β-Defensins 2 and 3 Together Promote Resistance toPseudomonas aeruginosaKeratitis

Abstract

Defensins play an important role in both innate and adaptive immunity due to their antimicrobial, regulatory, and chemotactic effects. Nonetheless, the role of murine β-defensins (mBD) 3 and 4, the murine homologs of human β-defensins (hBD) 2 and 3, remains unknown in Pseudomonas aeruginosa keratitis. This study explored their role in corneal infection and potential synergy with mBD2, a defensin associated with better outcome in this disease. Immunostaining and real-time RT-PCR data demonstrated that mBD3 and mBD4 expression was inducible and differentially regulated in the infected cornea of resistant BALB/c vs susceptible C57BL/6 (B6) mice. Knockdown studies using small interfering RNA treatment indicated that mBD3, but not mBD4, is required in ocular defense. Moreover, in vivo studies demonstrated individual and combined effects of mBD2 and mBD3 that modulate bacterial load, polymorphonuclear neutrophil (PMN) infiltration, and production of IFN-γ, MIP-2, IL-1β, TNF-α, inducible NO synthase (iNOS), TLR2, TLR4, MyD88, and NF-κB. Most notably, bacterial load was increased at 5 days postinfection by silencing either mBD2 or mBD3, but it was elevated at both 1 and 5 days postinfection when silencing both defensins. PMN infiltration was increased at 1 day postinfection by silencing both defensins or mBD3, but not mBD2 alone. iNOS expression was elevated by silencing mBD2, but it was reduced after silencing mBD3 or both defensins. Additionally, cell sources of mBD2 (macrophages, PMN and fibroblasts) and mBD3 (PMN) in corneal stroma were identified by dual label immunostaining after infection. Collectively, the data provide evidence that mBD2 and mBD3 together promote resistance against corneal infection.

Induction of human β-defensin-2 in pulmonary epithelial cells byLegionella pneumophila: involvement of TLR2 and TLR5, p38 MAPK, JNK, NF-κB, and AP-1

“…Furthermore, we demonstrated that recombinant hBD-2 has a strong antimicrobial effect on L. pneumophila, and silencing endogenous hBD-2 in alveolar epithelium increased intracellular replication of this pathogen. Our data is supported by observations of Wu and colleagues (43) who showed, with RNAi against mBD3 (homolog to hBD-2) in mice, an increased bacterial load of Pseudomonas aeruginosa in corneal infection. Thus, hBD-2 might be essential in controlling bacteria replication in the course of infections.…”

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

“…Genotypes for Cnlp Ϫ/Ϫ , WT, and KO mBD3 mice were confirmed by standard PCR analysis done on DNA isolated from tail clips. In vivo knockdown of mBD4 was achieved using a method previously described by Wu et al using siRNA purchased from Santa Cruz Biotechnology (24). Briefly, 5 l of 8 M mBD4 siRNA or scrambled control siRNA was injected subconjunctivally into the right eye of C57BL/6 mice 1 day prior to fungal challenge.…”

“…The absorbance was measured for 90 min at 450 nm and plotted in comparison to a standard curve generated using purified MPO (Calbiochem, San Diego, CA) on the same plate (33). Results are expressed as relative units of MPO activity per cornea (1 MPO unit is proportional to 2 ϫ 10 5 infiltrating neutrophils) (23,24). HRT imaging.…”

“…Silencing of mBD4 and its effect on disease progression. As there is no mBD4 knockout mouse line available, to examine the role of this AMP in the progression of F. solani keratitis in vivo, knockdown using siRNA was performed in C57BL/6 mice as described by Wu et al (24). Relative quantitative RT-PCR and immunostaining were performed at 3 days p.i.…”

“…Recent in vivo studies in a murine model of bacterial keratitis have shown that deficiency (either by transient silencing by short interfering RNA [siRNA] or gene knockout [KO]) of mouse ␤-defensin 2 (mBD2) or mBD3 (homolog of human ␤-defensin 2) or the cathelicidin cathelin-related antimicrobial peptide (CRAMP) (homolog of human LL37) results in more severe infection and tissue damage (23)(24)(25)(26). Further, exposure to Candida albicans significantly upregulated the expression of CRAMP (27), and mice deficient in CRAMP were more susceptible to Candida keratitis than wild-type (WT) mice (28).…”