Role of the Fas Pathway in<i>Pseudomonas aeruginosa</i>Keratitis

Abstract

PURPOSE: The role of the Fas pathway was tested in Pseudomonas aeruginosa-infected mouse cornea by contrasting the responses of FasL(-/-) and wild-type (WT) mice. METHODS: TUNEL staining, real-time RT-PCR, immunostaining, and ELISA assay were used. RESULTS: Compared with WT (resistant) mice, BALB/c FasL(-/-) exhibited significantly elevated bacterial counts and polymorphonuclear leukocyte numbers at 1 and 3 days postinfection (p.i.) and worse outcomes from disease. Similar bacterial challenges in C57BL/6 FasL(-/-) compared with WT mice also led to worsened disease as evidenced by earlier corneal perforation in the susceptible mouse strain. Intense TUNEL staining of apoptotic cells was seen earlier (1 day vs. 3 days) p.i. in BALB/c WT than in knockout mice, This earlier apoptotic pattern correlated with increased expression of caspases 3, 8, and 9 and BAX and with decreased expression of the antiapoptotic molecule Bcl-2. Furthermore, expression levels of the proinflammatory molecule TNF-alpha and its receptor, MIP-2, inducible nitric oxide synthase (iNOS), and nitrite also were significantly elevated in the infected cornea of BALB/c FasL(-/-) compared with WT mice. In vitro, LPS-stimulated Mphi from BALB/c FasL(-/-) mice expressed significantly less caspase 3 and 9, BAX, and IL-10 and more TNF-alpha, MIP-2, and IL-1beta than did cells from WT mice. CONCLUSIONS: Fas-FasL interaction in the cornea balances the host innate immune response to improve disease outcome by promoting earlier apoptosis and regulating proinflammatory cytokines/chemokines and nitric oxide (nitrite) production. Dysregulation of this interaction contributes to bystander tissue damage, enhancing nutrients for bacterial growth and worsened disease outcome after P. aeruginosa infection.