Administration of a bacterial lysate to the airway compartment is sufficient to inhibit allergen-induced lung eosinophilia in germ-free mice

Abstract

The nexus between eosinophils and microbes is attracting increasing attention. We previously showed that airway administration of sterile microbial products contained in dust collected from traditional dairy farms virtually abrogated bronchoalveolar lavage (BAL) eosinophilia and other cardinal asthma phenotypes in allergen-sensitized specific pathogen-free (SPF) mice. Interestingly, comparable inhibition of allergen-induced BAL eosinophilia and promotion of airway barrier integrity were found upon administration of a sterile, pharmacological-grade bacterial lysate, OM-85, to the airway compartment of allergen-sensitized SPF mice. Here, we asked whether intrinsic properties of airway-delivered microbial products were sufficient to inhibit allergic lung inflammation or whether these effects were mediated by reprogramming of the host microbiota. We compared germ-free (GF) mice and offspring of GF mice associated with healthy mouse gut microbiota and maintained under SPF conditions for multiple generations (Ex-GF mice). These mice were treated intranasally with OM-85 and evaluated in the ovalbumin and Alternaria models of allergic asthma focusing primarily on BAL eosinophilia. Levels of allergen-induced BAL eosinophilia were comparable in GF and conventionalized Ex-GF mice. Airway administration of the OM-85 bacterial lysate was sufficient to inhibit allergen-induced lung eosinophilia in both Ex-GF and GF mice, suggesting that host microbiota are not required for the protective effects of bacterial products in these models and local airway exposure to microbial products is an effective source of protection. OM-85-dependent inhibition of BAL eosinophilia in GF mice was accompanied by suppression of lung type 2 cytokines and eosinophil-attracting chemokines, suggesting that OM-85 may work at least by decreasing eosinophil lung recruitment.

Keywords: eosinophilia; germ-free mice; lung; microbes.

Fig. 1.

GF mice associated with healthy gut microbiota from birth (Ex-GF mice) exhibit attenuated AHR in 2 allergic asthma models. GF (black symbols) and Ex-GF (gray symbols) BALB/c mice were sensitized and challenged with OVA (A) or Alternaria alternata extracts (B). Shown are invasive measurements of total airway resistance (Rrs) from 2 independent experiments for each model. Numbers of mice/group are indicated in the figure captions. Symbols and bars denote mean and SEM, respectively. Statistical differences between dose-response curves of total airway resistance in GF and Ex-GF mice were assessed by linear mixed-effects models. ALT = Alternaria.

Fig. 2.

Airway administration of a bacterial lysate but not early association with healthy mouse gut microbiota inhibits allergen-induced BAL eosinophilia in GF mice BAL cellularity was measured in GF (black bars) and Ex-GF (gray bars) BALB/c mice sensitized and challenged with OVA (left and center) or Alternaria alternata (right). Additional groups of mice (hatched bars) received allergen + 9 (OVA model) or 14 (Alternaria [ALT] model) i.n. OM-85 treatments (1 mg/mouse/treatment). Shown are total numbers of BAL eosinophils (Eos)/mouse (mean and SEM) from 2 independent experiments for each model. Numbers of mice/group are indicated in the figure captions. Statistical differences between groups were assessed by an unpaired, 2-tailed t test after evaluating the normality of sample value distributions. Bolded P values remained significant after Bonferroni correction (P < 0.016).

Fig. 3.

Airway administration of a bacterial lysate but not early association with healthy mouse gut microbiota inhibits Alternaria (ALT)-induced expression of type 2 cytokines and eosinophil-attracting chemokines in the lung. mRNA levels of type 2 cytokines (Il5 [A], Il13 [B]) and eosinophil chemoattractants (Ccl11 [C], Ccl24 [D]) were measured by reverse-transcription qPCR in the lungs of GF (black bars) and Ex-GF (gray bars) BALB/c mice sensitized and challenged with ALT. Additional groups of mice (hatched bars) received ALT + 14 i.n. administrations of OM-85 (1 mg/mouse/treatment). Shown are cytokine and chemokine levels (mean and SEM) from 2 independent experiments. Numbers of mice/group are indicated in the figure captions. Statistical differences between groups were assessed by an unpaired, 2-tailed t test after evaluating the normality of sample value distributions. Bolded P values remained significant after Bonferroni correction (P < 0.016).