Mucosal administration of flagellin protects mice from Streptococcus pneumoniae lung infection

Abstract

Streptococcus pneumoniae is a major cause of pneumonia in infants and the elderly. Innate defenses are essential to the control of pneumococcal infections, and deficient responses can trigger disease in susceptible individuals. Here we showed that flagellin can locally activate innate immunity and thereby increase the resistance to acute pneumonia. Flagellin mucosal treatment improved S. pneumoniae clearance in the lungs and promoted increased survival of infection. In addition, lung architecture was fully restored after the treatment of infected mice, indicating that flagellin allows the reestablishment of steady-state conditions. Using a flagellin mutant that is unable to signal through Toll-like receptor 5 (TLR5), we established that TLR5 signaling is essential for protection. In the respiratory tract, flagellin induced neutrophil infiltration into airways and upregulated the expression of genes coding for interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), CXCL1, CXCL2, and CCL20. Using depleting antibodies, we demonstrated that neutrophils are major effectors of protection. Further, we found that B- and T-cell-deficient SCID mice clear S. pneumoniae challenge to the same extent as immunocompetent animals, suggesting that these cell populations are not required for flagellin-induced protection. In conclusion, this study emphasizes that mucosal stimulation of innate immunity by a TLR not naturally engaged by S. pneumoniae can increase the potential to cure pneumococcal pneumonia.

FIG. 1.

Flagellin protects BALB/c mice against a lethal respiratory challenge with S. pneumoniae. BALB/c mice (n = 8) were infected i.n. with 4 × 10⁵ CFU of S. pneumoniae (Sp) serotype 1 in saline alone (filled squares) or in saline supplemented either with 1 μg flagellin (FliC) (open circles) or with 1 μg trypsin-digested flagellin (FliC/T) (filled circles). (A) The survival of mice was monitored daily. The survival of the FliC-treated group was statistically significantly different from that of the saline group and the FliC/T-treated group. The results are representative of 1 out of 3 experiments. (B and C) CFU counts were determined in the lungs (B) or spleens (C) of mice (n = 6) at days 1, 2, and 7 after challenge with S. pneumoniae alone (filled squares) or S. pneumoniae plus flagellin (open circles). The solid lines represent means, and the dashed lines represent the CFU detection limit. The daggers indicate that all animals in the group were dead by day 7. Significant differences between groups (P < 0.05) are indicated by asterisks. Results are representative of 1 out of 2 experiments.

FIG. 2.

Therapeutic effect of FliC after S. pneumoniae challenge. BALB/c mice were infected intranasally with 4 × 10⁵ CFU of S. pneumoniae (filled squares); 24 h after challenge, 1 μg of FliC was administered i.n. to one group of animals (open circles). CFU counts were determined in the lungs (A) or spleens (B) at day 2 and day 7 after challenge with S. pneumoniae. Solid and dashed lines represent means and the CFU detection limit, respectively. The dagger indicates that all animals in the group were dead by day 7. Significant differences between groups (P < 0.05) are indicated by asterisks. Results are representative of 1 out of 2 experiments.

FIG. 3.

TLR5 signaling is required for flagellin-mediated protection against S. pneumoniae infection. BALB/c mice (n = 8) were infected i.n. with 4 × 10⁵ CFU of S. pneumoniae (Sp) serotype 1 in saline either alone (filled square) or supplemented either with 1 μg FliC_Δ174-400 (open circles) or with 1 μg FliC_{Δ174-400/89-96*}, lacking the TLR5-signaling motif (filled circle). Survival of mice was recorded daily. The survival of the FliC_Δ174-400-treated group was statistically significantly different from that of the untreated and FliC_{Δ174-400/89-96*}-treated groups (P < 0.05). Results are representative of 1 out of 2 experiments.

FIG. 4.

Flagellin upregulates chemokine and cytokine gene expression in the lungs of infected animals. BALB/c mice (n = 5) were instilled i.n. either with saline, with 1 μg flagellin (FliC), or with 4 × 10⁵ CFU of S. pneumoniae (Sp) serotype 1 either in saline alone or in saline supplemented with 1 μg FliC. Mice were sacrificed 24 h after challenge, and the expression of the Cxcl1, Cxcl2, Ccl20, Il6, and Tnf genes was assessed by qRT-PCR on total lung RNA. Values were normalized to that of β-actin mRNA, and the results are expressed relative to mRNA levels in saline-treated mice. Data are means; error bars represent standard errors of the means. Significant differences between groups (P < 0.05) are indicated by asterisks;. Results are representative of 1 out of 3 experiments.

FIG. 5.

Flagellin promotes heavy cell infiltration and lung remodeling and induces rapid restoration of tissue architecture. Mouse lungs were obtained at day 1 or day 7 after intranasal instillation of saline (A and F), 4 × 10⁵ CFU of S. pneumoniae serotype 1 (D), S. pneumoniae plus 1 μg flagellin (B and C), or flagellin alone (E) and were stained with hematoxylin and eosin. B, bronchiole; BV, blood vessel. Thin arrows indicate edema; arrowheads indicate cellular infiltration. On day 1, edema and recruitment of inflammatory cells were evident in peribronchial and perivascular regions in flagellin-treated animals. Infected but untreated animals showed less inflammatory-cell infiltration and edema at 24 h than flagellin-treated and infected mice. Flagellin-treated animals showed resolution of inflammation by day 7. Total magnification, ×200 for panels and ×1,000 for insets. The images shown are representative of the analysis of 6 sections for each individual and 3 mice per experimental group.

FIG. 6.

Flagellin-mediated neutrophil recruitment is required for protection against pneumonia. (A) Kinetics of PMN infiltration. BALB/c mice (n = 3) were infected i.n. with 4 × 10⁵ CFU of S. pneumoniae (Sp) serotype 1 in saline alone (filled squares) or in saline supplemented with 1 μg flagellin (FliC) (open circles). Animals were sacrificed at the indicated time points after challenge. The total number of cells in bronchoalveolar lavage fluid was determined, and the number of neutrophils (defined as Ly6C⁺ Ly6G⁺ CD11b⁺ cells) was determined by flow cytometry. (B) Depletion of PMN abrogates FliC-mediated protection. BALB/c mice (n = 6) were injected with 100 μg of an anti-Gr-1 monoclonal antibody or a control antibody. Animals were infected i.n. 24 h later with 4 × 10⁵ CFU of S. pneumoniae serotype 1 in saline alone (squares) or in saline supplemented with 1 μg flagellin (FliC) (circles). Survival was recorded daily. The survival of the nondepleted FliC-treated group was statistically significantly different from that of depleted FliC-treated animals or nondepleted untreated animals (P < 0.05). (C) BALB/c mice were infected intranasally with 4 × 10⁵ CFU of S. pneumoniae (filled square); 24 h after challenge, 1 μg of FliC was administered i.n. to one group of animals (open circles). At selected time points after infection (days 1, 2, and 7), BAL was carried out for 3 mice per time point in order to perform FACS analysis. The total number of cells in BAL fluid was determined, and the number of neutrophils was determined by flow cytometry. The dagger marks the time point at which all animals in the group were dead. For panels A and C, data are means; error bars represent the standard errors of the means; and significant differences between groups (P < 0.05) are indicated by asterisks. The results are representative of 1 out of 2 experiments.

FIG. 7.

Flagellin-mediated protection against pneumonia does not depend on B and T lymphocytes. (A and B) BALB/c (solid symbols) or SCID (open symbols) mice (n = 6) were infected i.n. with 2 × 10⁷ CFU of S. pneumoniae serotype 1 in saline alone (squares) or in saline supplemented with 1 μg flagellin (FliC) (circles). Bacterial counts were determined in lung (A) and spleen (B) homogenates 36 h after challenge. Significant differences between groups (P < 0.05) are indicated by asterisks. Results are representative of 2 experiments. (C) Recruitment of PMN (defined as Ly6G⁺ Ly6C⁺ CD11b⁺ CD11c⁻ cells) into BAL fluid and lungs after i.n. instillation of 1 μg flagellin for SCID (open bars) or BALB/c (filled bars) mice (n = 5). Note that the numbers of PMN recruited in the two strains were not statistically significantly different. The results are presented as medians and standard errors of the means.