Increase in CpG DNA-induced inflammatory responses by DNA oxidation in macrophages and mice

Unmethylated CpG dinucleotide (CpG motif) is involved in the exacerbation of DNA-associated autoimmune diseases. We investigated the effect of DNA containing 8-hydroxydeoxyguanosine (oxo-dG), a representative DNA biomarker for oxidative stress in the diseases, on CpG motif-dependent inflammatory responses. ODN1668 and ODN1720 were selected as CpG-DNA and non-CpG DNA, respectively. Deoxyguanosine in the CpG motif (G9) or outside the motif (G15) of ODN1668 was substituted with oxo-dG to obtain oxo(G9)-1668 and oxo(G15)-1668, respectively. Oxo(G15)-1668 induced a significantly higher amount of tumor necrosis factor (TNF)-α from RAW264.7 macrophage-like cells than ODN1668, whereas oxo(G9)-1668, oxo(G8)-1720, or oxo(G15)-1720 hardly did. CpG DNA-induced TNF-α production was significantly increased by addition of oxo(G8)-1720 or oxo(G15)-1720, but not of ODN1720. This oxo-dG-containing DNA-induced increase in TNF-α production was also observed in primary cultured macrophages isolated from wild-type mice, but not observed in those from Toll-like receptor (TLR)-9 knockout mice. In addition, TNF-α production by ligands for TLR3, TLR4, or TLR7 was not affected by oxo-dG-containing DNA. Then, the footpad swelling induced by subcutaneous injection of ODN1668 into mice was increased by coinjection with oxo(G8)-1720, but not with ODN1720. These results indicate that oxo-dG-containing DNA increases the CpG motif-dependent inflammatory responses, which would exacerbate DNA-related autoimmune diseases.     

Role of bacterial DNA in macrophage activation by group B streptococci

Bacterial DNA (CpG DNA) induces macrophage activation and the production of inflammatory mediators, including tumor necrosis factor (TNF) and nitric oxide (NO) by these cells. However, the role of bacterial DNA in the macrophage response to whole bacteria is unknown. We used overlapping strategies to estimate the relative contribution of bacterial DNA to the upregulation of TNF and NO production in macrophages stimulated with antibiotic-treated group B streptococci (GBS). Selective inhibitors of the bacterial DNA/TLR9 pathway (chloroquine, an inhibitory oligonucleotide, and DNase I) consistently inhibited GBS-induced TNF secretion by 35-50% in RAW 264.7 macrophages and murine splenic macrophages, but had no effect on inducible nitric oxide synthase (iNOS) accumulation or NO secretion. Similarly, splenic and peritoneal macrophages from mice lacking TLR9 expression secreted 40% less TNF than macrophages from control mice after GBS challenge but accumulated comparable amounts of iNOS protein. Finally, studies in both RAW 264.7 cells and macrophages from TLR9-/- mice implicated GBS DNA in the upregulation of interleukins 6 (IL-6) and 12 (IL-12) but not interferon-beta (IFNbeta), a key intermediary in macrophage production of iNOS/NO. Our data suggest that the bacterial DNA/TLR9 pathway plays an important role in stimulating TNF rather than NO production in macrophages exposed to antibiotic-treated GBS, and that TLR9-independent upregulation of IFNbeta production by whole GBS may account for this difference.     

Plasmid DNA activates murine macrophages to induce inflammatory cytokines in a CpG motif-independent manner by complex formation with cationic liposomes

Plasmid DNA (pDNA) is very important in non-viral gene therapy and DNA vaccination. Unmethylated CpG motifs in bacterial DNA, but not in vertebrate DNA, are known to trigger an inflammatory response, which inhibits gene expression while improving immunological consequences. In this report, we investigated the cytokine secretion induced by pDNA/cationic liposome complexes using murine macrophages. Naked CpG DNA induced tumor necrosis factor-alpha (TNF-alpha) secretion from the macrophages, but DNA without CpG motif did not, demonstrating that the cytokine induction was mediated by CpG motifs. pDNA complexed with cationic liposomes, but not the cationic liposomes alone, produced a significant amount of TNF-alpha from the macrophages. Surprisingly, methylated pDNA and calf thymus DNA complexed with the cationic liposomes were also able to induce TNF-alpha production, indicating that these responses were not dependent on CpG motifs. Taken together, the present study demonstrated that for the first time DNA can stimulate murine macrophages in a CpG motif-independent manner when it is complexed with the cationic liposomes.     

广叶绣球菌β-D-葡聚糖对巨噬细胞RAW264.7免疫调节作用受体TLR4和TLR2的影响

确定广叶绣球菌β-D-葡聚糖对巨噬细胞RAW264.7的免疫调节作用受体,探索广叶绣球菌β-D-葡聚糖的免疫调节机制。采用MTT法测定不同浓度广叶绣球菌β-D-葡聚糖对巨噬细胞RAW264.7增殖活力的影响,筛选出促进巨噬细胞增殖能力最强的浓度。用筛选出的β-D-葡聚糖浓度作用巨噬细胞RAW264.7;TLR4抗体和TLR2抗体分别作用巨噬细胞RAW264.7 1h,再用含有β-D-葡聚糖的细胞培养液培养。收集细胞培养上清和细胞,检测细胞培养上清中NO、IL-6、TNF-α、IFN-β的生成量;提取细胞内总RNA,采用RT-PCR测定巨噬细胞TLR4 mRNA表达量;提取巨噬细胞总蛋白,采用蛋白免疫印迹western blot测定TLR4的蛋白表达。广叶绣球菌β-D-葡聚糖能够促进巨噬细胞RAW264.7增殖,增加NO、IL-6、TNF-α、IFN-β的生成量,提高TLR4 mRNA表达和蛋白表达,差异极显著（P<0.01）。TLR4抗体作用细胞后,NO、IL-6、TNF-α、IFN-β的生成量明显下降,差异极显著（P<0.01）。TLR2抗体作用细胞后,NO、IL-6、TNF-α、IFN-β的生成量下降,但差异不显著。广叶绣球菌β-D-葡聚糖可以通过细胞表面受体TLR4激活信号转导通路,增强下游细胞因子的释放,从而调节巨噬细胞RAW264.7的免疫功能。TLR2可能不是广叶绣球菌β-D-葡聚糖的免疫受体。     

Molecular cloning and characterization of a novel anti-TLR9 intrabody

Toll-like receptor 9 (TLR9) is a component of the innate immune system, which recognizes the DNA of both pathogens and hosts. Thus, it can drive autoimmune diseases. Intracellular antibodies expressed inside the ER block transitory protein functions by inhibiting the translocation of the protein from the ER to its subcellular destination. Here, we describe the construction and characterization of an anti-TLR9 ER intrabody (αT9ib). The respective single-chain Fv comprises the variable domains of the heavy and light chain of a monoclonal antibody (mAb; 5G5) towards human and murine TLR9. Co-expression of αT9ib and mouse TLR9 in HEK293 cells resulted in co-localization of both molecules with the ER marker calnexin. Co-immunoprecipitation of mouse TLR9 with αT9ib indicated that αT9ib interacts with its cognate antigen. The expression of αT9ib inhibited NF-κB-driven reporter gene activation upon CpG DNA challenge but not the activation of TLR3 or TLR4. Consequently, TLR9-driven TNFα production was inhibited in RAW264.7 macrophages upon transfection with the αT9ib expression plasmid. The αT9ib-encoding open reading frame was integrated into an adenoviral cosmid vector to produce the recombinant adenovirus (AdV)-αT9ib. Transduction with AdVαT9ib specifically inhibited TLR9-driven cellular TNFα release. These data strongly indicate that αT9ib is a very promising experimental tool to block TLR9 signaling.     

Size-dependent attenuation of TLR9 signaling by gold nanoparticles in macrophages

Gold nanoparticles (GNPs), which are generally thought to be bio-inert and non-cytotoxic, have become one of the most ideal nanomaterials for medical applications. Once engulfed by phagocytes, the immunological effects of GNPs are still of concern and require detailed investigation. Therefore, this study explored the immunological significance of GNPs on TLR-mediated innate immunity in murine macrophages. GNP causes specific inhibition of TLR9 (CpG oligodeoxynucleotides; CpG-ODNs) signal in macrophages. The impaired CpG-ODN-induced TNF-α production is GNP concentration- and size-dependent in murine Raw264.7 cells: a GNP of 4 nm in size is more potent than a GNP of 11, 19, 35, or 45 nm in size. Consistent with cytokine inhibition, the CpG-ODN-induced phosphorylation of NF-κB and JNK as well as NF-κB activation are suppressed by GNPs. GNPs accumulate in lysosomes after phagocytosis and also increase TLR9-associated lysosomal cathepsin expression and activities, but this is irrelevant to TLR9 inhibition by GNPs in our studies. In addition, GNPs affected TLR9 translocation in response to CpG-ODNs and to phagosomes. Further exploring how GNPs inhibited TLR9 function, we found that GNPs could bind to high-mobility group box-1 (which is involved in the regulation of TLR9 signaling) inside the lysosomes. The current studies demonstrate that size-dependent inhibition of TLR9 function by GNP may be attributed to its binding to high-mobility group box-1.     

Siglec-9 enhances IL-10 production in macrophages via tyrosine-based motifs

We examined whether Siglec-9 modulates cytokine production in the macrophage cell line RAW264. Cells expressing Siglec-9 produced low levels of tumor necrosis factor (TNF)-α upon stimulation with lipopolysaccharide, peptidoglycan, unmethylated CpG DNA, and double-stranded RNA. On the other hand, interleukin (IL)-10 production was strongly enhanced in Siglec-9-expressing cells. Similar activities were also exhibited by Siglec-5. However, the up-regulation of IL-10 as well as the down-regulation of TNF-α was abrogated when two tyrosine residues in the cytoplasmic tail of Siglec-9 were mutated to phenylalanine. A membrane proximal ITIM mutant of Siglec-9 did not enhance IL-10 production but partly inhibited TNF-α production, indicating diverse regulation mechanisms of TNF-α and IL-10. Siglec-9 also enhanced the production of IL-10 in the human macrophage cell line THP-1. These results demonstrate that Siglec-9 enhances the production of the anti-inflammatory cytokine IL-10 in macrophages.     

Fucoidan对巨噬细胞RAW264.7的免疫调节作用

目的 观察褐藻多糖硫酸酯（Fucoidan）对巨噬细胞RAW264.7体外吞噬活性、细胞因子TNF-α和IL-6分泌,以及Toll样受体4（TLR4）mRNA表达的影响。方法 实验分对照组,Fucoidan高、中、低剂量组（浓度分别是200、400和800 μg/mL）。药物处理6~48 h后,MTT法检测RAW264.7细胞活力;中性红比色法检测细胞吞噬活性;ELISA法检测培养上清中TNF-α和IL-6的分泌水平;实时定量PCR检测Toll样受体4（TLR4）mRNA表达量。结果 与对照组相比,Fucoidan显著增强RAW264.7细胞代谢活力和吞噬能力（P＜0.01）,增加TNF-α和IL-6的分泌,上调TLR4的表达,呈剂量依赖关系。结论 Fucoidan可上调TLR4表达,增强巨噬细胞代谢和吞噬活性,增加TNF-α和IL-6的分泌,具有潜在的调节免疫作用。     

Molecular mechanism for adiponectin-dependent M2 macrophage polarization: link between the metabolic and innate immune activity of full-length adiponectin

The anti-inflammatory effects of globular adiponectin (gAcrp) are mediated by IL-10/heme oxygenase 1 (HO-1)-dependent pathways. Although full-length (flAcrp) adiponectin also suppresses LPS-induced pro-inflammatory signaling, its signaling mechanisms are not yet understood. The aim of this study was to examine the differential mechanisms by which gAcrp and flAcrp suppress pro-inflammatory signaling in macrophages. Chronic ethanol feeding increased LPS-stimulated TNF-α expression by Kupffer cells, associated with a shift to an M1 macrophage polarization. Both gAcrp and flAcrp suppressed TNF-α expression in Kupffer cells; however, only the effect of gAcrp was dependent on IL-10. Similarly, inhibition of HO-1 activity or siRNA knockdown of HO-1 in RAW264.7 macrophages only partially attenuated the suppressive effects of flAcrp on MyD88-dependent and -independent cytokine signatures. Instead, flAcrp, acting via the adiponectin R2 receptor, potently shifted the polarization of Kupffer cells and RAW264.7 macrophages to an M2 phenotype. gAcrp, acting via the adiponectin R1 receptor, was much less effective at eliciting an M2 pattern of gene expression. M2 polarization was also partially dependent on AMP-activated kinase. flAcrp polarized RAW264.7 macrophages to an M2 phenotype in an IL-4/STAT6-dependent mechanism. flAcrp also increased the expression of genes involved in oxidative phosphorylation in RAW264.7 macrophages, similar to the effect of flAcrp on hepatocytes. In summary, these data demonstrate that gAcrp and flAcrp utilize differential signaling strategies to decrease the sensitivity of macrophages to activation by TLR4 ligands, with flAcrp utilizing an IL-4/STAT6-dependent mechanism to shift macrophage polarization to the M2/anti-inflammatory phenotype.     

Cross-linking of SIGNR1 activates JNK and induces TNF-α production in RAW264.7 cells that express SIGNR1

In this study, we evaluated the signaling ability of SIGNR1 in murine macrophage-like RAW264.7 cells that stably expressed FLAG-tagged SIGNR1 (SIGNR1-FLAG). Cross-linking of SIGNR1-FLAG expressed on the cells by an anti-FLAG antibody induced JNK phosphorylation without induction of phosphorylation of ERK1/2 and p38 MAP kinase, and led to phosphorylations of Src family kinases (SFKs) and Akt. The SIGNR1-FLAG molecules in the cells were found in lipid raft-enriched membrane fractions, and the tyrosine kinases Lyn, Hck, and Fgr co-precipitated with SIGNR1-FLAG in the lipid raft fractions. The antibody-induced JNK phosphorylation was inhibited by inhibitors of SFKs and tyrosine kinases. Furthermore, cross-linking of SIGNR1 led to production of TNF-α, and the JNK inhibitor inhibited the antibody-induced TNF-α production. These results show that cross-linking of SIGNR1 triggers phosphorylation of SFKs, which leads to activation of the JNK pathway and induction of TNF-α production in macrophage-like RAW264.7 cells.     

Immunomodulatory beta-glucan from Lentinus edodes activates mitogen-activated protein kinases and nuclear factor-kappaB in murine RAW 264.7 macrophages

Lentinan, a cell wall β-glucan from the fruiting bodies of Lentinus edodes, is well known to be a biological defense modifier, but the signal transduction pathway(s) induced by Lentinan have not been elucidated. In this study, we extracted Lentinan (LNT-S) by ultrasonication from Lentinus edodes and report that, in murine RAW 264.7 macrophages, LNT-S glucan activated NF-κB p65 and triggered its nuclear translocation as determined by Western blotting. Moreover, LNT-S enhanced NF-κB-luciferase activity in the Dual-Luciferase gene system assay. Its upstream signaling molecules, MAPKs such as ERK1/2 and JNK1/2, were shown to be activated by assessing the level of phosphorylation in a time- and concentration-dependent manner, but its downstream proinflammatory enzyme, inducible NOS, was not observed. The data evaluated using a TNF-α ELISA kit and Griess reagent further demonstrated that no proinflammatory mediators such as TNF-α and NO were produced by LNT-S stimulation in RAW 264.7 cells. In contrast, LPS significantly induced inducible NOS expression and increased NO and TNF-α production, which are associated with activation of the NF-κB p65/p50 heterodimer complex. It is possible that LNT-S did not activate NF-κB p65/p50, and the activation of NF-κB p65 was not sufficient to stimulate cytokine production. These data demonstrate that LNT-S glucan carries out its immunomodulating activity by activating MAPK signaling pathways without secretion of TNF-α and NO.     

IFN-gamma primes RAW264 macrophages and human monocytes for enhanced oxidant production in response to CpG DNA via metabolic signaling: roles of TLR9 and myeloperoxidase trafficking

Macrophages and monocytes are activated by CpG DNA motifs to produce NO, which is enhanced dramatically by IFN-gamma. We hypothesize that synergistic cellular responses to IFN-gamma and CpG DNA are due to cross-talk between metabolic signaling pathways of leukocytes. Adherent RAW264.7 macrophages and human monocytes exhibited NAD(P)H autofluorescence oscillation periods of approximately 20 s. IFN-gamma increased the oscillatory amplitude, which was required for CpG DNA-mediated metabolic changes. These alterations in metabolic dynamics required the appropriate combinations of murine/human TLR9 and murine/human-specific CpG DNA. Other factors that also promoted an increase in metabolic oscillatory amplitude could substitute for IFN-gamma. Because recent studies have shown that the metabolic frequency is coupled to the hexose monophosphate shunt, and the amplitude is coupled to the peroxidase cycle, we tested the hypothesis that myeloperoxidase (MPO) participates in IFN-gamma priming for oxidant production. MPO inhibitors blocked cell responses to IFN-gamma and CpG DNA. In the absence of IFN-gamma exposure, the effects of CpG DNA could be duplicated by MPO addition to cell samples. Moreover, monocytes from MPO knockout mice were metabolically unresponsive to IFN-gamma and CpG DNA. NAD(P)H frequency doubling responses due to CpG DNA were blocked by an inhibitor of the hexose monophosphate shunt. Because NAD(P)H participates in electron trafficking to NO and superoxide anions, we tested oxidant production. Although CpG DNA alone had no effect, IFN-gamma plus CpG enhanced NO and reactive oxygen metabolite release compared with IFN-gamma treatment alone. We suggest that amplitude and frequency modulation of cellular metabolic oscillations contribute to intracellular signaling synergy.     

Cutting edge: a TLR9 cytoplasmic tyrosine motif is selectively required for proinflammatory cytokine production

Compartmentalization of nucleic acid sensing TLR9 has been implicated as a mechanism to prevent recognition of self nucleic acid structures. Furthermore, recognition of CpG DNA in different endosomal compartments leads to the production of the proinflammatory cytokine TNF-α, or type I IFN. We previously characterized a tyrosine-based motif at aa 888-891 in the cytoplasmic tail of TLR9 important for appropriate intracellular localization. In this article, we show that this motif is selectively required for the production of TNF, but not IFN. In response to CpG DNA stimulation, the proteolytically processed 80-kDa fragment is tyrosine phosphorylated. Although Y888 is not itself phosphorylated, the structure of this motif is necessary for both TLR9 phosphorylation and TNF-α production in response to CpG DNA. We conclude that bifurcation in TLR9 signaling is regulated by a critical tyrosine motif in the cytoplasmic tail.     

Ras participates in CpG oligodeoxynucleotide signaling through association with toll-like receptor 9 and promotion of interleukin-1 receptor-associated kinase/tumor necrosis factor receptor-associated factor 6 complex formation in macrophages

CpG oligodeoxynucleotides (ODN) activate immune cells to produce immune mediators by Toll-like receptor 9 (TLR9)-mediated signal transduction, which activates mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) through the MyD88/IRAK/TRAF6 kinases cascade. However, the precise mechanisms of CpG ODN activation of immune cells have not been fully elucidated. The small GTP-binding protein Ras mediates MAPK activation in response to a variety of stimuli. Up to now, it is not clear whether Ras plays a role in CpG ODN signaling. In the present study, we found that the dominant-negative version of Ras (RasN17) and specific Ras inhibitor, FTI-277, inhibited CpG ODN-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by murine macrophage cell line RAW264.7. While overexpression of wild-type Ras enhanced CpG ODN-induced extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and NF-kappaB activation, overexpression of RasN17 inhibited CpG ODN-induced ERK, JNK, and NF-kappaB activation. RasN17 overexpression also inhibited CpG ODN-induced IRAK1/TRAF6 complex formation. Further studies revealed that CpG ODN activated Ras in a time- and dose-dependent manner, and Ras associated with TLR9 in a CpG ODN-dependent manner. Most interestingly, activation of Ras preceded the association of Ras with TLR9, giving rise to a possibility that Ras activation might not be dependent on the interaction between Ras and TLR9. Our data demonstrate for the first time that Ras can be activated by CpG ODN in macrophages, and Ras is involved in CpG ODN signaling as an early event by associating with TLR9 and promoting IRAK1/TRAF6 complex formation, and MAPK and NF-kappaB activation.     

Autocrine abscisic acid plays a key role in quartz-induced macrophage activation

Inhalation of quartz induces silicosis, a lung disease where alveolar macrophages release inflammatory mediators, including prostaglandin-E(2) (PGE(2)) and tumor necrosis factor α (TNF-α). Here we report the pivotal role of abscisic acid (ABA), a recently discovered human inflammatory hormone, in silica-induced activation of murine RAW264.7 macrophages and of rat alveolar macrophages (AMs). Stimulation of both RAW264.7 cells and AMs with quartz induced a significant increase of ABA release (5- and 10-fold, respectively), compared to untreated cells. In RAW264.7 cells, autocrine ABA released after quartz stimulation sequentially activates the plasma membrane receptor LANCL2 and NADPH oxidase, generating a Ca(2+) influx resulting in NFκ B nuclear translocation and PGE(2) and TNF-α release (3-, 2-, and 3.5-fold increase, respectively, compared to control, unstimulated cells). Quartz-stimulated RAW264.7 cells silenced for LANCL2 or preincubated with a monoclonal antibody against ABA show an almost complete inhibition of NFκ B nuclear translocation and PGE(2) and TNF-α release compared to controls electroporated with a scramble oligonucleotide or preincubated with an unrelated antibody. AMs showed similar early and late ABA-induced responses as RAW264.7 cells. These findings identify ABA and LANCL2 as key mediators in quartz-induced inflammation, providing possible new targets for antisilicotic therapy.