Matrix Metalloproteinase-13 as a Target for Suppressing Corneal Ulceration Caused by Pseudomonas aeruginosa Infection

Bacterial keratitis is a sight-threatening disease that is most commonly associated with extended contact lens wear [1]. Pseudomonas aeruginosa is the common causative agent of keratitis [2]. To cause keratitis, microbes must traverse the epithelial layer and cross the basement membrane to reach the stroma [3, 4]. Pseudomonas aeruginosa keratitis usually presents as a rapidly progressing condition, with distinct inflammatory epithelial edema and stromal ulceration, that can lead to significant stromal tissue destruction and loss of vision [5]. These consequences are largely caused by the host's inflammatory responses, although bacterial toxins and exoproducts may contribute to P. aeruginosa keratitis [5]. Hence, it is of importance to identify pathogenic factors and to delineate their mechanisms of action, which may lead to the development of new therapeutic strategies for treating bacterial keratitis. Innate immunity is the first line of defense against a wide range of pathogens at mucosal surfaces. Studies from our laboratory demonstrated that the activation of Toll-like receptor 5 (TLR5) with flagellin prior to pathogen inoculation significantly improves the clinical outcomes of microbial keratitis in C57BL/6 mouse corneas [6, 7]. This protection is related to its ability to enhance innate responses, as manifested by dampened proinflammatory cytokine expression and augmented antimicrobial peptide production in response to infection, particularly in corneal epithelial cells [7–9]. To comprehensively identify genes altered by flagellin pretreatments, we recently performed a whole genome complementary DNA microarray and found that matrix metalloproteinase-13 (MMP13) is one of the most profoundly affected genes at 6 hours after infection, and flagellin-pretreatment significantly dampens infection-induced MMP13 expression. Hence, a decrease in MMP13 activity might contribute to flagellin-induced protection against P. aeruginosa keratitis [10, 11]. MMP13 belongs to a large family of zinc-dependent neutral endopeptidases that are collectively capable of degrading extracellular matrix. MMP13 is one of the collagenases that degrade native collagen fibrils in vivo and is thought to execute the rate-limiting function in extracellular matrix reorganization, which is essential for morphogenesis and tissue remodeling [12] and corneal wound healing [13]. While all collagenases cleave type I, II, and III collagens, MMP13 (collagenase-3) cleaves collagen type IV, X, and XIV, as well [12, 14, 15]. Because of its exceptionally wide substrate specificity, MMP13 expression is limited to physiological situations in which rapid and effective remodeling of collagenous extracellular matrix is required [16, 17]. In the cornea, MMP13 has been shown to be expressed only at the basal layer of healing corneal epithelium [13, 18], suggesting its potential involvement in remodeling the underlying basement membrane. Moreover, overexpression and/or activation of MMP13 has been linked to the excessive degradation of extracellular matrix in osteoarthritic cartilage, rheumatoid synovium, chronic cutaneous ulcers, intestinal ulcerations, and chronic periodontitis [19–23]. As such, there are many MMP13-specific inhibitors designed to treat osteoarthritis and rheumatoid arthritis without the side effects often associated with many nonselective MMP inhibitors [24, 25]. These inhibitors may also have the potential to be used for ameliorating infection-induced tissue damage and ulceration. In this study, we investigated MMP13 expression in C57BL/6 mouse corneas in response to P. aeruginosa infection and demonstrated that its inhibition decreased the severity of keratitis and significantly reduced the bacterial burden. We also used an MMP13 inhibitor (MMP13i) as an adjunctive therapy to antibiotics used to treat P. aeruginosa keratitis. Our data suggest that increased MMP13 activity contributes to basement membrane breaching and P. aeruginosa stromal invasion and that MMP13i might be used as an adjuvant therapy to reduce basement membrane damage and the stromal destruction associated with microbial keratitis.