Effect of PEX, a noncatalytic metalloproteinase fragment with integrin-binding activity, on experimental Chlamydophila pneumoniae infection

Abstract

Chlamydophila pneumoniae is a pathogen that is involved in acute and chronic respiratory infections and that is associated with asthma and coronary artery diseases. In this study, we evaluated the effects of PEX, a noncatalytic metalloproteinase fragment with integrin-binding activity, against experimental infections caused by C. pneumoniae. Moreover, we investigated the relationships between C. pneumoniae and alpha(v)beta(3) integrin functions in order to explain the possible mechanism of action of PEX both in vitro and in vivo. For the in vitro experiments, HeLa cells were infected with C. pneumoniae and treated with either PEX or azithromycin. The results obtained with PEX were not significantly different (P > 0.05) from those achieved with azithromycin. Similar results were also obtained in a lung infection model. Male C57BL/J6 mice inoculated intranasally with 10(6) inclusion-forming units of C. pneumoniae were treated with either PEX or azithromycin plus rifampin. Infected mice treated with PEX showed a marked decrease in C. pneumoniae counts versus those for the controls; this finding did not differ significantly (P > 0.05) from the results observed for the antibiotic-treated group. Integrin alpha(v)beta(3) plays an important role in C. pneumoniae infection. Blockage of integrin activation led to a significant inhibition of C. pneumoniae infection in HeLa cells. Moreover, CHO(DHFR) alpha(v)beta(3)-expressing cells were significantly (P < 0.001) more susceptible to C. pneumoniae infection than CHO(DHFR) cells. These results offer new perspectives on the treatment of C. pneumoniae infection and indicate that alpha(v)beta(3) could be a promising target for new agents developed for activity against this pathogen.

FIG. 1.

Inhibitory effects of human PEX and azithromycin on the infectivity of C. pneumoniae. HeLa cells were inoculated with 1.5 × 10⁶ IFU/well and were treated with either human PEX or azithromycin at various concentrations. The number of IFU per field was evaluated. The results of four different experiments are shown (mean ± SD). ⧫, human PEX; ▾, bovine albumin; ▪, azithromycin.

FIG. 2.

Effect of α_vβ₃ soluble integrins or α_vβ₃ integrin-blocking antibodies on C. pneumoniae infection. HeLa cells were inoculated with 1.5 × 10⁶ IFU/well and were treated with either α_vβ₃ soluble integrins or α_vβ₃ integrin-blocking antibodies at various concentrations. After 72 h of incubation, the number of IFU per field was evaluated. The results of four different experiments are shown (mean ± SD). ⧫, Ab against α_vβ₃; □, Dako P0399 aspecific Ab; ▪, soluble integrin α_vβ₃; ▾, bovine albumin.

FIG. 3.

Infection of HeLa cells with C. pneumoniae elementary bodies preincubated with α_vβ₃ soluble integrins. HeLa cells were inoculated with 1.5 × 10⁶ IFU/well that had been pretreated for 30 min with various concentrations of α_vβ₃ soluble integrin. After 72 h of incubation, the number of IFU per field was evaluated. The results of four different experiments are shown (mean ± SD). ○, control; ▪, soluble integrin α_vβ₃; ▾, bovine albumin.

FIG. 4.

Infection of HeLa cells with C. pneumoniae elementary bodies incubated with α_vβ₃ soluble integrins pretreated with PEX. HeLa cells were inoculated with 1.5 × 10⁶ IFU/well that had been pretreated for 30 min with α_vβ₃ soluble integrin (100 ng/ml) that had been coincubated with PEX (100 ng/ml). After 72 h of incubation, the number of IFU per field was counted. The results of two experiments are shown (mean ± SD). *, P < 0.001 versus the results for the control; #, P < 0.001 versus the results for α_vβ₃ soluble integrin; white bars, control; black bars, soluble integrin α_vβ₃; gray bars, soluble integrin α_vβ₃ pretreated with PEX.

FIG. 5.

Differential susceptibility to C. pneumoniae infection of CHO_DHFR and CHO_DHFR α_vβ₃-transfected cell monolayers. CHO_DHFR and CHO_DHFR α_vβ₃-transfected cells were inoculated with 1.5 × 10⁶ IFU/well and treated with either α_vβ₃ integrin-blocking antibodies or aspecific antibodies at various concentrations. After 72 h of incubation, the number of IFU per field was evaluated. These data are representative of those from four similar experiments (mean ± SD). ▪, CHO_DHFR plus Ab against α_vβ₃; ▴, CHO_DHFR plus aspecific Ab; ▾, CHO_DHFR α_vβ₃-transfected cells plus Ab against α_vβ₃; □, CHO_DHFR α_vβ₃-transfected cells plus aspecific Ab.

FIG. 6.

Binding of C. pneumoniae to purified integrin α_vβ₃. ELISA plates coated with α_vβ₃ or BSA (100 ng/ml per well) were incubated with increasing amounts of C. pneumoniae IFU. The amount of the microorganism added to the plate is plotted against the optical density at 450 nm (OD₄₅₀) reading obtained for the bound C. pneumoniae. The arithmetic means and SDs of two independent experiments performed in duplicate are shown. ▪, α_vβ₃ integrin-coated wells; ▴, BSA-coated wells.

FIG. 7.

Effect of treatment on isolation of C. pneumoniae by infectivity assay from lung tissue homogenates of infected mice 7 days after infection. Mice (age, 6 weeks) were inoculated with C. pneumoniae (1.0 × 10⁶ IFU/mouse); treated with placebo (PBS; ▪), azithromycin plus rifampin (Azi+Rif; ▴), or PEX (▾); and killed 7 days after inoculation. Negative lungs were considered to have a reading of 0 IFU/lung. Note that each datum point represents one mouse. Data are the sum of two different experiments.