Sensing of interleukin-1 cytokines during Streptococcus pneumoniae colonization contributes to macrophage recruitment and bacterial clearance

Abstract

Streptococcus pneumoniae (the pneumococcus), a leading cause of bacterial disease, is most commonly carried in the human nasopharynx. Colonization induces inflammation that promotes the organism's growth and transmission. This inflammatory response is dependent on intracellular sensing of bacterial components that access the cytosolic compartment via the pneumococcal pore-forming toxin pneumolysin. In vitro, cytosolic access results in cell death that includes release of the proinflammatory cytokine interleukin-1β (IL-1β). IL-1 family cytokines, including IL-1β, are secreted upon activation of inflammasomes, although the role of this activation in the host immune response to pneumococcal carriage is unknown. Using a murine model of pneumococcal nasopharyngeal colonization, we show that mice deficient in the interleukin-1 receptor type 1 (Il1r1(-/-)) have reduced numbers of neutrophils early after infection, fewer macrophages later in carriage, and prolonged bacterial colonization. Moreover, intranasal administration of Il-1β promoted clearance. Macrophages are the effectors of clearance, and characterization of macrophage chemokines in colonized mice revealed that Il1r1(-/-) mice have lower expression of the C-C motif chemokine ligand 6 (CCL6), correlating with reduced macrophage recruitment to the nasopharynx. IL-1 family cytokines are known to promote adaptive immunity; however, we observed no difference in the development of humoral or cellular immunity to pneumococcal colonization between wild-type and Il1r1(-/-) mice. Our findings show that sensing of IL-1 cytokines during colonization promotes inflammation without immunity, which may ultimately benefit the pneumococcus.

FIG 1

Pneumococcal infection results in IL-1β release in vitro. (A and B) Bone marrow-derived macrophages (BMMs) were infected with wild-type (WT) or pneumolysin-deficient (ply−) S. pneumoniae strains or left uninfected (Un). Where indicated, BMMs were treated with 20 μM cytochalasin D (CytD) to block phagocytosis. Supernatants were collected at 24 h postinfection. (A) Western blot of supernatants for the presence of caspase-1 (Casp-1) and interleukin-1 beta (IL-1β). BMMs primed with 0.5 μg/ml lipopolysaccharide (LPS) and stimulated with ATP were used as a positive control (PC). Representative images are shown. (B) Amounts of IL-1β in supernatants were quantified by ELISA. Results are from 2 independent experiments. Error bars represent ± standard error of the mean (SEM). Significance was determined by one-way analysis of variance (ANOVA) with Newman-Keuls posttest. ***, P < 0.001.

FIG 2

Expression of IL-1 cytokines is upregulated during S. pneumoniae colonization and is dependent on TLR2. (A and B) Wild-type (black bars) or TLR2-deficient (Tlr2^−/− [white bars]) mice were intranasally inoculated with 10⁷ CFU of S. pneumoniae (Spn). At 14 days postcolonization, the upper respiratory tracts were lavaged with RLT RNA lysis buffer. Gene expression relative to PBS (mock)-inoculated mice was measured by quantitative RT-PCR for Il1a (A) and Il1b (B). Results are from at least 2 independent experiments (n > 10 mice). Error bars represent ± SEM. Significance was determined by the Mann-Whitney U test. *, P < 0.05; ***, P < 0.001. ns, not significant.

FIG 3

Sensing of IL-1 family cytokines is important for bacterial clearance and macrophage recruitment. (A to E) Wild-type (WT [black]) or IL-1 receptor-deficient (Il1r1^−/− [gray]) mice were intranasally inoculated with 10⁷ CFU of S. pneumoniae, and at the time points indicated, PBS lavages of the upper respiratory tract were obtained. (A) Bacterial numbers of colonizing pneumococci were quantified by plating of nasal lavages. The dashed line indicates the limit of detection. Results are from 2 to 4 independent experiments (n ≥ 10 mice per group). Error bars represent ± SEM. Significance was determined by Kruskal-Wallis test with Dunn's posttest. **, P < 0.01; ns, not significant. (B to E) Cellular infiltrates in the nasal lavage samples from 5 mice were measured by flow cytometry. Quantification of neutrophils (Ly6G⁺, CD11b⁺) and monocytes/macrophages (F4/80⁺, CD11b⁻) after 3 (B and C) and 14 (D and E) days of colonization is shown. The total events available for capture were used due to the low cell counts that are commonly seen in nasal lavage samples. Results are from 4 independent experiments. Error bars represent ± SEM. Significance was determined by Student's t test or Mann-Whitney U test as appropriate. *, P < 0.05; ns, not significant. (F and G) WT (F) or Il1r1^−/− (G) mice were inoculated with 10⁷ CFU of a pneumolysin-deficient S. pneumoniae strain and intranasally administered recombinant IL-1β (open circles) or vehicle control (PBS [closed circles]) every other day for 14 days. Nasal lavage samples were obtained, and numbers of pneumococci were measured by plating serial dilutions. The dashed line indicates the limit of detection. Results are from 2 experiments (n ≥ 4 mice per group). Error bars represent ± SEM. Significance was determined by Kruskal-Wallis test with Dunn's posttest or Student's t test as appropriate. *, P < 0.05; ns, not significant.

FIG 4

Il1r1^−/− mice have altered C-C chemokine profiles during S. pneumoniae colonization. (A and B) Wild-type (WT [black bars]) or IL-1 receptor-deficient (Il1r1^−/− [gray bars]) mice were inoculated with 10⁷ CFU of S. pneumoniae. At 3 (A) and 14 (B) days postcolonization, RLT RNA lysis buffer lavage samples from the upper respiratory tract were obtained, and gene expression of Ccl2, Ccl6, Ccl7, and Ccl8 was measured by quantitative RT-PCR. Values are relative to PBS (mock)-inoculated mice. Results are from 2 to 4 independent experiments (n ≥ 9 mice). Error bars represent ± SEM. Significance was determined by Mann-Whitney U test. *, P < 0.05; **, P < 0.01. ns, not significant.

FIG 5

Sensing of IL-1 does not alter adaptive immunity to the pneumococcus. (A and B) Wild-type (WT [black bars]) or IL-1 receptor-deficient (Il1r1^−/− [gray bars]) mice were intranasally colonized with S. pneumoniae (Spn) for 14 days. (A) RLT RNA lysis buffer lavage samples from the respiratory tract were obtained, and expression of Il17a was measured by quantitative RT-PCR. Values are reported as fold change relative to PBS (mock)-inoculated mice. Significance was determined by Student's t test. ns, not significant. (B) Total anti-pneumococcal serum IgG levels were measured by ELISA. Values are expressed as geometric mean titer (GMT). Significance was determined by Kruskal-Wallis test with Dunn's posttest. ns, not significant. (C) WT (black circles) or Il1r1^−/− (gray circles) mice were inoculated with 10⁷ CFU of S. pneumoniae. For primary (1°) colonization, mice were sacrificed at day 3 postcolonization, PBS nasal lavage samples were obtained, and bacterial density was measured by plating. For secondary (2°) colonization, mice were allowed 8 weeks to clear the pneumococci and then rechallenged with an isogenic S. pneumoniae strain distinguishable by antibiotic resistance. Nasal lavage samples were obtained at day 4 post-secondary colonization, and bacterial density was quantified by plating. The dashed line indicates the limit of detection. Results are from 2 to 5 independent experiments (n ≥ 10 mice). Error bars represent ± SEM. Significance was determined by the Kruskal-Wallis test with Dunn's posttest. **, P < 0.01; ***, P < 0.001.