Journal: PLoS Pathogens
Article Title: Clearance of Pneumococcal Colonization in Infants Is Delayed through Altered Macrophage Trafficking
Figure Lengend Snippet: Depleting the microbiota limits infant Ccl2 expression and accelerates pneumococcal clearance. (A) Breeding pairs were placed on sterile filtered tap water or water containing 5 antibiotics (Abx) for at least 5 days prior to giving birth, and continued until the infant mice were 10d old. Mice were sacrificed, and nasal lavages obtained with PBS. DNA was isolated from lavage fluid, and qPCR used to measure the number of copies of 16S bacterial ribosomal DNA. (B-C) Adult mice were maintained on sterile filtered tap water or water containing 5 antibiotics for at least 2 weeks. Breeding pairs were provided water as above. Mice were sacrificed, and nasal lavages obtained with RLT lysis buffer. RNA was isolated and reverse-transcribed into cDNA, and qRT-PCR used to measure relative expression of Ccl2 (B) and Ccl7 (C) in the nasopharynx. (D) Infant mice were exposed to tap water or antibiotics as in the previously described protocol. Treatments were continued until infant mice were 6d old, then all mice were given tap water to drink. 24 hrs later, mice were inoculated with pneumococci. At 7 dpi, mice were sacrificed and nasal lavages fixed and stained for flow cytometry to measure the number of macrophages present in the airway lumen. (E-F) Infant mice were treated as in (D). At 14d (E) and 7d (F) post-inoculation, mice were sacrificed. Nasal lavages were obtained and plated on media containing neomycin and catalase to measure pneumococcal density in the URT. (G) Ccl2 expression was measured in the nasopharynx of tap- or antibiotic-water exposed conventionally-reared (Conv), or germ-free infant mice. Data are represented as mean +/- SEM. Horizontal lines indicate median values. n.s., not significant. * = p
Article Snippet: qRT-PCR RNA was obtained from URT epithelium by lavage with RLT buffer (Qiagen) with 1% β-mercaptoethanol, or from cultured peritoneal macrophages by lysing cells in RLT buffer with 1% β-mercaptoethanol and frozen at -80°C until used.
Techniques: Expressing, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction, Lysis, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry