Increased levels of human beta-defensins mRNA in sexually HIV-1 exposed but uninfected individuals

Abstract

Protection against HIV-1 infection in exposed seronegative (ESN) individuals likely involves natural resistance mechanisms that have not been fully elucidated. Human beta defensins (HBD) are antimicrobial peptides found primarily in mucosae, the main ports of HIV entry. HBD-2 and 3 mRNA are induced by HIV-1 in human oral epithelial cells and exhibit strong anti-HIV-1 activity; in addition, polymorphisms in the DEFB1 gene, which encodes HBD-1, have been associated with resistance/susceptibility to different infections, including HIV-1. Here, we have assessed the association of HBD expression with the ESN phenotype. Peripheral blood and vaginal/endocervical and oral mucosal samples were taken from 47 ESN, 44 seropositive (SP) and 39 healthy controls (HC). HBD-1, 2 and 3 mRNA copy numbers were quantified by real time RT-PCR and A692G/G1654A/A1836G polymorphisms in the DEFB1 gene were detected by restriction fragment length polymorphisms and confirmed by nucleotide sequencing. ESN expressed significantly greater mRNA copy numbers of HBD-2 and 3 in oral mucosa than HC; p=0.0002 and p=0.007, respectively. mRNA copy numbers of HBD-1, 2 and 3 in vaginal/endocervical mucosa from ESN and HC were similar. Homozygosity for the A692G polymorphism was significantly more frequent in ESN (0.39) than in SP (0.05) (p=0.0002). In summary, ESN exhibited enhanced mucosal expression of the innate defense genes HBD-2 and 3; however, additional studies are required to verify these results and the potential association of the A692G polymorphism to the relative resistance of ESN to HIV-1 infection.

Fig. (1). Copy numbers of HBD mRNAs in vaginal and endocervical mucosa from HC, ESN and SP individuals

Standard curves were constructed using plasmids diluted (10¹-10⁹) containing segments of the HBD-1, 2, 3 and CK19 genes. The corresponding copy numbers were calculated using the equation: 1 μg of 1000-bp DNA × 9.1 × 10¹¹ molecules [50]. Standard curves were generated using the relationship of known number of input templates to the cycle threshold. The real time PCR generated a different cycle threshold (CT) for each dilution; the CT was defined as the PCR cycle number at which the mean fluorescence increased 10 SD above baseline. CT is inversely proportional to the log of the input copy equivalent. The number of HBD mRNA copies was expressed by each 1000 CK19 mRNA copies observed in vaginal and endocervical mucosa. (A) Number of HBD-1 mRNA copies. (B and C) Copy numbers of mRNA of HBD-2 and 3 respectively.

Fig. (2). Copy numbers of HBDs mRNA in oral mucosa from HC, ESN and SP individuals

(A) Number of HBD-2 mRNA copies per 1000 CK19 mRNA copies observed in oral mucosa. (B) Copy numbers of HBD-3 mRNA.