Nod2 sensing of lysozyme-digested peptidoglycan promotes macrophage recruitment and clearance of S. pneumoniae colonization in mice

Abstract

Streptococcus pneumoniae colonizes the mucosal surface of the human upper respiratory tract. A colonization event is gradually cleared through phagocytosis by monocytes/macrophages that are recruited to the airway lumen. Here, we sought to define the bacterial and host factors that promote monocyte/macrophage influx and S. pneumoniae clearance using intranasal bacterial challenge in mice. We found that the recruitment of monocytes/macrophages required their expression of the chemokine receptor CCR2 and correlated with expression of the CCR2 ligand CCL2. Production of CCL2 and monocyte/macrophage recruitment were deficient in mice lacking digestion of peptidoglycan by lysozyme (LysM) and cytosolic sensing of the products of digestion by Nod2. Ex vivo macrophages produced CCL2 following bacterial uptake, digestion by LysM, and sensing of peptidoglycan by Nod2. Sensing of digested peptidoglycan by Nod2 also required the pore-forming toxin pneumolysin. The generation of an adaptive immune response, as measured by anti-pneumococcal antibody titers, was also LysM- and Nod2-dependent. Together, our data suggest that bacterial uptake by professional phagocytes is followed by LysM-mediated digestion of S. pneumoniae-derived peptidoglycan, sensing of the resulting products by Nod2, release of the chemokine CCL2, and CCR2-dependent recruitment of the additional monocytes/macrophages required for the clearance of an S. pneumoniae colonization event.

Figure 1. Monocyte/macrophage recruitment impacts early clearance of S. pneumoniae and requires CCR2 and Nod2.

Mice of the indicated genetic backgrounds were inoculated intranasally with 10⁷ CFU S. pneumoniae. Upper respiratory tract lavages were used to measure colonization density (CFU/ml) and the composition of the cellular infiltrate by flow cytometry. (A) Colonization density of WT (C57BL/6) or Ccr2^–/– mice at day 7 after inoculation with strain TIGR4 or strain 23F. The dashed line indicates the limit of detection. Horizontal lines indicate mean values. *P < 0.05, **P < 0.01, Mann-Whitney U test. (B) Flow cytometry was used to detect total numbers of monocytes/macrophages (F4/80⁺, CD11b^–) and neutrophils (Ly6G⁺, CD11b⁺) in nasal lavages during colonization of WT (C57BL/6), Nod2^–/–, or Ccr2^–/– mice with strain 23F. Each point represents the average number of events ± SD in 3 experiments each with 5 mice.

Figure 2. Lysozyme releases Nod2 ligands and pneumolysin from S. pneumoniae.

(A) Lysis of heat-killed bacterial samples with human lysozyme (L) or mutanolysin (M) as measured by optical density (OD at 600 nm). (B) HEK293T cells were transiently transfected with an NF-κB–luciferase expression vector and either a Nod2-expressing vector or empty vector control. Transfected cells were stimulated with heat-killed bacteria digested with human lysozyme or mutanolysin or with undigested bacteria (–). Values are expressed as fold increase in relative luciferase units in Nod2-expressing cells compared with empty vector controls ± SD. *P < 0.05, **P < 0.01, Wilcoxon matched-pairs test. (C) Western blot detecting pneumolysin (Ply) released by strain TIGR4 or TIGR4 pgdA^–adr^– following incubation with human lysozyme or no treatment (–). (D) Peritoneal macrophages were isolated from WT (FVB/NJ) or LysM^–/– mice and infected with strain TIGR4 or TIGR4 pgdA^–adr^–. Macrophages were stained by immunofluorescence with monoclonal type 4 antisera (red, S. pneumoniae) and DAPI (blue, DNA). Insets show fluorescence images in the red channel. Fluorescence images were overlaid onto Nomarski light images; original magnification, ×600.

Figure 3. S. pneumoniae is digested by LysM within phagocytes, resulting in Nod2-dependent sensing and cytokine production.

(A) WT (C57BL/6, white bars) or Nod2^–/– (black bars) bone marrow macrophages were infected with strain TIGR4, strain TIGR4 pgdA^–adr^–, or media control for 4 hours, and secreted IL-6 was measured by ELISA. (B) WT (C57BL/6) or Nod2^–/– bone marrow macrophages were stimulated with heat-killed strain TIGR4, media control, or MDP for 24 hours, and secreted IL-6 was measured by ELISA. Cytochalasin D (cyt D) was added prior to stimulation to prevent uptake. (C) WT (C57BL/6) or Nod2^–/– peritoneal macrophages were stimulated with heat-killed strain TIGR4, heat-killed strain TIGR4 pgdA^–adr^–, or media control, and secreted IL-6 was measured by ELISA. (D) WT (FVB/NJ, white bars) or LysM^–/– (gray bars) peritoneal macrophages were stimulated with heat-killed strain TIGR4 or heat-killed strain TIGR4 pgdA^–adr^–, and secreted IL-6 was measured by ELISA. Values are relative to WT cells treated with TIGR4 ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t test.

Figure 4. Nod2 and LysM are required for the monocyte/macrophage influx in response to pneumococcal colonization.

Mice of the indicated genetic backgrounds were inoculated intranasally with 10⁷ CFU of S. pneu­moniae. Upper respiratory tract lavages were obtained 3 days after inoculation, and the composition of the cellular infiltrate was determined by flow cytometry. Numbers of monocytes/macrophages (F4/80⁺, CD11b^–) and neutrophils (Ly6G⁺, CD11b⁺) are shown. (A) WT (C57BL/6) or Nod2^–/– mice were inoculated with strain TIGR4 or TIGR4 pgdA^–adr^–. (B) WT (FVB/NJ) or LysM^–/– mice were inoculated with strain TIGR4. Each bar represents the average the number of events ± SD in 4 experiments, each with 5 mice. *P < 0.05, Mann-Whitney U test.

Figure 5. Expression of CCL2 requires pore formation by pneumolysin and sensing by Nod2.

(A) Upper respiratory tract lavages were obtained 3 days after inoculation with the indicated strain, using RNA lysis buffer. RNA was isolated and reverse transcribed, and CCL2 expression levels were measured by quantitative RT-PCR relative to GAPDH controls. (B) IL-17A expression levels were measured by quantitative PCR relative to GAPDH controls. (C) CCL2 expression levels were measured by quantitative PCR relative to GAPDH controls. Values are relative to WT (C57BL/6) mice infected with the parental strain ± SD (n > 10 mice). Baseline values reflect mice mock-infected with PBS. *P < 0.05, **P < 0.01, unpaired t test. (D) WT (C57BL/6) peritoneal macrophages were stimulated with heat-killed preparations of the indicated strains, and secreted CCL2 was measured by ELISA. *P < 0.05, unpaired t test.

Figure 6. Nod2-dependent sensing occurs within macrophages, which initiates the immune response to pneumococcal colonization.

(A) Bone marrow macrophages from WT (C57BL/6, white bars) or Nod2^–/– (black bars) mice were stimulated with heat-killed strain TIGR4, and levels of secreted CCL2 were measured by ELISA. Values are relative to WT mice stimulated with strain TIGR4 ± SD. (B) Peritoneal cells were isolated from WT (C57BL/6) or Nod2^–/– mice and stimulated with opsonized heat-killed TIGR4, and secreted CCL2 was measured by ELISA. Cytochalasin D was added prior to stimulation to prevent uptake. (C) Peritoneal cells were isolated from WT (FVB/NJ) or LysM^–/– mice and stimulated with opsonized heat-killed strain TIGR4, and secreted CCL2 was measured by ELISA. **P < 0.01, ***P < 0.001, unpaired t test. (D) Intracellular CCL2 was detected by flow cytometry following stimulation of peritoneal cells isolated from WT (C57BL/6) or Nod2^–/– mice and stimulated with opsonized heat-killed strain TIGR4. Macrophage (F4/80⁺) and neutrophil (Ly6G⁺) populations are shown. Lines indicate CCL2 fluorescence intensity on the x axis (FL-1 signal) for duplicate cell samples. Filled peaks denote binding by CCL2 isotype control antibodies.

Figure 7. Nod2 and LysM promote the generation of adaptive immune responses.

Mice were inoculated intranasally with 10⁷ CFU of the indicated strain or PBS. Twenty-one days after inoculation, mice were sacrificed, serum was isolated, and levels of anti-pneumococcal serum IgG were determined by ELISA. Values are expressed as geometric mean titers (GMT) ± SD (n > 10 mice). (A) WT (C57BL/6, white bars), Nod2^–/– (black bars), or Tlr2^–/– (dark gray bars) mice were inoculated with strain TIGR4. (B) WT (C57BL/6) or Nod2^–/– mice were inoculated with strain 23F. (C) WT (FVB/NJ, white bars) or LysM^–/– (light gray bars) mice were inoculated with strain TIGR4. *P < 0.05, **P < 0.01, ***P < 0.001, Mann-Whitney U test.

Figure 8. Nod2 and TLR2 contribute to clearance of pneumococcal colonization.

WT (C57BL/6), Nod2^–/–, Tlr2^–/–, and Tlr2^–/–Nod2^–/– double-knockout mice were inoculated intranasally with 10⁷ CFU of strain TIGR4. Upper respiratory tract lavages were obtained at 21 days after inoculation and used to measure colonization density (CFU/ml). Dashed lines indicate the limit of detection. Horizontal lines indicate mean values. *P < 0.05, Mann-Whitney U test.