NOD1 and NOD2 regulation of pulmonary innate immunity to Legionella pneumophila

Abstract

The role of nucleotide-binding oligomerization domain-1 (NOD1) and nucleotide-binding oligomerization domain-2 (NOD2), cytoplasmic receptors which detect bacterial cell wall molecules, in pulmonary innate immune responses is poorly understood. We determined that both NOD1 and NOD2 detect heat-killed Legionella and stimulate NF-κb and IFN-β promoter activity using an in vitro luciferase reporter system. We next infected NOD1- and NOD2-deficient animals with aerosolized Legionella pneumophila. At 3 days post infection, Nod1(-/-) mice had impaired bacterial clearance compared to WT controls. In addition, at 4 h and 24 h, Nod1(-/-) mice had impaired neutrophil recruitment to the alveolar space. In contrast, increased lung neutrophils were seen in the Nod2(-/-) animals at 24 h. Analysis of cytokine production at 4 h post infection revealed a significant decrease in proinflammatory cytokines in the Nod1(-/-) animals when compared to WT animals. In contrast, increased 4-h proinflammatory cytokines were seen in the Nod2(-/-) animals. Furthermore, the lungs of both Nod1(-/-) and Nod2(-/-) mice had significantly increased pro-inflammatory cytokine levels at 24 h, suggesting possible suppressive roles for later stages of infection. Together, our data suggest that although both NOD1 and NOD2 can detect Legionella, these receptors modulate the in vivo pulmonary immune response differently.

Figure 1

Heat-killed Legionella is detected by NOD1 and NOD2. HEK293 cells were transfected with expression vectors containing human NOD1 (A, C) and NOD2 (B, D) (grey) or empty vector (EV) controls (black). Firefly luciferase reporter constructs for the NF-κB (ELAM-pGL2) (A, B) and IFN-β promoters (IFNβ-pGL2) (C, D) were simultaneously transfected. Cells were then exposed to NOD1 ligand (Tri-DAP, 1 μg/mL), NOD2 ligand (MDP, 1 μg/mL), or heat-killed flagellin-deficient Lp (Lp FlaA, Corby strain) at decreasing heat-killed MOI as determined by OD 600 nm. HEK cells were incubated overnight (16 h) and then lysed. Transfection efficiency was compared between wells by co-transfecting the expression vector for Renilla luciferase. Data are mean ± SD, n = 3, representative of three experiments. Lp: Legionella pneumophila, RLU: relative light units.

Figure 2

NOD1-deficient animals show impaired clearance of Legionella pneumophila in lungs at 3 days. Mice were infected with aerosolized L. pneumophila (Philadelphia strain) and then euthanized at 4 h (A), 24 h (B), 72 h (C), and 240 h (D). Bacterial CFU were determined by serial dilution of tissue homogenates plated on buffered yeast charcoal extract agar. Nod1^–/– knockout mice (■), WT controls (•), Nod2^–/– knockout mice (▲). Initial deposition was approximately 1 × 10⁶ Lp CFU per lung. A Student's t-test was used to determine significance. Bars represent mean ± SEM range; results combined from three separate experiments with four mice per time point (n = 12 total) except for the 720-h time point, which was one experiment (n = 4).

Figure 3

NOD1 and NOD2 regulate neutrophil recruitment to the lung in Lp-infected mice. Bronchoalveolar lavages were performed on mice infected with Legionella pneumophila and euthanized at the indicated time points: 4 h (A, D), 24 h (B, E), and 72 h (C, F). Total neutrophil (PMN) (D, E, F) and monocyte (Mono) (A, B, C) counts were compared in WT (•), Nod1^–/– (■) and Nod2^–/– animals (▲). A Student's t-test was used to determine significance as represented on the graph. Bars represent mean ± SEM. Combined results from three separate experiments with four mice per time point (n = 12).

Figure 4

NOD1-deficient mice have impaired inflammatory cell infiltrates at 24 h. Lungs from WT, NOD1- and NOD2-deficient mice were fixed in paraformaldehyde and stained in H and E. Representative slides for 24 h (A) and 72 h (B) are shown. Ten high-power fields for each lung were examined and the percentage of airspace involvement was scored by a pathologist blinded to condition. Student's t-test was used to determine significance. Scores are shown in Table 1 (mean ± SEM, n = 6).

Figure 5

Cytokine production in lung homogenates of Lp-infected mice. WT (white), Nod1^–/– (black), and Nod2^–/– (grey) mice were infected with Lp and at 4 and 24 h after infection lungs were homogenized. Total levels of TNFα (A,D), IL-6 (B,E), IL-1β (C,F), mKC (G,J), IL-18 (H,K), and MCP-1 (I,L) were measured. To combine experiments, Nod1^–/– and Nod2^–/– cytokine levels were normalized to average of WT controls for each cytokine. *p<0.05 compared with WT, **p≤0.01 compared with WT. Statistical significance was determined by performing Student's t-test. Data combined from three separate experiments with n = 12 total for each group except Nod2^–/– animals, which had n = 11. Bars represent mean ± SEM.