OPN impairs the bactericidal activity of AMPs.

<p>(A) Interference of OPN with the bactericidal activity of AMPs was investigated using viable counts with <i>S</i>. <i>pneumoniae</i> and <i>P</i>. <i>aeruginosa</i>. Bacteria were grown to mid-logarithmic phase and incubated with AMPs alone (3 μM) or AMPs pre-incubated with OPN at ratio of 1:1 for one hour at 37°C. OPN caused inhibition of the bactericidal activity of all AMPs investigated except for LL-37. The histograms represent mean and standard deviation from three separate experiments. Two-way ANOVA with Sidak’s multiple comparisons test was used for statistical analysis. *<i>P</i>≤0.05, and ****<i>P</i> ≤ 0.0001. (B) Scanning electron microscopy images shows the morphology of bacteria after incubation with AMPs alone or AMPs pre-incubated with OPN (1:1). The AMPs alone permeabilized the membrane and caused leakage of intracellular contents indicating killing of the bacteria, which was confirmed in a parallel viable counts assay. Upon co-incubation of AMPs and OPN, the bacteria remained intact, except in the case of LL-37 and to some extent in the cases of <i>P</i>. <i>aeruginosa</i> (OPN with lactoferrin, lysozyme, and midkine). The scale bars in bottom figures in the right panels are 5 μm.</p

OPN expression and co-localization with antimicrobial proteins in COPD lung tissues.

<p>(A) Multiple cells in the bronchiolar epithelium expressed OPN (brown-colored DAB). (I) Goblet cells, the inset shows OPN expression in the goblet cell mucus. (II) Bronchial submucosal glands (Br = bronchial epithelium. (III) OPN expression in some basal cells (arrowhead). (IV) Flask-shaped cells (arrowhead). Primary antibodies from non-immunized animals resulted in loss of labeling (not shown). Scale bars: (I) = 15 μm; (II) = 70 μm; (III, IV) = 20 μm. (B) Immunohistochemical staining for OPN and the AMPs lactoferrin, lysozyme, and SLPI performed on parallel sections of lung tissue obtained from a patients with COPD (GOLD stage IV). Immunoreactivity is visualized by a brown-colored DAB staining in the bronchiolar epithelium and also in the airway lumen of COPD lungs. OPN, lactoferrin, and SLPI are all detected both in bronchiolar epithelium (Ep) and in cellular debris and mucus of the lumen (Lu) while lysozyme is present only in the lumen and to a lesser extent in the airway epithelium. The latter is explained by its preferential expression in the submucosal glands of large airways (not shown). Cell nuclei are counterstained with Mayer’s hematoxylin (blue stain). The scale bar in the right panel of the bottom figure is 100 μm.</p

Impairment of the bactericidal activity of AMPs from the OPN-fragment VSS60 that is generated by elastase of <i>P</i>. <i>aeruginosa</i>.

<p>(A) Amino acid sequence of full length OPN and the VSS60-peptide (highlighted in green) which is generated by elastase of <i>P</i>. <i>aeruginosa</i>. The full-length OPN has an RGD-motif providing a binding site for integrins (highlighted in orange). (B) To investigate whether the VSS60-peptide retains the AMP-neutralizing properties of full-length OPN, <i>P</i>. <i>aeruginosa</i> were incubated in either buffer alone, with AMPs (3 μM), or AMPs pre-incubated with VSS60 peptide at a molecular ratio of 1:1 before addition to bacteria and incubated for one hour at 37°C. The antimicrobial activity was determined using viable counts. The VSS60-peptide reduced the bactericidal activity of SLPI, hBD-3, and TSLP. A similar but lower inhibitory activity compare with full length OPN. The histograms represent mean and standard deviation from three separate experiments. Two-way ANOVA with Sidak’s multiple comparisons test was used for statistical analysis. *<i>P</i>≤0.05, and ****<i>P</i> ≤ 0.0001. (C) Biophysical properties of OPN and peptide VSS60 derived from OPN showing number of amino acids, molecular weight, net charge and pI.</p

OPN does not influence the muramidase and protease inhibitory functions of lysozyme and SLPI respectively.

<p>(A) Effect of OPN on muramidase activity of lysozyme. Lysozyme was pre-incubated with or without OPN at equimolar concentrations and fluorogenic substrate was added to the mixture and incubated for 1 h at 37°C. The lysozyme activity was determined by the development of fluorescence, which is represented as relative fluorescence units (RFU). (B) Neutrophil elastase inhibitory property of SLPI was investigated in presence of OPN. Equimolar concentrations of OPN and SLPI were pre-incubated for 1 h at 37°C. This mixture was incubated with human neutrophil elastase (NE) (0.05 U/ml) for 20 min at RT. The NE activity was determined by a chromogenic substrate solution by recording the absorbance at 405 nm. The above experiments suggest that OPN cannot influence the enzymatic activities of lysozyme and SLPI.</p

Binding of OPN to antimicrobial proteins.

<p>(A) Surface plasmon resonance (SPR) sensorgrams illustrating interactions between different AMPs (analyte) and immobilized OPN (ligand). The curves were obtained after injection of different concentration of the AMPs (31.25, 62.5, 125, 250, and 500 μM respectively) and analysis shows binding incidence with association and dissociation curves between lactoferrin, SLPI, midkine, hBD-3, TSLP, and OPN. No significant binding to OPN was observed for lysozyme and LL-37. (B) The association rate constants (k_on), the dissociation rate constants (k_off), and the equilibrium binding constants (K_D) as calculated from the SPR. The association kinetics between OPN and neither lysozyme nor LL-37 could be determined (n. d.). (C) ELISA-based analysis of interaction between AMPs and OPN to confirm the binding pattern obtained using SPR. A 96 well plate was coated with 0.25, 1 and 2 μg of AMPs. Thereafter, the wells were incubated with 2 μg OPN, washed, followed by detection of bound OPN with antibodies. The histogram represents mean absorbance at 405 nm with standard deviation for each AMP. The incubations were performed in triplicates.</p