Early life antibiotic-driven changes in microbiota enhance susceptibility to allergic asthma

Abstract

Allergic asthma rates have increased steadily in developed countries, arguing for an environmental aetiology. To assess the influence of gut microbiota on experimental murine allergic asthma, we treated neonatal mice with clinical doses of two widely used antibiotics--streptomycin and vancomycin--and evaluated resulting shifts in resident flora and subsequent susceptibility to allergic asthma. Streptomycin treatment had little effect on the microbiota and on disease, whereas vancomycin reduced microbial diversity, shifted the composition of the bacterial population and enhanced disease severity. Neither antibiotic had a significant effect when administered to adult mice. Consistent with the 'hygiene hypothesis', our data support a neonatal, microbiota-driven, specific increase in susceptibility to experimental murine allergic asthma.

Figure 1

Neonatal vancomycin treatment exacerbates allergic asthma. (A) Total cellular infiltrates from bronchoalveolar lavage (BAL) of control or antibiotic-treated mice challenged with ovalbumin (OVA) or PBS. (B) Eosinophil numbers in the BAL quantified by cytospin. (C) Serum OVA-specific IgE responses measured by enzyme-linked immunosorbent assay. (D) Airway hyperresponsiveness, measured by changes in resistance (R) in response to increasing doses of methacholine administered intravenously. (E) Total pathological scores and representative haematoxylin- and eosin-stained lung sections. Scale bar, 300 μm. All assessments were made on day 26. The data are shown as means of 5–7 mice per group±s.e.m. and represent at least two independent experiments. Statistics shown are based on comparisons to OVA-challenged controls. Antibiotic-treated animals in B–E were treated neonatally. ^*P<0.05, ^**P<0.01; Abx, antibiotics; ND, none detected; NS, not significant; Strep, streptomycin; Vanco, vancomycin.

Figure 2

Antibiotic treatment alters gut bacterial communities. (A) Total bacteria in faeces of control or antibiotic-treated mice quantitated by SYBR green. These data are shown as means of 3–5 mice per group±s.e.m. and represent two independent experiments. ^*P<0.05 relative to control; NS, not significant. (B) Bacterial communities from ileum and faeces of naive or ovalbumin (OVA)-challenged control and antibiotic-treated mice were compared using principal coordinate analysis (PCO). (C) Family level phylogenetic classification of 16S rRNA gene frequencies in faeces collected from naive or OVA-challenged control and antibiotic-treated animals. Those indicated with a classification level other than family level (f) could only be identified confidently to the level indicated. Classification scheme: k, kingdom; p, phylum; c, class; o, order; f, family. Each bar represents one mouse. Rare taxa were removed from the legend, but still included in the graph. Antibiotic-treated animals in A and B were treated neonatally. Abx, antibiotics; Strep, streptomycin; Vanco, vancomycin.

Figure 3

Vancomycin treatment reduces CD4⁺CD25⁺Foxp3⁺ regulatory T-cell accumulation in the colon but not in the lung. At 7 weeks of age, the percentage of CD25⁺Foxp3⁺ cells within the CD45⁺CD4⁺ cell population in the colon (A) or lung (B) of naive mice treated with neonatal antibiotics was analysed. The data shown are means of three mice per group±s.e.m. and represent three independent experiments. Vanco, vancomycin; Strep, streptomycin. ^**P<0.01.