TLR4 deficiency reduces pulmonary resistance to Streptococcus pneumoniae in gut microbiota-disrupted mice

Abstract

Streptococcus pneumoniae is a clinically important pathogen responsible for significant morbidity and mortality worldwide. Disruption of the host gut microbiota by antibiotics reduces the pulmonary resistance to S. pneumoniae. The aim of our study was to determine the potential role of TLR4 in the reduced pulmonary resistance to S. pneumoniae following gut microbiota disruption. Wild-type and TLR4-deficient mice were given broad-spectrum antibiotics for 3 weeks by oral gavage to disrupt the gut microbiota, and subsequently inoculated intra-nasally with S. pneumoniae. The extent of the decline in pulmonary resistance in both animal groups was evaluated in terms of the overall survival and pulmonary bacterial clearance. Both survival and pulmonary clearance of S. pneumoniae were lower in the TLR4-deficient mice with disrupted gut microbiota, compared to their intestinally healthy counterparts after pneumococcal infection. However, the degree of decline was much lower in the TLR4-deficient mice compared to the wild-type mice. Our findings indicate that impaired TLR4 function might be the basis of the reduced pulmonary resistance to S. pneumoniae caused by gut microbiota disruption.

Fig 1. Gut microbiota disruption decreases the pulmonary resistance to S. pneumoniae.

Wild-type mice were given broad-spectrum antibiotics or autoclaved saline. Three days after cessation of administration, mice were inoculated intra-nasally with 2.3 × 10⁸ CFU of S. pneumoniae. (A) The survival was observed for 7 days (n = 20 per group). (B) Bacterial load, and (C) TNF-α and (D) IL-1β concentration per mg lung tissue were determined 6 hours and 12 hours after infection (n = 6 per group). CFU, colony-forming units. Data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, analyzed with the log-rank test or unpaired Student’s t test.

Fig 2. Histopathology of the lung in wild-type mice 6 hours and 12 hours after pneumococcal infection.

Representative pictures of H & E stained lung tissues: (A) The picture of the lung tissue in gut microbiota-undisrupted uninfected mice (n = 6 per group). (B and C) Pictures of the lung tissue in gut microbiota-undisrupted mice 6 hours and 12 hours after infection (n = 6 per group). (D) The picture of the lung tissue in gut microbiota-disrupted uninfected mice (n = 6 per group). (E and F) Pictures of the lung tissue in gut microbiota-disrupted mice 6 hours and 12 hours after infection (n = 6 per group). Original magnification, × 20; scale bar = 2 mm.

Fig 3. TLR4 deficiency reduces host resistance to S. pneumoniae.

Wild-type and TLR4-deficient mice were intra-nasally inoculated with 2.3 × 10⁸ CFU of S. pneumoniae. (A) The survival was observed for 7 days (n = 20 per group). (B) Bacterial load, and (C) TNF-α and (D) IL-1β concentration per mg lung tissue were determined 6 hours and 12 hours after infection (n = 6 per group). CFU, colony-forming units. Data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, analyzed with the log-rank test or unpaired Student’s t test.

Fig 4. Histopathology of the lung in TLR4-deficient mice 6 hours and 12 hours after pneumococcal infection.

Representative pictures of H & E stained lung tissues: (A) The picture of the lung tissue in gut microbiota-undisrupted uninfected mice (n = 6 per group). (B and C) Pictures of the lung tissue in gut microbiota-undisrupted mice 6 hours and 12 hours after infection (n = 6 per group). (D) The picture of the lung tissue in gut microbiota-disrupted uninfected mice (n = 6 per group). (E and F) Pictures of the lung tissue in gut microbiota-disrupted mice 6 hours and 12 hours after infection (n = 6 per group). Original magnification, × 20; scale bar = 2 mm.

Fig 5. Gut microbiota disruption does not obviously affect the pulmonary resistance to S. pneumoniae in TLR4-deficient mice.

TLR4-deficient mice were treated with broad-spectrum antibiotics or autoclaved saline, and infected intra-nasally with 2.3 × 10⁸ CFU of S. pneumoniae. (A) The survival was observed for 7 days (n = 20 per group). (B) Bacterial load, and (C) TNF-α and (D) IL-1β concentration per mg lung tissue were determined 6 hours and 12 hours after infection (n = 6 per group). CFU, colony-forming units. Data are presented as means ± SD. Analyzed with the log-rank test or unpaired Student’s t test.