CpG DNA Stimulation Research Articles

CpG DNA stimulates autoreactive immature B cells in the bone marrow

Polyclonal activation of developing B cells is an injurious process, because most of these cells are nontolerant and express autoreactive receptors. CpG DNA is a polyclonal activator of mature B cells, but its effect on developing B cells is unclear. We tested whether developing, nontolerant B cells are responsive to mitogenic stimulation by CpG DNA and whether such a stimulus can interfere with the establishment of central tolerance. We found that developing B cells express Toll-like receptor 9 and undergo a polyclonal response to CpG DNA stimulation, as revealed by proliferation and differentiation to antibody-producing cells. In vitro and ex vivo experiments revealed that stimulation with CpG DNA protects immature B cells from negative selection imposed by apoptosis and receptor editing and results in the production of autoantibodies. Finally, we found that in vivo administration of CpG DNA activates immature B cells in the bone marrow and suppresses the expression of recombination-activating genes in a mouse model of central tolerance and receptor editing. These results suggest that mitogenic signals provided by CpG DNA stimulate nontolerant immature B cells in the bone marrow and have the potential to interfere with central tolerance.

Cloning and characterization of equine Toll-like receptor 9 (52.9)

Abstract Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLRs have recently emerged as key PRRs that detect microbial infection and trigger antimicrobial host defense responses. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells. The molecular ligand for TLR9 is unmethylated bacterial CpG DNA. Following cell activation though TLR9, dendritic cells mature and secrete cytokines that initiate naïve T helper lymphocytes to polarize to a Th1 bias. Th1 directed activity is essential for host control of intracellular infection. We cloned and characterized equine TLR9. The deduced equine TLR9 protein has more than 80% homology to that of human, monkey, bovine, canine, feline, porcine, and sheep based on our sequence analysis. We found equine TLR9 expression in spleen, lymph nodes, and peripheral blood leukocytes based on quantitative real time PCR. Flow cytometric analysis revealed that equine TLR9 was expressed in CD4+ T, CD8+ T, and B lymphocytes, particularly in MHC class II expressing cells. Equine TLR9 expression was enhanced by phytohemagglutinin or CpG DNA stimulation. From these data we suggest that the TLR9 ligand, CpG DNA, is a potential equine vaccine adjuvant signaling an antigen-specific Th1 bias for pronounced host immunity.

CpG DNA activation and plasma-cell differentiation of CD27− naive human B cells

AbstractUnmethylated CpG DNA activation of naive CD27− B cells has been reported to require B-cell–receptor (BCR) cross-linking. We describe a culture system using CpG DNA with sequential steps for T-cell–independent activation of naive CD19+CD27− human peripheral blood B cells that induces efficient CD138+ plasma-cell differentiation. CD27+ and CD27− B cells were cultured in a 3-step system: (1) days 0 to 4: CpG, IL-2/10/15; (2) days 4 to 7: IL-2/6/10/15 and anti-CD40L; (3) days 7 to 10: IL-6/15, IFN-α, hepatocyte growth factor, and hyaluronic acid. Both CD27+ and CD27− B cells up-regulated intracytoplasmic TLR-9 following CpG DNA activation. CD27− B-cell activation required cell-cell contact. Both naive and memory B cells progressed to a plasma-cell phenotype: CD19lowCD20lowCD27+CD38+HLA-DRlow. Seventy percent of the CD27−-derived CD138+ cells demonstrated productive V chain rearrangements without somatic mutations, confirming their origin from naive precursors. Plasma cells derived from CD27+ B cells were primarily IgG+, while those from CD27− B cells were IgM+. Our results indicate that under certain conditions, naive B cells increase TLR-9 expression and proliferate to CpG DNA stimulation without BCR signaling. In addition to its immunologic significance, this system should be a valuable method to interrogate the antigenic specificity of naive B cells.

Contribution of Interferon-β to the Murine Macrophage Response to the Toll-like Receptor 4 Agonist, Lipopolysaccharide

Interferon-beta (IFN-beta) has been identified as the signature cytokine induced via the Toll-like receptor (TLR) 4, "MyD88-independent" signaling pathway in macrophages stimulated by Gram-negative bacterial lipopolysaccharide (LPS). In this study, we analyzed the responses of macrophages derived from wild-type (IFN-beta(+/+)) mice or mice with a targeted mutation in IFN-beta (IFN-beta(-/-)) to the prototype TLR4 agonist, Escherichia coli LPS. A comparison of basal and LPS-induced gene expression (by reverse transcription-PCR, real-time PCR, and Affymetrix microarray analyses) resulted in the identification of four distinct patterns of gene expression affected by IFN-beta deficiency. Analysis of a subset of each group of differentially regulated genes by computer-assisted promoter analysis revealed putative IFN-responsive elements in all genes examined. LPS-induced activation of intracellular signaling molecules, STAT1 Tyr-701, STAT1 Ser-727, and Akt, but not p38, JNK, and ERK MAPK proteins, was significantly diminished in IFN-beta(-/-) versus IFN-beta(+/+) macrophages. "Priming" of IFN-beta(-/-) macrophages with exogenous recombinant IFN-beta significantly increased levels of LPS-induced gene expression for induction of monocyte chemotactic protein 5, inducible nitric-oxide synthase, IP-10, and IL-12 p40 mRNA, whereas no increase or relatively small increases were observed for IL-1beta, IL-6, monocyte chemotactic protein 1, and MyD88 mRNA. Finally, IFN-beta(-/-) mice challenged in vivo with LPS exhibited increased survival when compared with wild-type IFN-beta(+/+) controls, indicating that IFN-beta contributes to LPS-induced lethality; however, not to the extent that one observes in mice with more complete pathway deficiencies (e.g. TLR4(-/-) or TRIF(-/-) mice). Collectively, these findings reveal unanticipated regulatory roles for IFN-beta in response to LPS in vitro and in vivo.

Maturation of murine bone marrow-derived dendritic cells with poly(I:C) produces altered TLR-9 expression and response to CpG DNA

Although poly(I:C) and LPS induced differential dendritic cell (DC) cytokine profiles and toll-like receptor (TLR) expression, all were capable of causing phenotypic and functional DC maturation. Both LPS and poly(I:C) downregulated TLR-4/MD-2 expression on DCs. Although poly(I:C) highly upregulated their cell surface TLR-9 expression, LPS upregulated the intracellular TLR-9 expression. LPS-treated DCs could not produce IL-12p70 in response to subsequent both LPS- and CpG DNA-stimulation. On the other hand, poly(I:C)-treated DCs retained to produce IL-12p70 by subsequent CpG DNA-stimulation, while subsequent LPS-stimulation did not induce IL-12p70 production. Chloroquine, inhibitor of endosomal maturation, completely inhibited cytokine production of LPS-treated DCs as well as unstimulated control in response to subsequent CpG DNA-stimulation, while it failed to delete the IL-12p40 and IL-10 production in poly(I:C)-treated DCs. These data suggest that poly(I:C) may induce a novel DC phenotype that preserves the capacity of cytokine production to subsequent CpG DNA-stimulation.

CpG DNA enhances macrophage cell spreading by promoting the Src-family kinase-mediated phosphorylation of paxillin

Macrophages are an important component of the innate immune response to infection by microbial pathogens. The activation of macrophages by pathogens is largely mediated by Toll-like receptors (TLRs). Bacterial DNA, which contains unmethylated CpG dinucleotide motifs, is specifically recognised by TLR9 and triggers the activation of a complex network of intracellular signalling pathways that orchestrates the ensuing inflammatory responses of macrophages to the pathogen. Here, we have established that CpG DNA promotes reorganisation of the actin cytoskeleton and enhances cell spreading by primary mouse bone marrow macrophages. CpG DNA stimulation resulted in an approximately 70% increase in cell size. Notably, CpG DNA-induced cell spreading was dependent on the activity of Src-family kinases. Tyrosine phosphorylation of several proteins was increased in a Src-family kinase-dependent manner following CpG DNA stimulation of bone marrow macrophages, including the cytoskeletal protein paxillin. Paxillin was phosphorylated both in vitro and in vivo by the Src-family kinase Hck. Significantly, paxillin from CpG DNA-stimulated bone marrow macrophages had a greater capacity to bind the SH2 domain of the adapter protein Crk than did paxillin from unstimulated bone marrow macrophages. Furthermore, phosphorylation of paxillin by Hck created a binding site for Crk. We propose that the formation of paxillin-Crk complexes may mediate the cytoskeletal changes that underlie the increased cell spreading of macrophages following their activation by CpG DNA.

DNA‐PKcs, but not Toll‐like receptor 9, is required for activation of Akt by CpG‐DNA

CpG-DNA and its related synthetic CpG oligodeoxynucleotides (CpG-ODNs) play an important role in immune cell survival. It has been suggested that Akt is one of the CpG-DNA responsive serine/threonine kinases; however, the target protein of CpG-DNA that leads to Akt activation has not been elucidated. Here, we report that stimulation of bone marrow-derived macrophages (BMDMs) from mice lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) or mice harboring a point mutation in DNA-PKcs gene results in defective phosphorylation and activation of Akt by CpGDNA. Unexpectedly, loss of the Toll-like receptor 9 has a minimal effect on Akt activation in response to CpG-DNA. Further in vitro analysis using purified DNA-PK and recombinant Akt proteins reveals that DNA-PK directly induces phosphorylation and activation of Akt. In addition, in BMDMs, DNA-PKcs associates with Akt upon CpG-DNA stimulation and triggers transient nuclear translocation of Akt. Thus, our findings establish a novel role for DNAPKcs in CpG-DNA signaling and define a CpG-DNA/DNAPKcs/Akt pathway.

TLR9 expression is related to immune activation but is impaired in individuals with chronic immune activation

Millions of individuals in developing countries are infected with helminths and other chronic infectious diseases, such as HIV-1, which lead to persistent immune activation and unbalanced immune state. We have suggested that the capacity of chronically immune activated individuals to protect themselves, cope with infections, and mount protective immunity following vaccination, is highly impaired. Here we examined the expression of toll-like receptor 9 (TLR9), as an essential component in the recognition of immunostimulating bacterial CpG-DNA motifs, in different subsets of human peripheral blood mononuclear cells (PBMC) obtained from chronically immune activated and non-activated individuals. TLR9 expression was correlated to immune cell activation and was upregulated following phytohemagglutinin or anti-CD3 activation. PBMC obtained from chronically immune activated individuals had a different overall pattern of TLR9 expression, including reduced upregulation of this receptor following additional immune activation, and diminished responsiveness to CpG-DNA stimulation, in comparison to non-activated individuals. These differences may partly account for the reduced capacity of chronically immune activated individuals to mount effective immune responses and strengthen the notion that the host immune background should be considered in the design and trial of potential adjuvants and vaccines.

Synergistic action of immunostimulatory DNA and Fcγ receptor IIB-crosslinking on B-cell phenotype and function

CpG DNA functions via the toll-like receptor-9 (TLR-9) receptor, inducing B cell proliferation and promoting immunoglobulin production. B cell responses to CpG DNA-containing immune complexes could be important in chronic autoimmunity and immune responses to bacterial components. Therefore, we investigated the potential synergy of CpG DNA-stimulation with FcγR clustering (CFR) on splenic B cell activity. CFR-induced splenocyte proliferation was significantly increased compared to treatment with CpG DNA alone. While the levels of interleukin-10 (IL-10) were increased in CpG DNA-treated splenocyte cultures, particularly following FcγRII/III-clustering, CFR treatment reduced IL-6 levels. B-cell maturation in culture was enhanced by CFR. Indeed, the frequency of IgG expressing cells after stimulation with CpG DNA was increased and was even higher after CFR stimulation. Furthermore, the frequency of plasma cell precursors was markedly increased by stimulation with CFR. Late splenic B cell subsets, transitional type 2 (T2) and mature (M) B cells, responded strongly to CpG DNA with proliferation and the response was enhanced by FcγR-clustering. Immature transitional type 1 (T1) B cells showed distinctly lower proliferative response to CpG DNA and very small effects of FcγR-clustering, despite similar expression of Fcγ-receptors by all B cell subsets. In conclusion, these data show synergistic impact of CpG DNA and simultaneous FcγR-clustering on B cell proliferation and differentiation.

DNA-PKcs, but not TLR9, is required for activation of Akt by CpG-DNA

CpG-DNA and its related synthetic CpG oligodeoxynucleotides (CpG-ODNs) play an important role in immune cell survival. It has been suggested that Akt is one of the CpG-DNA-responsive serine/threonine kinases; however, the target protein of CpG-DNA that leads to Akt activation has not been elucidated. Here, we report that ex vivo stimulation of bone marrow-derived macrophages (BMDMs) from mice lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) results in defective phosphorylation and activation of Akt by CpG-DNA. Unexpectedly, loss of the Toll-like receptor 9 has a minimal effect on Akt activation in response to CpG-DNA. Further in vitro analysis using purified DNA-PK and recombinant Akt proteins reveals that DNA-PK directly induces phosphorylation and activation of Akt. In addition, in BMDMs, DNA-PKcs associates with Akt upon CpG-DNA stimulation and triggers transient nuclear translocation of Akt. Thus, our findings establish a novel role for DNA-PKcs in CpG-DNA signaling and define a CpG-DNA/DNA-PKcs/Akt pathway.

Suppressor of Cytokine Signaling (SOCS) Proteins Indirectly Regulate Toll-like Receptor Signaling in Innate Immune Cells

Suppressor of cytokine signaling (SOCS) proteins constitute a class of negative regulators for Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. These intracellular proteins are induced by cytokine signaling, but they can also be induced by stimulation of Toll-like receptors (TLR). It has even been suggested that SOCS proteins are important negative regulators of TLR signaling. Here we have elucidated the nature of the regulatory role of SOCS in TLR signaling. Induction of SOCS-3 and cytokine-inducible Src homology 2-containing protein (CIS) by TLR stimulation was strictly dependent on MyD88 but showed differing needs in case of SOCS-1. However, induction of SOCS proteins by TLR ligands was independent of type I interferon. In macrophages overexpressing SOCS, we were not able to observe an inhibitory effect of SOCS-1, SOCS-2, SOCS-3, or CIS on prototypical TLR target genes such as tumor necrosis factor-alpha. However, we found that TLR-2, TLR-3, TLR-4, and TLR-9 stimulation induced interferon-beta (IFN-beta), which is able to exert auto- and paracrine signaling, leading to the activation of secondary genes like IP-10. SOCS-1 and, to a lesser extent, SOCS-3 and CIS were able to inhibit this indirect signaling pathway following TLR stimulation, whereas neither MAP kinase nor NF kappa B signaling were affected. However, STAT-1 tyrosine phosphorylation following TLR triggering was severely impaired by SOCS-1 overexpression. Thus, our data suggest that SOCS proteins induced by TLR stimulation limit the extent of TLR signaling by inhibiting type I IFN signaling but not the main NF kappa B pathway.

Rapid immune activation by CpG motifs in bacterial DNA. Systemic induction of IL-6 transcription through an antioxidant-sensitive pathway.

Unmethylated CpG dinucleotides (CpG motif) in bacterial DNA or synthetic oligodeoxynucleotides (CpG DNA) rapidly activate murine B cells to secrete IL-6 and IgM, as well as to proliferate. Within 30 min after CpG DNA stimulation in vivo, IL-6 mRNA levels were increased in liver, spleen, and thymus cells. Serum IL-6 protein was markedly increased within 1 h of stimulation. Treatment of a B cell line with CpG DNA led to an increase in the transcriptional activity of the IL-6 promoter. This CpG DNA-induced IL-6 production was not mediated via either a protein kinase C (PKC)-, protein kinase A (PKA)-, or nitric oxide (NO.)-dependent pathway but was inhibited by an antioxidant. In addition, the level of intracellular reactive oxygen species was increased within 20 min after CpG DNA, but not control non-CpG DNA, treatment. These results suggest that CpG DNA-induced IL-6 production is mediated through a reactive oxygen intermediate-dependent pathway. CpG DNA-mediated IL-6 production was enhanced by simultaneous signals delivered through the Ag receptor. The addition of neutralizing Abs against IL-6 to B cell cultures along with CpG oligodeoxynucleotides essentially abolished the CpG DNA-induced increased IgM secretion but had no significant effect on the B cell proliferation induced by the CpG motif. Our results suggest that the induction of IL-6 expression in response to CpG motifs in bacterial DNA may be an important immune defense mechanism that facilitates a rapid response to microbial infection.

IFN-gamma promotes IL-6 and IgM secretion in response to CpG motifs in bacterial DNA and oligodeoxynucleotides.

Lymphocyte recognition of characteristic structural features in microbial DNA may contribute to immune defense by promoting protective immune responses. The dinucleotide CpG is significantly under-represented in vertebrate DNA and is usually methylated. In contrast, CpG dinucleotides are generally present at the expected frequency in bacterial DNA and are unmethylated. These unmethylated CpG motifs induce B cells to secrete IL-6 and IgM, and can induce NK and CD4+ T cells to produce the immunoregulatory Th1 cytokine, IFN-gamma. IFN-gamma inhibits IgM secretion that is triggered by a different bacterial product, LPS. The present study demonstrates that in contrast to its antagonistic interaction with LPS, IFN-gamma causes a dose-dependent increase in the level of IgM secretion induced by CpG DNA. Like IgM secretion, B cell secretion of IL-6 more than doubles after the addition of exogenous IFN-gamma. Mice with disrupted IFN-gamma genes produced less than half as much IL-6 and IgM in response to CpG DNA, supporting the hypothesis that CpG-induced IFN-gamma production contributes to the B cell response. In contrast to its promotion of IL-6 and IgM secretion, IFN-gamma did not significantly affect the spleen cell proliferation activated by CpG motifs. These results indicate that IFN-gamma produced by T and NK cells after CpG DNA stimulation contributes to the B cell production of IL-6 and the subsequent Ig production. These studies provide further evidence that the immune system responds to CpG motifs in bacterial DNA by activating a coordinated set of humoral and cellular responses.