Identification of immunologic and pathologic parameters of death versus survival in respiratory tularemia

Abstract

Francisella tularensis can cause severe disseminated disease after respiratory infection. The identification of factors involved in mortality or recovery following induction of tularemia in the mouse will improve our understanding of the natural history of this disease and facilitate future evaluation of vaccine candidate preparations. BALB/c mice were infected intranasally with the live vaccine strain (LVS) of F. tularensis subsp. holarctica and euthanized at different stages of disease to analyze the induction of immune molecules, gross anatomical features of organs, bacterial burdens, and progression of the histopathological changes in lung and spleen. Tissue-specific interleukin-6 (IL-6), macrophage inflammatory protein 2, and monocyte chemotactic protein 1 were immune markers of mortality, while anti-LVS immunoglobulin M and IL-1beta were associated with survival. Moribund mice had enlarged spleens and lungs, while surviving mice had even more prominent splenomegaly and normal-appearing lungs. Histopathology of the spleens of severely ill mice was characterized by disrupted lymphoid follicles and fragmented nuclei, while the spleens of survivors appeared healthy but with increased numbers of megakaryocytes and erythrocytes. Histopathology of the lungs of severely ill mice indicated severe pneumonia. Lungs of survivors at early time points showed increased inflammation, while at late times they appeared healthy with peribronchial lymphoid aggregates. Our results suggest that host immune factors are able to affect bacterial dissemination after respiratory tularemia, provide new insights regarding the pathological characteristics of pulmonary tularemia leading to systemic disease, and potentially identify immune markers associated with recovery from the disease.

FIG. 1.

Survival and weight loss curves following pulmonary infection with F. tularensis LVS. Mice were inoculated via the intranasal route with 10⁴ CFU of the LVS strain, and survival and body weight were monitored over time. (A) All mice showed disease signs by day 3 or 4, and approximately 75% became moribund and died between days 5 and 8, while a small percentage (∼25%) overcame the illness and recovered. In this representative experiment, a total of 22 mice were included, 5 of which were survivors. (B) Moribund mice showed a body weight loss of approximately 25% (n = 12; filled circles) compared to uninfected controls (n = 4; open triangles). Survivors also lost body weight concurrently with the appearance of clinical signs but regained it upon recovery (n = 3; filled inverted triangles). Differences in body weight between moribund mice and survivors were statistically significant at day 8 postinfection (*, P < 0.05). The error bars represent standard deviations.

FIG. 2.

Antibody and chemokine responses in lungs and spleens of mice infected with LVS. Following intranasal infection with LVS, mice (three to six animals per group) were humanely sacrificed at a terminal disease stage or at different time points after recovering from symptoms. The results are shown in light gray for spleens and dark gray for lungs. Organs from mice injected with PBS were used as negative controls. Data are presented as the quantity (in ng or pg) of antibody or chemokine per milliliter at different stages of disease, and the error bars represent standard deviations. Differences between moribund mice and survivors were either significant (*, P < 0.05) or highly significant (**, P < 0.01). ND, not detectable.

FIG. 3.

Cytokine levels in lungs and spleens of mice infected with LVS. Following intranasal infection with LVS, supernatants from spleens (light-gray histograms) and lungs (dark-gray histograms) from moribund and survivor mice (three to six animals per group) were collected and analyzed for cytokine production. Organs from mice injected with PBS were used as negative controls. The data are reported as amounts (in pg) of cytokine per milliliter at different stages of disease, and the error bars represent standard deviations. Differences between moribund mice and survivors were either significant (*, P < 0.05) or highly significant (**, P < 0.01). ND, not detectable.

FIG. 4.

Changes in lung and spleen gross anatomy after infection with LVS. (A) Spleens collected from representative moribund mice appeared enlarged and pale (right) compared to those of uninfected controls (left). (B) Lungs from representative moribund mice did not collapse and showed some color variation (right) compared to those from uninfected controls (left). (C) Splenomegaly in representative survivors at early (day 7; center) and late (day 50; right) time points in comparison to normal spleens from uninfected control mice (left). (D) Lung tissue from survivors at both early (day 7; center) and late (day 50; right) times appeared normal in size and color, similar to those of uninfected controls (left). Bars = 5 mm.

FIG. 5.

Analysis of organ weight and enumeration of bacterial loads in tissue and blood after induction of tularemia. Following intranasal infection, mice (n = 3 to 6) were sacrificed at different time intervals. Lungs and spleens were dissected, weighed, and homogenized, and samples were serially diluted to determine LVS CFU by culture. The data are reported as weight per milligram and CFU per milligram of either spleen (A and C) or lung (B and D) tissue over time, and the error bars represent standard deviations (SD). (E) In a separate experiment, bacterial loads were also evaluated in blood from all mice (n = 25) collected at different time intervals (24 to 96 h). Also shown are CFU values detected in the blood of ∼75% of the mice that became moribund at day 7 (n = 18) and the ∼25% of survivor mice at days 7, 15, and 50 (n = 7). The data are expressed as CFU per milliliter, and the error bars represent SD. Differences in organ weight (mg) were either significant (*, P < 0.05) or highly significant (**, P < 0.01) in comparison to uninfected controls. “Survivor” data in panels A and B include organ weights determined at days 7, 15, and 50 postinfection (no differences were observed between the time points).

FIG. 6.

Hematoxylin and eosin staining of formalin-fixed splenic tissue. Spleens were removed at different stages of disease. (A) Spleen from an uninfected control with no pathological changes. (B) Spleen from a representative moribund animal sacrificed at day 7 with lymphoid follicle disruption. The inset shows infiltration of fragmented nuclei, some neutrophils, and the absence of megakaryocytes. (C and D) Spleens from survivors sacrificed at early (day 7) (C) and late (day 50) (D) time points showed normal lymphoid follicle architecture. The insets demonstrate the presence of prominent megakaryocytes, as indicated by the arrows. The sections were photographed with a 10× objective, with a 50× objective for the insets.

FIG. 7.

Hematoxylin and eosin staining of formalin-fixed pulmonary tissue. (A) No alterations were observed in the lung tissue of uninfected control mice. (B) Seven days after infection, moribund mice presented with pneumonia characterized by diffuse edema, neutrophil infiltration, and fibrin deposits. The inset shows a large area of diffuse edema. (C) Survivors sacrificed at day 7 postinfection presented with severe cellular infiltration but no fibrin deposits or edema. Neutrophil infiltration is evident in the inset. (D) Survivors sacrificed at day 50 no longer showed pathological changes in the lung, and lymphoid aggregates were evidenced, as shown by the arrows. The sections were photographed with a 10× objective, with a 50× objective for the insets.