Expression and function of macrophage migration inhibitory factor (MIF) in melioidosis

Abstract

Background: Macrophage migration inhibitory factor (MIF) has emerged as a pivotal mediator of innate immunity and has been shown to be an important effector molecule in severe sepsis. Melioidosis, caused by Burkholderia pseudomallei, is an important cause of community-acquired sepsis in Southeast-Asia. We aimed to characterize the expression and function of MIF in melioidosis.

Methodology and principal findings: MIF expression was determined in leukocytes and plasma from 34 melioidosis patients and 32 controls, and in mice infected with B. pseudomallei. MIF function was investigated in experimental murine melioidosis using anti-MIF antibodies and recombinant MIF. Patients demonstrated markedly increased MIF mRNA leukocyte and MIF plasma concentrations. Elevated MIF concentrations were associated with mortality. Mice inoculated intranasally with B. pseudomallei displayed a robust increase in pulmonary and systemic MIF expression. Anti-MIF treated mice showed lower bacterial loads in their lungs upon infection with a low inoculum. Conversely, mice treated with recombinant MIF displayed a modestly impaired clearance of B. pseudomallei. MIF exerted no direct effects on bacterial outgrowth or phagocytosis of B. pseudomallei.

Conclusions: MIF concentrations are markedly elevated during clinical melioidosis and correlate with patients' outcomes. In experimental melioidosis MIF impaired antibacterial defense.

Figure 1. MIF plasma and leukocyte mRNA levels are elevated in melioidosis patients and correlate with poor outcome.

In patients (▴, n = 34) with melioidosis, strongly increased plasma concentrations of plasma MIF were present on admission when compared to healthy controls (▪, n = 32) (A). Patients who went on to die from melioidosis had higher MIF plasma concentrations on admission than patients who survived (B). MIF mRNA was strongly upregulated in the peripheral blood leukocytes of melioidosis patients compared to healthy controls (C). B2M: β2 microglobulin. ** p<0.01; *** p<0.001.

Figure 2. Increased MIF concentrations during experimental melioidosis in mice.

Pulmonary MIF expression was strongly increased 72 hours after intranasal inoculation with B. pseudomallei (A). Plasma MIF concentrations remained constant during the first two days of infection, followed by a steep increase over time (B). Data are means ± SEM of 5 mice per group at each time point. ** p<0.01 versus t = 0.

Figure 3. Immunostaining for MIF in the lungs of mice.

Positive immunostaining for MIF in lung tissue was observed in non-infected control animals of bronchial epithelial cells and alveolar macrophages (A). 48 hours after infection with B. pseudomallei there was a marked increase in immunostaining of the epithelial submucosa, bronchial epithelial cells and inflammatory cells, most notably of alveolar macrophages (B). Magnification ×10, insets ×20.

Figure 4. Effect of recombinant MIF on bacterial clearance.

Mice, treated with control buffer (white bars) or recombinant MIF (grey bars) and inoculated with 2.5×10² CFU B. pseudomallei intranasally, were analysed for bacterial outgrowth in the lungs (A), liver (B) and blood (C) 48 hours later. Data represent mean ± SEM of n = 8 mice per group; ** p<0.01.

Figure 5. Effect of anti-MIF antibodies on bacterial clearance.

Mice were treated with non-immune IgG control (white bars) or anti-MIF antibodies (grey bars) and inoculated with 2.5×10² CFU B. pseudomallei intranasally after which bacterial outgrowth in the lungs (A), liver (B) and blood (C) was analysed 48 hours later. Data represent mean ± SEM of n = 8 mice per group.

Figure 6. Effect of anti-MIF treatment on bacterial clearance is dependent on the inoculum size.

Mice were treated with non-immune IgG control (white bars) or anti-MIF antibodies (grey bars) and inoculated with 5×10¹ CFU (A) or 5×10² CFU (B) B. pseudomallei intranasally after which bacterial outgrowth in the lungs, liver and blood was analysed 48 hours later. No bacterial outgrowth in the liver and no bacteremia was observed in any of the mice inoculated with 5×10¹ CFU B. pseudomallei. Data represent mean ± SEM of n = 8 mice per group; * p<0.05.

Figure 7. Effect of treatment with anti-MIF antibodies on survival of mice infected with B. pseudomallei.

Mice were injected intraperitoneally with 2 mg of anti-MIF (straight line with downward arrow) or non-immune control IgG (dashed line with upward arrow) 2 hours before inoculation with 5.0×10² CFU of B. pseudomallei intranasally; n = 12 mice per group; * p<0.05.