Deficiency of mannose-binding lectin greatly increases susceptibility to postburn infection with Pseudomonas aeruginosa

Abstract

Burn injury disrupts the mechanical and biological barrier that the skin presents against infection by symbionts like the Pseudomonas aeruginosa, a Gram-negative bacteria. A combination of local factors, antimicrobial peptides, and resident effector cells form the initial response to mechanical injury of the skin. This activity is followed by an inflammatory response that includes influx of phagocytes and serum factors, such as complement and mannose-binding lectin (MBL), which is a broad-spectrum pattern recognition molecule that plays a key role in innate immunity. A growing consensus from studies in humans and mice suggests that lack of MBL together with other comorbid factors predisposes the host to infection. In this study we examined whether MBL deficiency increases the risk of P. aeruginosa infection in a burned host. We found that both wild-type and MBL null mice were resistant to a 5% total body surface area burn alone or s.c. infection with P. aeruginosa alone. However, when mice were burned then inoculated s.c. with P. aeruginosa at the burn site, all MBL null mice died by 42 h from septicemia, whereas only one-third of wild-type mice succumbed (p = 0.0005). This result indicates that MBL plays a key role in containing and preventing a systemic spread of P. aeruginosa infection following burn injury and suggests that MBL deficiency in humans maybe a premorbid variable in the predisposition to infection in burn victims.

FIGURE 1

Increased mortality in MBL null mice following P. aeruginosa (P.a.) infection after burn injury. WT, MBL null mice, and MBL null mice that were reconstituted with rhMBL were subjected to burn alone, infection alone, or infection postburn and survival was followed as described in Materials and Methods. WT (n = 13) mice, MBL null (n = 13) mice, and reconstituted MBL null (n = 6) mice for the infection postburn, WT and MBL null (n = 8) mice for burn alone, and WT and MBL null (n = 4) mice for infection alone were used. Two experiments were combined. ***, p < 0.001; *, p < 0.05.

FIGURE 2

Increased bacterial load in MBL null mice at 20 h after burn injury and s.c. infection. Bacterial load was determined in blood and organ homogenates as described in Materials and Methods. Symbols represent individual mice for WT (n = 8), MBL null (n = 9), and reconstituted MBL null (n = 9) mice. For skin homogenate, WT (n = 5), MBL null (n = 6), and reconstituted MBL null (n = 5) mice were used. Bars indicate mean. **, p < 0.01; *, p < 0.05.

FIGURE 3

MBL-deficient blood fails to restrict growth of P. aeruginosa. a, Growth of P. aeruginosa was analyzed in ex vivo killing assay. Hirudin-treated whole blood or plasma from mice or whole blood from human donors was incubated with P. aeruginosa, and CFU per milliliter was determined at 120 min after the incubation at 37°C. Bars indicate mean ± SD of six results (three experiments in duplicates). **, p < 0.01. One representative result of two repeated experiments is shown. b, P. aeruginosa express ligands for MBL binding. Binding of P. aeruginosa to human MBL and mouse MBL-A and MBL-C was determined using suspension phase binding assay as described in Materials and Methods. Result is presented as a percentage of bound MBL. Bars indicate mean ± SD of four binding experiments. c, Binding of Cy3-MBL to P. aeruginosa was examined by FACS. Shaded area and solid line indicate bacteria alone and binding of Cy3-MBL to bacteria, respectively. S. aureus was used as a positive control.

FIGURE 4

IL-6 and TNF-α levels following infection after burn. Levels of IL-6 and TNF-α were determined in plasma and in homogenates of skin, lung, and liver as described in Materials and Methods. Symbols represent individual mice. Two experiments were combined, and mice used were WT (n = 8), MBL null (n = 9), and reconstituted MBL null (n = 9) mice. Bars indicate mean. *, p < 0.05; **, p < 0.01.

FIGURE 5

Decreased cytokine levels in MBL null mice following burn injury. Levels of TNF-α and IL-6 were determined in plasma and skin homogenates at 20 h after burn as described in Materials and Methods. Symbols represent individual mice. Two experiments were combined, and WT (n = 6) and MBL null (n = 5) mice were used. Bars indicate mean. *, p < 0.05; **, p < 0.01.