozen in liquid nitrogen. The diagnosis in all patients was based on standard criteria using clinical, endoscopic, and histopathologic findings.22 All patients gave written informed consent before colonoscopy was performed. The study was approved by the clinical ethics committee at Nanfang Hospital.

Mutation Assay

Frozen biopsies were disrupted mechanically and genomic DNA was extracted using MiniBEST Universal Genomic DNA Extraction kit version 5.0 (TaKaRa) according to the manufacturer’s protocol. Genotyping of genomic DNA for mutations of 22 IBD-associated SNPs was performed using MassARRAY MALDI-TOF System (SEQUENOM) according to the manufacturer’s protocol. The primer sequences were shown in Table S1 (Supplemental Digital Content 1, http://links.lww.com/JCG/A459).

Real-time Quantitative RT-PCR

Frozen biopsies were disrupted mechanically and total RNA was extracted using the RNAiso Plus Kit (TaKaRa), then cDNA synthesis was performed using the PrimeScript RT reagent Kit (TaKaRa), and finally real-time PCR was performed in triplicate using the LightCycler 480 System (Roche) according to the manufacturer’s protocol. Briefly, each 20-μl PCR reaction sample contained 5 μl cDNA corresponding to 25 ng RNA, 0.5 μM of each primer and 1× LightCycler 480 SYBR Green I Master (Roche). Samples were loaded into the LightCycler 480 Multiwell Plate 96 (Roche) and incubated for initial denaturation at 95°C for 10 min followed by 45 cycles, each cycle consisting of 95°C for 10s, 60°C for 10s, and 72°C for 20s. The primer sequences were shown in Table S1 (Supplemental Digital Content 1, http://links.lww.com/JCG/A459). 18SrRNA were used as internal controls. The 2−ΔCT method was used to analyze real-time PCR data.

Statistical Analysis

The results are shown as the mean±SEM. Statistical significance was determined by 1-way analysis of variance with the Tukey multiple comparisons under equal variances or with the Dunnett T3’s multiple comparisons under unequal variances; P<0.05 was considered statistically significant.

RESULTS

SNP Mutation Analyses

Analyses of 118 specimens from 16 controls, 42 UC and 60 CD patients failed to show mutant genotypes at rs2066844, rs2066845, rs2066847, rs11209026, rs4151651, rs6930777, but showed mutant genotypes at rs11805303, rs12212067, rs2241880, rs2542151, rs7234029, rs35261698, rs2172252, rs3197999, rs77005575, rs9268832, rs3129891, rs10885394, rs10885395, and rs3814570. However, homozygous mutant rs77005575 genotype CC and rs3129891 genotype AA were not found in controls. In addition, mutant genotypes at rs2066842 and rs3115674 were only found in CD patients and controls, respectively (Supplementary Data, Supplemental Digital Content 2, http://links.lww.com/JCG/A460).

Colonic Gene Expression of HD5 and HD6 was Decreased in UC and CD Patients Carrying rs3129891 Mutant Genotypes

In total, 27 of 42 UC patients were positive for mutant genotypes AG and AA at rs3129891 (21 heterozygotes and 6 homozygotes). In inflamed colonic tissues, the mRNA expression of HD5 and HD6 was significantly lower in rs3129891 mutant heterozygotes than in patients carrying homozygous wild genotype GG, with reduced expression being more pronounced in mutant homozygotes. In noninflamed colonic tissues, the mRNA expression of HD5 and HD6 was significantly lower in rs3129891 mutant homozygotes than in patients carrying homozygous wild genotype GG (Figs. 1A, B). In contrast with HD5 and HD6, the mRNA expression of lysozyme and SPLA2 was not significantly influenced by rs3129891 genotypes in both inflamed and noninflamed colonic tissues (Figs. 1C, D). These results indicate that rs3129891 genotypes can modulate enteric α-defensins production in colonic tissues of UC patients.

FIGURE 1
FIGURE 1:
Effect of genotypes at rs3129891 on colonic mRNA expression of HD5 (A), HD6 (B), lysozyme (C), and SPLA2 (D) in UC patients. Relative mRNA levels were determined using real-time PCR and normalized to 18SrRNA. Data are shown as the mean±SEM. Ctrl indicates control; HD5, human α-defensin 5; HD6, human α-defensin 6; NS, not significant (number of patients); SPLA2, secretory phospholipase A2; UC, ulcerative colitis. *P<0.05, **P<0.01, ***P<0.001.

In total, 27 of 60 CD patients were positive for mutant genotypes AG and AA at rs3129891 (21 heterozygotes and 6 homozygotes). In both inflamed and noninflamed colonic tissues, the mRNA expression of HD5 and HD6 was significantly lower in rs3129891 mutant homozygotes than in mutant heterozygotes and patients carrying homozygous wild genotype GG. However, there were no significant differences in the mRNA expression of HD5 and HD6 in inflamed tissues between patients carrying rs3129891 mutant genotype AG and wild genotype GG (Figs. 2A, B). In contrast with HD5 and HD6, the mRNA expression of lysozyme and SPLA2 was not significantly influenced by rs3129891 genotypes in both inflamed and noninflamed colonic tissues (Figs. 2C, D). These results indicate that rs3129891 genotypes can modulate enteric α-defensins production in colonic tissues of CD patients.

FIGURE 2
FIGURE 2:
Effect of genotypes at rs3129891 on colonic mRNA expression of HD5 (A), HD6 (B), lysozyme (C), and SPLA2 (D) in CD patients. Relative mRNA levels were determined using real-time PCR and normalized to 18SrRNA. Data are shown as the mean±SEM. CD indicates Crohn’s disease; ctrl, control; HD5, human α-defensin 5; HD6, human α-defensin 6; NS, not significant (number of patients); SPLA2, secretory phospholipase A2. *P<0.05, **P<0.01, ***P<0.001.

Colonic Gene Expression of HD5 and HD6 was Decreased in UC Patients Carrying rs77005575 Mutant Genotypes

In total, 30 of 42 UC patients were positive for mutant genotypes CT and CC at rs77005575 (21 heterozygotes and 9 homozygotes). In inflamed colonic tissues, the mRNA expression of HD5 and HD6 was significantly lower in rs77005575 mutant heterozygotes than in patients carrying homozygous wild genotype TT, with reduced expression being more pronounced in mutant homozygotes. In noninflamed colonic tissues, there was a tendency toward diminished mRNA expression of HD5 and HD6 in patients carrying rs77005575 mutant genotypes when compared with patients carrying homozygous wild genotype TT, but this difference did not attain statistical significance (Figs. 3A, B). In contrast with HD5 and HD6, the mRNA expression of lysozyme and SPLA2 was not significantly influenced by rs77005575 genotypes in inflamed and noninflamed colonic tissues (Figs. 3C, D). These results indicate that rs77005575 genotypes can modulate enteric α-defensins production in inflamed colonic tissues of UC patients.

FIGURE 3
FIGURE 3:
Effect of genotypes at rs77005575 on colonic mRNA expression of HD5 (A), HD6 (B), lysozyme (C), and SPLA2 (D) in UC patients. Relative mRNA levels were determined using real-time PCR and normalized to 18SrRNA. Data are shown as the mean±SEM. Ctrl indicates control; HD5, human α-defensin 5; HD6, human α-defensin 6; NS, not significant (number of patients); SPLA2, secretory phospholipase A2; UC, ulcerative colitis. **P<0.01, ***P<0.001.

In total, 30 of 60 CD patients were positive for mutant genotypes CT and CC at rs77005575 (21 heterozygotes and 9 homozygotes). In inflamed colonic tissues, the mRNA expression of HD5 and HD6 was higher in rs77005575 mutant homozygotes when compared with patients carrying homozygous wild genotype TT, but this increase was not statistically significant (Figs. 4A, B). In addition, there was a tendency toward increased mRNA expression of SPLA2 in patients carrying rs77005575 mutant genotypes, but this difference did not reach statistical significance (Fig. 4D). In noninflamed colonic tissues, there were no genotype-specific differences in the mRNA expression of HD5, HD6, lysozyme, or SPLA2 (Figs. 4A–D). These results indicate that rs77005575 genotypes do not influence the expression of enteric antimicrobial peptides in CD patients.

FIGURE 4
FIGURE 4:
Effect of genotypes at rs77005575 on colonic mRNA expression of HD5 (A), HD6 (B), lysozyme (C), and SPLA2 (D) in CD patients. Relative mRNA levels were determined using real-time PCR and normalized to 18SrRNA. Data are shown as the mean±SEM. CD indicates Crohn’s disease; ctrl, control; HD5, human α-defensin 5; HD6, human α-defensin 6; NS, not significant (number of patients); SPLA2, secretory phospholipase A2.

Colonic Gene Expression of HD5, HD6, Lysozyme, and SPLA2 in CD Patients was Not Affected by NOD2 rs2066842 Mutant Genotypes

Six of 60 CD patients were positive for mutant genotype CT at NOD2 rs2066842. However, in both inflamed and noninflamed colonic tissues, there were no genotype-specific differences in the mRNA expression of HD5, HD6, lysozyme, or SPLA2 between patients carrying rs2066842 mutant genotype CT and wild genotype CC (Figs. S1A–S1D, Supplemental Digital Content 1, http://links.lww.com/JCG/A459). This result indicates that this genetic variation at rs2066842 does not influence the expression of enteric antimicrobial peptides in CD patients.

DISCUSSION

This study assessed which genetic variations are associated with mucosal expression of antimicrobial peptides in colonic CD and UC patients. Our results show that genetic variations at rs3129891 and rs77005575 are associated with reduced expression of enteric α-defensins in colonic IBD patients. We found that compared with rs3129891 wild homozygotes, the colonic mRNA expression of HD5 and HD6 was decreased in colonic CD and UC patients carrying rs3129891 mutant genotypes, especially in mutant homozygotes. In addition, their mRNA expression was also decreased in UC patients carrying rs77005575 heterozygous mutant genotype, with decreased expression being more pronounced in rs77005575 mutant homozygotes as compared with rs77005575 wild homozygotes. However, genetic variations at rs77005575 did not influence the colonic mRNA expression of α-defensins in CD patients.

In contrast with enteric α-defensins, mucosal expression of the other 2 antimicrobial peptides lysozyme and SPLA2 were not influenced by genetic variations at rs3129891 and rs77005575. In addition, although genetic variations at rs2241880, rs9268832, rs11805303, rs12212067, rs2542151, rs7234029, rs35261698, rs2172252, rs3197999, rs10885394, rs10885395, and rs3814570 were found in colonic IBD patients, these mutations did not significantly influence colonic mucosal expression of antimicrobial peptides (Table S2, Supplemental Digital Content 1, http://links.lww.com/JCG/A459). These results suggest that colonic mucosal expression of α-defensins could be influenced mainly through genetic variations at rs3129891 and rs77005575.

Previous studies have demonstrated that the Wnt-signaling pathway transcription factor Tcf-4 is a critical regulator of α-defensin expression.23,24 We would like to know whether genetic variations at rs3129891 and rs77005575 influence enteric α-defensin expression through the Wnt/Tcf-4 pathway. Nevertheless, we found that there were no genotype-specific differences in the gene expression of Tcf-4 with respect of genotypes at rs3129891 and rs77005575 (Table S3, Supplemental Digital Content 1, http://links.lww.com/JCG/A459). Thus, possible underlying mechanisms by which these genetic variations modulate enteric α-defensin production need to be further explored and studied.

Interestingly, although NOD2 mutations (SNP8, rs2066844; SNP12, rs2066845; SNP13, rs2066847) are significantly associated with ileal involvement in CD patients of European descent,17 they were not found in our study. However, our study shows that genetic variations at rs3129891 are significantly associated with colonic involvement in CD patients. These findings suggest that ileal and colonic CD may have different hereditary basis. Nevertheless, this difference could also be explained as genetic heterogeneity between divergent populations, as the 3 most common NOD2 variants (rs2066844, rs2066845, rs2066847) have a large effect on CD risk in Europeans while having risk-allele frequency of 0 in East Asians.15

In summary, our results show that genetic variations at rs3129891 and rs77005575 are associated with reduced expression of enteric α-defensins in colonic IBD patients. In view of an important role of the host-microbe balance at the intestinal mucosa in IBD etiology, we reasoned that a decrease in enteric α-defensins could weaken antimicrobial defenses of the intestinal mucosa, which may in principle lead to loss of tolerance against the luminal microorganisms and susceptibility to bacterial invasion. Ultimately, these alterations could trigger and sustain mucosal inflammation in IBD. Taking into consideration the substantial heterogeneity in individual response to therapy for IBD,25 we speculate that for colonic IBD patients carrying genetic variations at rs3129891 and rs77005575, restoring the host-microbe balance at their intestinal mucosa may be superior to broadly inhibiting inflammation and adaptive immunity.

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Keywords:

inflammatory bowel disease; human enteric antimicrobial peptides; human enteric α-defensins; single-nucleotide polymorphisms

Supplemental Digital Content

Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc.