Therapeutic effect of artemisinin on lupus nephritis mice and its mechanisms

In this study, we investigated the therapeutic effect of artemisinin (Art) on lupus nephritis mice and its mechanisms by comparing the differences between lupus nephritis (LN) mice given Art and control mice in molecular biology, immunohistochemistry, and histopathology. The results showed that Art could remarkably relieve the symptoms, decrease the level of urine protein/24 h, and alleviate pathological renal lesions. The differences among the four groups in the expression of the NF-κBp65 protein, nuclear factor-κB (NF-κB) activity, and the expression of transforming growth factor-β1 (TGF-β1) mRNA in renal tissue suggested that Art can lower the serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and inhibit the expression of the NF-κBp65 protein and NF-κB and TGF-β1 mRNA in the renal tissues of LN mice. These results proved that it is reliable and effective to use Art to treat LN mice, and its therapeutic mechanisms should closely be related to the fact that Art can obviously decrease the serum levels of TNF-α and IL-6 and down-regulate the expression of the NF-κBp65 protein and NF-κB and TGF-β1 mRNA in renal tissues.     

Propofol Administration Modulates AQP-4 Expression and Brain Edema After Traumatic Brain Injury

The increased intracranial pressure caused by brain edema following traumatic brain injury (TBI) always leads to poor patient prognosis. Aquaporin-4 (AQP-4) plays an important role in edema formation and resolution, which may provide a novel therapeutic target for edema treatment. In this present study, we found that propofol treatment, within a short time, after TBI significantly reduced brain edema in a controlled cortical injury rat model and suppressed in vivo expression of AQP-4. The ameliorating effect of propofol was associated with attenuated expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). In addition, the regulatory effect of propofol on AQP-4 expression was investigated in cultured astrocytes. Results showed that propofol could block the stimulatory effect of IL-1β and TNF-α on AQP-4 expression in cultured astrocytes. We also found that both NFκB and p38/MAPK pathways were involved in IL-1β and TNF-α-induced AQP-4 expression and that propofol functions as a dual inhibitor of NFκB and p38/MAPK pathways. In conclusion, treatment with propofol, within a short time, after TBI attenuates cerebral edema and reduces the expression of AQP-4. Propofol modulates acute AQP-4 expression by attenuating IL-1β and TNF-α expression and inhibiting IL-1β and TNF-α induced AQP-4 expression.     

Baicalein inhibits IL-1β- and TNF-α-induced inflammatory cytokine production from human mast cells via regulation of the NF-κB pathway

Background

Human mast cells are multifunctional cells capable of a wide variety of inflammatory responses. Baicalein (BAI), isolated from the traditional Chinese herbal medicine Huangqin (Scutellaria baicalensis Georgi), has been shown to have anti-inflammatory effects. We examined its effects and mechanisms on the expression of inflammatory cytokines in an IL-1β- and TNF-α-activated human mast cell line, HMC-1.

Methods

HMC-1 cells were stimulated either with IL-1β (10 ng/ml) or TNF-α (100 U/ml) in the presence or absence of BAI. We assessed the expression of IL-6, IL-8, and MCP-1 by ELISA and RT-PCR, NF-κB activation by electrophoretic mobility shift assay (EMSA), and IκBα activation by Western blot.

Results

BAI (1.8 to 30 μM) significantly inhibited production of IL-6, IL-8, and MCP-1 in a dose-dependent manner in IL-1β-activated HMC-1. BAI (30 μM) also significantly inhibited production of IL-6, IL-8, and MCP-1 in TNF-α-activated HMC-1. Inhibitory effects appear to involve the NF-κB pathway. BAI inhibited NF-κB activation in IL-1β- and TNF-α-activated HMC-1. Furthermore, BAI increased cytoplasmic IκBα proteins in IL-1β- and TNF-α-activated HMC-1.

Conclusion

Our results showed that BAI inhibited the production of inflammatory cytokines through inhibition of NF-κB activation and IκBα phosphorylation and degradation in human mast cells. This inhibitory effect of BAI on the expression of inflammatory cytokines suggests its usefulness in the development of novel anti-inflammatory therapies.     

Astragaloside IV protects against focal cerebral ischemia/reperfusion injury correlating to suppression of neutrophils adhesion-related molecules

Inflammation injury plays a key role in the process of cerebral injury induced by ischemia/reperfusion (I/R). Thus, we studied the potential of astragaloside IV, one of the major and active components of the astragalus membranaceous, to protect rat against cerebral inflammation injury elicited by focal cerebral ischemia and reperfusion and related protective mechanisms. The rat model was induced by intraluminal occlusion of the right middle cerebral artery with reperfusion. Animals received astragaloside IV (10 or 20 mg/kg) injections when reperfusion was began to. Neurobehavioral evaluation and infarct assessment were studied. Myeloperoxidase (MPO) and tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were measured by enzyme-linked immunosorbent assay (ELISA). The rates of CD11b/CD18-positive neutrophils were analyzed via flow cytometry. Intercellular adhesion molecule-1 (ICAM-1) and nuclear factor κB (NF-κB) were measured by immunohistochemistry and Western blot. Astragaloside IV improved neurological outcome and reduced infarct volume at 24 h after reperfusion. The protective effect was achieved by preventing neutrophils accumulation in the brain parenchyma demonstrated by significantly reducing the concentration of MPO in brain tissue. Astragaloside IV exerts the protection through remarkably decreasing the percentage of CD11b/CD18-positive neutrophils and down-regulating the expression of intercellular adhesion molecule-1 (ICAM-1), which is partly achieved by strongly attenuating the production of TNF-α and IL-1β and inhibiting level of nuclear factor-κB (NF-κB). We propose an anti-inflammatory mechanism evoked by astragaloside IV by suppression of neutrophils adhesion-related molecules, which exerts neuroprotection against I/R injury.     

Salidroside promotes rat spinal cord injury recovery by inhibiting inflammatory cytokine expression and NF-κB and MAPK signaling pathways

Spinal cord injury (SCI) is a public health problem in the world. The SCI usually triggers an excessive inflammatory response that brings about a secondary tissue wreck leading to further cellular and organ dysfunction. Hence, there is great potential of reducing inflammation for therapeutic strategies of SCI. In this study, we aim to investigate if Salidroside (SAD) exerts an anti-inflammatory effect and promotes recovery of motor function on SCI through suppressing nuclear factor-κB (NF-κB) and the mitogen-activated protein kinase (MAPK) pathways. In vitro, real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) were used to examine the inhibitory effect of SAD on the expression and release of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) activated by lipopolysaccharide (LPS) in astrocytes. In addition, SAD was found to inhibit NF-κB, p38 and extracellular-regulated protein kinases (ERK) signaling pathways by western blot analysis. Further, in vivo study showed that SAD was able to improve hind limb motor function and reduce tissue damage accompanied by the suppressed expression of inflammatory cytokines IL-1β, IL-6, and TNF-α. Overall, SAD could reduce the inflammatory response and promote motor function recovery in rats after SCI by inhibiting NF-κB, p38, and ERK signaling pathways.     

Toll-like receptor 4 contributes to uterine activation by upregulating pro-inflammatory cytokine and CAP expression via the NF-κB/P38MAPK signaling pathway during pregnancy

Evidence indicates that inflammatory response is significant during the physiological process of human parturition; however, the specific signaling pathway that triggers inflammation is undefined. Toll-like receptors (TLRs) are key upstream gatekeepers that control inflammatory activation before preterm delivery. Our previous study showed that TLR4 expression was significantly increased in human pregnancy tissue during preterm and term labor. Therefore, we explore whether TLR4 plays a role in term labor by initiating inflammatory responses, therefore promoting uterine activation. The results showed that expression of TLR4, interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), CC chemokine ligand 2 (CCL-2), and uterine contraction-associated proteins (CAPs) was upregulated in the human and mice term labor (TL) group compared with the not-in-labor (TNL) group, and the TLR4 level positively correlated with CAP expression. In pregnant TLR4-knockout (TLR4^−/−) mice, gestation length was extended by 8 hr compared with the wild-type group, and the expression of IL-1β, IL-6, TNF-α, CCL-2, and CAPs was decreased in TLR4^−/− mice. Furthermore, nuclear factor-κB (NF-κB) and P38MAPK activation is involved in the initiation of labor but was inhibited in TLR4^−/− mice. In uterine smooth muscle cells, the expression of inflammatory cytokines and CAPs decreased when the NF-κB and P38MAPK pathway was inhibited. Our data suggest that TLR4 is a key factor in regulating the inflammatory response that drives uterine activation and delivery initiation via activating the NF-κB/P38MAPK pathway.     

Genipin Inhibits LPS-Induced Inflammatory Response in BV2 Microglial Cells

Genipin, an aglycon of geniposide, has been reported to have anti-inflammatory effect. However, the anti-inflammatory activity of genipin on LPS-stimulated BV2 microglial cells has not been reported. In this study, we investigated the molecular mechanisms responsible for the anti-inflammatory activity of genipin both in vivo and in vitro. The levels of TNF-α, IL-1β, NO and PGE₂ were detected by ELISA. The expression of Nrf2, HO-1, and NF-κB were detected by western blot analysis. In vivo, genipin significantly attenuated LPS-induced memory deficit in the Morris water maze and passive avoidance tasks. Genipin also inhibited LPS-induced TNF-α and IL-1β expression in brain tissues. In vitro, our results showed that genipin inhibited LPS-induced TNF-α, IL-1β, NO and PGE₂ production in a concentration-dependent manner. Genipin also suppressed LPS-induced NF-κB activation. In addition, the expression of Nrf2 and HO-1 were up-regulated by treatment of genipin. Furthermore, the inhibition of genipin on inflammatory mediator production was attenuated by transfection with Nrf2 siRNA. In conclusion, genipin inhibited LPS-induced inflammatory response by activating Nrf2 signaling pathway in BV2 microglia.     

Eupatilin alleviates inflammatory response after subarachnoid hemorrhage by inhibition of TLR4/MyD88/NF-κB axis

Early brain injury (EBI) is associated with the adverse prognosis of subarachnoid hemorrhage (SAH) patients. The key bioactive component of the Chinese herbal medicine Artemisia asiatica Nakai (Asteraceae) is eupatilin. Recent research reports that eupatilin suppresses inflammatory responses induced by intracranial hemorrhage. This work is performed to validate whether eupatilin can attenuate EBI and deciphers its mechanism. A SAH rat model was established by intravascular perforation in vivo. At 6 h after SAH in rats, 10 mg/kg eupatilin was injected into the rats via the caudal vein. A Sham group was set as the control. In vitro, BV2 microglia was treated with 10 μM Oxyhemoglobin (OxyHb) for 24 h, followed by 50 μM eupatilin treatment for 24 h. The SAH grade, brain water content, neurological score, and blood-brain barrier (BBB) permeability of the rats were measured 24 h later. The content of proinflammatory factors was detected via enzyme-linked immunosorbent assay. Western blot analysis was conducted to analyze the expression levels of TLR4/MyD88/NF-κB pathway-associated proteins. In vivo, eupatilin administration alleviated neurological injury, and decreased brain edema and BBB injury after SAH in rats. Eupatilin markedly reduced the levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α), and suppressed the expression levels of MyD88, TLR4, and p-NF-κB p65 in the SAH rats' cerebral tissues. Eupatilin treatment also reduced the levels of IL-1β, IL-6, and TNF-α, and repressed the expression levels of MyD88, TLR4, and p-NF-κB p65 in OxyHb-induced BV2 microglia. Additionally, pyrrolidine dithiocarbamate or resatorvid enhanced the suppressive effects of eupatilin on OxyHb-induced inflammatory responses in BV2 microglia. Eupatilin ameliorates SAH-induced EBI via modulating the TLR4/MyD88/NF-κB pathway in rat model.     

幽门螺杆菌感染激活NF-κB信号通路促进胃癌SGC-7901细胞炎症因子释放

为了探讨幽门螺杆菌对胃癌SGC-7901细胞炎症因子释放的影响,本研究将幽门螺杆菌感染SGC-7901细胞后,采用细胞计数盒(CCK-8)检测SGC-7901细胞活力,酶联免疫吸附实验(ELISA)检测炎症因子TNF-α、IL-1β以及IL-8的水平,Real-time PCR检测细胞TNF-α、IL-1β以及IL-8 m RNA的表达,蛋白免疫印迹法(Western blotting)检测NF-κB信号通路相关蛋白NF-κB p65蛋白表达以及IκBα磷酸化水平。研究结果表明,幽门螺杆菌感染后,SGC-7901细胞活力显著增加;幽门螺杆菌感染明显上调SGC-7901细胞TNF-α、IL-1β以及IL-8 mRNA的表达;本研究还进一步发现幽门螺杆菌感染显著增加SGC-7901细胞TNF-α、IL-1β以及IL-8的水平;此外,幽门螺杆菌处理的SGC-7901细胞,其NF-κB p65的蛋白表达以及IκBα磷酸化水平均显著上调。本研究的结论初步表明,幽门螺杆菌感染促进胃癌SGC-7901细胞炎症因子的释放,其机制可能涉及激活NF-κB信号通路。     

NF-κB inhibitory action of resveratrol: A probable mechanism of neuroprotection in experimental diabetic neuropathy

Resveratrol has shown array of biological actions, and is under clinical development for various disease conditions. The etiology of diabetic neuropathy revolves around oxidative stress, AGE formation, lipid peroxidation etc. All these stimulate inflammatory processes and NF-κB cascade is considered as one of the major players of inflammatory response. Activation of NF-κB results in elevated levels of inflammatory mediators. COX-2 and TNF-α activity have also been correlated with inflammatory damage in the pathophysiology of diabetic neuropathy (DN). Therefore we investigated the effect of resveratrol on NF-κB inflammatory cascade, COX-2, TNF-α and IL-6 levels in experimental DN.We found that resveratrol protected against various functional and behavioral deficits in diabetic neuropathy in line with our earlier published reports. In this study we found that the resveratrol treatment decreased the expression of p65 and IκB-α in treated rats. Treatment also ameliorated the elevated levels of TNF-α, IL-6 and COX-2. Resveratrol treatment produced significant decrease in nerve MDA levels in treated animals which may also be contributing to reduction in neuro-inflammation. This study confirms the NF-κB inhibitory activity and anti-inflammatory activity of resveratrol which may contribute to neuroprotection in diabetic neuropathy apart from its antioxidant effect.     

JNK-IN-8, a c-Jun N-terminal kinase inhibitor,improves functional recovery through suppressing neuroinflammation in ischemic stroke

C-Jun N-terminal kinase (JNK) is a pivotal MAPK (mitogen-activated protein kinase), which activated by ischemia brain injury and plays a fairly crucial function in cerebral ischemic injury. Emerging studies demonstrated that JNK-IN-8 (a JNK inhibitor with high specificity) regulates traumatic brain injury through controlling neuronal apoptosis and inflammation. However, the function of JNK-IN-8 in ischemic stroke and the mechanisms underlying of JNK-IN-8 about neuroprotection are not well understood. In this work, male rats were treated with JNK-IN-8 after transient middle cerebral artery occlusion, and then the modified improved neurological function score (mNSS), the foot-fault test (FFT), interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) levels were assessed. We found that JNK-IN-8-treated rats with MCAO exerted an observable melioration in space learning as tested by the improved mNSS, and showed sensorimotor functional recovery as measured by the FFT. JNK-IN-8 also played anti-inflammatory roles as indicated through decreased activation of microglia and decreased IL-6, IL-1β, and TNF-α expression. Furthermore, JNK-IN-8 suppressed the activation of JNK and nuclear factor-κB (NF-κB) signaling as indicated by the decreased level of phosphorylated-JNK and p65. All data demonstrate that JNK-IN-8 inhibits neuroinflammation and improved neurological function by inhibiting JNK/NF-κB and is a promising agent for the prevention of ischemic brain injury.     

Reduced hepatic injury in Toll-like receptor 4-deficient mice following D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure

Liver transplantation is the only therapy of proven benefit in fulminant hepatic failure (FHF). Lipopolysaccharide (LPS), D-galactosamine (GalN)-induced FHF is a well established model of liver injury in mice. Toll-Like Receptor 4 (TLR4) has been identified as a receptor for LPS. The aim of this study was to investigate the role of TLR4 in FHF induced by D-GalN/LPS administration in mice. Wild type (WT) and TLR4 deficient (TLR4ko) mice were studied in vivo in a fulminant model induced by GalN/LPS. Hepatic TLR4 expression, serum liver enzymes, hepatic and serum TNF-α and interleukin-1β levels were determined. Apoptotic cells were identified by immunohistochemistry for caspase-3. Nuclear factor-kappaβ (NF-κ β) and phosphorylated c-Jun hepatic expression were studied using Western blot analysis. All WT mice died within 24 hours after administration of GalN/LPS while all TLR4ko mice survived. Serum liver enzymes, interleukin-1β, TNF-α level, TLR4 mRNA expression, hepatic injury and hepatocyte apoptosis all significantly decreased in TLR4ko mice compared with WT mice. A significant decrease in hepatic c-Jun and IκB signaling pathway was noted in TLR4ko mice compared with WT mice. In conclusion, following induction of FHF, the inflammatory response and the liver injury in TLR4ko mice was significantly attenuated through decreased hepatic c-Jun and NF-κB expression and thus decreased TNF-α level. Down-regulation of TLR4 expression plays a pivotal role in GalN/LPS induced FHF. These findings might have important implications for the use of the anti TLR4 protein signaling as a potential target for therapeutic intervention in FHF.     

Short-term high fat feeding increases organ injury and mortality after polymicrobial sepsis

The purpose of this study was to examine the effect of short-term high fat feeding on the inflammatory response in polymicrobial sepsis. Male C57BL/6 mice at 6 weeks of age were randomized to a high-fat diet (HFD) (60% kcal fat) or control diet (CD) (16% kcal fat) for 3 weeks. After 3 weeks of feeding, sepsis was induced by cecal ligation and puncture (CLP) and animals were monitored for survival. In a separate experiment, after 3 weeks of feeding mice underwent CLP and were sacrificed at various time points thereafter. Tissue was collected for biochemical studies. Mice fed a HFD gained more weight and had a greater fat mass compared to CD-fed mice. Mice on a HFD had a lower probability of survival and more severe lung injury compared with CD-fed mice following sepsis. Myeloperoxidase (MPO) activity, an indicator of neutrophil infiltration, was increased in the lung and liver after CLP in HFD-fed mice compared with CD (P < 0.05). The plasma cytokines tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were increased in both groups after CLP, however, TNF-α and IL-6 levels were lower in HFD mice at 3 h after CLP compared with CD and consistent with lung, but not liver, messenger RNA (mRNA) expression. Leptin levels were higher in HFD-fed mice at 18 h after sepsis compared to baseline levels (P < 0.05). Polymicrobial sepsis increased hepatic nuclear factor-κB (NF-κB) activation in HFD-fed mice after CLP vs. CD-fed mice. Short duration high fat feeding increases mortality and organ injury following polymicrobial sepsis. These effects correspond to changes in NF-κB.     

Picroside II Attenuates CCI-Induced Neuropathic Pain in Rats by Inhibiting Spinal Reactive Astrocyte-Mediated Neuroinflammation Through the NF-κB Pathway

Reactive astrocyte-mediated neuroinflammatory responses in the spinal dorsal horn have been reported to play a pivotal role in pathological pain. Chronic constriction injury (CCI) enhances the activation of nuclear factor kappa B (NF-κB), which is involved in neuropathic pain (NP). Picroside II (PII), a major active component of Picrorhiza scrophulariiflora, has been investigated for its anti-oxidative, anti-inflammatory, and anti-apoptotic activities. Here, we explored the analgesic effects of PII on a model of CCI-induced NP and investigated the levels of the GFAP protein and the mRNA and protein levels of pro-inflammatory cytokines in the spinal cord, including interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). CCI significantly induced mechanical allodynia and thermal hyperalgesia. Intraperitoneal administration of PII remarkably reversed the CCI-induced mechanical allodynia and thermal hyperalgesia and reduced the mRNA and protein levels of IL-1β, IL-6, and TNF-α in the spinal cord. Additionally, according to the in vitro data, the PII treatment inhibited LPS-induced increases in the mRNA and protein levels of IL-1β, IL-6, and TNF-α and suppressed the NF-κB pathway by inhibiting the phosphorylation of NF-κB/p65 and the degradation of inhibitor of NF-κB (IκB) in astrocytes without toxicity to astrocytes. Overall, the analgesic effect of PII correlated with the inhibition of spinal reactive astrocyte-mediated neuroinflammation through the NF-κB pathway in rats with NP.     

Interleukin-1β and tumor necrosis factor-α augment acidosis-induced rat articular chondrocyte apoptosis via nuclear factor-kappaB-dependent upregulation of ASIC1a channel

The acute-phase proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) demonstrate high-level expression and pleiotropic biological effects, and contribute to the progression and persistence of rheumatoid arthritis (RA). Acid hydrarthrosis is also an important pathological characteristic of RA, and the acid-sensing ion channel 1a (ASIC1a) plays a critical role in acidosis-induced chondrocyte cytotoxicity. However, the roles of IL-1β and TNF-α in acid-induced apoptosis of chondrocytes remain unclear. Rat adjuvant arthritis and primary articular chondrocytes were used as in vivo and in vitro model systems, respectively. ASIC1a expression in articular cartilage was increased and highly colocalized with nuclear factor (NF)-κB expression in vivo. IL-1β and TNF-α could upregulate ASIC1a expression. These cytokines activated mitogen-activated protein kinase and NF-κB pathways in chondrocytes, while the respective inhibitors of these signaling pathways could partially reverse the ASIC1a upregulation induced by IL-1β and TNF-α. Dual luciferase and gel-shift assays and chromatin immunoprecipitation-polymerase chain reaction demonstrated that IL-1β and TNF-α enhanced ASIC1a promoter activity in chondrocytes by increasing NF-κB DNA-binding activities, which was in turn prevented by the NF-κB inhibitor ammonium pyrrolidinedithiocarbamate. IL-1β and TNF-α also decreased cell viability but enhanced LDH release, intracellular Ca²⁺ concentration elevation, loss of mitochondrial membrane potential, cleaved PARP and cleaved caspase-3/9 expression, and apoptosis in acid-stimulated chondrocytes, which effects could be abrogated by the specific ASIC1a inhibitor psalmotoxin-1 (PcTX-1), ASIC1a-short hairpin RNA or calcium chelating agent BAPTA-AM. These results indicate that IL-1β and TNF-α can augment acidosis-induced cytotoxicity through NF-κB-dependent up-regulation of ASIC1a channel expression in primary articular chondrocytes.     

美洛昔康对Aβ诱导Alzheimer’s disease大鼠脑内炎症损伤的影响

目的：研究美洛昔康对β-淀粉样蛋白（Aβ）诱导的阿尔茨海默病（AD）模型大鼠脑内炎症损伤的保护作用,并探讨其抑制炎症作用的机制。方法：Aβ1-40海马注射建立AD大鼠模型。免疫组化法观察大鼠海马核因子κBp65（NF-κBp65）和星形胶质细胞（AS）胶质纤维酸性蛋白（GFAP）表达变化;Western-blot法测定大鼠皮层组织GFAP的表达;ELISA法检测大鼠皮层组织肿瘤坏死因子-α（TNF-α）水平变化;RT-PCR法检测大鼠海马组织白细胞介素-1β（IL-1β）mRNA的表达情况。结果：美洛昔康能抑制AD大鼠海马NF-κBp65和GFAP的表达;降低大鼠皮层TNF-α的含量;抑制AD大鼠海马IL-1βmRNA的表达。结论：美洛昔康通过减少AD模型大鼠海马、皮层组织GFAP表达,抑制AS的增生,降低NF-κBp65的活性,减少炎症因子TNF-α和IL-1β的水平,减轻脑内炎症反应。     

Organic dust augments nucleotide-binding oligomerization domain expression via an NF-{kappa}B pathway to negatively regulate inflammatory responses

Nucleotide-binding oligomerization domain 2 (NOD2) is involved in innate immune responses to peptidoglycan degradation products. Peptidoglycans are important mediators of organic dust-induced airway diseases in exposed agriculture workers; however, the role of NOD2 in response to complex organic dust is unknown. Monocytes/macrophages were exposed to swine facility organic dust extract (ODE), whereupon NOD2 expression was evaluated by real-time PCR and Western blot. ODE induced significant NOD2 mRNA and protein expression at 24 and 48 h, respectively, which was mediated via a NF-κB signaling pathway as opposed to a TNF-α autocrine/paracrine mechanism. Specifically, NF-κB translocation increased rapidly following ODE stimulation as demonstrated by EMSA, and inhibition of the NF-κB pathway significantly reduced ODE-induced NOD2 expression. However, there was no significant reduction in ODE-induced NOD2 gene expression when TNF-α was inhibited or absent. Next, it was determined whether NOD2 regulated ODE-induced inflammatory cytokine production. Knockdown of NOD2 expression by small interfering RNA resulted in increased CXCL8 and IL-6, but not TNF-α production in response to ODE. Similarly, primary lung macrophages from NOD2 knockout mice demonstrated increased IL-6, CXCL1, and CXCL1, but not TNF-α, expression. Lastly, a higher degree of airway inflammation occurred in the absence of NOD2 following acute (single) and repetitive (3 wk) ODE exposure in an established in vivo murine model. In summary, ODE-induced NOD2 expression is directly dependent on NF-κB signaling, and NOD2 is a negative regulator of complex, organic dust-induced inflammatory cytokine/chemokine production in mononuclear phagocytes.