Human β-Defensin 2 Mutations Are Associated With Asthma and Atopy in Children and Its Application Prevents Atopic Asthma in a Mouse Model

Abstract

Asthma and allergies are complex, chronic inflammatory diseases in which genetic and environmental factors are crucial. Protection against asthma and allergy development in the context of farming environment is established by early animal contact, unpasteurized milk consumption and gut microbiota maturation. The human β-defensin 2 (hBD-2) is a host defense peptide present almost exclusively in epithelial tissues, with pronounced immunomodulatory properties, which has recently been shown to ameliorate asthma and IBD in animal models. We hypothesized that adequate hBD-2 secretion plays a role in the protection against asthma and allergy development and that genetic variations in the complex gene locus coding for hBD-2 may be a risk factor for developing these diseases, if as a consequence, hBD-2 is insufficiently produced. We used MALDI-TOF MS genotyping, sequencing and a RFLP assay to study the genetic variation including mutations, polymorphisms and copy number variations in the locus harboring both genes coding for hBD-2 (DEFB4A and DEFB4B). We administered hBD-2 orally in a mouse model of house dust mite (HDM)-asthma before allergy challenge to explore its prophylactic potential, thereby mimicking a protective farm effect. Despite the high complexity of the region harboring DEFB4A and DEFB4B we identified numerous genetic variants to be associated with asthma and allergy in the GABRIELA Ulm population of 1,238 children living in rural areas, including rare mutations, polymorphisms and a lack of the DEFB4A. Furthermore, we found that prophylactic oral administration of hBD-2 significantly curbed lung resistance and pulmonary inflammation in our HDM mouse model. These data indicate that inadequate genetic capacity for hBD-2 is associated with increased asthma and allergy risk while adequate and early hBD-2 administration (in a mouse model) prevents atopic asthma. This suggests that hBD-2 could be involved in the protective farm effect and may be an excellent candidate to confer protection against asthma development.

Keywords: asthma; atopy; defensin; hBD-2; prevention.

Figure 1

DEFB4 locus on chromosome 8p23.1 with surrounding genes and pseudogenes (A) and location of the 40 selected SNPs for genotyping in GABRIELA Ulm (B). (A) Red dots: A-copies of the β-defensin genes; orange dots: B-copies of the β-defensin genes; red and orange dots with black circle: DEFB4 genes. Blue dots: unknown genes; yellow dots: FAM90A-pseudogenes; purple dots: PRR23-genes and OR7E-pseudogenes; brown dots: SPAG11-genes; green dots: HSPD1-pseudogenes; grey dots: ZNF705-genes. The hatched rectangle in the middle represents a not yet sequenced 50 Kb area, which presents like a mirror in this cluster. (B) positions of the selected SNPs for genotyping are shown as black vertical lines with numbers in relation to the position of DEFB4A and DEFB4B. Circled numbers represent SNPs that were successfully genotyped. Black boxes refer to exonic regions, white boxes to UTRs, hatched boxes to putative promoter regions.

Figure 2

Presence of DEFB4A in healthy (n=78) and diseased (n=122) subjects after RFLP assay for DEFB4A and DEFB4B. The diseased group include 41 non-atopic asthmatics, 41 atopic asthmatics and 40 atopics. *p=0.012 (chi-square test).

Figure 3

Prophylactic administration of hBD-2 in BALB/c mice and grouping according to sensitization and treatment. Challenge was carried out 1 h after the last dose of saline or vehicle on day 14. AHR, airway hyper-reactivity; BALF, bronchoalveolar lavage fluid; CFA, Complete Freund’s Adjuvant; hBD-2, human beta-defensin 2; HDM, house dust mite; i.n., intra-nasal; p.o., per oral; s.c., subcutaneous.

Figure 4

Lung resistance (A) and compliance (B) in 6 mice per group 48 h after saline or HDM challenge. Saline: Mice without treatment and challenged with saline only. Vehicle/HDM: Mice prophylactically treated with vehicle and challenged with HDM. hBD-2/HDM: Mice prophylactically treated with hBD-2 and challenged with HDM. *significant difference (ANOVA with post-hoc t-test; p<0.05) between vehicle treated and hBD-2–treated mice; ^#significant difference (ANOVA with post-hoc t-test; p<0.05) between saline-challenged and HDM-challenged mice. hBD-2, human beta-defensin 2; HDM, house dust mite.

Figure 5

Total and differential cell counts (eosinophils, neutrophils, macrophages, and lymphocytes) in bronchoalveolar lavage fluid (A) and concentration of inflammatory cytokines (TNFα, IL-4, IL-5, IL-6, IL-9, IL-13, and IL-33) in lung homogenates of the 3 treatment groups (n=12 per group) (B). Saline: mice sensitized with saline + CFA only (group 1). vehicle/HDM: mice sensitized with HDM at day 0, prophylactic treatment with vehicle at days 12 to 14, and challenge with HDM at day 14 (group 2). hBD-2/HDM: mice sensitized with HDM at day 0, prophylactic treatment with hBD-2 at days 12 to 14, and challenge with HDM at day 14 (group 3). *p < 0.05 when compared to other groups (ANOVA with post-hoc t-test,). hBD-2, human beta-defensin 2; HDM, house dust mite.