Identification of early interactions between Francisella and the host

Abstract

The adaptive immune response to Francisella tularensis is dependent on the route of inoculation. Intradermal inoculation with the F. tularensis live vaccine strain (LVS) results in a robust Th1 response in the lungs, whereas intranasal inoculation produces fewer Th1 cells and instead many Th17 cells. Interestingly, bacterial loads in the lungs are similar early after inoculation by these two routes. We hypothesize that the adaptive immune response is influenced by local events in the lungs, such as the type of cells that are first infected with Francisella. Using fluorescence-activated cell sorting, we identified alveolar macrophages as the first cell type infected in the lungs of mice intranasally inoculated with F. novicida U112, LVS, or F. tularensis Schu S4. Following bacterial dissemination from the skin to the lung, interstitial macrophages or neutrophils are infected. Overall, we identified the early interactions between Francisella and the host following two different routes of inoculation.

FIG 1

Lung gating scheme. Single cells were discriminated from doublets by plotting side scatter height (SSC-H) versus side scatter area (SSC-A). Cells were selected by plotting SSC-A versus forward scatter area (FSC-A). F4/80⁻ and F4/80⁺ cells were gated on by plotting FSC-A versus F4/80. From the F4/80⁺ gate, alveolar macrophages (AMs) were discriminated from interstitial macrophages (IMs) by plotting CD11c versus CD11b. Of the F4/80⁻ cells, dendritic cells were identified by plotting CD11c versus CD11b and neutrophils were identified by plotting CD11b versus GR-1. For each gate, the percentage of the parent gate is indicated in bold (for example, AMs are 4.6% of the cells within the F4/80⁺ gate).

FIG 2

LVS infects myeloid cells following intranasal inoculation. B6 mice were intranasally inoculated with 1 × 10⁴ CFU of LVS. Four hours postinfection, mice were sacrificed and lungs were removed and digested into a single-cell suspension. Cells were stained with CD45-APC, and then CD45⁺ cells were enriched using magnetic beads. (A) Representative flow cytometry analysis of CD45 enrichment. (B) CD45⁺ and CD45⁻ populations were directly plated on chocolate agar, and the colonies were counted 72 h later. We counted 123 total CFU among 4 mice. Data are weighted by the total number of CFU and presented as the percentage of CFU within a population from 4 infected mice in 2 independent experiments.

FIG 3

Alveolar macrophages are the primary infected cell type in the lungs after intranasal inoculation with Francisella. B6 mice were intranasally inoculated with 1 × 10⁴ CFU of U112, LVS, or Schu S4. Four hours postinoculation, mice were sacrificed and lungs were removed and digested into a single-cell suspension and stained for sorting. Alveolar macrophages, interstitial macrophages, dendritic cells, and other cell populations were sorted and directly plated on chocolate agar. Resulting colonies were counted 24 to 72 h later. Data are weighted by the total number of CFU and presented as the percentage of CFU within a population from 2 mice (U112; 139 total CFU), 6 mice (LVS; 132 total CFU), or 3 mice (Schu S4; 398 total CFU) from 1 (U112), 3 (LVS), or 2 (Schu S4) independent experiments. A Kruskal-Wallis test was used to determine whether the distribution of infected cells was significantly different. Significant differences were as follows: for U112, not significant (P = 0.1767); for LVS, P ≤ 0.01; and for Schu S4, P ≤ 0.05.

FIG 4

Alveolar macrophages are infected with LVS at the highest frequency. B6 mice were intranasally inoculated with 1 × 10⁴ CFU of LVS. Four hours postinoculation, mice were sacrificed and lungs were removed and digested into a single-cell suspension and stained for sorting. Alveolar macrophages, interstitial macrophages, dendritic cells, and other cell populations were sorted and directly plated on chocolate agar. The total number of sorted cells for each population was recorded during the sort. Resulting colonies were counted 72 h later. Data are represented as the number of CFU per 10⁵ sorted cells for each population from 6 mice in 3 independent experiments.

FIG 5

Interstitial macrophages and neutrophils are the primary cell types infected with U112 or LVS in the lungs after intradermal inoculation. B6 mice were intradermally inoculated with 5 × 10⁵ CFU of U112 or LVS in 50 μl of PBS at the base of the tail. Forty-eight hours postinoculation, mice were sacrificed and lungs were removed and digested into a single-cell suspension and stained for sorting. Alveolar macrophages, interstitial macrophages, dendritic cells, and neutrophil populations were sorted and directly plated on chocolate agar. Resulting colonies were counted 24 to 72 h later. Data are weighted by the total number of CFU and presented as the percentage of CFU within a population from 4 mice (U112; 9,344 total CFU) or 2 mice (LVS; 537 total CFU) from 1 experiment per strain. A Kruskal-Wallis test was used to determine whether the distribution of infected cells was significantly different. The distributions were not significantly different for U112 (P = 0.1184) or LVS (P = 0.1116).

FIG 6

Interstitial macrophages and neutrophils are infected with U112 and LVS at the highest frequencies. B6 mice were intranasally inoculated with 1 × 10⁴ CFU of U112 or LVS. Four hours postinoculation, mice were sacrificed and lungs were removed and digested into a single-cell suspension and stained for sorting. Alveolar macrophages, interstitial macrophages, dendritic cells, and other cell populations were sorted and directly plated on chocolate agar. The total number of sorted cells for each population was recorded during the sort. Resulting colonies were counted 24 or 72 h later. Data are represented as the number of CFU per 10⁵ sorted cells for each population from 4 (U112) or 2 (LVS) mice in 1 experiment per strain.