Divergent role of gamma interferon in a murine model of pulmonary versus systemic Klebsiella pneumoniae infection

Abstract

Klebsiella pneumoniae is a leading cause of both community-acquired and nosocomial gram-negative-bacterial pneumonia. A further clinical complication of pulmonary K. pneumoniae infection is dissemination of bacteria from the lung into the peripheral blood, resulting in bacteremia concurrent with the localized pulmonary infection. Here, we report studies detailing the divergent role of gamma interferon (IFN-gamma) in pulmonary versus systemic K. pneumoniae infection. Intratracheal inoculation of IFN-gamma knockout mice resulted in significantly increased mortality compared to that observed for wild-type infected animals. Increased mortality correlated with a 100-fold increase in pulmonary bacteria within 2 days postinfection and upregulation of lung-associated interleukin-10 (IL-10) mRNA. Interestingly, IFN-gamma knockout mice had a twofold reduction in plasma aminospartate transferase activity, indicating diminished liver injury following peripheral blood bacterial dissemination. To study the host response towards blood-borne bacteria in the absence of the ongoing pulmonary infection, intravenous inoculation studies were initiated. IFN-gamma knockout mice were no more susceptible to intravenous infection than their wild-type counterparts. The consistent observation in IFN-gamma knockout mice was for improved survival correlating with increased clearance of blood- and liver-associated bacteria. Intravenous inoculation resulted in a two- to threefold increase in hepatic IL-10 production 24 and 48 h postinfection. Liver injury was also significantly reduced in IFN-gamma knockout mice. These data indicate that IFN-gamma secretion is a critical mediator in the resolution of localized gram-negative pulmonary pneumonia. Surprisingly, host responses towards systemic infection with the same bacteria appear to be IFN-gamma independent.

FIG. 1.

Increased mortality following intratracheal K. pneumoniae inoculation in IFN-γ KO mice. IFN-γ KO and C57BL/6 wild-type control mice were intratracheally inoculated with 2 × 10³ CFU of K. pneumoniae, and survival was observed over the course of 7 days. At this dose of bacteria, 85% of control mice survive through day 7, compared to only 38% of IFN-γ KO mice. Survival curves were generated from two independent experiments with a total of 20 animals per group and were statistically different (P < 0.005) by log-rank analysis.

FIG. 2.

Impaired pulmonary bacterial clearance in IFN-γ KO mice following intratracheal K. pneumoniae infection. Infected IFN-γ KO and wild-type control mice were analyzed on days 1 and 2 postinfection for bacterial burden in total lung homogenates. Total lung CFU were significantly higher in mice lacking IFN-γ on day 1 (P < 0.05) and day 2 (P < 0.01) postinfection. CFU data are expressed as mean + standard error of the mean (error bars) from two independent experiments representing 15 to 18 total animals.

FIG. 3.

Lung RT-PCR analyses of IFN-γ KO mice 2 days post-intratracheal K. pneumoniae inoculation. Total lung RNA was isolated and RT-PCR was performed for the indicated cytokine mRNA. IFN-γ KO and C57BL/6 control mice expressed similar levels of TNF-α mRNA following pulmonary infection. Interestingly, lung IL-10 message was increased in the absence of IFN-γ. As expected, IFN-γ message was upregulated in control animals while being absent in the genetically deficient mice.

FIG. 4.

IFN-γ KO mice display decreased liver cellular injury in spite of similar bacterial numbers in the peripheral circulation following dissemination of pulmonary infection. (A) Peripheral blood (per milliliter of blood) was collected from animals 2 days post-intratracheal infection. Bacterial numbers were similar in both groups of mice, with a consistent trend for higher numbers in IFN-γ KO mice. CFU data are expressed as means + standard errors of the means (error bars) from two independent experiments representing 15 to 18 total mice. (B) Peripheral blood was collected as described in Materials and Methods, and plasma AST levels were determined on day 2 postinfection. Plasma AST levels were significantly lower (P < 0.05) in IFN-γ KO animals.

FIG. 5.

Survival following intravenous K. pneumoniae infection is independent of IFN-γ production. IFN-γ KO and C57BL/6 wild-type control mice were intravenously inoculated with 5 × 10⁴ bacteria and survival was observed over the course of 8 days. While not reaching the level of statistical significance (P = 0.09), the consistent trend in all experiments was for improved survival in IFN-γ KO mice compared to C57BL/6 controls. Survival curves were generated from two independent experiments with a total of 18 animals per group.

FIG. 6.

Unimpaired bacterial clearance in IFN-γ KO mice following intravenous K. pneumoniae inoculation. Bacterial clearance from blood (A) and liver (B) of intravenously infected IFN-γ KO and C57BL/6 mice was determined as described. Six hours postinfection, IFN-γ KO mice had a modest but statistically significant reduction in bacterial burden in blood and liver (P < 0.05). By 48 h, there was a consistent trend towards improved clearance of peripheral blood bacteria in IFN-γ KO mice compared to wild-type animals in all experiments (P = 0.07 [Fisher's exact test]). Liver bacterial clearances by 24 and 48 h postinfection were similar in both IFN-γ KO and wild-type infected animals. CFU data were generated from two to three independent experiments with a total of 14 to 25 animals per group.

FIG. 7.

Diminished liver cellular injury in IFN-γ KO mice following intravenous K. pneumoniae infection. Plasma AST levels were determined 1 day postinfection from IFN-γ KO and wild-type mice. Mice lacking IFN-γ had significantly reduced levels of plasma AST compared to control mice (351 versus 871 U/ml, respectively; P < 0.05). It is worth noting that both groups of mice had equivalent bacterial numbers in liver at this time point. AST levels were determined from two independent experiments with a total of 15 animals per group.

FIG. 8.

Improved liver pathology in IFN-γ KO mice 1 day post-intravenous K. pneumoniae infection. Liver sections from C57BL/6 (A, C, and E) and IFN-γ KO (B, D, and F) mice were prepared and stained with H&E (A and B) or TUNEL (C to F) and then examined at low-power (40×) (A and B) and intermediate-power (200×) (C to F) magnifications. Lesions of hepatocyte cellular injury and necrosis were smaller in number and individual size in IFN-γ KO mice when compared to wild-type animals (compare panels A and B). Without exception, all sites of overt tissue injury seen on the H&E-stained sections also stained TUNEL positive (E and F). TUNEL staining, however, was more intense in wild-type liver sections compared to IFN-γ KO mice. TUNEL staining in the absence of terminal deoxynucleotidyl transferase (C and D) confirmed the specificity of the TUNEL reaction.

FIG. 9.

Elevated liver IL-10 production in IFN-γ KO mice following intravenous bacterial infection. Total liver homogenates were prepared from IFN-γ KO and C57BL/6 infected animals at the indicated time points following intravenous inoculation, and levels of IL-10 were determined by ELISA. IL-10 levels were significantly elevated in IFN-γ KO mice compared to wild-type infected animals and uninfected, baseline levels (dotted line). Data are expressed as means + standard errors of the means (error bars) from two independent experiments with a total of 10 to 12 animals per group.