Protection against Pseudomonas aeruginosa lung infection in mice by recombinant OprF-pulsed dendritic cell immunization

Abstract

Background: The Pseudomonas aeruginosa major constitutive outer membrane porin protein F (OprF) has been shown to be a protective antigen and was previously used to activate an immunological response in a mouse model of lung pneumonia. The purpose of our study was to demonstrate the ability of mouse dendritic cells pulsed with purified or recombinant OprF to protect mice against P. aeruginosa infection and inflammation.Both native (n-OprF), isolated and purified from PAO1 bacterial strain, and recombinant (histidin-conjugated) OprF (His-OprF), obtained by cloning of the oprF gene into the pET28a expression vector, were used to stimulate dendritic cells in vitro before adoptive transfer into prospective recipient mice with P. aeruginosa pulmonary infection.

Results: Similar to n-OprF, His-OprF activated dendritic cells in vitro, inducing the costimulatory molecule expression as well as cytokine production. Upon adoptive transfer in vivo, porin-pulsed dendritic cells (DCs) induced Th1-mediated resistance to infection and associated inflammatory pathology caused by either the PAO1 strain or a clinically-isolated mucoid strain.

Conclusions: This study highlights the pivotal contribution of DCs to vaccine-induced protection against P. aeruginosa infection and associated inflammation.

Figure 1

Activation of murine dendritic cells by OprF. Purified splenic dendritic cells (DCs) were pulsed with LPS (10 μg/ml), native (n) or recombinant (His) OprF at different concentrations for 18 hrs before the assessment of costimulatory molecule expression (A) and cytokine production (B and C). FACS analysis was done by staining with FITC and PE-conjugated mAbs to costimulatory molecules. Number represent percent of positive cells. Cytokine levels were determined in the culture supernatants by cytokine-specific ELISA. * Indicates P < .05 (cytokine production by LPS- or porin-pulsed versus unpulsed (-) DCs). ** Indicate P < .05 (cytokine production by n-OprF-pulsed tlr4^-/- DCs versus n-OprF-pulsed WT DCs only and His-OprF-pulsed DCs versus n-OprF-pulsed DCs).

Figure 2

OprF-pulsed DCs protect mice from infection with the PAO1 strain. Splenic 10⁵ dendritic cells (DCs), either unpulsed (-) or pulsed as in legend to figure 1, were administered into recipient mice intraperitoneally a week before the intranasal injection of 3 × 10⁷ P. aeruginosa PAO1 strain. (A) Resistance to infection was assessed in terms of CFU at different days after the infection and (B) cytokine production in lung homogenates and culture supernatants of total cells from TLNs stimulated with plate bound anti-CD3e (2 μg/ml) and anti-CD28 (2 μg/ml) for 72 hours. Results are expressed as mean ± SE. * Indicates P < .05, mice receiving pulsed versus unpulsed (-) DCs. In C, - and + alone indicate uninfected and infected mice, respectively.

Figure 3

Lung sections from uninfected mice. Lung sections were hematoxylin-eosin stained. A - magnification ×10. B - magnification ×40.

Figure 4

Lung sections of mice vaccinated with OprF-pulsed DCs and infected with PAO1 strain. Histopathology at 7 days after infection. Lung sections A-B from infected mice show the involvement of bronchioles and of the alveolar space by an inflammatory infiltrate predominantly consisting of neutrophils filling most of bronchioles (red arrow: bronchial epithelium; blue arrow: neutrophilic infiltrate); the lungs sections from mice vaccinated with n-OprF-pulsed DCs (C-D) and His-OprF-pulsed DCs (E-F) show a great reduction of inflammatory cell recruitment. Lung sections were hematoxylin-eosin stained. A-C-E magnification ×10. B-D-F magnification ×40.

Figure 5

OprF-pulsed DCs protect mice from infection with the clinical isolate. Splenic DCs were pulsed and administered as in legend to figure 1. Mice were infected intranasally with 3 × 10⁷ P. aeruginosa mucoid strain. (A) Resistance to infection and (B) cytokine production in lung homogenates and culture supernatants of TLNs were assessed as in legend to Figure 2. * Indicates P < .05 (mice receiving pulsed versus unpulsed (-) DCs). In C - and + alone indicate uninfected and infected mice, respectively.

Figure 6

Lung sections of mice vaccinated with OprF-pulsed DCs and infected with clinical isolate. Lung sections A-B representing histologic pictures of pneumonia similar to those described in fig. 4 are shown (red arrow: bronchial epithelium; blue arrow: neutrophilic infiltrate). Lung sections from mice vaccinated with n-OprF-pulsed DCs (C-D) and His-OprF-pulsed DCs (E-F) show a lung in which inflammatory cell recruitment was greatly reduced. Lung sections were hematoxylin-eosin stained. A-C-E magnification ×10. B-D-F magnification ×40.