RESUMO
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-ß subuit (REHbßP) from the epididymal fluid of rabbit, Oryctologus cuniculus. The demonstration of a protective role of REHbßP in epididymal epithelial cells (EPEC's) led us to investigate: (1) the identification of LPS interactive domain in REHbßP, and (2) whether the REHbßP 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 REHbßP or neutral peptide, nREHbßP. 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 REHbßP. Similar results were obtained on NF-κB protein and hBD-1 mRNA expression. Confocal microscopy studies revealed that REHbßP 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 REHbßP. REHbßP was found to be localized on the acrosome of rabbit spermatozoa, suggesting its role in sperm protection beside sperm function. In conclusion, REHbßP may have the potential to develop as a therapeutic agent for reproductive tract infections (RTI's).
RESUMO
Recently the cDNA coding for anti-lipopolysaccharide factor (ALF) has been identified from the Indian mud crab, Scylla serrata and has been named S. serrata anti-lipopolysaccharide factor (SsALF). SsALF protein sequence demonstrated the presence of two highly conserved cystine residues between which the putative lipopolysaccharide (LPS) binding domain is known to be located. In this study, we have designed and synthesized a 24 amino acid linear (lSsALF24) and a cyclic (cSsALF24) peptides based on this putative LPS binding domain and demonstrated the ability of these peptides to bind to LPS. The peptides were active against vaginal pathogens demonstrated by MIC, CFU and phagocytosis assays. cSsALF24 did not show toxicity to human vaginal epithelial cells (HeLa-S3), macrophages and rabbit erythrocytes even at high concentration (64.64 µM). Flow cytometry results demonstrated that cSsALF24 peptide suppressed LPS induced phagocytosis of FITC labeled E. coli. HeLa cells were stimulated with LPS (10 µg/ml) alone for 6 h or after two washings with PBS, treated for 1 h with cSsALF24 (64.64 µM). After washing, the cells were cultured for 24 h in fresh media. The spent media as well as cells were collected for the determination of cytokine/chemokine levels such as interleukin-6 (IL-6) interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1) and interleukin-1α (IL-1α) using ELISA and RT-PCR respectively. Similar results were obtained with LPS stimulated cells treated with c/nSsALF24 or unstimulated cells treated with c/nSsALF24. The expression of cytokine/chemokines and mRNA's coding these proteins were unaffected in c/nSsALF24 treated cells. In contrast, in LPS stimulated cells, the expression levels of these molecules were up-regulated via the induction of nuclear factor kappa-B (NF-kB) levels. However, the expression of these pro-inflammatory markers was decreased in LPS stimulated cells following the treatment with cSsALF24, attributing anti-inflammatory potential of the peptide. Collectively, these findings suggest that cSsALF24 might regulate the vaginal epithelial cell immune responses indirectly through modulation of LPS-TLR4 binding in NF-kB pathway.
