NOD2 Negatively Regulates Organic Dust-Induced Inflammation in Monocytes/Macrophages

Abstract

RATIONALE: Chronic organic dust exposure in the agricultural industry results in significant airway disease. Since we have recently demonstrated high concentration of peptidoglycan in large animal farming environments, we examined in vitro, the role of the intracellular peptidoglycan breakdown product sensor, nucleotide oligomerization domain (NOD)2, in regulating organic dust-induced inflammation.METHODS: Organic dust extract (ODE) from swine operations was utilized. Adherent THP-1 monocytes were transfected with NOD2 or non-targeting siRNA by HiPerfect system. Transfected cells were subsequently stimulated with and without 1% ODE and as a control, the NOD2 agonist, muramyl dipetide (MDP), for 24 hours. Transfection efficiency was evaluated by real-time RT-PCR. Next, lung macrophages isolated from wild type and NOD2 knockout mice were ex vivo stimulated with 1% ODE for 24 hours. Cell-free supernatants for mediators were evaluated by ELISA.RESULTS: Relative mRNA expression of NOD2 in MDP- and ODE-stimulated NOD2 knockdown cells was significantly reduced to 32.8% ± 9.6% and 34.85% ± 8.6%, respectively. As anticipated, TNFα and CXCL8 secretion was diminished in NOD2 knockdown cells stimulated with MDP (p < 0.05; N = 3). However, stimulation of NOD2 knockdown cells with ODE resulted in significantly enhanced CXCL8 and IL-6 secretion as compared to control, but no change in TNFα (N = 3). Confirming these findings, murine NOD2-/- lung macrophages demonstrated significantly enhanced KC and MIP-2 (human CXCL8 homologs), IL-6, but not TNFα secretion, with ODE stimulation as compared to wild type lung macrophages (p < 0.05; N = 4 mice/group).CONCLUSIONS: The peptidoglycan breakdown product intracellular sensor, NOD2, appears to negatively regulate organic dust-induced macrophage inflammation in a selective manner. RATIONALE: Chronic organic dust exposure in the agricultural industry results in significant airway disease. Since we have recently demonstrated high concentration of peptidoglycan in large animal farming environments, we examined in vitro, the role of the intracellular peptidoglycan breakdown product sensor, nucleotide oligomerization domain (NOD)2, in regulating organic dust-induced inflammation. METHODS: Organic dust extract (ODE) from swine operations was utilized. Adherent THP-1 monocytes were transfected with NOD2 or non-targeting siRNA by HiPerfect system. Transfected cells were subsequently stimulated with and without 1% ODE and as a control, the NOD2 agonist, muramyl dipetide (MDP), for 24 hours. Transfection efficiency was evaluated by real-time RT-PCR. Next, lung macrophages isolated from wild type and NOD2 knockout mice were ex vivo stimulated with 1% ODE for 24 hours. Cell-free supernatants for mediators were evaluated by ELISA. RESULTS: Relative mRNA expression of NOD2 in MDP- and ODE-stimulated NOD2 knockdown cells was significantly reduced to 32.8% ± 9.6% and 34.85% ± 8.6%, respectively. As anticipated, TNFα and CXCL8 secretion was diminished in NOD2 knockdown cells stimulated with MDP (p < 0.05; N = 3). However, stimulation of NOD2 knockdown cells with ODE resulted in significantly enhanced CXCL8 and IL-6 secretion as compared to control, but no change in TNFα (N = 3). Confirming these findings, murine NOD2-/- lung macrophages demonstrated significantly enhanced KC and MIP-2 (human CXCL8 homologs), IL-6, but not TNFα secretion, with ODE stimulation as compared to wild type lung macrophages (p < 0.05; N = 4 mice/group). CONCLUSIONS: The peptidoglycan breakdown product intracellular sensor, NOD2, appears to negatively regulate organic dust-induced macrophage inflammation in a selective manner.