Figure 1.
B. pertussis induces a longer course of infection than B. parapertussis.
BALB/c mice were infected intranasally with 5×106 CFU of Bordetella strains in 20 µl of PBS, or mock infected (as detailed in the Methods section). Bacterial load in the lungs was assessed at the time points shown above. 3 mice were used at each time point. This graph represents data from two independent experiments.
Table 1.
Antibodies used to stain dendritic cells.
Table 2.
Antibodies used to stain T cells.
Figure 2.
Leukocyte recruitment to the lungs of mice infected with Bordetella strains.
(A) Frozen sections of lungs from BALB/c mice infected with B. pertussis, B. parapertussis, BpΔPTX, or uninfected controls at 5 and 25 days p.i. were H&E stained, and ten independent fields per infection were photographed under 400× magnification. Three independent experiments were performed. (B) Frozen lung sections from mice infected with B. pertussis, B. parapertussis, BpΔPTX, or uninfected controls at 5 and 25 days p.i. were stained sequentially with F4/80 (green) for macrophages, followed by CD45R/B220 (red) for B cells. Alternatively, the slides were sequentially incubated with CD3 (green) for T cells, followed by Gr-1 (red) for neutrophils. Slides were counterstained with DAPI for cell nuclei. Staining with isotype control mAbs revealed no false positive cells (not depicted). Ten separate fields were photographed from each treatment at 100× magnification on a confocal microscope.
Figure 3.
Trafficking receptor expression on emTh cells in blood and lungs from B. pertussis-, B. parapertussis-, and BpΔPTX- infected mice at 5 and 25 days p.i.
(A) Mononuclear cells were obtained from the peripheral blood and from perfused lungs. Cells were gated for CD4+, and further gated for emTh cells (CD44+/CD45RBlow). The frequency of emTh cells expressing trafficking receptors was assessed in the blood (B, D) and lungs (C, E), and normalized to the respective cells in uninfected control mice (1.0 by definition). The bar charts represent compiled data with error bars showing the SEM of four independent experiments. In each independent experiment, six mice were used per treatment group. *: p<0.05, no asterisk indicates p<0.05 as analyzed by a two-tailed student's t-test.
Figure 4.
Functional effects of α4 integrins on migration and adhesion of blood leukocytes from B. pertussis and B. parapertussis infections in vitro.
(A) Migration of blood leukocytes from B. pertussis and B. parapertussis infections at 5 days p.i. through vascular cell adhesion molecule 1 (VCAM-1)-coated or BSA-coated transwells, in response to stromal cell-derived factor-1 alpha (SDF-1α) or control medium. The number of leukocytes derived from B. parapertussis cells through VCAM-1 towards SDF-1α is 100% by definition. Error bars represent the SEM of three independent experiments. In each experiment, four mice were used per treatment group. A two-tailed student's t-test was performed; *: p<0.05. (B) Adhesion of blood leukocytes at 5 days p.i. to 96-well plates pre-coated with VCAM-1 (10 µg/ml) or BSA, from B. pertussis and B. parapertussis infections. After 30 minutes, adherents were stained with crystal violet. Values represent mean A595 of the solubilized dyes. Error bars represent the SEM of six independent observations. A two-tailed student's t-test was performed; *: p<0.05 between B. pertussis and B. parapertussis infection groups.
Figure 5.
Characterization of lung CD11c+ cells during infection with B. pertussis and B. parapertussis.
(A) Enumeration of lung CD11c+ cells during the early phase of infection with Bordetella sp. Mononuclear cells were isolated from lungs and enriched for DCs (CD11c+) as detailed. After column enrichment, the total number of CD11c+ cells per mouse was estimated using a hemocytometer. (B) CD11c+ enriched lung mononuclear cells were gated on singlets and then on CD11c+ cells (DCs). The frequency of CD11c+ cells expressing maturation markers was assessed at 5 days p.i. (C) and at 25 days p.i. (D), and normalized to the respective cells in uninfected control mice (1.0 by definition). In addition, the frequency of lung CD11c+ cells expressing CCR7 and CXCR4 at 5 days p.i. was measured (E). The bar charts (C, D, E) represent compiled data with error bars showing the SEM of four independent experiments. In each experiment, six mice were used per treatment group. *: p<0.05, and no asterisk indicates p<0.05 as analyzed by a two-tailed student's t-test.
Figure 6.
Functional analyses of lung DCs derived from Bordetella infections.
(A) Transwell CCR7 dependent migration of lung DCs. 5×105 isolated lung DCs from Bordetella infections were placed in the transwell upper chamber and allowed to migrate through a 5 µm polycarbonate membrane in the presence or absence of chemokines. Migrated cells were harvested, stained for CD11c & MHC-II, and enumerated by flow cytometry. Chemotaxis index data are calculated as indicated in Materials and Methods. Depicted are combined data of three experiments. In each experiment, five mice were used per treatment group. *: p<0.05 as analyzed by a two-tailed student's t-test. (B) Enumeration of CD11c+ cells in the mediastinal lymph nodes. The mediastinal lymph nodes were harvested, stained with CD11c and CD45, and enumerated with Trubeads by flow cytometry. The absolute number of CD11c+ cells from each treatment was normalized to that from B. pertussis (1.0 by definition) as detailed in Materials and Methods. The bar chart represents compiled data with error bars showing the SEM of three independent experiments. In each experiment, three mice were used per treatment group. *: p<0.05, and no asterisk indicates p<0.05 as analyzed by the one sample two-tailed student's t-test.
Figure 7.
α4β7 and α4β1 imprinting on allogeneic Thy1.1+/CD4+/CD38++ cells.
Lung DCs from 5 days p.i. with one of four infection types—B. pertussis, B. parapertussis, BpΔPTX, or uninfected controls—were co-cultured for 4 days with naïve allogeneic Thy1.1+/CD4+ splenocytes at a 1∶5 ratio. (A) Lung DCs from Bordetella infections were used to stimulate purified naïve Thy1.1+/CD4+ splenocytes labeled with CFSE. Histograms depict CFSE dilution profiles. Approximately four cell cycles of division are observed in each system as estimated by FlowJo proliferation analysis. Low mRMS values obtained in every allogeneic system confirmed the accuracy of the analysis. (B) Co-cultured cells were gated on lymphocytes, followed by gating on singlets, and then on Thy1.1+/CD4+/CD38++ cells. (C) Proportion of Thy1.1+/CD4+/CD38++ cells expressing α4β7 or α4β1 following co-culture with lung DCs derived from Bordetella infections was measured and normalized to the respective cells from the uninfected control co-culture (1.0 by definition). The bar charts represent compiled data with error bars showing the SEM of three independent experiments. In each experiment, five mice were used per treatment group. *: p≤0.05, no asterisk: p<0.05.