Prmt2 regulates the lipopolysaccharide-induced responses in lungs and macrophages

Precise control of the LPS stimulation in the lung modulates inflammation and airway hyperresponsiveness involving the well-known TLR4/NF-κB pathway. As a consequence, the expression and secretion of proinflammatory cytokines is tightly regulated with the recruitment of neutrophils. Changes in the LPS-induced responses have been observed in the Prmt2-Col6a1 monosomic model, suggesting the presence of dosage-sensitive genes controlling LPS pathway in the mouse. In this article, we report that the Prmt2 regulates the LPS-induced lung responses in lungs and macrophages. We demonstrate that Prmt2 gene dosage influences the lung airway hyperresponsiveness, the recruitment of neutrophils, and the expression of proinflammatory cytokines, such as IL-6 and TNF-α. In addition, Prmt2 loss of function also altered the nuclear accumulation of NF-κB in stimulated macrophages. Prmt2 should be considered as a new member of the NF-κB pathway controlling LPS-induced inflammatory and lung responses in a dosage-dependent manner, certainly through regulating nuclear accumulation of NF-κB as shown already in fibroblasts.     

Essential role of NF-κB and AP-1 transcription factors in TNF-α-induced TSLP expression in human airway smooth muscle cells

Human airway smooth muscle (HASM) cells are a rich source of inflammatory mediators that may propagate the airway inflammatory responses. Recent studies from our laboratory and others demonstrate that HASM cells express the proallergic cytokine thymic stromal lymphopoietin (TSLP) in vitro and in vivo. Compelling evidence from in vitro studies and animal models suggest that the TSLP is a critical factor sufficient and necessary to induce or maintain the allergic airway inflammation. Despite of an immense interest in pathophysiology of TSLP in allergic inflammation, the triggers and mechanisms of TSLP expression remain inadequately understood. In this study, we found that TNF-α upregulates the TSLP mRNA and induces high levels of TSLP protein release in primary human ASM cells. Interestingly, TNF-α induced the TSLP promoter activity (P < 0.05; n = 4) in HASM that was mediated by upstream NF-κB and activator protein-1 (AP-1) binding sites. Mutation in NF-κB and AP-1 binding sites completely abrogated the effect of TNF-α-mediated TSLP promoter activity and so did the expression of a dominant-negative mutant construct of IκB kinase. Furthermore, the peptide inhibitors of IκB kinase or NF-κB inhibited the TNF-α-induced TSLP protein release (P < 0.05; n = 3) in HASM. Collectively, our data suggest a novel important biological role for NF-κB pathway in TNF-α-induced TSLP expression in HASM and recommend this as a prime target for anti-inflammatory drugs.     

Inhibition of Aldose Reductase Prevents Experimental Allergic Airway Inflammation in Mice

Background

The bronchial asthma, a clinical complication of persistent inflammation of the airway and subsequent airway hyper-responsiveness, is a leading cause of morbidity and mortality in critically ill patients. Several studies have shown that oxidative stress plays a key role in initiation as well as amplification of inflammation in airways. However, still there are no good anti-oxidant strategies available for therapeutic intervention in asthma pathogenesis. Most recent studies suggest that polyol pathway enzyme, aldose reductase (AR), contributes to the pathogenesis of oxidative stress–induced inflammation by affecting the NF-κB-dependent expression of cytokines and chemokines and therefore inhibitors of AR could be anti-inflammatory. Since inhibitors of AR have already gone through phase-III clinical studies for diabetic complications and found to be safe, our hypothesis is that AR inhibitors could be novel therapeutic drugs for the prevention and treatment of asthma. Hence, we investigated the efficacy of AR inhibition in the prevention of allergic responses to a common natural airborne allergen, ragweed pollen that leads to airway inflammation and hyper-responsiveness in a murine model of asthma.

Methods and Findings

Primary Human Small Airway Epithelial Cells (SAEC) were used to investigate the in vitro effects of AR inhibition on ragweed pollen extract (RWE)-induced cytotoxic and inflammatory signals. Our results indicate that inhibition of AR prevents RWE -induced apoptotic cell death as measured by annexin-v staining, increase in the activation of NF-κB and expression of inflammatory markers such as inducible nitric oxide synthase (iNOS), cycloxygenase (COX)-2, Prostaglandin (PG) E₂, IL-6 and IL-8. Further, BALB/c mice were sensitized with endotoxin-free RWE in the absence and presence of AR inhibitor and followed by evaluation of perivascular and peribronchial inflammation, mucin production, eosinophils infiltration and airway hyperresponsiveness. Our results indicate that inhibition of AR prevents airway inflammation and production of inflammatory cytokines, accumulation of eosinophils in airways and sub-epithelial regions, mucin production in the bronchoalveolar lavage fluid and airway hyperresponsiveness in mice.

Conclusions

These results suggest that airway inflammation due to allergic response to RWE, which subsequently activates oxidative stress-induced expression of inflammatory cytokines via NF-κB-dependent mechanism, could be prevented by AR inhibitors. Therefore, inhibition of AR could have clinical implications, especially for the treatment of airway inflammation, a major cause of asthma pathogenesis.     

Magnesium isoglycyrrhizinate suppresses LPS-induced inflammation and oxidative stress through inhibiting NF-κB and MAPK pathways in RAW264.7 cells

Magnesium Isoglycyrrhizinate (MgIG), a novel molecular compound extracted from licorice root, has exhibited greater anti-inflammatory activity and hepatic protection than glycyrrhizin and β-glycyrrhizic acid. In this study, we investigated the anti-inflammatory effect and the potential mechanism of MgIG on Lipopolysaccharide (LPS)-treated RAW264.7 cells. MgIG down-regulated LPS-induced pro-inflammatory mediators and enzymes in LPS-treated RAW264.7 cells, including TNF-α, IL-6, IL-1β, IL-8, NO and iNOS. The generation of reactive oxygen species (ROS) in LPS-treated RAW264.7 cells was also reduced. MgIG attenuated NF-κB translocation by inhibiting IKK phosphorylation and IκB-α degradation. Simultaneously, MgIG also inhibited LPS-induced activation of MAPKs, including p38, JNK and ERK1/2. Taken together, these results suggest that MgIG suppresses inflammation by blocking NF-κB and MAPK signaling pathways, and down-regulates ROS generation and inflammatory mediators.     

Enterovirus 71 2C protein inhibits TNF-α-mediated activation of NF-κB by suppressing IκB kinase β phosphorylation

Enterovirus 71 (EV71), a single, positive-stranded RNA virus, has been regarded as the most important neurotropic enterovirus after the eradication of the poliovirus. EV71 infection can cause hand, foot, and mouth disease or herpangina. Cytokine storm with elevated levels of proinflammatory and inflammatory cytokines, including TNF-α, has been proposed to explain the pathogenesis of EV71-induced disease. TNF-α-mediated NF-κB signaling pathway plays a key role in inflammatory response. We hypothesized that EV71 might also moderate host inflammation by interfering with this pathway. In this study, we tested this hypothesis and identified EV71 2C protein as an antagonist of TNF-α-mediated activation of NF-κB signaling pathway. Expression of 2C protein significantly reduced TNF-α-mediated NF-κB activation in 293T cells as measured by gene reporter and gel mobility shift assays. Furthermore, overexpression of TNFR-associated factor 2-, MEK kinase 1-, IκB kinase (IKK)α-, or IKKβ-induced NF-κB activation, but not constitutively active mutant of IKKβ (IKKβ SS/EE)-induced NF-κB activation, was inhibited by 2C protein. These data together suggested that the activation of IKKβ is most likely targeted by 2C; this notion was further strengthened by immunoblot detection of IKKβ phosphorylation and IκBα phosphorylation and degradation. Coimmunoprecipitation and colocalization of 2C and IKKβ expressed in mammalian cells provided compelling evidence that 2C interacts with IKKβ. Collectively, our data indicate that EV71 2C protein inhibits IKKβ activation and thus blocks NF-κB activation.     

AGGF1 is a novel anti-inflammatory factor associated with TNF-α-induced endothelial activation

Endothelial activation contributes to the development of vascular inflammation and subsequent vascular diseases, particularly atherosclerosis. AGGF1, a new member of angiogenic factors with a FHA and a G-patch domain, has been shown critical for the regulation of vascular differentiation and angiogenesis. In this study, we found that various inflammatory cytokines strongly induced the expression of AGGF1 in endothelial cells (ECs) and identified AGGF1 as a novel anti-inflammatory factor both in vivo and in vitro. Overexpression of AGGF1 significantly repressed the expression of pro-inflammatory molecules such as E-Selectin, ICAM-1, and IL-8 and the adhesion of monocytes onto ECs activated by TNF-α. Conversely, the knockdown of AGGF1 resulted in the increased expressions of these pro-inflammatory molecules and the enhanced monocyte-EC interaction. We further demonstrated that AGGF1 potently attenuated TNF-α triggered NF-κB pathway, as indicated by the decreased promoter activity, nuclear distribution and phosphorylation of NF-κB p65 subunit as well as the increased protein level of IκBα. This inhibitory effect of AGGF1 was further proved through blocking the phosphorylation of ERK induced by TNF-α. Finally, we showed that the FHA domain of AGGF1 was required for its anti-inflammatory effect. Thus, our findings for the first time demonstrate that AGGF1 suppresses endothelial activation responses to TNF-α by antagonizing the ERK/NF-κB pathway, which makes AGGF1 a promising therapeutic candidate for the prevention and treatment of inflammatory diseases.     

Tumor necrosis factor alpha-induced inflammation is increased but apoptosis is inhibited by common food additive carrageenan

Tumor necrosis factor (TNF)-α, a homotrimeric, pleiotropic cytokine, is secreted in response to inflammatory stimuli in diseases such as rheumatoid arthritis and inflammatory bowel disease. TNF-α mediates both apoptosis and inflammation, stimulating an inflammatory cascade through the non-canonical pathway of NF-κB activation, leading to increased nuclear RelB and p52. In contrast, the common food additive carrageenan (CGN) stimulates inflammation through both the canonical and non-canonical pathways of NF-κB activation and utilizes the adaptor molecule BCL10 (B-cell leukemia/lymphoma 10). In a series of experiments, colonic epithelial cells and mouse embryonic fibroblasts were treated with TNF-α and carrageenan in order to simulate the possible effects of exposure to dietary CGN in the setting of a TNF-α-mediated inflammatory disease process. A marked increase in secretion of IL-8 occurred, attributable to synergistic effects on phosphorylated NF-κB-inducing kinase (NIK) in the non-canonical pathway. TNF-α induced the ubiquitination of TRAF2 (TNF receptor-associated factor 2), which interacts with NIK, and CGN induced phosphorylation of BCL10, leading to increased NIK phosphorylation. These results suggest that TNF-α and CGN in combination act to increase NIK phosphorylation, thereby increasing activation of the non-canonical pathway of NF-κB activation. In contrast, the apoptotic effects of TNF-α, including activation of caspase-8 and PARP-1 (poly(ADP-ribose) polymerase 1) fragmentation, were markedly reduced in the presence of CGN, and CGN caused reduced expression of Fas. These findings demonstrate that exposure to CGN drives TNF-α-stimulated cells toward inflammation rather than toward apoptotic cell death and suggest that CGN exposure may compromise the effectiveness of anti-TNF-α therapy.     

Flavonoids of <Emphasis Type="Italic">Rosa roxburghii</Emphasis> Tratt offers protection against radiation induced apoptosis and inflammation in mouse thymus

The present study evaluated the protective effect of the natural compound flavonoids of Rosa roxburghii Tratt (FRT) against γ-radiation-induced apoptosis and inflammation in mouse thymus cells in vivo and in vitro. Thymus cells and mice were exposed to ⁶⁰Co γ-ray at a dose of 6 Gy. The radiation treatment induced significant cell apoptosis and inflammation. Radiation increased the expressions of cleaved caspase 3/8–10, AIF, and PARP-1, and FRT could mitigate their activation and inhibit subsequent apoptosis in the thymus both in vitro or in vivo. Irradiation increased the mRNA expression of ICAM-1/VCAM-1, IL-1α/IL-6 and TNF-α/NF-κB. Our results also indicated that FRT alleviated gene expression of some inflammatory factors such as ICAM-1/VCAM-1, TNF-α/NF-κB, but not IL-1α/IL-6. Irradiation increased the protein expression levels of ICAM-1/VCAM-1, IL-1α/IL-6 and TNF-α/NF-Κb, and our results also indicated that FRT alleviated protein level expression of certain inflammatory factors such as ICAM-1, IL-1α/IL-6, TNF-α/NF-κB, but not VCAM-1. Our results suggested that FRT enhanced radioprotection at least partially by regulating caspase 3/8–10, AIF, and PARP-1 to reduce apoptosis and by regulating ICAM-1, IL-1α/IL-6, TNF-α/NF-κB to reduce inflammation.     

Insulin-like growth factor-binding protein-3 (IGFBP-3) blocks the effects of asthma by negatively regulating NF-κB signaling through IGFBP-3R-mediated activation of caspases

Insulin-like growth factor-binding protein-3 (IGFBP-3) is a multifunctional protein known for modulating mitogenic and metabolic actions of IGFs as well as exerting a variety of biological actions not involving IGFs. Here, we show that IGFBP-3 blocks specific physiological consequences of asthma in an IGF-independent manner in vitro and in vivo. IGFBP-3 treatment effectively reduced all physiological manifestations of asthma examined in vivo (airway hyper-responsiveness, cellular and pathological changes in bronchoalveolar lavage fluid and lung tissue, and expression of numerous proinflammatory molecules). These unique IGFBP-3 effects were further confirmed in IGFBP-3-transgenic mice, thus strengthening the notion of IGFBP-3 actions within the respiratory system. Using human epithelial cells, we demonstrated the following: 1) IGFBP-3 blocks TNF-α-induced expression of proinflammatory molecules; 2) IGFBP-3 attenuates the TNF-α-induced migratory response of eosinophils; and 3) IGFBP-3 negatively regulates TNF-α-induced expression of the key NF-κB regulatory molecules IκBα and p65-NF-κB at the post-translational level. We identified that IGFBP-3 degrades IκBα and p65-NF-κB proteins through IGFBP-3 receptor (IGFBP-3R)-mediated activation of caspases thereby inhibiting TNF-α-induced activation of NF-κB signaling cascades. This unique IGFBP-3/IGFBP-3R action was further confirmed by demonstrating complete inhibition of IGFBP-3 action in the presence of caspase inhibitors as well as IGFBP-3R siRNAs. Non-IGF-binding IGFBP-3 mutants further proved the IGF-independent action of IGFBP-3. Our findings indicate that IGFBP-3 inhibits airway inflammation and hyper-responsiveness via an IGF-independent mechanism that involves activation of IGFBP-3R signaling and cross-talk with NF-κB signaling. The IGFBP-3/IGFBP-3R system therefore plays a pivotal role in the pathogenesis of asthma and can serve as a newly identified potential therapeutic target for this debilitating disease.     

Saikosaponin a inhibits lipopolysaccharide-oxidative stress and inflammation in Human umbilical vein endothelial cells via preventing TLR4 translocation into lipid rafts

Saikosaponin a (SSa), the major triterpenoid saponin derivatives from Radix bupleuri (RB), has been reported to have anti-inflammatory effects. The aim of this study was to investigate the effects of SSa on lipopolysaccharide (LPS)-induced oxidative stress and inflammatory response in human umbilical vein endothelial cells (HUVECs). HUVECs were stimulated with LPS in the presence or absence of SSa. The levels of TNF-α and IL-8 were detected by ELISA. The expression of COX-2 and iNOS, NF-κB and IκB protein were determined by Western blotting. To investigate the protective mechanisms of SSa, TLR4 expression was detected by Western blotting and membrane lipid rafts were separated by density gradient ultracentrifugation and analyzed by immunoblotting with anti-TLR4 antibody. The results showed that SSa dose-dependently inhibited the production of ROS, TNF-α, IL-8, COX-2 and iNOS in LPS-stimulated HUVECs. Western blot analysis showed that SSa suppressed LPS-induced NF-κB activation. SSa did not affect the expression of TLR4 induced by LPS. However, translocation of TLR4 into lipid rafts and oligomerization of TLR4 induce by LPS was inhibited by SSa. Furthermore, SSa disrupted the formation of lipid rafts by depleting cholesterol. Moreover, SSa activated LXRα-ABCA1 signaling pathway, which could induce cholesterol efflux from lipid rafts. Knockdown of LXRα abrogated the anti-inflammatory effects of SSa. In conclusion, the effects of SSa is associated with activating LXRα-ABCA1 signaling pathway which results in disrupting lipid rafts by depleting cholesterol and reducing translocation of TLR4 to lipid rafts and oligomerization of TLR4, thereby attenuating LPS mediated oxidative and inflammatory responses.     

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.     

Regulation of inflammation by DAPK

Death-associated protein kinase (DAPK) is a tumor suppressor and negatively regulates several activation signals. Consistent with its potential anti-inflammatory activity, DAPK promotes the formation of IFN-γ-activated inhibitor of translation (GAIT) complex that suppresses the translation of selected inflammatory genes. DAPK has been found to inhibit tumor necrosis factor-α (TNF-α)- or lipopolysaccharides (LPS)-induced NF-κB activation and pro-inflammatory cytokine expression. Inflammation is always associated with T cell activation, while DAPK attenuates T cell activation by a selective suppression in T cell receptor-triggered NF-κB activation. Recent studies, however, also reveal a contribution of DAPK to pro-inflammatory processes. DAPK is shown to mediate pro-inflammatory signaling downstream of TNF-α, LPS, IL-17, or IL-32. In addition, DAPK is required for the full formation of NLRP3 inflammasome, essential for the generation of IL-1β and IL-18. These results suggest the complicated role of DAPK in the regulation of inflammation that is likely dependent on cell types and environmental cues.     

人参皂苷Rg1对游离脂肪酸诱导非酒精性脂肪肝细胞炎症的改善作用及机制研究

该研究探讨人参皂苷Rg1对非酒精性脂肪性肝细胞炎症反应的作用及其分子机制。用1 mmol/L游离脂肪酸处理HepG2和L02细胞24 h,再用20μg/mL或40μg/mL人参皂苷Rg1处理6 h;设置对照组、模型组、低剂量Rg1组、高剂量Rg1组。全自动生化仪检测各组细胞上清谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)的含量;酶联免疫吸附法测定细胞上清IL-1β、IL-6、TNF-α。RT-qPCR及Western blot检测NF-κB通路相关基因及蛋白的改变。免疫荧光染色观察NF-κB核转移;Western blot检测各组胞质与胞核内的NF-κB P65蛋白的表达。与对照组相比,模型组培养上清炎症指标明显增加(P<0.05);Rg1能降低炎症指标的表达(P<0.05)。Rg1能减少游离脂肪酸诱导的NF-κB磷酸化及其下游IL-1β、IL-6、TNF-α的表达,减少NF-κB P65从胞质向胞核的转移(P<0.05)。Rg1可通过抑制NF-κB活化减少NASH细胞模型炎症反应,为非酒精性脂肪性肝炎的治疗提供了可能的靶点。