Noncapsulated Klebsiella pneumoniae bearing mannose-containing O antigens is rapidly eradicated from mouse lung and triggers cytokine production by macrophages following opsonization with surfactant protein D

Abstract

To better understand the relationship between the surface polysaccharides of pulmonary pathogens and components of the lung innate immune system, we employed selected serotypes of Klebsiella pneumoniae expressing distinct capsular polysaccharides and/or O antigen in a murine model of K. pneumoniae infection. In addition, we examined the effect of surfactant protein D (SP-D) on the cytokine response of human monocyte-derived macrophages to these serotypes in vitro. Noncapsulated mannose-containing O3 serotypes (K50/n and K55/n), which react efficiently with SP-D in vitro, triggered high levels of interleukin-1beta (IL-1beta) and IL-6 production. In vivo, they were more efficiently cleared from the lungs of mice but not from macrophage-depleted mice. They also were more efficiently internalized by alveolar macrophages in vivo. In contrast, galactose-containing O1 serotypes (K2/n and K21a/n), which interact poorly with SP-D, exhibited significantly lower cytokine production and less efficient pulmonary clearance and were ineffectively internalized by alveolar macrophages. These findings are consistent with in vitro results showing that production of IL-1beta and IL-6 mRNA and IL-6 protein by human macrophages exposed to mannose-bearing Klebsiella O serotypes is significantly increased by SP-D. Thus, survival of inhaled bacteria in the lung depends partially on the lipopolysaccharide structure of the bacteria and their interactions with innate immunity components. We speculate that an imbalance of host SP-D and therefore cytokine levels may result in high susceptibility of the host to the pathogen.

FIG. 1.

Survival of noncapsulated K. pneumoniae strains in the lungs of BALB/c mice (normal and immunosuppressed). Normal and CP-treated BALB/c mice were inoculated with 4 × 10⁴ CFU/mouse of the K50/n, K55/n, K2/n, and K21a/n Klebsiella strains. Mice were sacrificed 72 h after bacterial inoculation, the lungs were homogenized, and the CFU were counted. The controls consisted of mock infections without bacteria. The results are expressed as means and standard deviations calculated from three different experiments (five animals per group). Statistical differences (as determined by an ANOVA test) between the O1 and O3 serotypes were found for normal (P < 0.024) and CP-treated (P < 0.02) mice.

FIG. 2.

Survival of noncapsulated K. pneumoniae strains in the lungs of alveolar macrophage-depleted BALB/c mice. To deplete alveolar macrophages, 50 μl of either clodronate- or PBS-containing liposomes was inoculated intranasally 48 h before bacterial administration. Clodronate-containing liposome-treated mice and PBS-containing liposome-treated BALB/c mice were inoculated with 4 × 10⁴ CFU/mouse of the K50/n, K55/n, K2/n, and K21a/n Klebsiella strains. Mice were sacrificed 72 h after bacterial inoculation, the lungs were homogenized, and the CFU were counted. The controls consisted of mock infections without bacteria. The results are expressed as means and standards deviation calculated from two different experiments (sixanimals per group). Statistical differences (as determined by an ANOVA test) between the O1 and O3 serotypes were found for PBS-containing liposome-treated mice (P < 0.024). Also, statistical differences (as determined by the Student t test) were found between clodronate-containing liposome-treated mice and PBS-containing liposome-treated mice inoculated with the K50/n mannose-bearing O3-antigen K. pneumoniae strain (P = 0.001) and between clodronate-containing liposome-treated mice and PBS-containing liposome-treated mice inoculated with the K55/n mannose-bearing O3-antigen K. pneumoniae strain (P = 0.04). No differences were found between clodronate-containing liposome-treated mice and PBS-containing liposome-treated mice inoculated with O1-antigen K. pneumoniae strains.

FIG. 3.

Production of IL-1β and IL-6 mRNA by splenocytes from mice infected with noncapsulated K. pneumoniae strains with different O-antigen structures: RT-PCR analysis of spleen cells (three spleens per group) from normal BALB/c mice infected with 4 × 10⁴ CFU/mouse of noncapsulated Klebsiella strains. The results are expressed as average ratios of the optical density (OD) of cytokine to the optical density of β-actin (averages and standard errors). The data represent the results of three experiments, measured by the TINA computer program. The statistical differences for IL-1β mRNA production (as determined by an ANOVA test) between the O3-bearing strains are significantly greater than those between the O1-bearing strains (P < 0.04), and the statistical differences for IL-6 mRNA production (as determined by an ANOVA test) between the O3-bearing strains are significantly greater than those between the O1-bearing strains (P < 0.00023).

FIG. 4.

Production of IL-6 protein by lung tissue from mice infected with noncapsulated K. pneumoniae strains with different O-antigen structures as measured by a specific sandwich ELISA in the lungs of BALB/c mice (lungs of two mice per group). The data represent the results of three separate experiments. The controls consisted of mock infections without bacteria. The statistical differences for IL-6 protein production (as determined by an ANOVA test) between the O3-bearing strains are significantly greater than the differences between the O1-bearing strains (P < 0.0012).

FIG. 5.

Expression of IL-1β mRNA by human MoDM triggered by SP-D-coated noncapsulated K. pneumoniae. Noncapsulated K. pneumoniae strains (10⁸ CFU/ml) were incubated with human macrophages with and without precoating with human SP-D (10 μg/ml). Cytokine mRNA expression was analyzed by RT-PCR. The results are expressed as the ratios of the optical density (OD) of cytokine to the optical density of β-actin (averages and standard errors of three experiments). Statistical differences (as determined by Student's two-tailed t test) were found for IL-1β mRNA production by MoDM exposed to the K50/n (P = 0.00004) and K55/n (P = 0.000025) O3-bearing K. pneumoniae strains with SP-D and for IL-1β mRNA production by MoDM exposed to the K50/n and K55/n O3-bearing K. pneumoniae strains without SP-D.

FIG. 6.

Expression of IL-6 mRNA by human MoDM triggered by SP-D-coated noncapsulated K. pneumoniae. Noncapsulated K. pneumoniae strains (10⁸ CFU/ml) were incubated with human macrophages with and without precoating with human SP-D (10 μg/ml). Cytokine mRNA expression was analyzed by RT-PCR. The results are expressed ratios of the optical density (OD) of cytokine to the optical density of β-actin (averages and standard errors of three experiments). Statistical differences (as determined by Student's two-tailed t test) were found for IL-6 mRNA production by MoDM exposed to the K50/n O3-bearing K. pneumoniae strain with and without SP-D (P = 0.035) and for IL-6 mRNA production by MoDM exposed to the K55/n O3-bearing K. pneumoniae strain with and without SP-D (P = 0.00038).

FIG. 7.

IL-6 protein production by human MoDM triggered by SP-D-coated noncapsulated K. pneumoniae. Noncapsulated K. pneumoniae strains (10⁸ CFU/ml) were incubated with human macrophages with and without precoating with human SP-D (10 μg/ml). Cytokine protein production was measured by a specific sandwich ELISA. The results are expressed as averages and standard errors of three separate experiments. Statistical differences (as determined by Student's two-tailed t test) were found for IL-6 protein production by MoDM exposed to the K50/n O3-bearing K. pneumoniae strain (P = 0.0037) and for IL-6 protein production by MoDM exposed to the K55/n O3-bearing K. pneumoniae strain (P = 0.0134).