Post-exposure therapeutic efficacy of COX-2 inhibition against Burkholderia pseudomallei

Abstract

Burkholderia pseudomallei is a Gram-negative, facultative intracellular bacillus and the etiologic agent of melioidosis, a severe disease in Southeast Asia and Northern Australia. Like other multidrug-resistant pathogens, the inherent antibiotic resistance of B. pseudomallei impedes treatment and highlights the need for alternative therapeutic strategies that can circumvent antimicrobial resistance mechanisms. In this work, we demonstrate that host prostaglandin E2 (PGE2) production plays a regulatory role in the pathogenesis of B. pseudomallei. PGE2 promotes B. pseudomallei intracellular survival within macrophages and bacterial virulence in a mouse model of pneumonic melioidosis. PGE2-mediated immunosuppression of macrophage bactericidal effector functions is associated with increased arginase 2 (Arg2) expression and decreased nitric oxide (NO) production. Treatment with a commercially-available COX-2 inhibitor suppresses the growth of B. pseudomallei in macrophages and affords significant protection against rapidly lethal pneumonic melioidosis when administered post-exposure to B. pseudomallei-infected mice. COX-2 inhibition may represent a novel immunotherapeutic strategy to control infection with B. pseudomallei and other intracellular pathogens.

Figure 1. B. pseudomallei rapidly induces COX-2 and PGE₂ production by macrophages.

A) Bone-marrow derived macrophages (BMDM) were treated with viable B. thailandensis (Bt), B. pseudomallei (Bp) or heat-inactivated Bp (iBp) at MOI 1 and COX-2 mRNA expression was measured by RT-PCR. B) COX-2 enzyme was detected by Western blot in BMDM infected with Bp at MOI 1. C) BMDM were treated with Bp at MOI 0.1 or 1 and iBp at MOI 1 and PGE₂ was measured in culture supernatants by ELISA. The data represent biological triplicates per time point. Error bars represent the standard deviation (SD). Statistical significance was determined using a two way ANOVA with Bonferroni post-test. *p<0.05, *** p<0.001. Data is representative of two independent experiments.

Figure 2. PGE₂ promotes B. pseudomallei intracellular survival.

BMDM were incubated in the presence or absence of NS398 (100 µM) +/− PGE₂ (1 µM) for 30 minutes then infected with Bps at MOI 1 A) Percent intracellular survival of Bps and B) corresponding nitrite levels in BMDM supernatants. The data represent biological triplicates per time point. Error bars represent the SEM. Statistical significance was determined using a two way ANOVA with Bonferroni post-test. *p<0.05, *** p<0.001. Data is representative of two independent experiments.

Figure 3. Arginase 2 enhances B. pseudomallei survival in macrophages.

BMDM were incubated in the presence or absence of NS398 (100 µM) +/− PGE₂ (1 µM) for 30 minutes, then infected with Bps at MOI 1 for 4 h A) Fold-change in mRNA expression for iNOS, arginase 1 (Arg1) and Arg2 in response to Bps was measured by RT-PCR. Error bars represent the SEM. B) Intracellular survival of Bps in BMDM pre-treated with 100 µM nor-NOHA for 30 minutes and C) corresponding nitrite production by Bps-infected cells in the presence or absence of nor-NOHA. The data represent biological triplicates per time point. Error bars represent the SEM. Statistical significance was determined using a two way ANOVA with Bonferroni post-test. *** p<0.001. Data is representative of two independent experiments.

Figure 4. Lung PGE₂ increases in a time-dependent manner after pulmonary challenge with B. pseudomallei.

BALB/c mice were infected intranasally with 3×10³ cfu of Bps and serially sacrificed between 0 (pre-challenge) and 72 h post-infection (n = 3 per timepoint). Animal weight was recorded daily and PGE₂ was measured in total lung homogenates by ELISA. Error bars represent the SEM. *p<0.05 compared to 0 h timepoint as determined by one way ANOVA. Data is representative of two independent experiments.

Figure 5. COX-2 inhibition provides significant protection against lethal pulmonary melioidosis.

BALB/c mice (n = 8 per group) were infected with 3×10³ cfu (4 LD₅₀) of Bps 1026b intranasally. Three h post-exposure, mice were administered 15 mg/kg of COX-2 inhibitor (NS398) or DMSO (Mock treatment) intraperitoneally, then again daily for two consecutive days. Survival was monitored for 10 days. Statistical significance was determined using Kaplan Meier analysis. p<0.0001. Data is representative of two independent bacterial challenge experiments.

Figure 6. Arg2 is expressed in the lungs of B. pseudomallei-infected mice and decreases upon COX-2 inhibition.

Arg1 and Arg2 expression was examined by Western blot in lung homogenates of uninfected and Bps-infected mice (n = 3 per group) treated with NS398 or mock control. Mouse liver extract and mouse Arg2-transfected 293T cell lysate were used as positive controls for Arg1 and Arg2 respectively. β-actin was used as a loading control and for normalization in densitometry analysis using the ImageJ program: http://rsb.info.nih.gov/ij/. Statistical significance was determined using one-way ANOVA with Bonferroni post test. *** p<0.001.