Abstract
Purpose: :
Multifunctional antimicrobial peptides (AMPs) such as defensins and cathelicidins form an integral part of the innate immune system and protect the ocular surface against infection. However, their role in defense against Fusarium solani (F. solani) keratitis remains elusive. Here we determined the role of murine β-defensins (mBD3, -4) and the cathelicidin CRAMP following F. solani challenge in-vivo.
Methods: :
Corneas of C57BL/6, and B6 background mBD3 deficient (mBD3 KO), mBD3 wild type (mBD3 WT) and CRAMP deficient (CRAMP KO) mice were infected with F. solani following a scratch injury. Disease progression was graded at day 1, 3 and 7 post-infection (PI) using a slit-lamp biomicroscope. Polymorphonuclear neutrophil (PMN) recruitment, fungal load and AMP mRNA expression by RT-PCR was determined in corneas harvested at each time point. Involvement of mBD4 was evaluated at day 3 PI by siRNA knockdown in C57BL/6 mice.
Results: :
Expression of mBD 2, -3, -4, -5, -6, -14 and CRAMP was increased (range 2.3- 6.5 log2 fold) at day 1 and 3 PI in C57BL/6 infected corneas. The mean-clinical-score (MCS) of infected C57BL/6 eyes increased significantly (p<0.05) at day 1 to 4.7 ± 0.58, peaked at day 3 to 6.2 ± 0.4 and was returning to baseline (1.2 ± 0.4, p<0.12) at day 7 PI (n=4). mBD3 KO mice showed a significantly higher MCS of 6.0 ± 0.72 and 5.9 ± 0.72 compared to 3.7 ± 0.36 and 3.5 ± 0.72 (p<0.002) for their WT counterparts at days 3 and 7 PI respectively (n=8 mice). MCS for CRAMP KO mice was significantly higher (p<0.0005) than B6 controls at days 3 and 7 PI with scores of 5.6 ± 1.2 vs 11.6 ± 0.25 and 10.9 ± 0.58 versus 2.9 ± 0.56 respectively (n=8 mice). siRNA application resulted in a 60% knockdown of mBD4. Mice treated with siRNA had a significantly higher MCS of 10.4 ± 0.5 versus 6.7 ± 1.16 (n=5 mice). PMN infiltration and fungal load data paralled the MCS data with infected KO/siRNA mice demonstrating significant increases (p<0.05) in PMN count and fungal load at days 3 and 7 PI.
Conclusions: :
F.solani induces upregulation of AMP mRNA expression. Furthermore, mBD3, -4 and CRAMP deficient mice exhibited enhanced disease severity and progression, increased PMN recruitment and delayed pathogen elimination. Taken together these data indicate that mBD3, -4 and CRAMP play a key role in innate defense against F.solani keratitis.
Keywords: fungal disease • keratitis • cornea: epithelium