Figure 1.
Experimental protocol for the study.
Mice were sensitized by i.p. administration of 2 µg ovalbumin admixed with 100 µl alum at day 0 and 14. At day 21, the mice were treated by i.n. administration (50 µl volume) of PBS, live A. baumannii (∼108 CFU) or formalin-fixed (ff) A. baumannii (∼108 CFU). At day 28, mice were intranasally challenged with 100 µg OVA in 50 µl PBS or 50 µl PBS alone, as described in Material and Methods. Five days after challenge (day 33), mice were sacrificed for sample collection.
Figure 2.
Inhibition of airway eosinophilia in OVA-sensitized mice by live A. baumannii.
Mice were sensitized by i.p. administration of OVA/alum on days 0 and 14, then treated with A. baumannii or PBS on day 21, and intranasally challenged with OVA on day 28, as described in Fig. 1. Five days after the i.n. OVA challenge, mice were euthanized, and their lungs were lavaged. Upper panel: Total and differential cell counts in the bronchoalveolar lavage (BAL) fluid were enumerated on cytospin preparations. Each bar represents the mean total number of respective types of cells in the BAL fluid ± SD (n = 5). The data presented represent 1 of at least 2 separate experiments with similar results. ***P<0.001. Lower panel: The BAL cells from OVA-sensitized, PBS-challenged mice (A) consist of predominantly alveolar macrophages whereas the BAL cells from PBS-treated, OVA-challenged mice (B) consist of mainly eosinophils (arrows) with a donut- or horseshoe-shaped nucleus and a pink granular cytoplasm. The majority of BAL cells from A. baumannii-treated, OVA-challenged mice (C) are large alveolar macrophages (arrowheads) with a foamy cytoplasm. HemaStat-3 staining, bar = 100 µm.
Figure 3.
Representative lung histopathology from OVA-sensitized mice treated with A. baumannii.
The mice were sensitized and treated as described in Figure 2 and killed 5 days after the OVA challenge. Note the severe pulmonary inflammation in the areas adjacent to various sized airways in PBS-treated, OVA-sensitized/challenged mouse (arrows, top right panel) and the presence of large numbers of mucus-producing goblet cells (dark purple) (bottom right panel) whereas the inflammation, goblet cell hyperplasia and mucus production were relatively minor in the lungs of mice treated with A. baumannii (top left and bottom left panels). B = bronchus. Top panels stained with H&E; bottom panels stained with periodic acid-Schiff, Bar = 100 µm.
Figure 4.
Inhibition of airway eosinophilia in OVA-sensitized mice by intranasal treatment with live or formalin-fixed A. baumannii (ffAb).
Mice were sensitized by i.p. administration with OVA/alum on days 0 and 14. Sensitized mice were i.n. treated with either 1×108 CFU live or formalin-fixed A. baumannii on day 21 and i.n. challenged with OVA on day 28. Cells in the bronchoalveolar lavage (BAL) fluid were collected 5 days after OVA challenge and differential cell types were enumerated on cytospin preparations. Each bar represents the mean total numbers of respective types of cells in the BAL fluid ± SD (n = 5). The data presented represent 1 of at least 2 separate experiments with similar results. ***P<0.001.
Figure 5.
OVA-specific serum IgG1, IgG2a, and IgE levels in OVA-sensitized mice.
Mice were OVA-sensitized as described in Figure 2 and treated with PBS and challenged with PBS (OVA/PBS/PBS),,treated with A. baumannii and challenged with OVA (OVA/A. baumannii/OVA), treated with formalin-fixed A. baumanii and challenged with OVA (OVA/ffAb/OVA), or treated with PBS and challenged with OVA (OVA/PBS/OVA). Groups of 5 mice were euthanized five days after i.n. OVA challenge, and serum was collected. The OVA-specific IgG1, IgG2a and IgE levels were measured using ELISA. Each data point represents the mean OD value ± SD of five mice in each group.
Figure 6.
Cytokine responses in the lung and BAL fluid of OVA-sensitized mice following OVA challenge.
(A) Real-time PCR analysis of cytokine mRNA expression in lung tissues in OVA-sensitized and A. baumannii treated mice following i.n. OVA challenge. Mice were sensitized and treated as described in Figure 2 and were euthanized 5 days after i.n. OVA challenge. The lungs were collected for RNA extraction. Relative levels of cytokine mRNA expression were determined by real-time RT-PCR analysis as described in Materials and methods. Mouse β-2 microglobulin RNA was measured and used to calculate relative expression using the formula Rel Exp = 2−(ΔΔCT). Results shown are the average and ranges (error bars) of relative expression values determined using cDNA from A. baumannii- or PBS-treated, OVA challenged mice in relation to the corresponding expression levels in PBS challenged mice (n = 5 for all groups). (B) Effect of A. baumannii infection on cytokine levels in BAL fluid and lung homogenates in OVA-sensitized mice following i.n. OVA challenge. Mice were sensitized and treated as described in Figure 2 and were euthanized 5 days after i.n. OVA challenge. The levels of indicated cytokines in BAL fluid and in the lung homogenate supernatants were measured on a Luminex 100IS system using the Milliplex MAP mouse cytokine/chemokine detection kit (Millipore). Each bar represents the mean pg cytokine/mL ± SD (n = 5). The data are representative of two to three independent experiments. *P<0.05 compared to the PBS-treated group.
Figure 7.
Cytokine responses to in vitro re-stimulation of tracheobronchial lymph node (TBLN) cells from A. baumannii-treated mice.
Groups of OVA-sensitized C57BL/6 mice were i.n. treated with either A. baumannii or PBS 7 days before i.n. OVA challenge. Mice were killed 5 days after the challenge and their TBLNs were collected and used for in vitro culture to determine cytokine production in response to OVA stimulation. Single cell suspensions (4×106 cells/mL) were re-stimulated in vitro for 48 h with either OVA (1 mg/mL) or culture medium only. The cytokine levels in the supernatants were determined by Luminex. Data are presented as mean concentration (pg/ml) ± SD (n = 5), and are representative of two independent experiments.
Figure 8.
A. baumannii infection inhibits airway eosinophilia in OVA-challenged TLR-4−/− and IFN-γ−/− mice.
Knock out (KO) and corresponding wild-type (WT) mice were sensitized i.p. with OVA/alum on days 0 and 14 and treated with live A. baumannii or PBS as described in Fig. 2. Mice were i.n. challenged with OVA on day 28. Cells in the bronchoalveolar lavage (BAL) fluid were collected 5 days after OVA challenge and different cell types were enumerated on cytospin preparations. Each bar represents the mean total number of respective types of cells per mouse lung ± SD (n = 5). *P<0.05 and ***P<0.001.
Figure 9.
Regulatory T cells are not increased in BAL fluid after A. baumannii treatment of OVA-sensitized mice.
BAL from A. baumannii-treated and PBS-treated, allergic mice were collected at 5 days after i.n. OVA challenge and the percentage of regulatory T cells was calculated by flow cytometry as determined by CD4+CD25+Foxp3+ staining. A representative dot plot from an A. baumannii-treated (left panel) and a sham-treated (right panel) mouse illustrates the percentage of CD4+Foxp3+ Treg cells gated on total lymphocytes in the BAL fluid.