Chronic stimulation of Nod2 mediates tolerance to bacterial products

Abstract

The Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (Nod) families of proteins are critical for bacterial recognition, and, acutely, this frequently leads to proinflammatory responses. Polymorphisms in Nod2 (CARD 15) are associated with an increased likelihood of developing Crohn's disease. However, it is not yet clear how Nod2 dysfunctions lead to defects in human intestinal immune homeostasis. Studies to date have focused on functions after acute, rather than chronic, Nod2 stimulation. However, the intestine is an environment of chronic bacterial product exposure with tolerance to luminal flora. We therefore hypothesized that long-term Nod2 stimulation contributes to down-regulation of inflammatory responses from innate immune receptors. We found that pretreatment with muramyl dipeptide (MDP), a ligand for Nod2, significantly decreased production of the proinflammatory cytokines TNF-alpha, IL-8, and IL-1beta upon Nod2, TLR4, and TLR2 restimulation in primary human monocyte-derived macrophages from a large cohort of individuals. Importantly, TNF-alpha-induced production of proinflammatory cytokines remained intact in these same cells. MDP-stimulated macrophages from Crohn's disease-relevant Leu1007insC Nod2 homozygote individuals were deficient in their ability to cross-tolerize to subsequent treatment with TLR2 and TLR4 ligands. We show that acute Nod2 stimulation induced IRAK-1 activation, and that chronic MDP treatment down-regulated IRAK-1 activation upon Nod2 or TLR4 restimulation. In a subset of individuals, chronic Nod2 stimulation induced expression of the IRAK-1 inhibitory protein IRAK-M. Significantly, intestinal macrophages exhibit tolerance to MDP per production of inflammatory cytokines. These results illustrate a role for chronic stimulation of Nod2 in mediating tolerance to bacterial products.

Fig. 1.

Prolonged pretreatment with MDP induces self-tolerance and cross-tolerance to lipid A but not to TNF-α stimulation in primary monocyte-derived macrophages. Human primary macrophages from control individuals were left untreated or stimulated with 100 μg/ml MDP for the indicated time periods, washed, and restimulated for an additional 24 h with 100 μg/ml MDP (A) or 0.1 μg/ml synthetic lipid A (B). Supernatants were assayed for TNF-α. Controls include 100 μg/ml MDP pretreatment for each of the indicated time periods followed by washes alone. The average is shown by a black line. The data are representative of 4 of 4 (A) or 4 of 12 (B) individuals. (C) Macrophages from control individuals were left untreated or stimulated with 100 μg/ml MDP for 48 h, washed, and restimulated for an additional 24 h with 10 ng/ml TNF-α. Supernatants were assayed for IL-8. The data are representative of 4 of 18 individuals. tx, treatment; M, MDP; L, lipid A; T, TNF-α.

Fig. 2.

MDP induces self-tolerance and cross-tolerance to TLR2 and TLR4 pathways in monocyte-derived macrophages from control individuals but not Leu1007insC Nod2 homozygotes. Human primary macrophages from control individuals (A) or Leu1007insC Nod2 homozygous individuals (B) were left untreated or stimulated with 100 μg/ml MDP for 48 h, washed, and restimulated for an additional 24 h with 100 μg/ml MDP, 10 μg/ml Pam₃Cys, or 0.1 μg/ml synthetic lipid A. The supernatants were assayed for TNF-α. The average is shown as a black line. The data are representative of eight (A) or five individuals (B). (C) Graph summarizing the tolerance induction in human primary macrophages from control individuals. Shown for comparison is the failure of tolerance induction in macrophages from Leu1007insC Nod2 homozygous individuals. The data are represented as the percent cytokine secretion by MDP-pretreated cells upon restimulation compared with that of nonpretreated cells (represented by the dotted line at 100%) plus SEM. Numbers below the bars indicate the number of individuals assessed. tx, treatment; M, MDP; P, Pam₃Cys; L, lipid A. Significance compared with non-MDP-pretreated cells was calculated by using the t test. Each condition for control individuals showed P < 1 × 10⁻⁵ (††).

Fig. 3.

IRAK-1 kinase activity is down-regulated after chronic Nod2 stimulation. (A) Human primary macrophages from control individuals were stimulated with 100 μg/ml MDP for 48 h or left untreated, washed, and restimulated for an additional 40 min with 100 μg/ml MDP or 0.1 μg/ml synthetic lipid A. Cell lysates were immunoprecipitated with anti-IRAK-1 antibody, and phosphorylation of myelin basic protein (MBP) by the immunoprecipitated complexes was analyzed. IRAK-1 expression served as the loading control. Overall, MDP pretreatment reduced IRAK-1 kinase activity in 17 of 24 (70%) or 10 of 21 (48%) individuals after MDP or lipid A restimulation, respectively. (B) Graph summarizing the reduction of IRAK-1 activation after chronic stimulation of Nod2 in human macrophages. The data are representative of the individuals that demonstrated down-regulation of IRAK-1 kinase activity compared with nonpretreated cells (represented by the dotted line at 100%). Significance for each treatment as compared with non-MDP-pretreated cells was calculated by using the t test. ††, P < 1 × 10⁻⁵. tx, treatment; M, MDP; L, lipid A.

Fig. 4.

Monocyte-derived macrophages up-regulate IRAK-M upon prolonged treatment with MDP, and silencing of IRAK-M expression reduces Nod2-mediated self-tolerance. (A) Human primary monocyte-derived macrophages were left unstimulated or treated with 100 μg/ml MDP for 48 h, and cell lysates were analyzed by Western blotting for the expression of IRAK-M. GAPDH served as the loading control. Cells from three representative individuals are shown. (B) Human primary macrophages from control individuals were stimulated with 100 μg/ml MDP for 24 h or left untreated, then transfected with siRNA for IRAK-M or control siRNA. Cells were lysed 24 h later and analyzed by Western blotting for IRAK-M expression. GAPDH served as the loading control. Data are representative of eight individuals. (C) Human primary macrophages from control individuals were stimulated with 100 μg/ml MDP for 24 h or left untreated, transfected with scrambled siRNA or siRNA for IRAK-M, and restimulated 24 h later for an additional 24 h with 100 μg/ml MDP or 0.1 μg/ml lipid A. The supernatants were assayed for IL-8. The dotted line represents the normalized level of cytokine secretion by scrambled siRNA-transfected, non-MDP-pretreated cells acutely stimulated for 24 h with MDP or lipid A (= 100%). The data are representative of individuals that demonstrated a >15% reversal in Nod2-mediated self-tolerance or cross-tolerance to lipid A. tx, treatment; M, MDP; L, lipid A. IL-8 secretion of MDP-pretreated scrambled and siIRAK-M-transfected cells was compared, and significance was calculated by using the t test. ***, P < 0.001.

Fig. 5.

Intestinal monocyte-derived cells are tolerant to MDP stimulation but phagocytose bacteria. (A) Human peripheral (n = 17) or intestinal (n = 6) monocyte-derived cells from control individuals were stimulated for 16 h with 100 μg/ml MDP or 0.1 μg/ml lipid A. Supernatants were collected and assayed for TNF-α by ELISA. M, MDP; L, lipid A. Significance was calculated by using the t test comparing untreated cells to those treated with MDP or lipid A. ††, P < 1 × 10⁻⁵. (B) Freshly isolated intestinal cells were cultured for 16 h and incubated with heat-killed FITC-labeled E. coli, and monocyte-derived cells were analyzed by flow cytometry. The shaded area represents unlabeled bacteria, and the solid line represents FITC-labeled E. coli. Data are representative of six individuals.