Pulmonary inflammation induced by Pseudomonas aeruginosa lipopolysaccharide, phospholipase C, and exotoxin A: role of interferon regulatory factor 1

Abstract

Chronic pulmonary infection with Pseudomonas aeruginosa is common in cystic fibrosis (CF) patients. P. aeruginosa lipopolysaccharide (LPS), phosholipase C (PLC), and exotoxin A (ETA) were evaluated for their ability to induce pulmonary inflammation in mice following intranasal inoculation. Both LPS and PLC induced high levels of tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta-6, gamma interferon (IFN-gamma), MIP-1 alpha MIP-2 in the lungs but did not affect IL-18 levels. ETA did not induce TNF-alpha and was a weak inducer of IL-1 beta, IL-6, macrophage inflammatory protein 1 alpha (MIP-1 alpha), and MIP-2. Remarkably, ETA reduced constitutive lung IL-18 levels. LPS was the only factor inducing IFN-gamma. LPS, PLC, and ETA all induced cell infiltration in the lungs. The role of interferon regulatory factor-1 (IRF-1) in pulmonary inflammation induced by LPS, PLC, and ETA was evaluated. When inoculated with LPS, IRF-1 gene knockout (IRF-1 KO) mice produced lower levels of TNF-alpha, IL-1 beta, and IFN-gamma than did wild-type (WT) mice. Similarly, a milder effect of ETA on IL-1 beta and IL-18 was observed for IRF-1 KO than for WT mice. In contrast, the cytokine response to PLC did not differ between WT and IRF-1 KO mice. Accordingly, LPS and ETA, but not PLC, induced expression of IRF-1 mRNA. IRF-1 deficiency had no effect on MIP-1 alpha and MIP-2 levels and on cell infiltration induced by LPS, PLC, or ETA. Flow cytometric evaluation of lung mononuclear cells revealed strongly reduced percentages of CD8(+) and NK cells in IRF-1 KO mice compared to percentages observed for WT mice. These data indicate that different virulence factors from P. aeruginosa induce pulmonary inflammation in vivo and that IRF-1 is involved in some of the cytokine responses to LPS and ETA.

FIG. 1.

Increase in relative lung wet weight following administration of LPS, PLC, or ETA. Mice were inoculated with either LPS (10 μg/mouse), PLC (0.5 μg/mouse), or ETA (2 μg/mouse). Control mice received vehicle. At the indicated time points mice were bled and sacrificed, and body weight was recorded. Lungs were removed and weighed. Data are means ± SEM of five mice per group and are expressed as percentage of lung weight over body weight. ∗, P < 0.05; ∗∗∗, P < 0.001 versus respective control by factorial ANOVA.

FIG. 2.

Cellular infiltrate in the lungs following inoculation of LPS, PLC, or ETA. Mice were inoculated with either LPS (10 μg/mouse), PLC (0.5 μg/mouse), or ETA (2 μg/mouse). Control mice received vehicle. (Top) Mice were sacrificed either 6 h (LPS and PLC) or 24 h (ETA) following inoculation. Lungs were removed, and MPO levels were evaluated as described in Materials and Methods. (Bottom) Mice were sacrificed 24 h following inoculation, and BAL was performed as described in Materials and Methods. Data are means ± SEM of five mice per group. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 versus respective control by factorial ANOVA.

FIG. 3.

Induction of IRF-1 mRNA following inoculation of LPS, PLC, or ETA. Mice were inoculated with either LPS (10 μg/mouse), PLC (0.5 μg/mouse), or ETA (2 μg/mouse). Control mice received vehicle. One hour after inoculation, lungs were removed, total RNA was extracted, and RNase protection assay for IRF-1 was performed as described in Materials and Methods. Data are means ± SEM of five mice per group and are expressed as the percent increase of IRF-1 mRNA compared to control mice. ∗, P < 0.05 versus control by factorial ANOVA.

FIG. 4.

Cytokine levels in WT and IRF-1 KO mice following inoculation of LPS, PLC, or ETA. WT (open bars) and IRF-1 KO (filled bars) mice were inoculated with either LPS (10 μg/mouse), PLC (0.5 μg/mouse), or ETA (2 μg/mouse). Control mice received vehicle. Mice were sacrificed either 2 h (for TNF-α), 6 h (for IL-1β and IL-18) or 24 h (for IFN-γ) after inoculation, and cytokine levels were measured in the lung homogenates. Data are means ± SEM of five mice per group. ∗, P < 0.05; ∗∗, P < 0.01 versus respective WT by unpaired Student's t test.

FIG. 5.

Chemokine and MPO levels in WT and IRF-1 KO mice following inoculation of LPS, PLC, or ETA. WT (open bars) and IRF-1 KO (filled bars) mice were inoculated with either LPS (10 μg/mouse), PLC (0.5 μg/mouse), or ETA (2 μg/mouse). Control mice received vehicle. Mice were sacrificed 6 h after inoculation of LPS or PLC and 24 h after administration of ETA. Chemokine and MPO levels were measured in the lung homogenates. Data are means ± SEM of five mice per group. ∗∗, P < 0.01 versus respective WT by unpaired Student's t test.

FIG. 6.

Reduced CD8⁺ cells in the lungs of IRF-1 KO mice. LMC were isolated from untreated WT and IRF-1 KO mice. Cells were evaluated by flow cytometry for expression of CD4 and CD8. Results from one representative mouse per genotype are shown and are representative of four independent analyses performed.

FIG. 7.

Reduced NK cells in the lungs of IRF-1 KO mice. LMC were isolated from untreated WT and IRF-1 KO mice. Cells were evaluated by flow cytometry for expression of CD3 and NK1.1. Results from one representative mouse per genotype are shown and are representative of four independent analyses performed.

FIG. 8.

Increased percentage of macrophages in the lungs of IRF-1 KO mice. LMC were isolated from untreated WT and IRF-1 KO mice. Cells were evaluated by flow cytometry for expression of F4/80. Results from one representative mouse per genotype are shown and are representative of four independent analyses performed.