NOD2 and toll-like receptors are nonredundant recognition systems of Mycobacterium tuberculosis

Abstract

Infection with Mycobacterium tuberculosis is one of the leading causes of death worldwide. Recognition of M. tuberculosis by pattern recognition receptors is crucial for activation of both innate and adaptive immune responses. In the present study, we demonstrate that nucleotide-binding oligomerization domain 2 (NOD2) and Toll-like receptors (TLRs) are two nonredundant recognition mechanisms of M. tuberculosis. CHO cell lines transfected with human TLR2 or TLR4 were responsive to M. tuberculosis. TLR2 knock-out mice displayed more than 50% defective cytokine production after stimulation with mycobacteria, whereas TLR4-defective mice also released 30% less cytokines compared to controls. Similarly, HEK293T cells transfected with NOD2 responded to stimulation with M. tuberculosis. The important role of NOD2 for the recognition of M. tuberculosis was demonstrated in mononuclear cells of individuals homozygous for the 3020insC NOD2 mutation, who showed an 80% defective cytokine response after stimulation with M. tuberculosis. Finally, the mycobacterial TLR2 ligand 19-kDa lipoprotein and the NOD2 ligand muramyl dipeptide synergized for the induction of cytokines, and this synergism was lost in cells defective in either TLR2 or NOD2. Together, these results demonstrate that NOD2 and TLR pathways are nonredundant recognition mechanisms of M. tuberculosis that synergize for the induction of proinflammatory cytokines.

Figure 1. TLR2 and TLR4 Are Partially Responsible for Induction of Cytokine Production by M. tuberculosis

(A) CHO cells cotransfected with CD14 and TLR2 (CD14/TLR2, hatched bars) induced potent expression of CD25 on the cell membrane as measured by FACS analysis, after stimulation with both a sonicated M. tuberculosis (MTB-son, 1 μg/ml) or whole mycobacteria (MTB-whole, 1 × 10⁷/ml). In contrast, cells transfected with CD14 and TLR4 (CD14/TLR4, black bars) were only moderately activated by whole M. tuberculosis, but not by the sonicated material. LPS (1 μg/ml) and Pam3Cys (5 μg/ml) served as control stimuli for TLR4 and TLR2, respectively. As controls, cells transfected only with CD14 were used (white bars). (B and C) Stimulation of TNF production by M. tuberculosis in peritoneal macrophages of mice deficient for TLR2 (TLR2-KO) (B) or TLR4 (TLR4-def) (C). Groups of five mice were stimulated with the indicated reagents, and the experiment was repeated twice. Medium-stimulated cells resulted in cytokine concentrations below detection limit. Data are presented as mean ± SD (n = 5; *p < 0.05).

Figure 2. Blockade of Internalization of M. tuberculosis Impairs Recognition and Cytokine Production

Blockade of M. tuberculosis internalization by cytochalasin B (20 μg/ml) impairs TNF (A) and IL-10 (B) stimulation in human MNCs stimulated with sonicated M. tuberculosis (1 × 10⁶ microorganisms/ml), but not zymosan (1 μg/ml). Data are presented as mean ± SD (n = 5; *p < 0.05).

Figure 3. Stimulation of HEK Cells Transfected with NOD1 or NOD2

HEKs transfected with NOD2 were strongly activated by whole M. tuberculosis, in contrast to cells transfected with NOD1, which were only weakly activated by M. tuberculosis. MDP (100 nM) and TriDAP (100 nM) served as control stimuli for NOD2 and NOD1, respectively. Data are presented as fold increase over unstimulated cells (mean ± SD).

Figure 4. Stimulation of Cells from Mice Deficient in NOD1 or NOD2

Stimulation of cytokine production by sonicated M. tuberculosis in peritoneal macrophages of mice deficient for either NOD1 (A) or NOD2 (B). Groups of four mice were stimulated; medium-stimulated cells resulted in cytokine concentrations below detection limit. Data are presented as mean ± SD (*p < 0.05).

Figure 5. Human NOD2 Is a Receptor for M. tuberculosis

(A) MNCs isolated from four patients with Crohn's disease homozygous for the 3020insC NOD2 mutation (NOD2fs), five patients heterozygous for NOD2 mutations (NOD2het), five patients with the wild-type NOD2 allele (NOD2WT), and five healthy volunteers with wild-type NOD2 (control) were stimulated with 10 μg/ml sonicated M. tuberculosis. TNF (white bars) and IL-10 (black bars) were measured after 24 h of stimulation at 37 °C by specific RIA and ELISA, respectively. (B) MNCs isolated from four patients with Crohn's disease homozygous for the 3020insC NOD2 mutation (NOD2fs, black bars), five patients with the wild-type NOD2 allele (NOD2WT, gray bars), and five healthy volunteers with wild-type NOD2 (control), were stimulated with 1 μg/ml MDP or 10 ng/ml LPS. TNF were measured after 24 h of stimulation at 37 °C by specific RIA. Medium-stimulated cells resulted in cytokine concentrations below detection limit. Data are presented as mean ± SD (*p < 0.05).

Figure 6. NOD2 and TLR2 Have Synergistic Effects on Cytokine Production

(A) MNCs isolated from five healthy volunteers with wild-type NOD2 were costimulated with 1 μg/ml MDP and increasing concentrations of 19-kDa lipoprotein (indicated on the x-axis). TNF was measured after 24 h of stimulation at 37 °C by specific RIA. (B) The synergistic effects observed in the five control volunteers, as well as in five Crohn's disease patients heterozygous for NOD2 mutations (NOD2-het), and in five patients with the wild-type NOD2 allele (NOD2-WT), was lost in patients homozygous for the NOD2 3020insC mutation. Medium-stimulated cells resulted in cytokine concentrations below detection limit. Data are presented as mean ± SD (*p < 0.05).