Characterisation of antimicrobial peptides and toll-like receptors in amniotic membranes

• Main Author: Mathew, Manu
• Published: University of Nottingham 2016
• Subjects: 612
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.716469

PURPOSE- Antimicrobial peptides (AMPs) are cationic host defence peptides with microbicidal properties providing a "chemical barrier" in response to pathogens and various pathological states. They show promise as potential therapeutic agents. Amniotic membrane (AM) transplantation is indicated in ocular surface surgery procedure. However, little is known of the AMP profile and variability within the AM. In addition, the effects of processing, storage, and preoperative preparation on AMPs in the transplant ready amniotic membrane (TRAM) are not clear. This can alter the therapeutic potential of AM used for such procedures. In the present study, we first profiled the expression of AMPs on the amniotic membrane in healthy women delivering by caesarean section. This was followed by protein analysis of one a potent AMP namely HBD-3 in fresh AM versus TRAM. Methods- Amniotic membrane samples of healthy women delivering by elective caesarian section were studied. Conventional PCR (semi-quantitative RT- PCR) of 21 AMPs was performed. Subsequently, quantitative real-time polymerase chain reactions (QRT-PCR), confirmed the relative gene expression of eight AMPs, by RT- PCR in these healthy patients. Further, the AMPs were confirmed to be primarily localized to the epithelium by immunohistochemistry. Western blotting in fresh AM with spongy layer, AM alone and TRAM and antimicrobial activity of TRAM was also performed. Results- RT- PCR showed expression of 8AMPs namely HBD-1, HBD-2, HBD- 3, LL37, Leap-1, Leap-2 DEFB109 and RNase7 in variable patterns. QRT-PCR confirmed expression of HBD-3 (mean 1.11; +/- 0.05, SEM 0.02), LEAP-2 (mean 1.20; +/- 0.02, SEM 0.01), and DEFB109 (mean 1.07; +/- 0.06, SEM 0.03), in all five samples with relatively little variation in the normalized expression of these three genes. HBD-1, HBD-2 and RNase7 were also expressed but in fewer (2 /5, 4/5, 3/5) samples respectively. LL37 and Leap-1 were detected in only one sample (AM 51) and confirmed by QRT- PCR in another 10 fresh samples. All 10 TLRs were noted to be expressed by RT- PCR. QRT-PCR confirmed expression of TLR-1 (mean 1.32; +/- 0.03, SEM 0.01) and TLR-3 (mean 1.21; +/- 0.07, SEM 0.03) in all five samples. While TLR-6 (mean 1.16; +/- 0.03, SEM 0.01) and TLR-7 (mean 1.31; +/- 0.09, SEM 0.05) were present in most (4/5) samples, TLR 2 (mean 1.29; +/- 0.02, SEM 0.06),TLR-4 (mean 1.42; +/- 0.05, SEM 0.02), TLR-5 (mean 1.22; +/- 0.1, SEM 0.01) and TLR-10 (mean 1.19; +/- 0.03, SEM 0.07) were noted to be expressed in only 3/5 samples. While TLR 8 (mean 1.19; +/- 0.07, SEM 0.1) was noted in two samples, TLR 9 was expressed in only one sample. Western blotting was unable to detect the presence of these small peptides. Immunohistochemistry confirmed the presence of these 8 peptides in fresh and TRAM AM epithelium but not in spongy layer. Both fresh and TRAM were not noted to have any significant antimicrobial properties. Conclusion- In this study, we profiled the AMP expression on the AM surface. We detected the expression of novel AMPs, Leap-2 and DEFB109 constitutively in all AM samples, along with confirming the constitutive expression of HBD-3. We also found a variable expression of RNase7, 6-defensins HBD-1, HBD-2, and AMPs LL-37 and LEAP-1 on the AM surface. We found that, while AMPs are localized in the epithelium they are absent in compact and spongy layer of the amniotic membrane. There are inter-donor variations but no significant intra-donor variations within regions. While AMP genes are expressed by the AM surface, they may not reach therapeutic potential as noted by the absence of the antimicrobial properties of TRAM by both MIC and MBC. The expression of these small but important peptides (potentially all other soluble proteins at the AM surface) may be altered by current processing techniques. Leap-2 and DEFB109 alongside HBD-3 may have a role in developing the innate immune response in the foetus.