Satya S. Kolar

Role of host-defence peptides in eye diseases

The eye and its associated tissues including the lacrimal system and lids have evolved several defence mechanisms to prevent microbial invasion. Included among this armory are several host-defence peptides. These multifunctional molecules are being studied not only for their endogenous antimicrobial properties but also for their potential therapeutic effects. Here the current knowledge of host-defence peptide expression in the eye will be summarised. The role of these peptides in eye disease will be discussed with the primary focus being on infectious keratitis, inflammatory conditions including dry eye and wound healing. Finally the potential of using host-defence peptides and their mimetics/derivatives for the treatment and prevention of eye diseases is addressed.

Protective Role of Murine β-Defensins 3 and 4 and Cathelin-Related Antimicrobial Peptide in Fusarium solani Keratitis

ABSTRACT Antimicrobial peptides (AMPs), such as β-defensins and cathelicidins, are essential components of innate and adaptive immunity owing to their extensive multifunctional activities. However, their role in fungal infection in vivo remains elusive. In this study, we investigated the protective effect of murine β-defensin 3 (mBD3), mBD4, and the cathelicidin cathelin-related antimicrobial peptide (CRAMP) in a murine model of Fusarium solani keratitis. C57BL/6 mice showed significant corneal disease 1 and 3 days after infection, which was accompanied by enhanced expression of β-defensins and CRAMP. Disease severity was significantly improved 7 days after infection, at which time AMP expression was returning to baseline. Mice deficient in mBD3 (genetic knockout), mBD4 (short interfering RNA knockdown), or CRAMP (genetic knockout) exhibited enhanced disease severity and progression, increased neutrophil recruitment, and delayed pathogen elimination compared to controls. Taken together, these data suggest a vital role for AMPs in defense against F. solani keratitis, a potentially blinding corneal disease.

Contact lens care solution killing efficacy against Acanthamoeba castellanii by in vitro testing and live-imaging

In the past decade there has been an increased incidence of Acanthamoeba keratitis, particularly in contact lens wearers. The aim of this study was to utilize in vitro killing assays and to establish a novel, time-lapse, live-cell imaging methodology to demonstrate the efficacy of contact lens care solutions in eradicating Acanthamoeba castellanii (A. castellanii) trophozoites and cysts. Standard qualitative and quantitative in vitro assays were performed along with novel time-lapse imaging coupled with fluorescent dye staining that signals cell death. Quantitative data obtained demonstrated that 3% non-ophthalmic hydrogen peroxide demonstrated the highest percent killing at 87.4% corresponding to a 4.4 log kill. The other contact lens care solutions which showed a 72.9 to 29.2% killing which was consistent with 4.3-2.8 log reduction in trophozoite viability. Both analytical approaches revealed that polyquaternium/PHMB-based was the least efficacious in terms of trophicidal activity. The cysticidal activity of the solutions was much less than activity against trophozoites and frequently was not detected. Live-imaging provided a novel visual endpoint for characterizing the trophocidal activity of the care solutions. All solutions caused rapid rounding or pseudocyst formation of the trophozoites, reduced motility and the appearance of different morphotypes. Polyquaternium/alexidine-based and peroxide-based lens care system induced the most visible damage indicated by significant accumulation of debris from ruptured cells. Polyquaternium/PHMB-based was the least effective showing rounding of the cells but minimal death. These observations are in keeping with care solution biocides having prominent activity at the plasma membrane of Acanthamoeba.

Role of Pattern Recognition Receptors in the Modulation of Antimicrobial Peptide Expression in the Corneal Epithelial Innate Response to F. solani.

Purpose Fusarium solani (F. solani) keratitis is a potentially sight-threatening fungal infection of the cornea. Antimicrobial peptides (AMPs), such as human β-defensins (hBDs) and cathelicidins, essential components of the immune system, likely have a protective role against F. solani keratitis. We examined the role of pattern recognition receptors (PRRs), Dectin-1, and TLR2 in F. solani–induced modulation of AMP expression in vitro. Methods Human corneal epithelial cells (HCECs) were exposed to heat-inactivated F. solani or pathogen-associated molecular patterns (PAMPs) of F. solani (Zymosan or Zymosan Depleted) for 6, 12, or 24 hours following which AMP mRNA and protein levels were determined. Involvement of TLR2 and Dectin-1 was confirmed by using siRNA knock-down (TLR2 and Dectin-1) or chemical inhibitor BAY 61-3606 (Dectin-1). The functional significance of AMP upregulation was tested using culture supernatant from F. solani or PAMP-treated HCECs against F. solani in the presence of hBD2 or LL37 neutralizing antibody. Results We confirm that HCECs express Dectin-1 and TLR2. HCECs demonstrated upregulation of AMPs hBD2 and cathelicidin LL37 following exposure to heat-inactivated F. solani or PAMPs. TLR2 and Dectin-1 knockdown and BAY 61-3606 treatment decreased AMP mRNA upregulation confirming PRR involvement. The culture supernatant from F. solani or PAMP-treated HCECs showed substantial killing of F. solani and hBD2 or LL37 neutralizing antibody significantly decreased this effect implicating involvement of these AMPs. Conclusions These findings demonstrate that Dectin-1 and TLR2 have an important role in regulating F. solani-induced AMP expression in corneal epithelial cells.

Methods for In Vitro Analysis of Antimicrobial Activity and Toxicity of Anti-keratitis Peptides: Bacterial Viability in Tears, MTT, and TNF-α Release Assays

Ease of access to the cornea makes antimicrobial peptides (AMPs) ideal candidates for topical drug application. However, before bringing them to the clinic, it is fundamental to evaluate in vitro: (1) the ability of AMPs to kill bacteria in the presence of human tears, by counting the number of surviving bacteria on agar plates; (2) the potential cytotoxicity of AMPs to mammalian cells by a colorimetric method based on the production of a colored formazan crystals by metabolically active cells; and (3) the ability of AMPs to neutralize the toxic effect of the bacterial cell wall component, lipopolysaccharide (LPS), by measuring the level of the pro-inflammatory cytokine, TNF-α, released from LPS-activated macrophages, using a sandwich enzyme-linked immunosorbent assay.

Methods for In Vivo/Ex Vivo Analysis of Antimicrobial Peptides in Bacterial Keratitis: siRNA Knockdown, Colony Counts, Myeloperoxidase, Immunostaining, and RT-PCR Assays

Antimicrobial peptides (AMPs) are essential components of the innate immune response. They have direct killing ability as well as immunomodulatory functions. Here, we describe techniques to identify specific AMPs involved in the protection against microbial keratitis, a vision threatening infection of the cornea of the eye which is the most serious complication of contact lens wear. Specifically we detail the use of siRNA technology to temporarily knockdown AMP expression at the murine ocular surface in vivo and then describe ex vivo assays to determine the level of bacteria, relative number of neutrophils, and levels of cytokines, chemokines, and AMPs in infected corneas.

Designed Host Defense Peptides for the Treatment of Bacterial Keratitis

Purpose To limit corneal damage and potential loss of vision, bacterial keratitis must be treated aggressively. Innovation in antimicrobials is required due to the need for empirical treatment and the rapid emergence of bacterial resistance. Designed host defense peptides (dHDPs) are synthetic analogues of naturally occurring HDPs, which provide defense against invading pathogens. This study investigates the use of novel dHDPs for the treatment of bacterial keratitis. Methods The minimum inhibitory concentrations (MICs) were determined for dHDPs on both Gram-positive and -negative bacteria. The minimum biofilm eradication concentrations (MBEC) and in vitro time-kill assays were determined. The most active dHDP, RP444, was evaluated for propensity to induce drug resistance and therapeutic benefit in a murine Pseudomonas aeruginosa keratitis model. Results Designed HDPs were bactericidal with MICs ranging from 2 to >64 μg/mL and MBEC ranging from 6 to 750 μg/mL. In time-kill assays, dHDPs were able to rapidly reduce bacterial counts upon contact with as little as 2 μg/mL. RP444 did not induce resistance after repeated exposure of P. aeruginosa to subinhibitory concentrations. RP444 demonstrated significant efficacy in a murine model of bacterial keratitis as evidenced by a significant dose-dependent decrease in ocular clinical scores, a significantly reduced bacterial load, and substantially decreased inflammatory cell infiltrates. Conclusions Innovative dHDPs demonstrated potent antimicrobial activity, possess a limited potential for development of resistance, and reduced the severity of murine P. aeruginosa keratitis. These studies demonstrate that a novel dHDP may have potential to treat patients with sight-threatening bacterial keratitis.