Infected-host-cell repertoire and cellular response in the lung following inhalation of Francisella tularensis Schu S4, LVS, or U112

Abstract

Francisella tularensis causes systemic disease in humans and other mammals, with high morbidity and mortality associated with inhalation-acquired infection. F. tularensis is a facultative intracellular pathogen, but the scope and significance of cell types infected during disease is unknown. Using flow cytometry, we identified and quantified infected-cell types and assessed the impact of infection on cell populations following inhalation of F. tularensis strains U112, LVS, and Schu S4. Initially, alveolar macrophages comprised over 70% of Schu S4- and LVS-infected cells, whereas approximately 51% and 27% of U112-infected cells were alveolar macrophages and neutrophils, respectively. After 3 days, roughly half of Schu S4- and LVS- and nearly 80% of U112-infected cells were neutrophils. All strains infected CD11b(high) macrophages, dendritic cells, monocytes, and alveolar type II cells throughout infection. Macrophage, monocyte, and dendritic-cell populations were reduced during U112 infection but not Schu S4 or LVS infection. These results demonstrate directly that F. tularensis is a promiscuous intracellular pathogen in the lung that invades and replicates within cell types ranging from migratory immune cells to structural tissue cells. However, the proportions of cell types infected and the cellular immune response evoked by the human pathogenic strain Schu S4 differ from those of the human avirulent U112.

FIG. 1.

Identifying Francisella-infected lung cells. Lung cells infected with GFP⁺ Francisella are readily detectable by flow cytometry. Mice were intranasally inoculated with GFP⁺ Francisella strains, and on days 1 and 3 postinoculation, lungs were harvested and digested to produce single-cell suspensions which could be further analyzed. (A) Lung bacterial burden was determined for each strain at day 1 and day 3. (B) GFP⁺ lung cells were detected on day 1 following intranasal inoculation with GFP⁺ LVS (LVSgfp). SSC, side scatter. (C) High-GFP, low-GFP, and GFP-negative populations were collected and plated on coverslips for analysis by fluorescence microscopy. (D) The absolute numbers of GFP⁺ lung cells from mice infected with U112, LVS, or Schu S4 were determined on day 1 and day 3 postinoculation. Error bars represent standard deviations of the means (n = 3 to 6 mice). Statistical significance of difference between results on day 1 and day 3 was determined by unpaired two-tailed t test assuming unequal variance (*, P < 0.05).

FIG. 2.

Identifying cell types in the lung by flow cytometry. Cell types were identified based on differential expression of F4/80, CD11b, CD11c, and GR-1. Mouse lungs were digested with dispase, and cells were stained with fluorescently labeled cell-type-specific antibodies. (A, B) F4/80⁺ cells (A) that were CD11b^low (B) were classified as alveolar macrophages, while F4/80^high CD11b^high cells were classified as CD11b^high macrophages. (C) F4/80^low CD11c^high cells were classified as DCs and subdivided into CD11b^low/mid DCs and CD11b^high DCs. F4/80^low CD11c^low CD11b^mid cells were classified as monocytes. (D) F4/80^low CD11c^low CD11b^high GR-1^high cells were classified as neutrophils.

FIG. 3.

Lung cell types infected by Francisella following inhalation. Proportions of lung cell types infected with Francisella strain U112, LVS, or Schu S4 on day 1 and day 3 postinoculation.

FIG. 4.

Numbers of Francisella-infected cells following inhalation. Absolute numbers of infected alveolar macrophages (A), CD11b^high macrophages (B), CD11b^low/mid DCs (C), CD11b^high DCs (D), monocytes (E), neutrophils (F), and ATII cells (G) from mouse lungs on day 1 and day 3 following intranasal inoculation with U112, LVS, or Schu S4. Error bars represent standard deviations of the means (n = 3 to 6 mice). Statistical significance of difference between results on day 1 and day 3 was determined by unpaired two-tailed t test assuming unequal variance.

FIG. 5.

Effects of Francisella infection on cell numbers within lung populations. Absolute numbers of alveolar macrophages (A), CD11b^high macrophages (B), CD11b^low/mid DCs (C), CD11b^high DCs (D), monocytes (E), neutrophils (F), and ATII cells (G) from mouse lungs on day 1 and day 3 following intranasal inoculation with U112, LVS, or Schu S4. Error bars represent standard deviations of the means (n = 3 to 6 mice). Statistical significance of difference between day 1 and day 3 was determined by unpaired two-tailed t test assuming unequal variance.