Cyclooxygenase-2 enhances antimicrobial peptide expression and killing of Staphylococcus aureus

Abstract

Antimicrobial peptides such as human β-defensins (hBDs) and cathelicidins are critical for protection against infection and can be induced by activation of TLRs, a pathway that also activates cyclooxygenase(Cox)-2 expression. We hypothesized that Cox-2 is induced by TLR activation and is necessary for optimal AMP production, and that inhibitors of Cox-2 may therefore inhibit antimicrobial action. Normal human keratinocytes (NHEKs) stimulated with a TLR2/6 ligand, macrophage-activating lipopeptide-2, or a TLR3 ligand, polyinosinic-polycytidylic acid, increased Cox-2 mRNA and protein and increased PGE(2), a product of Cox-2. Treatment with a Cox-2 selective inhibitor (SC-58125) or Cox-2 small interfering RNA attenuated hBD2 and hBD3 production in NHEKs when stimulated with macrophage-activating lipopeptide-2, polyinosinic-polycytidylic acid, or UVB (15 mJ/cm(2)), but it did not attenuate vitamin D3-induced cathelicidin. SC-58125 also inhibited TLR-dependent NF-κB activation. Conversely, treatment with Cox-derived prostanoids PGD(2) or 15-deoxy-Δ(12,14)-PGJ(2) induced hBD3 or hBD2 and hBD3, respectively. The functional significance of these observations was seen in NHEKs that showed reduced anti-staphylococcal activity when treated with a Cox-2 inhibitor. These findings demonstrate a critical role for Cox-2 in hBD production and suggest that the use of Cox-2 inhibitors may adversely influence the risk for bacterial infection.

FIGURE 1

Cox inhibitors attenuate poly(I:C)-induced β-defensins. A, NHEKs were pretreated with aspirin (100 μM), SC-58125 (10 μM), NS-398 (10 μM), or SC-560 (10 μM) for 30 min and then treated with poly(I:C) (10 μg/ml) for 24 h. Poly(I:C) induces hBD2 mRNA expression ~75-fold over untreated when normalized to GAPDH levels (^#p < 0.05). Aspirin, SC-58125, and NS-398 significantly attenuated poly(I:C)-induced hBD2 mRNA expression (*p < 0.05). SC-560 failed to attenuate poly(I:C)-induced hBD2 mRNA expression. B, Poly(I:C)-induced hBD2 protein expression was attenuated by pretreating NHEKs with SC-58125 (10 μM) as demonstrated by fluorescence microscopy (original magnification ×400): vehicle (MFI = 0.3), poly(I:C) (MFI = 13.0), SC-58125 (MFI = 0.8), SC-58125 plus poly(I:C) (MFI = 13.9). C, NHEKs were pretreated with aspirin (100 μM), SC-58125 (10 μM), NS-398 (10 μM), or SC-560 (10 μM) for 30 min and then treated with poly(I:C) (10 μg/ml) for 24 h. Poly(I:C) induced hBD3 mRNA expression ~10-fold over untreated when normalized to GAPDH levels (^#p < 0.05). Aspirin, SC-58125, and NS-398 significantly attenuated poly(I:C)-induced hBD3 mRNA expression (*p < 0.05). SC-560 failed to attenuate poly(I:C)-induced hBD3 mRNA expression. D, hBD3 protein expression was attenuated by pretreating NHEKs with SC-58125 (10 μM) as demonstrated by fluorescence microscopy (original magnification ×400): vehicle (MFI = 106), poly(I:C) (MFI = 104), SC-58125 (MFI = 34), SC-58125 plus poly(I:C) (MFI = 54). A single NHEK stained for hBD3 is highlighted by the white box in the poly(I:C) panel and amplified to demostrate hBD3 localization. Cells in B and D were stained with defensin primary Abs and an Alexa Fluor 568 secondary Ab.

FIGURE 2

Cox-2 inhibitors attenuate TLR ligand-induced β-defensins. A, NHEKs were pre-treated with SC-58125 (10 μM) and then treated with 1,25-D3 (500 nM), MALP-2 (500 ng/ml), 1,25-D3 (500 nM) plus MALP-2 (500 ng/ml), or poly(I:C) (10 μg/ml) for 24 h. MALP-2, MALP-2 plus 1,25-D3, and poly(I:C) significantly induced hBD2 (^#p < 0.05), and SC-58125 significantly attenuated this induction (*p < 0.05). B, MALP-2, MALP-2 plus 1,25-D3, and poly(I:C) significantly induced hBD3 (^#p < 0.05), and SC-58125 significantly attenuated this induction (*p < 0.05). C, NHEKs were pre-treated with SC-58125 (10 μM) or NS-398 (10 μM) and then exposed to UVB (15 mJ/cm²). hBD mRNA expression was measured after 24 h. UVB induced hBD2 mRNA ~3-fold (^#p < 0.05). Cox-2 selective inhibitors significantly attenuated UVB-induced hBD2 mRNA (*p < 0.05). D, UVB induced hBD3 mRNA ~3-fold (^#p < 0.05). Cox-2 selective inhibitors significantly attenuated UVB-induced hBD3 mRNA (*p < 0.05). E, 1,25-D3, 1,25-D3 plus SC-58125, MALP-2 plus 1,25-D3, and MALP-2 plus 1,25-D3 plus SC-58125 significantly induced cathelicidin mRNA expression (^#p < 0.05). SC-58125 failed to inhibit 1,25-D3 or 1,25-D3 plus MALP-2–induced cathelicidin mRNA expression.

FIGURE 3

Cox-2 siRNA attenuates TLR ligand-induced β-defensins. NHEKs were transfected with 3 μM Cox-2 siRNA at 0 h and then again at 24 h. Twenty-fours after the last tranfection cells were harvested for Western blotting or treated with AMP-inducing stimuli for 24 h. A, Transfection of Cox siRNA constructs knocked-down Cox-2 protein by >99%. B, Poly(I:C) significantly induced hBD2 mRNA ~30-fold (^#p < 0.05). Cox-2 siRNA significantly attenuated poly(I:C)-induced hBD2 mRNA (*p < 0.05). C, Poly(I:C) significanlty induced hBD3 mRNA ~20-fold (^#p < 0.05). Cox-2 siRNA significantly attenuated poly(I:C)-induced hBD3 mRNA (*p < 0.05). D, UVB induced hBD2 mRNA ~6-fold (^#p < 0.05). Cox-2 siRNA significantly attenuated UVB-induced hBD2 mRNA (*p < 0.05). E, UVB induced hBD3 mRNA ~3-fold (^#p < 0.05). Cox-2 siRNA significantly attenuated UVB-induced hBD3 mRNA (*p < 0.05).

FIGURE 4

TLR ligands induce Cox-2 mRNA protein and activity. A, NHEKs were treated with MALP-2 (500 ng/ml) or poly(I:C) (10 μg/ml) for 24 h. MALP-2 and poly(I:C) significantly induced Cox-2 mRNA (*p < 0.05). B, MALP-2 and poly (I:C) failed to induce Cox-1 mRNA. C, MALP-2 (500 ng/ml) plus 1,25-D3 (500 nM), MALP-2 (500 ng/ml), and poly(I:C) (10 μg/ml) induced Cox-2 protein, but not Cox-1 protein. Western blot is representative of three separate experiments. D, NHEKs were pretreated with SC-58125 (10 μM) followed by treatment with 1,25-D3 (500 nM), MALP-2 (500 ng/ml), 1,25-D3 (500 nM) plus MALP-2 (500 ng/ml), or poly(I:C) (10 μg/ml) for 24 h. Supernatants were collected and PGE₂ was assayed by EIA for a measure of Cox activity. MALP-2 (500 ng/ml) plus 1,25-D3 (500 nM) plus MALP-2 (500 ng/ml) and poly(I:C) (10 μg/ml) induced PGE₂ release (^#p < 0.05). This release is attenuated with SC-58125 treatment (*p < 0.05). SC-58125 significantly attenuated basal level PGE₂ release as shown comparing the first two bars (black) with the third bar (gray) (*p < 0.05).

FIGURE 5

Cox-2–derived PGs induce β-defensins. NHEKs were stimulated with PGE₂, PGF_2a, iloprost (a stable analog of PGI₂), U-46619 (a thromboxane A₂ receptor agonist), PGD₂, or 15d-PGJ₂ for 24 h. A, 15d-PGJ₂ induced hBD2 mRNA expression ~100-fold (*p < 0.05). B, PGD₂ and 15d-PGJ₂ induced hBD3mRNA expression ~40-fold and 300-fold (*p < 0.05). C, 15d-PGJ₂ significantly induced hBD2 mRNA between 2 and 6 h (*p < 0.05). D, 15d-PGJ₂ significantly induced hBD3 mRNA between 2 and 6 h (*p < 0.05). E, Fluorescence microscopy of hBD2 immunostaining in vehicle-treated (MFI = 0.3) and 15d-PGJ₂–treated (MFI = 157) NHEKs. Cells were stained with a defensin primary Ab and an Alexa Fluor 568 secondary Ab. Original magnification ×400. Data are representative of three separate experiments. F, Fluorescence microscopy of hBD3 immunostaining in vehicle-treated (MFI = 107) and 15d-PGJ₂–treated (MFI = 167) NHEKs. Cells were stained with a defensin primary Ab and an Alexa Fluor 568 secondary Ab. Original magnification ×400. Data are representative of three separate experiments. Arrows point to granule localization of hBD3. G, NHEKs were treated with MALP-2 (500 ng/ml) or poly(I:C) (10 μg/ml) for 24 h. Supernatants were collected and PGD₂ was assayed by EIA. MALP-2 (500 ng/ml) and poly(I:C) (10 μg/ml) induced PGD₂ release ~4-fold (*p < 0.05).

FIGURE 6

Inhibition of Cox-2 attenuates TLR ligand-induced NF-κB activation. A, Fluorescence microscopy shows RelA/ p65 localization. NHEKs were pretreated with SC-58125 and then treated with MALP-2 or poly(I:C) for 60 min. SC-58125 attenuated RelA/p65 nuclear localization. Cells were stained with a p65 primary Ab and an Alexa Fluor 568 secondary Ab. Orignal magnification ×400. Results are representative of three separate experiments. B, Western blot of nuclear lysates of NHEKs pretreated with SC58125 for 30 min followed by treatment with MALP-2 or poly (I:C) for 60 min. MALP-2– and poly(I:C)-induced nuclear translocation of RelA/p65 was attenuated with SC58125. Lamin B1 was used as a nuclear lysate loading control. C, NHEKs were pretreated with BAY (1 μM) for 30 min followed by treatment with 15d-PGJ₂. 15d-PGJ₂ dose-dependently induced hBD2 mRNA (^#p < 0.05). BAY significantly attenuated 15d-PGJ₂–induced hBD2 mRNA (*p < 0.05). D, NHEKs were pretreated with BAY (1 μM) for 30 min followed by treatment with 15d-PGJ₂. 15d-PGJ₂ dose-dependently induced hBD3 mRNA (^#p < 0.05). BAY significantly attenuated 15d-PGJ₂–induced hBD3 mRNA (*p <0.05). E, NHEKs were pretreated with CAY (1 μM) for 30 min followed by treatment with 15d-PGJ₂. 15d-PGJ₂ dose-dependently induced hBD2 mRNA (^#p < 0.05). CAY significantly attenuated 15d-PGJ₂–induced hBD2 mRNA (*p < 0.05). F, NHEKs were pretreated with CAY (1 μM) for 30 min followed by treatment with 15d-PGJ₂. 15d-PGJ₂ dose-dependently induced hBD3 mRNA (^#p < 0.05). CAY significantly attenuated 15d-PGJ₂–induced hBD3 mRNA (*p < 0.05).

FIGURE 7

Cox-2 enhances S. aureus killing. NHEK lysates were incubated with 10⁶ bacteria at an MOI of 20 for 1, 3, or 5 h. Bacteria was quantified after 24 h. NHEKs pretreated with SC-58125 (10 μM) and then treated with poly(I:C) (10 μg/ml) showed a reduced ability to kill S. aureus strains (A) ΔmprF and (B) Sa113 when compared with NHEKs treated with poly(I:C) in the absence of SC-58125. NHEK lysates were incubated with 10⁷ bacteria at an MOI of 20 for 0.5, 1, or 3 h. Bacteria was quantified after 24 h. NHEKs treated with 15d-PGJ₂ (500 nM) showed a reduced ability to kill S. aureus strains (C) ΔmprF and (D) Sa113 when compared with vehicle-treated NHEKs. Results are representative of three separate experiments.