Development of allergic airway disease in mice following antibiotic therapy and fungal microbiota increase: role of host genetics, antigen, and interleukin-13

Abstract

Lending support to the hygiene hypothesis, epidemiological studies have demonstrated that allergic disease correlates with widespread use of antibiotics and alterations in fecal microbiota ("microflora"). Antibiotics also lead to overgrowth of the yeast Candida albicans, which can secrete potent prostaglandin-like immune response modulators, from the microbiota. We have recently developed a mouse model of antibiotic-induced gastrointestinal microbiota disruption that is characterized by stable increases in levels of gastrointestinal enteric bacteria and Candida. Using this model, we have previously demonstrated that microbiota disruption can drive the development of a CD4 T-cell-mediated airway allergic response to mold spore challenge in immunocompetent C57BL/6 mice without previous systemic antigen priming. The studies presented here address important questions concerning the universality of the model. To investigate the role of host genetics, we tested BALB/c mice. As with C57BL/6 mice, microbiota disruption promoted the development of an allergic response in the lungs of BALB/c mice upon subsequent challenge with mold spores. In addition, this allergic response required interleukin-13 (IL-13) (the response was absent in IL-13(-/-) mice). To investigate the role of antigen, we subjected mice with disrupted microbiota to intranasal challenge with ovalbumin (OVA). In the absence of systemic priming, only mice with altered microbiota developed airway allergic responses to OVA. The studies presented here demonstrate that the effects of microbiota disruption are largely independent of host genetics and the nature of the antigen and that IL-13 is required for the airway allergic response that follows microbiota disruption.

FIG. 1.

Experimental timeline for conidium challenge model of pulmonary hypersensitivity. BALB/C or IL-13^−/− mice were given oral cefoperazone (0.5 mg/ml) for 5 days (day −4 through day 0). At day 0, C. albicans (10⁷ CFU) was administered orally, and mice were challenged (days 2 and 9) by intranasal exposure to A. fumigatus (10⁷ conidia/mouse). Mice were analyzed at day 12 post-C. albicans inoculation.

FIG. 2.

Effect of antibiotic treatment on microbiota populations. At day 12 post-C. albicans inoculation, murine ceca were harvested, homogenized in sterile water, and plated on the following media for enumeration of cecal bacterial populations: VRBA for enteric bacteria, TSA II blood agar for total anaerobic bacteria, and SDA for yeast. Groups are as follows: untreated (conidium challenged, no antibiotic, no C. albicans inoculation) and Anb/Ca (conidium challenged, antibiotic treated, C. albicans oral inoculation). n = 6 to 7 mice per time point pooled from two separate experiments.

FIG. 3.

Effects of microbiota perturbation on pulmonary eosinophil recruitment, Th2 cytokine production by lung leukocytes, and serum IgE production in conidium-challenged mice. Both groups of mice were challenged with conidia. Mice were treated as diagrammed Fig. 1. Leukocytes were isolated from lungs by enzymatic digestion and mechanical dispersion. (a) Eosinophils were phenotyped by Wright-Giemsa staining of cytospun samples. Results are expressed as the mean number of leukocytes per mouse ± standard error of the mean. (b) Serum IgE concentrations were measured by ELISA. (c and d) For Th2 cytokine measurements, leukocytes were isolated from whole lungs and cultured for 24 h without additional stimulation. Supernatants were collected and assayed for IL-5 (c) and IL-13 (d) by ELISA. Results are expressed as the mean ± standard error of the mean. n = 7 to 8 mice pooled from two separate experiments. Groups are as follows: untreated (conidium challenged, no antibiotic, no C. albicans inoculation) and Anb/Ca (conidium challenged, antibiotic treated, C. albicans oral inoculation). *, P < 0.05 as determined by Student's t test.

FIG. 4.

Histological analysis of lungs of mice exposed to conidia. Mice were treated as outlined in Fig. 1. At day 12 post-C. albicans inoculation, lungs were harvested, fixed, sectioned, and stained with hematoxylin and eosin (H&E), which differentially stains leukocytes (a-d). Lung sections were also stained with PAS-hematoxylin, which stains mucus pink (e, f). Groups are as follows: untreated (conidium challenged, no antibiotic, no C. albicans inoculation) and Anb/Ca (conidium challenged, antibiotic treated, C. albicans oral inoculation).

FIG. 5.

Effects of microbiota perturbation on pulmonary eosinophil recruitment, Th2 cytokine production by lung leukocytes, and serum IgE production in conidium-challenged IL-13^−/− mice. Mice were treated as outlined in Fig. 1. Leukocytes were isolated from lungs by enzymatic digestion and mechanical dispersion. (a) Eosinophils were phenotyped by Wright-Giemsa staining of cytospun samples. Results are expressed as the mean number of leukocytes per mouse ± standard error of the mean. (b) Serum IgE concentrations were measured by ELISA. (c and d) For Th2 cytokine measurements, leukocytes were isolated from whole lungs and cultured for 24 h without additional stimulation. Supernatants were collected and assayed for IL-5 (c) and IL-13 (d) by ELISA. Results are expressed as means ± standard errors of the means. n = 7 to 8 mice pooled from two separate experiments. Both BALB/c and IL-13^−/− mice were challenged with A. fumigatus, treated with an antibiotic, and orally inoculated with C. albicans. n.d., not detected. *, P < 0.05 as determined by Student's t test.

FIG. 6.

Histological analysis of lungs of IL-13^−/− mice exposed to A. fumigatus. Mice were treated as outlined in Fig. 1. At day 12 post-C. albicans inoculation, lungs were harvested, fixed, sectioned, and stained with hematoxylin and eosin (H&E), which differentially stains leukocytes (a-c). Lung sections were also stained with PAS-methyl green, which stains mucus pink (d, e). Both BALB/c and IL-13^−/− mice were challenged with A. fumigatus, treated with an antibiotic, and orally inoculated with C. albicans.

FIG. 7.

Experimental timeline for OVA challenge model of pulmonary hypersensitivity. BALB/c mice were given oral cefoperazone (0.5 mg/ml) for 5 days (day −4 through day 0). At day 0, C. albicans (10⁷ CFU) was administered orally. Mice were challenged (days 2, 5, 9, 12, 16, and 19) with intranasal ovalbumin (50 μg/mouse). Mice were analyzed at day 21 post-C. albicans inoculation.

FIG. 8.

Effect of antibiotic treatment on cecal microbiota populations. At day 21, ceca were harvested and plated on the following media to enumerate cecal bacterial populations: VRBA for enteric bacteria, TSA II blood agar for total anaerobic bacteria, and SDA for yeast. n = 7 mice per time point pooled from two separate experiments. Groups are as follows: untreated (OVA challenged, no antibiotic, no C. albicans inoculation) and Anb/Ca (OVA challenged, antibiotic treated, C. albicans oral inoculation). *, P < 0.05 as determined by Student's t test.

FIG. 9.

Effects of microbiota perturbation on pulmonary eosinophil recruitment, Th2 cytokine production by lung leukocytes, and serum IgE production in OVA-challenged mice. Both groups of mice were challenged with OVA. Mice were treated as diagrammed in Fig. 7. Leukocytes were isolated from lungs by enzymatic digestion and mechanical dispersion. (a) Eosinophils were phenotyped by Wright-Giemsa staining of cytospun samples. Results are expressed as the mean number of leukocytes per mouse ± standard error of the mean. (b) Serum IgE concentrations were measured by ELISA. (c and d) For Th2 cytokine measurements, leukocytes were isolated from whole lungs and cultured for 24 h without additional stimulation. Supernatants were collected and assayed for IL-5 (c) and IL-13 (d) by ELISA. Results are means ± standard errors of the means. n = 7 mice pooled from two separate experiments. Groups are as follows: untreated (OVA challenged, no antibiotic, no C. albicans inoculation) and Anb/Ca (OVA challenged, antibiotic treated, C. albicans oral inoculation). *, P < 0.05 as determined by Student's t test; †, P < 0.05 as determined by the Mann-Whitney test.

FIG. 10.

Histological analysis of lungs of OVA-challenged mice. Mice were treated to disrupt the microbiota and were challenged intranasally with OVA as outlined in the experimental design (Fig. 7). At day 21 post-C. albicans inoculation, lungs were harvested, fixed, sectioned, and stained with hematoxylin and eosin (H&E), which differentially stains leukocytes (a-c). Lung sections were also stained with PAS-methyl green, which stains mucus pink (d, e). Groups are as follows: untreated (OVA challenged, no antibiotic, no C. albicans inoculation) and Anb/Ca (OVA challenged, antibiotic treated, C. albicans oral inoculation).