Osteopontin impairs host defense during established gram-negative sepsis caused by Burkholderia pseudomallei (melioidosis)

Abstract

Background: Melioidosis, caused by infection with Burkholderia (B.) pseudomallei, is a severe illness that is endemic in Southeast Asia. Osteopontin (OPN) is a phosphorylated glycoprotein that is involved in several immune responses including induction of T-helper 1 cytokines and recruitment of inflammatory cells.

Methodology and principal findings: OPN levels were determined in plasma from 33 melioidosis patients and 31 healthy controls, and in wild-type (WT) mice intranasally infected with B. pseudomallei. OPN function was studied in experimental murine melioidosis using WT and OPN knockout (KO) mice. Plasma OPN levels were elevated in patients with severe melioidosis, even more so in patients who went on to die. In patients who recovered plasma OPN concentrations had decreased after treatment. In experimental melioidosis in mice plasma and pulmonary OPN levels were also increased. Whereas WT and OPN KO mice were indistinguishable during the first 24 hours after infection, after 72 hours OPN KO mice demonstrated reduced bacterial numbers in their lungs, diminished pulmonary tissue injury, especially due to less necrosis, and decreased neutrophil infiltration. Moreover, OPN KO mice displayed a delayed mortality as compared to WT mice. OPN deficiency did not influence the induction of proinflammatory cytokines.

Conclusions: These data suggest that sustained production of OPN impairs host defense during established septic melioidosis.

Figure 1. OPN plasma levels are elevated in patients with melioidosis and correlate with mortality.

OPN plasma concentrations in (A) healthy controls versus melioidosis patients (n = 31 and n = 33, respectively), (B) survivors versus non-survivors (n = 19 and n = 14, respectively), and (C) patients at admission versus follow up sampling (n = 8). Data are expressed as means ± SEM, * P<0.05, ** P<0.01 and *** P<0.001.

Figure 2. Experimental murine melioidosis results in elevated OPN concentrations in plasma and lungs.

OPN concentrations in (A) plasma and (B) lung before, 24 and 72 h after intranasal infection with 10³ CFU of B. pseudomallei. Data are expressed as means ± SEM; n = 8 mice/group, * P<0.05, ** P<0.01 and *** P<0.001 as compared to t = 0. OPN concentrations in culture supernatants after incubation of (C) MH-S and (D) MLE-15 cells with medium or heat-killed B. pseudomallei (MOI 1∶10 and 1∶100) for 4 h. Data are expressed as means ± SEM; n = 4/group, * P<0.05 as compared to medium stimulation.

Figure 3. Decreased pulmonary bacterial growth in OPN KO mice.

WT (black bars) and OPN KO (white bars) mice were infected intranasally with 10³ CFU of B. pseudomallei and bacterial loads were determined 24 and 72 h after infection in (A) lung, (B) blood, and (C) spleen. Data are expressed as means ± SEM; n = 8 mice/group, ** P<0.01 as compared to WT mice. N.D. means not detectable.

Figure 4. Reduced lung pathology in OPN KO mice.

Representative lung pathology of WT (A, D) and OPN KO (B, E) mice 24 (A–C) and 72 (D–F) h after intranasal infection with 10³ CFU of B. pseudomallei. (G) Necrosis score and (H) the percentage of the inflamed area of the lung. (I) Myeloperoxidase levels in lung homogenates at 24 and 72 h after infection. The lung sections are representative for 8 mice per group per timepoint. H&E staining, original magnification 10×. Quantitative data are expressed as means ± SEM of 8 mice per group. * P<0.05 and ** P<0.01 as compared to WT.

Figure 5. OPN does not influence hepatocellular injury.

Representative liver pathology of WT (A) and OPN KO (B) mice 72 h after intranasal infection with 10³ CFU of B. pseudomallei. (C) Liver pathology scores (D) ALAT and (E) ASAT levels were similar at 72 h post infection. The liver sections are representative for 8 mice per group per timepoint. H&E staining, original magnification 10×. Quantitative data are expressed as means ± SEM of 8 mice per group.

Figure 6. OPN KO mice show delayed mortality.

Percentage survival of WT (closed symbols) and OPN KO (open symbols) mice after intranasal infection with 10³ CFU of B. pseudomallei. n = 14 per group. P value indicates the difference between groups.