Role of antibiotics and fungal microbiota in driving pulmonary allergic responses

Abstract

Over the past four decades, there has been a significant increase in allergy and asthma in westernized countries, which correlates with alterations in fecal microbiota (microflora) and widespread use of antibiotics (the "hygiene hypothesis"). Antibiotics also lead to overgrowth of the yeast Candida albicans, which can secrete potent prostaglandin-like immune response modulators. We have developed a mouse model of antibiotic-induced microbiota disruption that includes stable increases in gastrointestinal (GI) enteric bacteria and GI Candida levels with no introduction of microbes into the lungs. Mice are treated for 5 days with cefoperazone in the drinking water, followed by a single oral gavage of C. albicans. This results in alterations of GI bacterial populations and increased yeast numbers in the GI microbiota for at least 2 to 3 weeks and can drive the development of a CD4 T-cell-mediated allergic airway response to subsequent mold spore (Aspergillus fumigatus) exposure in immunocompetent mice without previous systemic antigen priming. The allergic response in the lungs is characterized by increased levels of eosinophils, mast cells, interleukin-5 (IL-5), IL-13, gamma interferon, immunoglobulin E, and mucus-secreting cells. In the absence of antibiotics, mice exposed to Aspergillus spores do not develop an allergic response in the airways. This study provides the first experimental evidence to support a role for antibiotics and fungal microbiota in promoting the development of allergic airway disease. In addition, these studies also highlight the concept that events in distal mucosal sites such as the GI tract can play an important role in regulating immune responses in the lungs.

Figures

FIG. 1.

Experiment timeline for the induction of allergic airway disease following antibiotic therapy and fungal microbiota increase. C57BL/6 mice were given oral cefoperazone (0.5 mg/ml) in their drinking water for 5 days (days −4 through 0). At day 0, C. albicans (107 CFU) was administered orally, and mice were challenged intranasally (days 2 and 9) with A. fumigatus conidia (107 conidia/mouse). Mice were harvested at day 12 posttreatment.

FIG. 2.

Effect of antibiotic treatment on microbiota populations. At various times, murine tissues were harvested, homogenized in sterile water, and plated with the following media to enumerate cecal bacterial populations: VRBA for enteric bacteria, TSA II blood agar in an anaerobic chamber for total anaerobic bacteria, and SDA for yeast. (a) C. albicans tissue deposition at day 1 postinfection. (b) Cecal bacterial populations before and after antibiotic treatment. (c) Cecal bacterial populations at day 12 postinoculation with C. albicans. n = 7 to 8 mice per time point pooled from two separate experiments. Asterisk, P < 0.01; n.d., not detected.

FIG. 3.

Effect of antibiotic therapy and fungal microbiota increase on the host response to intranasal conidia exposure. Mice were treated as described in the legend to Fig. 1 and analyzed at day 12 posttreatment. (a) Leukocytes were isolated from lungs by enzymatic digestion and mechanical dispersion. Lung eosinophils were phenotyped by Wright-Giemsa staining of cytospun samples. Results are expressed as the increase in the mean number of lung eosinophils per mouse ± the standard error of the mean compared to unchallenged, untreated mice (5 eosinophils/lung). (b) Lung mast cells were phenotyped by toluidine blue staining of histological sections. Results are expressed as mean number of leukocytes per 15 medium-power fields (200×); unchallenged, untreated mice showed <1 mast cell/medium-power field. (c) Total concentrations of IgE in serum were measured by using ELISA. Unchallenged, untreated mice produced <150 ng of IgE/ml. n = 7 to 9 mice per time point pooled from two separate experiments. Asterisks, P < 0.01 for group 3 compared to groups 1 and 2.

FIG. 4.

Effect of antibiotic therapy and fungal microbiota increase on lung leukocyte cytokine production in response to intranasal conidia exposure. Mice were treated as described in the legend to Fig. 1. At day 12 posttreatment, leukocytes were isolated from whole lungs and cultured (5 × 106 cells/ml) for 24 h without additional stimulation. Supernatants were collected and assayed by ELISA for IL-5 (a), IL-13 (b), and IFN-γ (c). Results are expressed as the increase in the levels of cytokine production compared to equivalent numbers of lung leukocytes from unchallenged, untreated mice (mean ± standard error of the mean). Lung leukocyte cultures from unchallenged, untreated mice produced <50 pg of IFNγ and IL-13/ml and <500 pg of IL-5/ml. n = 7 to 9 mice per time point. The experiments were repeated two times with similar results. Asterisks, P < 0.015 for group 3 compared to groups 1 and 2; double asterisks, P < 0.015 compared to group 1 only.

FIG. 5.

Effect of antibiotic therapy and fungal microbiota increase on lung inflammation and goblet cell metaplasia in response to intranasal conidia exposure. Mice were treated as described in the legend to Fig. 1. At day 12 posttreatment, lungs were harvested, fixed, sectioned, and stained with hematoxylin and eosin (H&E) (a to d) or PAS (e to h). In panel d, the arrow highlights the presence of numerous eosinophils in mice treated with antibiotics and colonized with GI C. albicans. In panel f, PAS stains mucus pink, indicating goblet cell metaplasia in the airway epithelium (arrow). Magnifications and groups are indicated on the photomicrographs.

FIG. 6.

Effect of CD4 T-cell depletion on the development of pulmonary hypersensitivity responses to conidia exposure in antibiotic-treated mice or mice with altered microbiota (Anb/Ca). All mice were treated with cefoperazone followed by C. albicans gavage and intranasal conidia challenge as described in the legend to Fig. 1. For CD4+-T-cell depletion, mice were injected with anti-CD4 monoclonal antibody (GK1.5) at days 0 and 7. n = 4 mice per group. Asterisks, P < 0.01 compared to undepleted challenged Anb/Ca mice (group 1).