Human NOD2 Recognizes Structurally Unique Muramyl Dipeptides from Mycobacterium leprae

Induction of IL-32 by M. leprae. (A) Purified human monocytes were cultured with live M. leprae (MOI of 10) or 10 μg/ml of either sonicated bacilli (mLEP-son), digested M. leprae peptidoglycan (mLEP-PG), M. leprae mycolyl-arabinogalactan-peptidoglycan (mAGP), synthetic MDP (syn-MDP), or inactive synthetic MDP (LL-syn-MDP), and IL-32 gene expression was measured. (B) Four enriched fractions of muropeptides derived from mLEP-PG (mLEP-PG F-16, -17, -18, and -19) were tested for their ability to induce IL-32 expression and compared to digested M. leprae peptidoglycan (mLEP-PG) and synthetic MDP (syn-MDP). Data are represented as mean fold change (FC) compared to the medium control (ctrl) ± SEM (n = 6). Statistical significance was calculated by two-tailed Student's t test. Asterisks indicate statistically significant differences compared to media control: *, P < 0.05; **, P < 0.01.

Chemical structures of synthetic MDP and M. leprae MDP. (A) Structural differences between the different MDPs are highlighted (box). The first amino acid residue of M. leprae MDP is Gly instead of l-Ala, and the d-Glu residue is amidated [mLEP-MDP(NH₂)] or nonamidated (mLEP-MDP). (B) mLEP-MDP(NH₂) and mLEP-MDP were synthesized enzymatically, and the molecular structures were confirmed by mass spectrometry: [M+H]⁺ of 493.2168 m/z belongs to syn-MDP, [M+H]⁺ of 480.1861 m/z has a calculated molecular formula of C₁₈H₂₉N₃O₁₂, which is consistent with the molecular formula of mLEP-MDP, and [M+H]⁺ of 479.2002 m/z has a calculated molecular formula of C₁₈H₃₀N₄O₁₁, which is also the molecular formula of mLEP-MDP(NH₂).

Cytokine response to structurally distinct MDPs. Purified human monocytes were activated by adding 1 μg/ml of syn-MDP, mLEP-MDP(NH₂), or mLEP-MDP. After 24 h, IL-32, IL-1β, and IL-6 induction was measured by mRNA expression as fold change (FC) compared to the medium control (A) and protein secretion (B). Data are represented as means ± SEM (n ≥ 6).

Induction of DC differentiation by different MDPs. Purified human monocytes were activated using syn-MDP, mLEP-MDP(NH₂), and mLEP-MDP, and DC differentiation was measured by flow cytometry for CD1b expression. A representative histogram (A) and the mean percentage of CD1b-positive cells ± SEM (B) are shown (n = 5).

Quantification of NOD2 signaling induced by structurally distinct MDPs. HEK-NOD2 reporter cells were used to quantitatively assess NOD2 activation by stimulating cells with structurally different MDP compounds [syn-MDP, mLEP-MDP-(NH₂), and mLEP-MDP] over a range of concentrations (0.001 to 1 μg/ml). Data are represented as mean arbitrary units (AU) ± SEM (n = 6).