Neutrophil infiltration into lungs of mice infected with Francisella is delayed. BALF was harvested and cytocentrifuged, and the resulting slides were stained with Diff-Quik to determine cell types. (A) Percentages of neutrophils in BALF obtained from mice (n = 3 for each group and each time point) infected with Francisella at 6 hpi and 1 and 3 dpi. (B) Total numbers of neutrophils entering the BALF calculated for the same time points. One asterisk, P < 0.05; two asterisks, P < 0.01; three asterisks, P < 0.005.

Kinetics of cytokines important for neutrophil chemotaxis is delayed in BALF of mice infected with Francisella. (A to D) BALF was harvested from mice (n = 3 per time point) infected with Francisella at 6 hpi and 1 and 3 dpi. Concentrations of (A) CXCL1 (Gro-α), (B) CXCL2 (MIP-2), (C) CXCL6 (GCP-2), and (D) granulocyte-macrophage colony-stimulating factor (GM-CSF) were determined using Luminex assays. The data are the averages for three infected mice per time point. One asterisk, P < 0.05; two asterisks, P < 0.01; three asterisks, P < 0.005. (E) Lung sections prepared from infected animals at indicated time points were incubated with anti-CXCL2, followed by rhodamine red X-conjugated anti-goat IgG, and were analyzed by in situ immunofluorescence microscopy. Nuclei of cells (blue) were visualized by staining with DAPI. Panel E1 is a representative image for mock-infected mice, showing a constitutive level of CXCL2 present in the lungs, especially in the bronchioles. Mice were infected with Francisella for 6 h (panel E2), 1 day (panel E3), or 3 days (panel E4). Magnification for all panels, ×800.

Kinetics of inflammasome-associated cytokines is delayed in vivo in BALF of mice infected with Francisella. BALF was harvested from Francisella-infected mice, and levels of IL-1β and IL-18 were measured using Luminex assays. (A) IL-1β levels in BALF of mice (n = 3) infected with Francisella at the indicated time points. (B) Concentrations of IL-18 in BALF. One asterisk, P < 0.05; three asterisks, P < 0.005. (C) Immunofluorescence microscopy performed to visualize the expression of IL-1β in vivo. Streptavidin-rhodamine red X was used to visualize IL-1β, while DAPI was used to visualize nuclei. Panel C1 is an image of a mock-infected control. Panels C2, C3, and C4 are images of F. tularensis subsp. novicida-infected mice at 6 hpi and 1 and 3 dpi, respectively. Magnification, ×400. (D) Immunofluorescence microscopy performed to analyze the kinetics of IL-18 in vivo. Panel D1 is an image of a mock-infected control. Panels D2, D3, and D4 show IL-18 expression in mice infected with F. tularensis subsp. novicida at 1 and 3 dpi, respectively. Magnification, ×400. (E) Expression of active caspase-1. Panel E1 shows expression of active caspase-1 in vivo in a mock-infected lung. Panels E2 and E3 show active caspase-1 expression at 6 hpi and 3 dpi, respectively. Panels E1 and E2 show images merged with differential interference contrast images. Magnification, ×1,000.

HMGB-1 is released from cells infected with Francisella both in vivo and in vitro. Immunofluorescence microscopy was performed to determine the localization of HMGB-1 in the lungs of infected mice and in BALF cells, as well as in infected J774 cells. HMGB-1 was visualized with Alexa 488 (green), while DAPI was used to observe the nuclei. Magnification for panels A1 to A4 and B1 to B4, ×800X; magnification for panels C1 to C4, ×1,000. (A) Images of similar areas in the lung (lung parenchyma and alveolar epithelium). Panel A1 is an image for the lung of a representative mock-infected mouse, while panels A2, A3, and A4 are images for mice infected with F. tularensis subsp. novicida at 6 hpi and 1 and 3 dpi, respectively. Panel A4 shows HMGB-1 expression in a lesion in the lung at 3 dpi. (B) HMGB-1 expression in cells isolated from the BALF. Panel B1 is an image for cells harvested from a mock-infected animal, while panels B2, B3, and B4 are images for cells harvested at 6 hpi and 1 and 3 dpi, respectively. (C) Representative micrographs of J774 cells infected with F. tularensis subsp. tularensis Schu S4 at an MOI of 50 at 0, 8, 24, and 72 hpi (panels C1 to C4, respectively). (D) Percentage of the region of interest positive for HMGB-1 in multiple tissue sections as determined by using IPLabs 3.7. Two asterisks, P < 0.01. (E) Representative Western blot for two independent experiments probed for the presence of HMGB-1 using equivalent amounts of whole-cell extracts of J774 cells infected with Schu S4 at an MOI of 50 at the following time points: 0 (mock infection), 2, 5, 18, 24, 48, and 72 hpi.

S100A9 expression is increased in the lungs of mice infected with F. tularensis subsp. novicida. (A) S100A9 expression assessed using immunofluorescence microscopy with lung sections from mice infected with F. tularensis subsp. novicida. Panel A1 shows S100A9 expression in mock-treated animals. Panels A2, A3, and A4 show S100A9 expression in F. tularensis subsp. novicida-infected lungs at 6 hpi and 1 and 3 dpi, respectively. All images are images of similar areas of the lung (lung parenchyma and alveolar epithelium). Magnification for all panels, ×400. (B) Percentage of the region of interest expressing S100A9 obtained from in situ immunofluorescence data for the lungs of mice infected with F. tularensis subsp. novicida using IPLabs 3.7 software. Two asterisks, P < 0.01. (C) Western blot demonstrating expression of S100A9 in lung homogenates obtained from mock-infected animals (lane M), as well as animals infected for 6, 24, and 72 hpi. The position of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as a loading control for the assay. The blot is a representative Western blot for two independent experiments.