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CpG-containing oligodeoxynucleotide promotes microglial cell uptake of amyloid ß 1-42 peptide by up-regulating the expression of the G-protein- coupled receptor mFPR2

Pablo Iribarren^*, Keqiang Chen^*, Jinyue Hu^*, Wanghua Gong, Edward H. Cho, Stephen Lockett, Badarch Uranchimeg and Ji Ming Wang^*

^* Laboratory of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland, USA;
Basic Research Program,
Image Analysis Laboratory, and
Developmental Therapeutics Program, Tumor Hypoxia Laboratory, SAIC-Frederick, Frederick, Maryland, USA

¹Correspondence: Laboratory of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute at Frederick, Bldg. 560, Room 31-40, Frederick, MD 21702-1201, USA. E-mail: wangji{at}mail.ncifcrf.gov

SPECIFIC AIMS

Inflammation contributes to the pathogenic process of Alzheimer’s disease (AD). We previously discovered that a human G-protein-coupled formyl peptide receptor-like 1 (FPRL1) and its mouse homologue mFPR2 mediate the chemotaxis and activation of mononuclear phagocytes in response to a variety of peptide agonists, including amyloid ß 1-42 (Aß₄₂), which is neurotoxic and a key mediator of inflammatory responses in AD. Since mFPR2 is up-regulated in mouse microglia by lipopolysaccharide (LPS), a Toll-like receptor 4 ligand, the aim of this study is to investigate whether TLR9, which is activated by CpG-containing oligodeoxynucleotide (ODN) as well as by potential agonists released by damaged host cells, may also enhance the expression and function of mFPR2, thereby affecting the progress of AD.

PRINCIPAL FINDINGS

1. CpG ODN enhances mFPR2 expression by microglial cells
We examined the capacity of CpG ODN, by interacting with TLR9, to regulate the expression and function of mFPR2 in mouse microglial cells. RT-PCR and real-time PCR showed that CpG ODN induced increases in the levels of mFPR2 mRNA in mouse microglial cell line N9 and primary microglial cells obtained from newborn mice. The induction of mFPR2 mRNA by CpG ODN was associated with the acquirement of potent chemotactic responses by microglial cells to mFPR2 peptide agonists, such as the bacterial formylated peptide fMLF and Aß₄₂ (Fig. 1 A–C). The effect of CpG ODN on microglial responses to mFPR2 agonists reached maximal levels at 24 h (Fig. 1A, D ). A control ODN, devoid of the capacity to activate TLR9, failed to induce the expression of functional mFPR2 in microglial cell (Fig. 1A ). In contrast to its effect on mFPR2, CpG ODN-treated microglial cells showed decreased chemotactic responses to the chemokine SDF-1 (Fig. 1) , suggesting that the effect of CpG ODN on expression of chemoattractant receptors in microglia is selective. Since CpG ODN-elicited TLR9 signaling requires acidification and maturation of endosomes, we tested whether chloroquine, a chemical that increases endosomal and lysosomal pH, may prevent TLR9 activation by CpG ODN in microglial cells. In fact, microglial cells treated with chloroquine failed to respond to CpG ODN by increased mFPR2 expression and function, confirming that TLR9 is critical for CpG ODN activation of microglia.

View larger version (42K): [in this window] [in a new window]   Figure 1. Induction of mFPR2 in microglia by CpG ODN. N9 cells (A) and primary mouse microglia (B) were incubated with different concentrations of CpG ODN or a control ODN for 24 h. The cells were then examined for migration in response to the mFPR2 agonists W peptide (1 µM) and fMLF (10 µM), as well as the chemokine SDF-1 (10 nM). C) Primary microglia were cultured in the presence of CpG ODN for 24 h. The cells were then examined for migration in response to Aß42 (10 µM) and SDF1 (10 nM). D) N9 cells were cultured in the presence of CpG ODN (1 µM) for different times, then examined for migration in response to W peptide (1 µM), fMLF (10 µM), and SDF-1 (10 nM). Results are expressed as chemotaxis index (CI) representing fold increase in cell migration in response to chemoattractants over the baseline migration (to medium). *Statistically significant (P<0.01) increase in cell migration compared with unstimulated cells. CpG: CpG ODN; Control: control ODN.

2. Requirement of p38 MAPK for induction of mFPR2 by CpG ODN in microglia
Activation of TLR9 recruits the adaptor protein MyD88, followed by phosphorylation of MAPKs and NFB translocation to stimulate gene transcription. We found that CpG ODN induced a rapid and potent phosphorylation of p38, with a relatively lower level of phosphorylation of ERK, MAPKs. This is in contrast to LPS, which stimulated a markedly increased phosphorylation of both ERK and p38 MAPKs in microglial cells. CpG ODN also induced phosphorylation of IB and Akt. Although phosphorylation of both p38 and ERK1/2 is induced by CpG ODN in microglia, p38 appeared to be critical for increased mFPR2 transcription since the p38 MAPK inhibitor SB202190, but not the MEK1/2 inhibitor PD98059, decreased CpG ODN-induced expression of mFPR2 mRNA. In addition, we observed that a selective inhibitor of IB phosphorylation, BAY 11-7082, decreased CpG ODN-induced expression of mFPR2 mRNA in microglial cells. These results suggest the involvement of p38 and NFB down-stream of TLR9 in CpG induction of mFPR2 in microglia.

3. Increased Aß₄₂ uptake by CpG ODN-activated microglia
Microglia are key phagocytic and immune effector cells in the brain. In AD models, these cells have been shown to ingest Aß₄₂, which may contribute to the clearance of Aß₄₂ or, alternatively, facilitate Aß₄₂ deposition depending on the amyloid burden and the duration of cell exposure. Since we had found that interaction of Aß₄₂ with FPRL1 in human macrophages resulted in Aß₄₂ internalization, we investigated the capacity of CpG ODN-activated mouse microglia to ingest Aß₄₂. We found a progressively increased Aß₄₂ accumulation in the cytoplasmic region of the cells stimulated for 24 h with CpG ODN (Fig. 2 A). Internalization of Aß₄₂ apparently was mediated by mFPR2 because preincubation of microglial cells with pertussis toxin (PTX) (Fig. 2B ), an inhibitor of Gi protein-coupled receptors including mFRP2, abrogated Aß₄₂ internalization by CpG ODN-activated microglia (Fig. 2B ). Moreover, an mFPR2 specific agonist, W peptide, considerably reduced Aß₄₂ internalization in CpG ODN-activated microglial cells (Fig. 2B ). Thus, mFPR2 expressed by CpG ODN-stimulated microglia plays a prominent role in mediating Aß₄₂ internalization.

View larger version (51K): [in this window] [in a new window]   Figure 2. Aß42 uptake by CpG ODN-activated microglia. A) N9 cells placed on chamber slides were cultured in the presence or absence of CpG ODN (1 µM) for 24 h at 37°C, then incubated with Aß42 (10 µM) at 37°C for different times. The slides were stained by using a monoclonal antibody anti-Aß42 (green) and propidium iodide (red) and evaluated by confocal microscopy. B) N9 cells placed on chamber slides were cultured in the presence or absence of CpG ODN (1 µM) for 24 h at 37°C. Cells were then incubated with cholera toxin (CTX) (500 ng/mL), pertussis toxin (PTX) (500 ng/mL), or W peptide (W pep) (1 µM) for 30 min before further incubation with Aß42 for 24 h. The slides were stained by using an anti-Aß42 monoclonal antibody (green) and propidium iodide (red), then evaluated by confocal microscopy. The results are presented as percentage of positive cells and mean fluorescence intensity (MFI) in histograms. M: medium; CpG: CpG ODN; Control: control ODN.

CONCLUSIONS AND SIGNIFICANCE

Aß₄₂ is a pathogenic factor of AD by its direct neurotoxicity and induction of proinflammatory responses in the brain. The identity of the cell surface receptor(s) for Aß₄₂ remains an issue of great interest because such a receptor may have the potential as a therapeutic target for the disease. Despite the reports on several cell surface molecules as putative Aß₄₂ receptors, the human formylpeptide receptor FPRL1 mediates monocyte chemotactic and neuronal cell toxic activities of Aß₄₂. The mouse analog of FPRL1, mFPR2, has been shown to also mediate myeloid cell chemotaxis and activation by Aß₄₂. Our present study showed that the activation of TLR9 in mouse microglia induced mFPR2, which in turn promoted inflammatory responses of the cells to Aß₄₂ and the capacity of the cells to uptake Aß₄₂ (Fig. 3 ). To our knowledge, this is the first direct visualization by confocal microscopy of the endocytosis of Aß₄₂ by microglial cells through G-protein-coupled receptor mFPR2. Our study thus implies the importance of TLR9 expressed in microglial cells in affecting the pathogenic process of AD and the potential of mFPR2 as a key molecule mediating microglial interaction with Aß₄₂.

View larger version (21K): [in this window] [in a new window]   Figure 3. Activation of TLR9 increases mFPR2-mediated microglial chemotaxis and Aß42 uptake. Microglial cells endocytose CpG ODN to activate TLR9 in endosomes, leading to phosphorylation of p38 MAPK. The activated TLR9 signaling cascade results in increased expression of the G-protein-coupled receptor mFPR2, which mediates cell chemotaxis and Aß42 uptake.

In addition to CpG ODN derived from infecting microorganisms, TLR9 in microglial cell may also be activated by ligands of other potential sources. For instance, targeted delivery of genes into adult nervous system has raised considerable interest in the treatment of neurodegenerative diseases. Although gene delivery vehicles are designed to reduce or avoid toxicity and immunogenicity, viral proteins, and genes as well as CpG DNA contained in plasmids may affect host immune responses. It is especially important when CpG containing vectors are to be considered in AD treatment because they are likely to induce the expression of mFPR2 or FPRL1 in microglial cells thereby enhancing proinflammatory responses triggered by Aß₄₂. Damaged host cells may also release agonist molecules that activate TLR9 in microglial cells and up-regulate mFPR2. On the other hand, mFPR2-mediated uptake of Aß₄₂ by microglia may benefit the degradation and clearance of Aß₄₂, as evidenced by our observation of progressive reduction of Aß₄₂ antigenic activity in microglial cells after prolonged exposure to Aß₄₂. Our study provides further insight into the important role of proinflammatory molecules in the development of AD and suggests molecular targets for the design of therapeutic agents.

FOOTNOTES

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