Association of a genetic polymorphism (-44 C/G SNP) in the human DEFB1 gene with expression and inducibility of multiple β-defensins in gingival keratinocytes

BACKGROUND: Human β-defensins (hBDs) are antimicrobial peptides with a role in innate immune defense. Our laboratory previously showed that a single nucleotide polymorphism (SNP) in the 5' untranslated region of the hBD1 gene (DEFB1), denoted -44 (rs1800972), is correlated with protection from oral Candida. Because this SNP alters the putative mRNA structure, we hypothesized that it alters hBD1 expression. METHODS: Transfection of reporter constructs and evaluation of antimicrobial activity and mRNA expression levels in keratinocytes from multiple donors were used to evaluate the effect of this SNP on constitutive and induced levels of expression. RESULTS: Transfection of CAT reporter constructs containing the 5' untranslated region showed that the -44 G allele yielded a 2-fold increase in CAT protein compared to other common haplotypes suggesting a cis effect on transcription or translation. The constitutive hBD1 mRNA level in human oral keratinocytes was significantly greater in cells from donors with the -44 GG genotype compared to those with the common CC genotype. Surprisingly, the hBD3 mRNA level as well as antimicrobial activity of keratinocyte extracts also correlated with the -44 G allele. Induced levels of hBD1, hBD2, and hBD3 mRNA were evaluated in keratinocytes challenged with Toll-like receptor 2 and 4 ligands, interleukin-1β, TNFα, and interferon-γ (IFNγ). In contrast to constitutive expression levels, IFNγ-induced keratinocyte hBD1 and hBD3 mRNA expression was significantly greater in cells with the common CC genotype, but there was no clear correlation of genotype with hBD2 expression. CONCLUSION: The DEFB1 -44 G allele is associated with an increase in overall constitutive antimicrobial activity and expression of hBD1 and hBD3 in a manner that is consistent with protection from candidiasis, while the more common C allele is associated with IFNγ inducibility of these β-defensins and is likely to be more protective in conditions that enhance IFNγ expression such as chronic periodontitis. These results suggest a complex relationship between genetics and defensin expression that may influence periodontal health and innate immune responses

Keratinocyte Expression of Human β Defensin 2 following Bacterial Infection: Role in Cutaneous Host Defense

Human β defensin 2 (hβD-2) is thought to play an important role in cutaneous immune defense. We hypothesized that (i) keratinocyte expression of hβD-2, measured by reverse transcription-PCR, would be upregulated in response to challenge with pathogenic bacteria, particularly highly adherent strains of Streptococcus pyogenes and Staphylococcus aureus, and (ii) hβD-2 would have potent antimicrobial activity against pathogenic but not commensal organisms. Expression of hβD-2 was induced consistently by S. aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa, whereas strains of S. pyogenes were poor and variable inducers of hβD-2. No correlation was found between levels of bacterial adherence and keratinocyte expression of hβD-2. S. pyogenes was significantly more sensitive to killing by hβD-2 than S. epidermidis. We conclude that the ability to induce hβD-2 expression in combination with sensitivity to its antimicrobial effects may contribute to the rarity of skin infections with the gram-negative bacterial organisms, whereas lack of stimulation of hβD-2 expression by S. pyogenes may be important in its ability to evade innate defenses and cause skin disease. Induction of expression of hβD-2 but relative tolerance to it may enable S. epidermidis to survive on the skin surface and modulate hβD-2 expression when the stratum corneum barrier is disrupted

Association of a genetic polymorphism (-44 C/G SNP) in the human <it>DEFB1 </it>gene with expression and inducibility of multiple β-defensins in gingival keratinocytes

Abstract Background Human β-defensins (hBDs) are antimicrobial peptides with a role in innate immune defense. Our laboratory previously showed that a single nucleotide polymorphism (SNP) in the 5' untranslated region of the hBD1 gene (DEFB1), denoted -44 (rs1800972), is correlated with protection from oral Candida. Because this SNP alters the putative mRNA structure, we hypothesized that it alters hBD1 expression. Methods Transfection of reporter constructs and evaluation of antimicrobial activity and mRNA expression levels in keratinocytes from multiple donors were used to evaluate the effect of this SNP on constitutive and induced levels of expression. Results Transfection of CAT reporter constructs containing the 5' untranslated region showed that the -44 G allele yielded a 2-fold increase in CAT protein compared to other common haplotypes suggesting a cis effect on transcription or translation. The constitutive hBD1 mRNA level in human oral keratinocytes was significantly greater in cells from donors with the -44 GG genotype compared to those with the common CC genotype. Surprisingly, the hBD3 mRNA level as well as antimicrobial activity of keratinocyte extracts also correlated with the -44 G allele. Induced levels of hBD1, hBD2, and hBD3 mRNA were evaluated in keratinocytes challenged with Toll-like receptor 2 and 4 ligands, interleukin-1β, TNFα, and interferon-γ (IFNγ). In contrast to constitutive expression levels, IFNγ-induced keratinocyte hBD1 and hBD3 mRNA expression was significantly greater in cells with the common CC genotype, but there was no clear correlation of genotype with hBD2 expression. Conclusion The DEFB1 -44 G allele is associated with an increase in overall constitutive antimicrobial activity and expression of hBD1 and hBD3 in a manner that is consistent with protection from candidiasis, while the more common C allele is associated with IFNγ inducibility of these β-defensins and is likely to be more protective in conditions that enhance IFNγ expression such as chronic periodontitis. These results suggest a complex relationship between genetics and defensin expression that may influence periodontal health and innate immune responses.</p