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

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-? subuit (REHb?P) from the epididymal fluid of rabbit, Oryctologus cuniculus. The demonstration of a protective role of REHb?P in epididymal epithelial cells (EPEC's) led us to investigate: (1) the identification of LPS interactive domain in REHb?P, and (2) whether the REHb?P 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 REHb?P or neutral peptide, nREHb?P. Effect of LPS and cytokines (IL-6 and IL-1?) 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 REHb?P. Similar results were obtained on NF-?B protein and hBD-1 mRNA expression. Confocal microscopy studies revealed that REHb?P 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 REHb?P. REHb?P was found to be localized on the acrosome of rabbit spermatozoa, suggesting its role in sperm protection beside sperm function. In conclusion, REHb?P may have the potential to develop as a therapeutic agent for reproductive tract infections (RTI's).

SUBMITTER: Reddy KV

PROVIDER: S-EPMC3312295 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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

Reddy K V R KV Sukanya D D Patgaonkar M S MS Selvaakumar C C

International journal of peptides 20120314

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) whethe ...[more]

PMID: 22505946

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Project description:BackgroundHost-derived (LL-37) and synthetic (WLBU-2) cationic antimicrobial peptides (CAPs) are known for their membrane-active bactericidal properties. LL-37 is an important mediator for immunomodulation, while the mechanism of action of WLBU-2 remains unclear.ObjectiveTo determine if WLBU-2 induces an early proinflammatory response that facilitates bacterial clearance in cystic fibrosis (CF).MethodsC57BL6 mice were given intranasal or intraperitoneal 1×10 (6) cfu/mL Pseudomonas aeruginosa (PA) and observed for 2h, followed by instillation of LL-37 or WLBU-2 (2-4mg/kg) with subsequent tissue collection at 24h for determination of bacterial colony counts and quantitative RT-PCR measurement of cytokine transcripts. CF airway epithelial cells (IB3-1, ?F508/W1282X) were cultured in appropriate media with supplements. WLBU-2 (25?M) was added to the media with RT-PCR measurement of TNF-? and IL-1? transcripts after 20, 30, and 60min. Flow cytometry was used to determine if WLBU-2 assists in cellular uptake of Alexa 488-labeled LPS.ResultsIn murine lung exposed to intranasal or intraperitoneal WLBU-2, there was a reduction in the number of surviving PA colonies compared to controls. Murine lung exposed to intraperitoneal WLBU-2 showed fewer PA colonies compared to LL-37. After 24h WLBU-2 exposure, PA-induced IL-1? transcripts from lungs showed a twofold decrease (p<0.05), while TNF-? levels were unchanged. LL-37 did not significantly change transcript levels. In IB3-1 cells, WLBU-2 exposure resulted in increased TNF-? and IL-1? transcripts that decreased by 60min. WLBU-2 treatment of IB3-1 cells displayed increased LPS uptake, suggesting a potential role for CAPs in inducing protective proinflammatory responses. Taken together, the cytokine response, LPS uptake, and established antimicrobial activity of WLBU-2 demonstrate its ability to modulate proinflammatory signaling as a protective mechanism to clear infection.ConclusionsThe immunomodulatory properties of WLBU-2 reveal a potential mechanism of its broad-spectrum antibacterial activity and warrant further preclinical evaluation to study bacterial clearance and rescue of chronic inflammation.

Dataset Information

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

Publications

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

Similar Datasets

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