Involvement of heat shock protein (Hsp)90 beta but not Hsp90 alpha in antiapoptotic effect of CpG-B oligodeoxynucleotide

Unmethylated CpG oligodeoxynucleotides (CpG ODNs) activate immune responses in a TLR9-dependent manner. In this study, we found that stimulation of mouse macrophages and dendritic cells with B-type CpG ODN (CpG-B ODN) increased the cellular level of heat shock protein (Hsp) 90beta but not Hsp90alpha and prevented apoptosis induced by serum starvation or staurosporine treatment. The CpG-B ODN-induced Hsp90beta expression depended on TLR9, MyD88, and PI3K. Inhibition of Hsp90beta level by expressing small-interfering RNA suppressed not only Hsp90beta expression but also PI3K-dependent phosphorylation of Akt and CpG-B ODN-mediated antiapoptosis. Additional studies demonstrated that as described by other group in mast cells, Hsp90beta but not Hsp90alpha was associated with Bcl-2. Inhibition of Hsp90beta suppressed the CpG-B ODN-induced association of Hsp90beta with Bcl-2 and impaired the inhibitory effect of CpG-B ODN in the release of cytochrome c and activation of caspase-3. This study thus reveals the involvement of Hsp90beta but not Hsp90alpha in CpG-B ODN-mediated antiapoptotic response and that Hsp90beta is distinct from Hsp90alpha in regulation of the cellular function of immune cells.     

Protein kinase D1: a new component in TLR9 signaling

Protein kinase D1 (PKD1) is expressed ubiquitously and regulates diverse cellular processes such as oxidative stress, gene expression, cell survival, and vesicle trafficking. However, the presence and function of PKD1 in monocytic cells are currently unknown. In this study, we provide evidence that PKD1 is involved in TLR9 signaling in macrophages. Class B-type CpG DNA (CpG-B DNA) induced activation of PKD1 via a pathway that is dependent on endosomal pH, TLR9, MyD88, and IL-1R-associated kinase 1 in macrophages. Upon CpG-B DNA stimulation, PKD1 interacted with the TLR9/MyD88/IL-1R-associated kinase/TNFR-associated factor 6 complex. Knockdown of PKD1 revealed that PKD1 is required for activation of NF-kappaB and MAPKs, and subsequent expression of cytokines in response to CpG-B DNA. Our findings identify PKD1 as a key signaling modulator in TLR9-mediated macrophage activation.     

CpG oligonucleotides induce an immune response of odontoblasts through the TLR9, MyD88 and NF-κB pathways

Odontoblasts are the first-line defense cells against invading microorganisms. Toll-like receptors (TLRs) play a crucial role in innate immunity, and TLR9 is involved in the recognition of microbial DNA. This study aimed to investigate whether odontoblasts can respond to CpG DNA and to determine the intracellular signaling pathways triggered by CpG DNA. We found that the mouse odontoblast-like cell line MDPC-23 constitutively expressed TLR9. Exposure to CpG ODN induced a potent proinflammatory response based on an increase of IL-6 and TNF-α expression. Pretreatment with an inhibitory MyD88 peptide or a specific inhibitor for TLR9, NF-κB or IκBα markedly inhibited CpG ODN-induced IL-6 and TNF-α expression. Moreover, the CpG ODN-mediated increase of κB-luciferase activity in MDPC-23 cells was suppressed by the overexpression of dominant negative mutants of TLR9, MyD88 and IκBα, but not by the dominant negative mutant of TLR4. This result suggests a possible role for the CpG DNA-mediated immune response in odontoblasts and indicates that TLR9, MyD88 and NF-κB are involved in this process.     

A combination of Lox-1 and Nox1 regulates TLR9-mediated foam cell formation

The formation of foam cells is the hallmark of early atherosclerotic lesions, and the uptake of modified low-density lipoprotein (LDL) by macrophage scavenger receptors is thought to be a key process in their formation. In this study, we examined the role of lectin-like oxLDL receptor-1 (Lox-1) and NADPH oxidase 1 (Nox1) in toll-like receptor 9 (TLR9)-mediated foam cell formation. TLR9 activation of Raw264.7 cells or mouse primary peritoneal macrophages by CpG ODN treatment enhanced Lox-1 gene and protein expression. In addition, CpG ODN-induced Nox1 mRNA expression, which in turn increased foam cell formation. The inhibition of CpG ODN-induced reactive oxygen species (ROS) generation by treatment with antioxidants, as well as with knockdown of Nox1 using siRNA, suppressed the formation of foam cells. The induction of Lox-1 and Nox1 by CpG ODN was regulated via the TLR9-p38 MAPK signaling pathway. CpG ODN also increased NFκB activity, and a potent inhibitor of NFκB that significantly blocked CpG-induced Nox1 expression, suggesting that Nox1 regulation is mediated through an NFκB-dependent mechanism. Taken together, these results suggest that a combination of Lox-1 and Nox1 plays a key role in the TLR9-mediated formation of foam cells via the p38 MAPK pathway.     

Activation of toll-like receptor-9 induces matrix metalloproteinase-9 expression through Akt and tumor necrosis factor-alpha signaling

CpG oligodeoxunucleotide (ODN) plays an important role in immune cell function. The present study examined whether temporal control of toll-like receptor (TLR)-9 by CpG ODN can regulate the expression of matrix metalloproteinase-9 (MMP-9). CpG ODN induced the release of tumor necrosis factor (TNF)-alpha and the expression of TNF receptor (TNFR)-II, but not of TNFR-I, in a time-dependent manner and stimulated significant, though delayed, MMP-9 expression. The endosomal acidification inhibitors, chloroquine or bafilomycin A, inhibited CpG ODN-induced TNF-alpha, TNFR-II, and MMP-9 expression. CpG ODN induced the phosphorylation of Akt, and the inhibition of Akt by LY294002 suppressed CpG ODN-induced TNF-alpha, TNFR-II, and MMP-9 expressions. Moreover, neutralizing TNF-alpha antibody significantly suppressed CpG ODN-induced MMP-9 expression, suggesting the involvement of TNF-alpha. These observations suggest that CpG ODN may play important roles in macrophage activation by regulating the expression of MMP-9 via a TLR-9/Akt/TNF-alpha-dependent signaling pathway.     

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.     

Pivotal role of ADP-ribosylation factor 6 in Toll-like receptor 9-mediated immune signaling

CpG oligodeoxynucleotide (CpG ODN) cellular uptake into endosomes, the rate-limiting step of Toll-like receptor 9 (TLR9) signaling, is critical in eliciting innate immune responses. ADP-ribosylation factor 6 (ARF6) is a member of the Ras superfamily, which is critical to a wide variety of cellular events including endocytosis. Here, we found that inhibition of ARF6 by dominant mutants and siRNA impaired CpG ODN-mediated responses, whereas cells expressing the constitutively active ARF6 mutant enhanced CpG ODN-induced cytokine production. Inhibition of ARF6 impaired TLR9 trafficking into endolysosomes, thereby inhibiting proceed functional cleavage of TLR9. Additional studies showed that CpG ODN uptake was increased in ARF6-activated cells but impaired in ARF6-defective cells. Furthermore, cells pretreated with CpG ODN but not GpC ODN had increased CpG ODN uptake due to CpG ODN-induced ARF6 activity. Further studies with ARF6-defective and ARF6-activated cells demonstrated that class III phosphatidylinositol 3-kinases (PI3K) was required for downstream ARF6 regulation of CpG ODN uptake. Together, our findings demonstrate that a novel class III PI3K-ARF6 axis pathway mediates TLR9 signaling by regulating the cellular uptake of CpG ODN.     

Reciprocal modulation of Toll-like receptor-4 signaling pathways involving MyD88 and phosphatidylinositol 3-kinase/AKT by saturated and polyunsaturated fatty acids

Toll-like receptor-4 (TLR4) can be activated by nonbacterial agonists, including saturated fatty acids. However, downstream signaling pathways activated by nonbacterial agonists are not known. Thus, we determined the downstream signaling pathways derived from saturated fatty acid-induced TLR4 activation. Saturated fatty acid (lauric acid)-induced NFkappaB activation was inhibited by a dominant-negative mutant of TLR4, MyD88, IRAK-1, TRAF6, or IkappaBalpha in macrophages (RAW264.7) and 293T cells transfected with TLR4 and MD2. Lauric acid induced the transient phosphorylation of AKT. LY294002, dominant-negative (DN) phosphatidylinositol 3-kinase (PI3K), or AKT(DN) inhibited NFkappaB activation, p65 transactivation, and cyclooxygenase-2 (COX-2) expression induced by lauric acid or constitutively active (CA) TLR4. AKT(DN) blocked MyD88-induced NFkappaB activation, suggesting that AKT is a MyD88-dependent downstream signaling component of TLR4. AKT(CA) was sufficient to induce NFkappaB activation and COX-2 expression. These results demonstrate that NFkappaB activation and COX-2 expression induced by lauric acid are at least partly mediated through the TLR4/PI3K/AKT signaling pathway. In contrast, docosahexaenoic acid (DHA) inhibited the phosphorylation of AKT induced by lipopolysaccharide or lauric acid. DHA also suppressed NFkappaB activation induced by TLR4(CA), but not MyD88(CA) or AKT(CA), suggesting that the molecular targets of DHA are signaling components upstream of MyD88 and AKT. Together, these results suggest that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4 and its downstream signaling pathways involving MyD88/IRAK/TRAF6 and PI3K/AKT and further suggest the possibility that TLR4-mediated target gene expression and cellular responses are also differentially modulated by saturated and unsaturated fatty acids.     

Aspergillus fumigatus-induced interleukin-8 synthesis by respiratory epithelial cells is controlled by the phosphatidylinositol 3-kinase, p38 MAPK, and ERK1/2 pathways and not by the toll-like receptor-MyD88 pathway

Previous studies have established that phagocytes are key cells of the pulmonary innate immune defense against A. fumigatus, an opportunistic fungus responsible of invasive pulmonary aspergillosis. Macrophages detect A. fumigatus via Toll-like receptors 2 and 4 (TLR2 and -4) and respond by the MyD88-NF-kappaB-dependent synthesis of inflammatory mediators. In the present study, we demonstrate that respiratory epithelial cells also sense A. fumigatus and participate in the host defense. Thus, the interaction of respiratory epithelial cells with germinating but not resting conidia of A. fumigatus results in interleukin (IL)-8 synthesis that is controlled by phosphatidylinositol 3-kinase, p38 MAPK, and ERK1/2. Using MyD88-dominant negative transfected cells, we also show that IL-8 production is not dependent on the TLR-MyD88 pathway, although the MyD88 pathway is activated by A. fumigatus and leads to NF-kappaB activation. Thus, our results provide evidence for the existence of two independent signaling pathways activated in respiratory epithelial cells by A. fumigatus, one that is MyD88-dependent and another that is My88-independent and involved in IL-8 synthesis.     

Hsp27 inhibits Bax activation and apoptosis via a phosphatidylinositol 3-kinase-dependent mechanism

Hsp27 inhibits mitochondrial injury and apoptosis in both normal and cancer cells by an unknown mechanism. To test the hypothesis that Hsp27 decreases apoptosis by inhibiting Bax, Hsp27 expression was manipulated in renal epithelial cells before transient metabolic stress, an insult that activates Bax, induces mitochondrial injury, and causes apoptosis. Compared with control, enhanced Hsp27 expression inhibited conformational Bax activation, oligomerization, and translocation to mitochondria, reduced the leakage of both cytochrome c and apoptosis-inducing factor, and significantly improved cell survival by >50% after stress. In contrast, Hsp27 down-regulation using RNA-mediated interference promoted Bax activation, increased Bax translocation, and reduced cell survival after stress. Immunoprecipitation did not detect Hsp27-Bax interaction before, during, or after stress, suggesting that Hsp27 indirectly inhibits Bax. During stress, Hsp27 expression prevented the inactivation of Akt, a pro-survival kinase, and increased the interaction between Akt and Bax, an Akt substrate. In contrast, Hsp27 RNA-mediated interference promoted Akt inactivation during stress. Hsp27 up- or down-regulation markedly altered the activity of phosphatidylinositol 3-kinase (PI3-kinase), a major regulator of Akt. Furthermore, distinct PI3-kinase inhibitors completely abrogated the protective effect of Hsp27 expression on Akt activation, Bax inactivation, and cell survival. These data show that Hsp27 antagonizes Bax-mediated mitochondrial injury and apoptosis by promoting Akt activation via a PI3-kinase-dependent mechanism.     

Role of MyD88 in phosphatidylinositol 3-kinase activation by flagellin/toll-like receptor 5 engagement in colonic epithelial cells

Bacterial flagellin, recognized by Toll-like receptor (TLR) 5, is suggested to be involved in colonic inflammation. However, the detailed signaling mechanisms mediated by flagellin/TLR5 engagement are not clear. Here we dissected the biochemical mechanism by which TLR5 engagement mediates phosphatidylinositol 3-kinase (PI3K) activation in colonic epithelial cells. We demonstrate that silencing TLR5 expression in nontransformed human colonic epithelial cells blocks flagellin-induced PI3K activation, indicating specific activation of PI3K by flagellin/TLR5 engagement. Moreover, we determine that TLR5 recruits the p85 regulatory subunit of PI3K to its cytoplasmic TIR domain in response to flagellin. However, the Src homology binding "YXXM" motif in the cytoplasmic TIR domain of TLR5 is not involved in p85 recruitment, implying that TLR5 indirectly recruits p85. Indeed, we demonstrate that the adaptor molecule MyD88 associates with TLR5 and silencing MyD88 expression blocks PI3K activation by disrupting the association between TLR5 and p85. Furthermore, we show that MyD88 associates with p85 in response to flagellin. Additionally, we determine that blocking PI3K activation reduces interleukin-8 production induced by flagellin in human colonic epithelial cells. Together, MyD88 bridges TLR5 engagement to PI3K activation in response to flagellin.     

Toll-like receptor 9 mediates CpG oligonucleotide-induced cellular invasion

Toll-like receptor 9 (TLR9) belongs to the innate immune system and recognizes microbial and vertebrate DNA. We showed previously that treatment with the TLR9-agonistic ODN M362 (a CpG sequence containing oligonucleotide) induces matrix metalloproteinase-13-mediated invasion in TLR9-expressing human cancer cell lines. Here, we further characterized the role of the TLR9 pathway in this process. We show that CpG oligonucleotides induce invasion in macrophages from wild-type C57/B6 and MyD88 knockout mice and in human MDA-MB-231 breast cancer cells lacking MyD88 expression. This effect was significantly inhibited in macrophages from TLR9 knockout mice and in human MDA-MB-231 breast cancer cells stably expressing TLR9 small interfering RNA or dominant-negative tumor necrosis factor receptor-associated factor 6 (TRAF6). Sequence modifications to the CpG oligonucleotides that targeted the stem loop and other secondary structures were shown to influence the invasion-inducing effect in MDA-MB-231 cells. In contrast, methylation of the cytosine residues of the parent CpG oligonucleotide did not affect the TLR9-mediated invasion compared with the unmethylated parent CpG oligonucleotide. Finally, expression of TLR9 was studied in clinical breast cancer samples and normal breast epithelium with immunohistochemistry. TLR9 staining localized in epithelial cells in both cancer and normal samples. The mean TLR9 staining intensity was significantly increased in the breast cancer cells compared with normal breast epithelial cells. In conclusion, our results suggest that TLR9 expression is increased in breast cancer and CpG oligonucleotide-induced cellular invasion is mediated via TLR9 and TRAF6, independent of MyD88. Further, our findings suggest that the structure and/or stability of DNA may influence the induction of TLR9-mediated invasion in breast cancer.     

Class I and III phosphatidylinositol 3'-kinase play distinct roles in TLR signaling pathway

PI3K involvement has been implicated in the TLR signal pathway. However, the precise roles of the different classes of PI3K in the pathway remain elusive. In this study, we have explored the functions of class I and class III PI3K in the TLR signal pathway using specific kinase mutants and PI3K lipid products. Our results reveal that class III PI3K specifically regulates CpG oligodeoxynucleotide (ODN)-induced cytokine and NO production as well as NF-kappaB activation, whereas class I PI3K regulates both CpG ODN- and LPS-induced IL-12 production and NF-kappaB activation. Additional studies of CpG ODN uptake with flow cytometric analysis show that class III PI3K, but not class I, regulates cellular CpG ODN uptake. Furthermore, experiments with MyD88-overexpressing fibroblast cells transfected with dominant-negative mutants of PI3K demonstrate that class III PI3K regulates CpG ODN-mediated signaling upstream of MyD88, while class I PI3K regulation is downstream of MyD88. These results suggest that class I and class III PI3K play distinct roles in not only the uptake of CpG ODN, but also responses elicited by CpG ODN and LPS.     

CpG-induced tyrosine phosphorylation occurs via a TLR9-independent mechanism and is required for cytokine secretion

Toll-like receptors (TLRs) recognize molecular patterns preferentially expressed by pathogens. In endosomes, TLR9 is activated by unmethylated bacterial DNA, resulting in proinflammatory cytokine secretion via the adaptor protein MyD88. We demonstrate that CpG oligonucleotides activate a TLR9-independent pathway initiated by two Src family kinases, Hck and Lyn, which trigger a tyrosine phosphorylation–mediated signaling cascade. This cascade induces actin cytoskeleton reorganization, resulting in cell spreading, adhesion, and motility. CpG-induced actin polymerization originates at the plasma membrane, rather than in endosomes. Chloroquine, an inhibitor of CpG-triggered cytokine secretion, blocked TLR9/MyD88-dependent cytokine secretion as expected but failed to inhibit CpG-induced Src family kinase activation and its dependent cellular responses. Knock down of Src family kinase expression or the use of specific kinase inhibitors blocked MyD88-dependent signaling and cytokine secretion, providing evidence that tyrosine phosphorylation is both CpG induced and an upstream requirement for the engagement of TLR9. The Src family pathway intersects the TLR9–MyD88 pathway by promoting the tyrosine phosphorylation of TLR9 and the recruitment of Syk to this receptor.     

A C-type CpG ODN accelerates wound healing via regulating fibroblasts and immune response

Abolished or delayed wound healing is a serious problem in clinical surgery, therefore, the new therapy for wound healing is needed. Synthetic oligodeoxynucleotides containing one or more CpG motifs (CpG ODN) has been reported to activate the immune system and improves skin wound healing. The aim of the present study was to evaluate the role of a new C-type CpG ODN in wound healing. We found that the CpG ODN promoted cell proliferation and collagen I production in human skin fibroblasts cells. Besides, we also investigated the effect of CpG ODN on the activation of immune cells. The macrophages and plasmacytoid dendritic cells (pDCs) were incubated with CpG ODN. CpG ODN activated macrophage and pDCs via regulating TLR9/MyD88/NF-κB pathway and TLR9/MyD88/IRF7 pathway, respectively. To further evaluate the effect of CpG ODN on wound healing in vivo a wound healing model was established in mice. The results showed that CpG ODN treatment accelerated wound healing in mice. CpG ODN increased cytokines secretion in wound skin and elevated the ratio of CD4 ⁺ and CD8 ⁺ T cells in the spleen. Our results showed that CpG ODN accelerated wound healing, which was partly due to the regulation of fibroblasts and immune response. The findings suggested that the CpG ODN might be a proper medicament for the treatment of wound healing.     

Involvement of DNA-PKcs in the Type I IFN Response to CpG-ODNs in Conventional Dendritic Cells in TLR9-Dependent or -Independent Manners

CpG-ODNs activate dendritic cells (DCs) to produce interferon alpha (IFNα) and beta (IFNβ). Previous studies demonstrated that Toll-like receptor 9 (TLR9) deficient DCs exhibited a residual IFNα response to CpG-A, indicating that yet-unidentified molecules are also involved in induction of IFNα by CpG-A. Here, we report that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) but not Ku70 deficient BMDCs showed defective IFNα and IFNβ responses to CpG-A or CpG-B. Loss of both DNA-PKcs and TLR9 further reduced the IFNα response to CpG-A. These DNA-PKcs and TLR9 effects were mediated by their downstream Akt/mTORC1 pathway and downstream events IRAK1 and IKKα. Loss of DNA-PKcs, TLR9, MyD88 or IRAK4 impaired phosphorylation of Akt(S473), S6K, S6, IRAK1, or IKKα in BMDCs in response to CpG-ODNs. The residual IFNα and IFNβ in DNA-PKcs-deficient BMDCs were partially responsible for the induction of IL-6 and IL-12 by CpG-ODNs and their stimulatory effect was blocked by IFNAR1 neutralizing antibodies. Further analysis indicated that CpG-ODN associated with DNA-PKcs and Ku70, and induced DNA-PKcs’s interaction with TRAF3. Intriguingly, DNA-PKcs but not Ku70 expression level was reduced in TLR9-deficient BMDCs. Taken together, our data suggest that DNA-PKcs is an important mediator in the type I IFN response to CpG-ODNs in TLR9-dependent or -independent fashions.     

Human B cells express functional TRAIL/Apo-2 ligand after CpG-containing oligodeoxynucleotide stimulation

CpG-containing oligodeoxynucleotides (CpG ODN) have broad-ranging immunostimulatory effects, including the generation of antitumor immune responses. Analysis of different CpG ODN have identified two classes: CpG-A ODN, which stimulate high levels of IFN-alpha production from plasmacytoid dendritic cells and weakly activate B cells, and CpG-B ODN, which strongly activate B cells but stimulate low production of IFN-alpha from plasmacytoid dendritic cells. Previously, we observed that CpG-B ODN (2006) induces TRAIL/Apo-2 ligand (Apo-2L)-mediated killing of tumor cells by CD14(+) PBMC. In this study, we extend our investigation of CpG ODN-induced TRAIL/Apo-2L expression and activity in PBMC to include CpG-A ODN. Of the two classes, IFN-alpha production and TRAIL/Apo-2L-mediated killing of tumor cells was greatest with CpG-A ODN. Surprisingly, CD3(+), CD14(+), CD19(+), and CD56(+) PBMC expressed high levels of TRAIL/Apo-2L following CpG-A ODN stimulation. When isolated, the CD19(+) PBMC (B cells) were able to kill tumor cells in a TRAIL/Apo-2L-dependent manner. As with CD14(+) PBMC, CD19(+) sorted B cells were capable of up-regulating TRAIL/Apo-2L expression when stimulated with IFN-alpha alone. Interestingly, agonist anti-CD40 mAb further enhanced the IFN-alpha-induced TRAIL/Apo-2L expression on CD19(+) B cells. These results are the first to demonstrate human B cell-mediated killing of tumor cells in a TRAIL/Apo-2L-dependent fashion.     

Stimulation of Proliferation and Migration of Mouse Macrophages by Type B CpG-ODNs Is F-Spondin and IL-1Ra Dependent

Macrophage proliferation and migration are important for many facets of immune response. Here we showed that stimulation of macrophages with type B CpG oligodeoxynucleotides (CpG-B ODNs) such as CpG-ODN 1668 increased the production of anti-inflammatory cytokine interleukin 1 receptor antagonist (IL-1Ra) in a TLR9- and MyD88-dependent manner. The CpG-B ODNs-induced IL-1Ra increased macrophage migration and promoted macrophage proliferation by down-regulating the expression of a cell cycle negative regulator, p27 to increase cell population in the S phase. The induction of IL-1Ra by CpG-B ODNs was F-spondin dependent. Knockdown of F-spondin and IL-1Ra decreased CpG-B ODNs-induced macrophage migration whereas overexpression of IL-1Ra increased migration of those cells. These findings demonstrated novel roles for F-spondin and IL-1Ra in CpG-B ODNs-mediated cell proliferation and migration of macrophages.