Wild-Mouse-Derived Gut Microbiome Transplantation in Laboratory Mice Partly Alleviates House-Dust-Mite-Induced Allergic Airway Inflammation

Abstract

Laboratory mice are instrumental for preclinical research but there are serious concerns that the use of a clean standardized environment for specific-pathogen-free (SPF) mice results in poor bench-to-bedside translation due to their immature immune system. The aim of the present study was to test the importance of the gut microbiota in wild vs. SPF mice for evaluating host immune responses in a house-dust-mite-induced allergic airway inflammation model without the influence of pathogens. The wild mouse microbiome reduced histopathological changes and TNF-α in the lungs and serum when transplanted to microbiota-depleted mice compared to mice transplanted with the microbiome from SPF mice. Moreover, the colonic gene expression of Gata3 was significantly lower in the wild microbiome-associated mice, whereas Muc1 was more highly expressed in both the ileum and colon. Intestinal microbiome and metabolomic analyses revealed distinct profiles associated with the wild-derived microbiome. The wild-mouse microbiome thus partly reduced sensitivity to house-dust-mite-induced allergic airway inflammation compared to the SPF mouse microbiome, and preclinical studies using this model should consider using both 'dirty' rewilded and SPF mice for testing new therapeutic compounds due to the significant effects of their respective microbiomes and derived metabolites on host immune responses.

Keywords: animal models; fecal microbiota transplantation; feral mice; gut microbiota; immunity; microbial metabolites; translatability; wild mice.

Figure 1

Compared with SPF-microbiome-associated mice, wild-mouse microbiome-associated mice showed reduced allergic airway inflammation: (A) Experimental design showing the timeline of antibiotic treatment (ABX) and microbiome transplantation (FMT) as well as house dust mite (HDM) sensitization and challenges before euthanization of male and female wild mice (wild) and specific pathogen-free (SPF) microbiome-associated BALB/c mice; (B) body weight; (C) bronchoalveolar fluid (BAL) cell counts; (D) numbers of eosinophils (CD11c-CD3-CD19-CD11b+Ly6G-SigF+) in the BAL fluid; (E) numbers of neutrophils (CD11c-CD3-CD19-CD11b+Ly6G+) in the BAL fluid; (F) numbers of T cells (CD11c-CD3+MHCII-) in the BAL fluid; (G) numbers of B cells (CD11c-CD19+MHCII+) in the BAL fluid; (H) lung concentration of TNF-α; (I) serum concentration of TNF-α; (J) serum total IgE concentrations; (K) histopathological inflammatory score in lung tissue, including representative images of H&E-stained sections (L,M); (N) percentage of mucin-positive bronchi, including representative images of PAS-stained sections (O,P), are shown for the wild (n = 13) and SPF (n = 8) microbiome-associated mice when euthanized at 7 weeks of age. All p values are given (significance < 0.05). The mean and SEM are shown.

Figure 2

Transplantation of wild-mouse-derived microbiome resulted in significantly increased expression of the Muc1 gene, which is related to mucosal barrier function. qPCR expression analysis of gut-barrier-related and immunoregulatory genes in the colon (A) and ileum (B) of wild (n = 13) and SPF (n = 8) microbiome-associated male and female BALB/c mice at 7 weeks of age after induction of HDM-induced allergic airway inflammation. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Red stars indicate significance after FDR correction. The mean and SD are shown.

Figure 3

Differential abundance analysis revealed distinct microbiome taxa, but not mycobiome, between wild- and SPF-microbiome-associated mice: (A) Chao1 alpha diversity analysis depicting the fecal microbiome population diversity among the wild- and SPF-microbiome-associated pregnant mothers at birth (wild, n = 5 and SPF, n = 4) and their male and female pups at weaning (wild, n = 13 and SPF, n = 8); (B) principal coordinates analysis plot of the 16S rRNA gene amplicon sequencing of fecal samples from mothers and pups based on the Bray–Curtis dissimilarity method at the feature level; (C) pairwise permutational MANOVA (PERMANOVA) test results showing the differences in beta diversity between the groups. The F value (referring to the test statistic that measures the ratio of the variance between group means to the variance within the groups), the R² value (representing the proportion of total variation in the data that is explained by the grouping variable), p values, and FDR-corrected p values are given for comparisons of the beta diversity observed in (B) of wild- and SPF-microbiome-associated mothers and pups as indicated; (D) heatmap visualizing the relative abundance of the top genera within each mouse fecal sample as well as in the donor cecum samples. The dendrogram cluster on the right side of the heatmap shows the abundance pattern of particular genera between sample groups; (E) bar chart showing the relative abundance of detected mycobiome taxa by ITS sequencing.

Figure 4

The wild and SPF microbiomes induced distinct metabolomic profiles in the cecal contents of the recipient mice: (A) score plot visualizing the distribution of samples based on their scores on the OPLS-DA components; (B) loadings plot showing the variable of importance for the projection (VIP) in component 1; (C) heatmaps showing differential levels of cecal metabolites between wild- and SPF-microbiome-associated pups at euthanization after HDM-induced airway inflammation; (D) differential concentrations of metabolites between wild- and SPF-microbiome-associated mice. An overview of the OPLS-DA model is shown in Figure S3. All p values are given (significance < 0.05). The mean and SEM are shown.