Effect of Rabbit Epididymal Antimicrobial Peptide, REHbβP, on LPS-Induced Proinflammatory Cytokine Responses in Human Vaginal Cells In Vitro.

K V R Reddy,C Selvaakumar,M S Patgaonkar,D Sukanya

doi:10.1155/2012/782019

Abstract

Antimicrobial peptides (AMP's) protect epithelial surfaces including epididymis against pathogens and play a key role in orchestrating various defensive responses. Recently, we have identified one such AMP, rabbit epididymal hemoglobin-β subuit (REHbβP) from the epididymal fluid of rabbit, Oryctologus cuniculus. The demonstration of a protective role of REHbβP in epididymal epithelial cells (EPEC's) led us to investigate: (1) the identification of LPS interactive domain in REHbβP, and (2) whether the REHbβP of rabbit origin mediates vaginal cellular immune responses of another species (human). HeLa-S3, human vaginal epithelial cells (hVECs) were exposed to LPS or the LPS-stimulated cells treated with REHbβP or neutral peptide, nREHbβP. Effect of LPS and cytokines (IL-6 and IL-1α) and chemokines (IL-8, MCP-1) levels was determined in the culture supernatants. In response to the LPS, hVECs synthesized these mediators and the levels were significantly higher than controls. This enhancing effect was ameliorated when the LPS-induced hVECs were treated with REHbβP. Similar results were obtained on NF-κB protein and hBD-1 mRNA expression. Confocal microscopy studies revealed that REHbβP attenuated the LPS-induced internalization of E. coli by macrophages. The chemotaxis studies performed using Boyden chamber Transwell assay, which showed elevated migration of U937 cells when the supernatants of LPS-induced hVECs were used, and the effect was inhibited by REHbβP. REHbβP was found to be localized on the acrosome of rabbit spermatozoa, suggesting its role in sperm protection beside sperm function. In conclusion, REHbβP may have the potential to develop as a therapeutic agent for reproductive tract infections (RTI's).

Highlights

A number pathogens can infect both male and female reproductive tracts in humans and animals [1]
The epididymis is anatomically connected to the urethra, so it is always at risk of ascending microbial invasion
We have demonstrated that LPS with concentrations of 10 μg/mL did not inhibit the viability human endocervical epithelial cells (End1/E6E7) [27]; this dose was selected for this study

Summary

Introduction

A number pathogens can infect both male and female reproductive tracts in humans and animals [1]. In a large proportion of infections, products such as lipopolysaccharide (LPS) and endotoxins are responsible. Identifying molecules that bind to LPS and neutralize its activity has clinical applications [3, 4]. The epididymis is anatomically connected to the urethra, so it is always at risk of ascending microbial invasion. It has been reported that in men the penile urethra is the entry for various STI-causing pathogens such as Neisseria gonorrhoeae and Chlamydia trachomatis, and urethritis is the most common clinical syndrome [5]. Infection originating from retrograde ascent of pathogens via the ejaculatory ducts, vas deferens, or the blood vessels supplying the epididymis is a common cause of acute epididymitis. Epididymitis is the most common intrascrotal inflammation and is a significant cause of urological consults [5, 6]

Objectives

Methods

Results

Conclusion