Purchase this article with an account.
Satya Sree N Kolar, Hasna Baidouri, Alison M McDermott; PAMPs, PRRs and AMPs in Human Corneal Epithelial Innate Response to Fusarium solani. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1886.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Antimicrobial peptides (AMPs), including defensins and cathelicidins are known to have antifungal activity against Fusarium solani (FS). We investigated the effect of fungal and PAMP challenge on AMP expression and the involvement of pattern recognition receptor (PRR), TLR2 in human telomerase corneal epithelial cells (hTCEpi).
hTCEpi cells were treated with or without heat inactivated FS, 10µg/ml of fungal derived PAMPs Zymosan (Z) or Zymosan depleted (ZD) alone or in combination for 6, 12 and 24h. Supernatants and cell lysates were used to determine protein or mRNA expression of AMPs hBD2 and LL37 via immunoblotting and RT-PCR. Micro-broth dilution assays were used to determine antifungal activity of PAMP treated cell culture supernatants. To address the involvement to TLR2 in fungal stimulated AMP expression, 10nM of specific siRNA was used to knock-down TLR2. Knock-down efficiency, AMP mRNA and protein expression was determined using PCR and immunoblotting.
Exposure of hTCEpi to heat inactivated FS for 6h caused significant upregulation of hBD2 and LL37 mRNA expression by 4.5-5.6 fold relative to control (p<0.0001; n=3). LL37 and hBD2 protein expression was significantly increased by 6 and 16.5 fold at 24h in FS treated cells (n=3). Z, ZD and their combination significantly increased hBD2 and LL37 mRNA expression by 10-45 fold (p<0.001, n=3) at 6h. AMP mRNA expression was not significantly different from control at 12 or 24h with any of the treatments (p>0.1). Culture supernatant from Z and ZD treated cells had greater anti-fungal activity than that from control cells (n=2). TLR2 siRNA knock-down efficiency was over 65%. Control scrambled siRNA followed by FS challenge resulted in a significant increase in hBD2 and LL37 expression (p<0.002). However, knock-down of TLR2 followed by FS challenge demonstrated no difference in expression compared to scrambled siRNA control (p>0.05).
hTCEpi respond to fungal challenge with increased LL37 and hBD2 production which may be responsible for the anti-fungal activity of culture media from PAMP treated cells. Z (TLR2 and dectin-1 agonist) and ZD (dectin-1 agonist only) gave comparable results and knock-down of TLR2 by siRNA did not significantly modulate FS induced AMP upregulation suggesting that this PRR is not involved in mediating the AMP response to FS exposure and that Dectin-1 is more likely involved.
This PDF is available to Subscribers Only