Streptomycin treatment alters the intestinal microbiome, pulmonary T cell profile and airway hyperresponsiveness in a cystic fibrosis mouse model

Abstract

Cystic fibrosis transmembrane conductance regulator deficient mouse models develop phenotypes of relevance to clinical cystic fibrosis (CF) including airway hyperresponsiveness, small intestinal bacterial overgrowth and an altered intestinal microbiome. As dysbiosis of the intestinal microbiota has been recognized as an important contributor to many systemic diseases, herein we investigated whether altering the intestinal microbiome of BALB/c Cftr(tm1UNC) mice and wild-type littermates, through treatment with the antibiotic streptomycin, affects the CF lung, intestinal and bone disease. We demonstrate that streptomycin treatment reduced the intestinal bacterial overgrowth in Cftr(tm1UNC) mice and altered the intestinal microbiome similarly in Cftr(tm1UNC) and wild-type mice, principally by affecting Lactobacillus levels. Airway hyperresponsiveness of Cftr(tm1UNC) mice was ameliorated with streptomycin, and correlated with Lactobacillus abundance in the intestine. Additionally, streptomycin treated Cftr(tm1UNC) and wild-type mice displayed an increased percentage of pulmonary and mesenteric lymph node Th17, CD8 + IL-17+ and CD8 + IFNγ+ lymphocytes, while the CF-specific increase in respiratory IL-17 producing γδ T cells was decreased in streptomycin treated Cftr(tm1UNC) mice. Bone disease and intestinal phenotypes were not affected by streptomycin treatment. The airway hyperresponsiveness and lymphocyte profile of BALB/c Cftr(tm1UNC) mice were affected by streptomycin treatment, revealing a potential intestinal microbiome influence on lung response in BALB/c Cftr(tm1UNC) mice.

Figure 1. Small intestinal bacterial load and microbiome of female BALB/c Cftr^tm1UNC mice and wild-type littermates, untreated and treated with streptomycin beginning in utero until death at 12 weeks of age.

(A) Bacterial load was measured using quantitative real-time PCR of the 16S rRNA of DNA isolated from 150mg of small intestinal contents. Average ± standard deviation is shown (n = 8–13 mice per group). *indicates a significant difference between groups, P < 0.05, by Student’s t-test. NS = non-significant. (B) Two dimensional non-metric multidimensional scaling (NMDS) of the Bray-Curtis dissimilarity between microbiome samples. (C) 16S rRNA gene frequencies of the most abundant operational taxonomic units (OTU) classified to the closest related taxon.

Figure 2. Architecture of the small intestine in female BALB/c Cftr^tm1UNC mice and wild-type littermates, untreated and treated with streptomycin beginning in utero until death at 12 weeks of age.

(A) Crypt to Villus axis height (CVA) was measured by image analysis of histological sections for 25 ileal CVAs per mouse. (B) Muscularis externa thickness was measured by image analysis of histological sections. (C) Representative ileal sections showing CF CVA distention and muscle thickness increase in BALB/c Cftr^tm1UNC mice. Hematoxylin and Eosin stain, magnification 200X. (D) Total goblet cells per CVA were measured by image analysis of histological sections for 25 ileal CVAs per mouse. (E) Representative ileal sections showing CF goblet cell number increase. Periodic acid-Schiff/Alcian Blue stain, magnification 400X. Data presented as the mean ± standard deviation (n = 7–9 mice per group). *indicates a significant difference between groups, P < 0.05, by Student’s t-test. NS = non-significant.

Figure 3. Left femur architecture of female BALB/c Cftr^tm1UNC mice untreated or treated with Streptomycin in utero until death at 12 weeks of age, and of wild-type littermates.

(A) Bone mineral density (BMD) (B) Bone volume to tissue volume (BV/TV) (C) Thickness of individual trabeculae (TrTh) (D) Number of trabeculae in a given area (TrNo). Average ± standard deviation (n = 4–5 mice per group). Bones from (E) WT untreated, (F) Cftr^tm1UNC untreated and (G) Cftr^tm1UNC streptomycin treated mice were dissected free of soft tissue, fixed and scanned on a SkyScan 1072 with three-dimensional reconstruction showing fewer, thinner trabeculae and bone volume in the Cftr^tm1UNC mice compared to WT mice. *indicates a significant difference between groups, P < 0.05, by Student’s t-test. NS = non-significant.

Figure 4. Airway hyperresponsiveness of female BALB/c Cftr^tm1UNC mice and wild-type littermates, untreated and treated with streptomycin beginning in utero until death at 12 weeks of age.

Tracheostimized mice received saline (zero) and increasing doses of aeronebulized methacholine and mechanics were assayed on a FlexiVent instrument. Average Rmax, defined as maximal resistance at each dose, ± SEM is shown (n = 9–12 mice per group). Vertical bar indicates a significant difference among groups as measured by repeated measures ANOVA. *indicates a significant difference between untreated Cftr^tm1UNC mice and untreated WT mice by Bonferroni post hoc test, P < 0.05. # indicates a significant difference between untreated Cftr^tm1UNC mice and streptomycin treated Cftr^tm1UNC mice by Bonferroni post hoc test, P < 0.05.

Figure 5. Correlation of airway hyperresponsiveness to total Lactobacillus levels.

(A) Abundance of total Lactobacillus within the small intestinal microbiome, based on sequences grouped by taxonomical assignment. Average ± standard deviation is shown (n = 5–13 mice per group). *indicates a significant difference between groups, P < 0.05, by Student’s t-test. (B) Correlation of total Lactobacillus abundance to airway hyperresponsiveness in streptomycin treated and untreated Cftr^tm1UNC mice. Pearson coefficient is shown.

Figure 6. T lymphocyte subsets in the lungs and mesenteric lymph nodes of female BALB/c Cftr^tm1UNC mice and wild-type littermates, untreated or treated with streptomycin beginning in utero until death at 12 weeks of age, as determined by flow cytometry.

Specific cytokine producing γδ T cells as a percent of total (A) lung or (D) mesenteric lymph node lymphocytes. Specific cytokine producing cells as a percent of total (B) lung or (E) mesenteric lymph node CD4+ lymphocytes; and as a percent of total (C) lung or (F) mesenteric lymph node CD8+ lymphocytes. IL13 producing γδ T cells and IL13 producing CD8+ lymphocytes were below detection levels in the lungs and mesenteric lymph nodes. Average ± standard deviation is shown (n = 8–14 mice per group). *indicates a significant difference between groups, P < 0.05, by Student’s t-test. NS = non-significant.