Toll-like receptor 2 has a prominent but nonessential role in innate immunity to Staphylococcus aureus pneumonia

Abstract

Staphylococcus aureus is an important cause of acute bacterial pneumonia. Toll-like receptor 2 (TLR2) recognizes multiple components of the bacterial cell wall and activates innate immune responses to gram-positive bacteria. We hypothesized that TLR2 would have an important role in pulmonary host defense against S. aureus TLR null (TLR2^-/-) mice and wild type (WT) C57BL/6 controls were challenged with aerosolized S. aureus at a range of inocula for kinetic studies of cytokine and antimicrobial peptide expression, lung inflammation, bacterial killing by alveolar macrophages, and bacterial clearance. Survival was measured after intranasal infection. Pulmonary induction of most pro-inflammatory cytokines was significantly blunted in TLR2^-/- mice 4 and 24 h after infection in comparison with WT controls. Bronchoalveolar concentrations of cathelicidin-related antimicrobial peptide also were reduced in TLR2^-/- mice. Lung inflammation, measured by enumeration of bronchoalveolar neutrophils and scoring of histological sections, was significantly blunted in TLR2^-/- mice. Phagocytosis of S. aureus by alveolar macrophages in vivo after low-dose infection was unimpaired, but viability of ingested bacteria was significantly greater in TLR2^-/- mice. Bacterial clearance from the lungs was slightly impaired in TLR2^-/- mice after low-dose infection only; bacterial elimination from the lungs was slightly accelerated in the TLR2^-/- mice after high-dose infection. Survival after high-dose intranasal challenge was 50-60% in both groups. TLR2 has a significant role in early innate immune responses to S. aureus in the lungs but is not required for bacterial clearance and survival from S. aureus pneumonia.

Keywords: Staphylococcus aureus; Alveolar macrophages; bacterial pneumonia; innate immunity; toll‐like receptor 2.

Figure 1

Lung cytokine responses to inhaled Staphylococcus aureus are blunted in TLR2^−/− mice. Cytokines were measured by microbead array or ELISA in lung homogenates harvested 4 h, 24 h, and 48 h after deposition of 4.5 × 10⁶ CFU/lung (intermediate dose; A–D, I–L) or 1.4 × 10⁷ CFU/lung (high dose; E–H, M–P). Data are mean ± SEM; n = 8 except for 48 h after intermediate dose and 4 h after high dose where n = 4; and represent the combined results of two separate experiments at each bacterial inoculum. *P < 0.05 versus WT; **P < 0.005 versus WT. Protein levels in uninfected lung homogenates were as follows (mean ± SEM, n = 4, all pg/mL): TNF‐α, 41 ± 9; IL‐1β, 311 ± 55; IL‐1α, 229 ± 27; MIP‐2 (CXCL2), 67 ± 43; KC (CXCL1), 264 ± 45; MCP‐1 (CCL2), 236 ± 97; IL‐6, 48 ± 7; GM‐CSF, 45 ± 13.

Figure 2

Reduced neutrophil response to inhaled Staphylococcus aureus in TLR2^−/− mice. Bronchoalveolar lavage (BAL) neutrophils (PMN) and mononuclear cells (MN) were determined at serial time points after inhalation of S. aureus at two different inocula: 4.5 × 10⁶ CFU/lung (intermediate dose; A, B), or 1.4 × 10⁷ CFU/lung (high dose; C, D). Data are mean ± SEM, n = 8, except for 48 h after intermediate dose and 4 h after high dose where n = 4; and represent the combined results of two separate experiments at each inoculum. *P < 0.05 versus WT; **P < 0.005 versus WT.

Figure 3

Reduced lung inflammation in TLR2^−/− mice with Staphylococcus aureus pneumonia. Lung tissue was harvested 4 h and 24 h after inhalation of S. aureus (6.6 × 10⁶ CFU/lung), sectioned, and stained with hematoxylin and eosin. Images of lesions representing the upper range of severity scores demonstrate less intense inflammation in TLR2^−/− mice.

Figure 4

Reduced lung inflammation in TLR2^−/− mice with Staphylococcus aureus pneumonia. Lung tissue was harvested 4 h and 24 h after inhalation of S. aureus (6.6 × 10⁶ CFU/lung). Hematoxylin and eosin stained sections were scored for A. Bronchial, B. Alveolar, and C. Vascular inflammation and necrosis on a 4 point scale. Each data point represents the composite score from examination of 4 widely spaced sections of lung from an individual mouse. *P < 0.05 versus WT.

Figure 5

Decreased release of cathelicidin‐related antimicrobial peptide (CRAMP) in TLR2^−/− mice. CRAMP was measured by ELISA in bronchoalveolar lavage fluid 24 h after inhalation of Staphylococcus aureus (deposition 6.6 × 10⁶ CFU/lung). Each data point represents an individual mouse. **P = 0.001

Figure 6

Increased viability of Staphylococcus aureus ingested by TLR2‐deficient alveolar macrophages (AM) in vivo. Bronchoalveolar cells (≥99% AM) were harvested 4 h after low‐dose airborne infection with S. aureus (deposition 4.3 × 10⁵ CFU/lung) (A). Cytocentrifuge samples were scored for the number of cell‐associated bacteria (B, mean ± SEM, n = 10). The cell pellets were lysed and quantitatively cultured (C). Lung homogenates were cultured 24 h after low‐dose infection (D). Data represent the combined results of two independent experiments. *P < 0.05; **P < 0.005.

Figure 7

Bacterial clearance after inhalation of Staphylococcus aureus is unimpaired in TLR2^−/− mice after intermediate or high‐dose infection. Bacterial burdens in lung and spleen were measured at serial time points after deposition of 4.5 × 10⁶ CFU/lung (intermediate dose; A, B) or 1.4 × 10⁷ CFU/lung (high dose; C, D). Data are mean ± SEM, n = 8, except for 48 h after intermediate dose and 4 h after high dose where n = 4; and represent the combined results of two separate experiments at each inoculum. **P < 0.005 versus WT.

Figure 8

Survival from Staphylococcus aureus pneumonia is unimpaired in TLR2^−/− mice. Anesthetized mice (n = 10 per group) were inoculated intranasally with S. aureus (5 × 10⁸ CFU). After 7 days surviving mice were euthanized. Left lungs and spleens were homogenized for quantitative culture.