Genetic identification of unique immunological responses in mice infected with virulent and attenuated Francisella tularensis

Abstract

Francisella tularensis is a category A select agent based on its infectivity and virulence but disease mechanisms in infection remain poorly understood. Murine pulmonary models of infection were therefore employed to assess and compare dissemination and pathology and to elucidate the host immune response to infection with the highly virulent Type A F. tularensis strain Schu4 versus the less virulent Type B live vaccine strain (LVS). We found that dissemination and pathology in the spleen was significantly greater in mice infected with F. tularensis Schu4 compared to mice infected with F. tularensis LVS. Using gene expression profiling to compare the response to infection with the two F. tularensis strains, we found that there were significant differences in the expression of genes involved in the apoptosis pathway, antigen processing and presentation pathways, and inflammatory response pathways in mice infected with Schu4 when compared to LVS. These transcriptional differences coincided with marked differences in dissemination and severity of organ lesions in mice infected with the Schu4 and LVS strains. Therefore, these findings indicate that altered apoptosis, antigen presentation and production of inflammatory mediators explain the differences in pathogenicity of F. tularensis Schu4 and LVS.

Figure 1. Time-course of lung and spleen bacterial burden in mice infected with F. tularensis LVS and Schu4

C57BL/6 mice (n = 4 per group) were inoculated i.n. with lethal doses of F. tularensis LVS (10⁴ CFU) or Schu4 (10² CFU), as described in Methods. Lung and spleen tissues were collected 12, 24, 48 and 120 hours after infection and homogenized in TRiZol or PBS for isolation or total RNA or CFU enumeration, error bars represent standard deviation of all 4 samples. (A) F. tularensis 16s rRNA detection in the lungs of mice infected with Schu4 and LVS. (B) F. tularensis 16s rRNA detection in the spleens of mice infected with Schu4 and LVS. Data show similar growth trends through 48 hours in the lung, whereas 120 hours post infection Schu4 shows statistically significantly higher numbers in both the lung and spleen. Data from each time point was subjected to students T-test, (*)=p<0.01, (**)=p<0.001.

Figure 2. Time-course of lung and spleen pathology in mice infected with F. tularensis LVS and Schu4

C57BL/6 mice (n = 4 per group) were inoculated i.n. with lethal doses of F. tularensis LVS (10⁴ CFU) or Schu4 (10² CFU), as described in Methods. Lung and spleen tissues were collected 48 hours or 120 hours after infection and processed with hematoxylin and eosin staining for histopathological examination. (A) Histology from the lung and spleen of control (uninfected) mice. (B) Histology from the lung and spleen 48 hours post-infection with Schu4 or LVS. (C) Histology from the lung and spleen 120 hours post-infection with Schu4 or LVS. Pathological changes at 48 hours after infection were mild in both the lungs and spleen and indistinguishable between F. tularensis LVS and Schu4 infected mice. At 120 hours after infection, more severe lesions were noted in the lungs and especially the spleens of F. tularensis Schu4 infected mice, compared to LVS infected mice. Image magnification was 40X for all images displayed.

Figure 3. Functional enrichment of global transcriptional response data

C57BL/6 mice (n = 2 per group) were inoculated via aerosol with lethal doses of F. tularensis LVS or Schu4 (10⁴ CFU), as described in Methods. Total RNA from the lung and spleen tissues was collected 12, 24, 48 and 120 hours post infection, converted to cDNA, labeled and hybridized on full mouse genome microarrays. (A) Ontology analysis showing select functional categories relevant to infection in the lung in response to infection with Schu4 or LVS. (B) Ontology analysis showing select functional categories relevant to infection in the spleen in response to infection with Schu4 or LVS. Genes with a p-value < 0.01 and differentially regulated > 1.5 fold were used for clustering and ontology analysis.