Bacterial keratitis remains a major cause of sight-limiting scarring and visual impairment, especially in extended-wear contact lens users,
45 46 47 despite the efficacy of broad spectrum antibacterial agents. Over 30 million people use contact lenses in the United States alone,
48 and 1 in 2500 daily wear contact lens users and 1 in 500 extended-wear contact lens users develop bacterial keratitis each year.
47 Traditionally, broad spectrum antibiotics (often ciprofloxacin) therapy is promptly instituted in keratitis cases, before obtaining results from culture specimens to identify a causative organism. Although antimicrobial treatment is often able to render a sterile cornea, it does not guarantee a clear visual axis, due to residual host-derived inflammation. The latter often may necessitate the use of corticosteroids to restore corneal clarity. In some cases, use of corticosteroids may have potential adverse effects, including delayed corneal wound healing.
49 50
In this study we tested and found that use of an ICE inhibitor only could reduce corneal disease when compared with placebo treatment. Although use of the ICE inhibitor alone did not fully stop inflammation or reduce its levels as much as ciprofloxacin, ICE inhibitor versus placebo-treated eyes did not perforate. In this regard, treatment with the ICE inhibitor was most efficacious in adjunctive therapy to complement the bacterial killing effects of ciprofloxacin and, together with the antibiotic, synergistically appeared to downregulate the host inflammatory response better than use of either of the agents alone. Treatment with the ICE inhibitor, VRT-043198, also contributed, indirectly, to lessening bacterial growth (as it had no ability to kill bacteria), perhaps because bacteria are not able to disseminate in a cornea in which damage is reduced. In addition, there may be other as yet untested mechanisms that contribute to bacterial clearance, for example, in ICE
−/− mice that endogenously lack caspase-1 versus wild-type mice, enhanced corneal epithelial cell apoptosis has been reported
51 . This too could serve to contain the bacterial infection and prevent stromal damage. In addition, ICE inhibitor (as well as ciprofloxacin) treatment reduced levels of IL-1β that may in turn decrease inducible nitric oxide synthase (iNOS).
52 This effect could then lead to a decrease in sustained nitric oxide (nitrite) production which is potentially toxic, dependent on its concentration and the microenvironment in which it is produced.
53 ICE inhibitor-treated mice showed significantly reduced levels of not only IL-1β, but MIP-2 (chemoattractants for PMN); reduced PMN infiltration and bacterial load compared to the placebo-treated group. Ciprofloxacin treatment significantly reduced levels of these cytokines with even greater effect, but most striking was the significant reduction of both cytokine levels and PMN number when the combination therapy of ICE inhibitor and ciprofloxacin was used. In addition, histopathological examination of the ICE inhibitor-treated group showed markedly reduced infiltrating cells with intact corneal epithelium, whereas all the corneas of the placebo-treated mice had perforated. Addition of the ICE inhibitor with topical antibiotic (ciprofloxacin) produced further improvement of corneal disease outcome, most evidenced by the reduction in inflammatory infiltrate in the anterior chamber and associated with the corneal endothelium (compare
Figs. 3C and 3D ).
Since the ICE inhibitor reduced the inflammatory infiltrate and prevented perforation (when compared with placebo) after bacterial corneal infection using a P. aeruginosa ATCC strain 19660, the next logical step was to begin to test at least one more P. aeruginosa strain, preferably a clinical isolate. Our results demonstrate that the ICE inhibitor versus placebo treatment lessens disease (no perforation) not only against a standard ATCC laboratory strain (19660), but also against a clinical isolate (KEI-1025). Obviously, it would be advisable to test additional clinical isolates as treatment outcome may differ, dependent on the strain of bacteria.
To further test the ICE inhibitor, a ciprofloxacin-resistant strain (19660CR) derived from the parent 19660 strain was produced and tested. The strain appeared normal in growth characteristics, and antibiotic resistance was maintained at least through three passages (unpublished results). Initial testing showed that clinical scores were significantly reduced in the ICE inhibitor versus placebo-treated corneas after infection with strain 19660CR. However, virulence of this mutant was decreased compared to the parent strain during in vitro generation. While this is not unusual and has been reported previously,
39 use of a more virulent ciprofloxacin-resistant strain (clinical isolate) and rigorous study of cytokine and chemokine levels will be needed to confirm our initial observations. Various studies
20 21 22 have shown a link between in vitro antibiotic resistance and clinical failure to respond to antibiotic in keratitis patients. Garg et al.
21 reported that of 141 culture-proven cases of
Pseudomonas keratitis, 22 cases were caused by isolates resistant to ciprofloxacin (mean MIC 43 mg/mL). Of the 19 (of 22) cases treated initially with ciprofloxacin, 15 (76.7%) worsened or showed no clinical improvement after 3 days of intensive therapy and required modification of antibiotic therapy, corneal grafting, or evisceration. Increasing incidence of antibiotic resistance of
Pseudomonas and failure to respond to antibacterial therapy leading to adverse outcomes provide strong reasons to search for new therapeutic strategies. An ICE inhibitor could be a novel therapeutic strategy for antibiotic-resistant
Pseudomonas keratitis cases.
Caspase-1 (ICE) also is required for processing of IL-18, as well as IL-1.
54 The former cytokine has been shown to be protective (via induction and tight regulation of IFN-γ) in a model of bacterial keratitis in the BALB/c mouse (whose cornea heals after similar bacterial infection).
12 55 Levels of IL-18 have not been determined in the B6 mouse cornea, but after infection, sustained levels of IL-12 (never detected in the BALB/c mouse cornea) are detected in the B6 mouse and it is IL-12 that regulates sustained IFN-γ production, contributing to corneal perforation in this model.
43 55 Because the IL-18 receptor and IL-1 receptor are very similar and signal through closely related, if not identical, pathways,
54 future experiments may be required to determine whether treatment with an ICE-inhibitor interferes with IL-18 induction of IFN-γ and how this is affected by combined treatment with the ICE inhibitor and ciprofloxacin.
In summary, these findings demonstrate that ICE inhibitor therapy can reduce P. aeruginosa-induced keratitis when compared with placebo treatment, that disease does progress, but corneas do not perforate. Evidence is also provided that combination treatment using an antibiotic such as ciprofloxacin together with the ICE inhibitor provides better disease outcome than antibiotic alone.