Access Type

Open Access Dissertation

Date of Award

1-1-2010

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Anatomy and Cell Biology

First Advisor

Linda D. Hazlett

Abstract

Corneal infection with P. aeruginosa results in corneal perforation in susceptible

B6, but not resistant BALB/c mice. This study explored their role mBD 1-4 in corneal

infection, and their potential synergy. Immunostaining and real-time RT-PCR data

demonstrated that their expression was either constitutive (mBD1 and mBD2) or

inducible (mBD3 and mBD4) in normal BALB/c and B6 corneas, and disparately

regulated in BALB/c vs B6 corneas after infection. Knock down studies using siRNA

treatment indicated that mBD2 and mBD3, but neither mBD1 nor mBD4, is required in

ocular defense. Moreover, in vivo studies demonstrated individual and combined effects

of mBD2 and mBD3 that modulate bacterial load, PMN infiltration, and production of

pro-inflammatory molecules (e.g., IFN-gamma, MIP-2, IL-1beta, TNF-alpha), iNOS, as well as TLR signaling molecules (e.g., TLR2, TLR4, MyD88) and transcription factor NF-kappaB. Most

notably, bacterial load was increased at 5 days p.i. by silencing either mBD2 or mBD3,

but was elevated at both 1 and 5 days p.i. when silencing both defensins. PMN

infiltration was increased at 1 day p.i. by silencing both defensins or mBD3, but not

mBD2 alone. iNOS expression was elevated by silencing mBD2, but reduced after

silencing mBD3 or both defensins. Additionally, cell sources of mBD2 and mBD3 in

corneal stroma were identified by dual label immunostaining after infection: PMN

produce both defensins, whereas macrophages and fibroblasts produce mBD2 but not mBD3.

Collectively, the data provide evidence that mBD2 and mBD3 together promote

resistance against corneal infection. The conclusions may be relevant to potential

treatment of other ocular diseases, in addition to P. aeruginosa keratitis.

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