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Originally published online as doi:10.1189/jlb.0907607 on February 25, 2008

Published online before print February 25, 2008
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(Journal of Leukocyte Biology. 2008;83:1467-1475.)
© 2008 by Society for Leukocyte Biology

Cooperation between NOD2 and Toll-like receptor 2 ligands in the up-regulation of mouse mFPR2, a G-protein-coupled Aβ42 peptide receptor, in microglial cells

Keqiang Chen*,{dagger}, Lingzhi Zhang{dagger}, Jian Huang{dagger}, Wanghua Gong{ddagger}, Nancy M. Dunlop{dagger} and Ji Ming Wang{dagger},1

* School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai, China;
{dagger} Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, and
{ddagger} Basic Research Program, SAIC-Frederick, National Cancer Institute-Frederick, Frederick, Maryland, USA

1Correspondence: Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute at Frederick, Building 560, Room 31-76, Frederick, MD 21702-1201, USA. E-mail: wangji{at}mail.ncifcrf.gov


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ABSTRACT
 
Human G-protein-coupled formyl peptide receptor-like 1 and its mouse homologue formyl peptide receptor 2 (mFPR2) mediate the chemotactic activity of a variety of pathogen and host-derived peptides, including amyloid β42, a key causative factor in Alzheimer’s disease. In mouse microglia, mFPR2 is up-regulated by pathogen-associated molecular patterns and proinflammatory cytokines, as shown, for instance, in our previous study using peptidoglycan (PGN) of Gram+ bacteria. As PGN and its components have been reported to use TLR2 and an intracellular receptor nucleotide-binding oligomerization domain 2 (NOD2), we investigated the capacity of palmitoyl-cys[(RS)-2, 3-di(palmitoyloxy)-propyl]-Ala-Gly-OH (PamCAG), a specific TLR2 ligand, and muramyl dipeptide (MDP), a NOD2 ligand, to cooperatively regulate the expression and function of mFPR2 in microglia. We found that MDP and PamCAG as well as another TLR2-specific agonist palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4), when used alone, each increased the expression of functional mFPR2 in microglial cells, and the combination of MDP and PamCAG or Pam3CSK4 exhibited an additive effect. Mechanistic studies revealed that MDP increased the levels of TLR2 expression on the microglial cell surface and enhanced the levels of MAPKs p-38, ERK1/2, and NF-{kappa}B activated by PamCAG. Our results suggest that TLR2 and NOD2 cooperate to up-regulate the expression of mFPR2 and therefore, may actively participate in the pathogenic processes of brain inflammation and neurodegenerative diseases.

Key Words: TLR2 • chemotaxis


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INTRODUCTION
 
Microglial cells are key sensors of pathological stimulants in the brain and accumulate at the sites of infection and inflammation by recognizing chemotactic mediators through cell surface receptors [1 ]. Recently, a G-protein-coupled amyloid β42(Aβ42) peptide receptor mouse homologue formyl peptide receptor 2 (mFPR2) has been shown to be expressed by activated microglial cells and implicated in the pathogenesis of Alzheimer’s disease (AD) [2 3 4 5 6 ]. The human homologue of mFPR2, formyl peptide receptor-like 1 (FPRL1), was originally identified as a low-affinity receptor for the bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalanine [7 , 8 ]. FPRL1 also contributes to the internalization of Aβ42 into the cytoplasmic compartment of macrophages, where Aβ42 forms fibrillary aggregates [8 , 9 ]. The expression of the FPRL1 counterpart mFPR2 by mouse primary microglia was enhanced when the cells were activated by ligands for TLRs such as LPS (TLR4), CpG (TLR9), and peptidoglycan (PGN; TLR2), as well as by the proinflammatory cytokines TNF-{alpha} and IFN-{gamma} [2 3 4 , 6 , 10 ]. In addition, the mFPR2 expression in microglia was synergistically increased by costimulation with CD40 ligand and IFN-{gamma} [10 ]. Thus, the expression of mFPR2 in microglial cells is tightly regulated by pathogen- and host-derived molecules associated with inflammatory and innate immune responses.

Nucleotide-binding oligomerization domain 2 (NOD2) contains three distinct regions: a leucine-rich pattern-recognition domain similar to TLRs, a nucleotide-binding domain that is essential for oligomerization and subsequent signaling, and effector motifs such as the caspase-recruitment domain [11 , 12 ]. NOD2 is involved in intracellular recognition of pathogens and their products, such as muramyldipeptide (MDP; N-acetylmuramyl-l-alanyl-d-isoglutamine) from bacteria [12 ]. In the CNS, ligation of TLRs and NOD2 by different pathogen-associated molecular patterns in microglial cells initiates intracellular signaling cascades, resulting in a proinflammatory phenotype.

To examine the potential, cooperative role of NOD2 and TLR2 in CNS inflammation and immune responses, we stimulated mouse microglial cells with a combination of ligands for NOD2 and TLR2 and measured the expression and function of mFPR2. We found that MDP and TLR2 ligands, palmitoyl-cys[(RS)-2, 3-di(palmitoyloxy)-propyl]-Ala-Gly-OH (PamCAG), palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4), and PGN, respectively, each up-regulated the expression of functional mFPR2 in microglial cells. In addition, by increasing cell surface TLR2 expression and the levels of activated MAPKs p-38, ERK1/2, and I{kappa}B{alpha}, MDP primes microglial cells and increases mFPR2 expression induced by TLR2 ligands.


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MATERIALS AND METHODS
 
Reagents and cells
Synthesized PamCAG was from Bachem (King of Prussia, PA, USA). Pam3CSK4, MDP, MDP control, PGN, and IL-10 were purchased from InvivoGen (San Diego, CA, USA). The mFPR2 agonist peptides MMK-1 and W-peptide (WKYMVm, W pep) were synthesized and purified at the Department of Biochemistry, Colorado State University (Fort Collins, CO, USA), according to the published sequence [13 ]. LPS were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Aβ42 peptide was from California Peptide Research (Napa, CA, USA). Antibodies specific for total ERK1/2, ERK1/2 phosphorylated at Tyr-204, phospho-p38 MAPK, total p38 MAPK, phospho-Akt, total Akt, phosphorylated I{kappa}B{alpha}, and total I{kappa}B{alpha} were purchased from Cell Signaling Technology (Beverly, MA, USA). SB202190, PD98059, BAY117082, and LY294002 were obtained from Calbiochem (San Diego, CA, USA). The murine microglial cell line N9 [14 , 15 ] was a kind gift from Dr. Paola Ricciardi-Castagnoli (Universita Degli Studi di Milano-Bicocca, Milan, Italy). The cells were grown in IMDM supplemented with 5% heat-inactivated FCS, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME. Primary murine microglial cells were isolated from 1-day-old newborn C57BL/6 mice or TLR2–/– mice [a kind gift from Drs. Shaobo Su and Rachel R. Caspi, Laboratory of Immunology, National Eye Institute, National Institutes of Health (NIH), Bethesda, MD, USA, and Dr. Shizuo Akira, Osaka University, Japan], as described [3 ]. Primary murine microglial cells were grown in DMEM supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 M HEPES, 2.5 µg/ml Fungizone, 100 µM nonessential amino acids, and 5 µg/ml insulin. The purity of the neonatal microglial cell population was determined by the expression of CD11b using flow cytometry as more than 95% [3 ].

Chemotaxis assays
Chemotaxis assays for microglial cells were performed with 48-well chemotaxis chambers and polycarbonate filters (8 µm pore size; NeuroProbe, Cabin John, MD, USA) as described previously [4 ]. The results were expressed as the mean ± SD of the chemotaxis index (CI), which represents the fold increase in the number of migrated cells, counted in three high-powered fields (x400), in response to chemoattractants over spontaneous cell migration (to control medium).

RT-PCR
Total RNA was extracted from cells with a RNeasy mini kit and depleted of contaminating DNA with RNase-free DNase (Qiagen, Valencia, CA, USA). For amplification of the mFPR2 gene, primers 5'-TCTACCATCTCCAGAGTTCTGTTGG-3' (sense) and 5'-TACATCTACCACAATGTGAACTA-3' (antisense) were designed to yield a 268-bp product. Mouse β-actin primers were used as control; the mouse β-actin primers were 5'-TGTGATGGTGGGAATGGGTCA-3' (sense) and 5'- TTTGATGTGACGCACGATTTCCC-3' (antisense), which yield a product of 514 bp. RT-PCR was performed with 0.5 µg (mFPR2) of total RNA for each sample (High Fidelity ProSTAR HF System, Stratagene, La Jolla, CA, USA), consisting of a 15-min RT at 42°C, a 1-min inactivation of Moloney murine leukemia virus RT at 95°C, 40 cycles of denaturing at 95°C (45 s), annealing at 55°C (45 s), extension at 72°C (1 min), and a final extension for 10 min at 72°C. All PCR products were resolved by 1.5% agarose gel electrophoresis and visualized with ethidium bromide staining. For quantitation, gels were scanned, and the pixel intensity for each band was determined using the ImageJ program (NIH Image, Bethesda, MD, USA) and normalized against the levels of β-actin.

Flow cytometry
The microglial N9 cell line was stimulated with MDP or PamCAG or in combination with PamCAG, then were examined for expression of TLR2 by labeling with PE-conjugated mAb (PharMingen, San Diego, CA, USA). All staining procedures were completed at 4°C in Dulbecco’s PBS containing 5 mM EDTA and 1% FCS. After extensive washing, the cells were analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA). The results are presented as percentage of positive cells and mean fluorescence intensity (MFI).

Western immunoblotting
N9 cells were grown in 60-mm dishes until subconfluency and then were cultured overnight in FCS-free medium. After treatment with MDP and TLR2 agonists, the cells were lysed with 1x SDS sample buffer [62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM DTT], sonicated for 15 s, and then heated at 100°C for 5 min. The cell lysate was centrifuged at 12,000 rpm (4°C) for 5 min, and the protein concentration of the supernatant was measured by the Micro bicinchoninic acid protein assay system (Pierce, Rockford, IL, USA). Western blotting of phosphorylated p38, ERK1/2, I{kappa}B{alpha}, and Akt was performed according to the manufacturer’s instructions using phospho-specific antibodies. Briefly, proteins were electrophoresed on 10% SDS-PAGE-precast gels (Invitrogen, Carisbad, CA, USA) under reducing conditions and transferred onto an ImmunoBlot polyvinylidene membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% nonfat milk and then were incubated with primary antibodies overnight at 4°C. After incubation with a HRP-conjugated secondary antibody, the protein bands were detected with a Super Signal chemiluminescent substrate (Pierce) and Biomax-MR film (Eastman Kodak Co., Rochester, NY, USA). For detection of total p38, ERK1/2, I{kappa}B{alpha}, and Akt, the membranes were stripped with Restore Western blot stripping buffer (Pierce), followed by incubation with specific antibodies.

Statistical analysis
All experiments were performed at least three times, and representative results were shown. For cell migration, the significance of the difference between test and control groups was analyzed with Student’s t-test aided by the Prism software (Prism Software Corp., Irvine, CA, USA), and P values equal to or less than 0.05 were considered statistically significant.


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RESULTS
 
The expression of functional mFPR2 by TLR2 ligand-stimulated murine microglia
We have reported previously that PGN, one of the TLR2 ligands, up-regulated the expression of functional mFPR2 in microglia [4 ]. As PGN may also activate NOD2, an intracellular pattern recognition receptor (PRR) [12 , 16 ], we examined the capacity of synthetic, specific TLR2 ligands, PamCAG, and Pam3CSK4 to promote the expression of mFPR2. The mouse microglial cell line N9 treated with PamCAG, Pam3CSK4, and PGN increased their expression of mFPR2 mRNA (Fig. 1A ) in association with cell migration to mFPR2 agonist peptides (Fig. 1B and 1C) . Maximal activity of PamCAG, Pam3CSK4, and PGN was observed at 20 µg/ml with significant effects at 5 µg/ml (Fig. 1B) . Similar observations were obtained with the use of primary microglial cells, as shown by up-regulation of mFPR2 mRNA by PamCAG and increased cell migration to a defined mFPR2 agonist peptide MMK-1 (Fig. 1D and 1E) .


Figure 1
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  		Figure 1. The expression and function of mFPR2 induced by TLR2-specific ligands in microglia. The N9 microglial cell line was treated with synthetic TLR2 agonists PamCAG (20 µg/ml) and Pam3CSK4 (20 µg/ml), as well as PGN (20 µg/ml) for 24 h. Total RNA was extracted and examined for mFPR2 mRNA expression by RT-PCR, and RT-PCR products were electrophoresed on an agarose gel and visualized with ethidium bromide staining (A). (B and C) N9 cells were stimulated with PamCAG (5, 20 µg/ml), Pam3CSK4 (5, 20 µg/ml), or PGN (5, 20 µg/ml) for 24 h and then were examined for migration in response to mFPR2 agonist peptides W-peptide (W-p; 10–6 M; B) and Aβ42 (75 µg/ml; C). The results of chemotaxis were expressed as CI representing fold increase in cell migration in response to chemoattractants over base-line migration (to medium). (D) Primary microglial cells from newborn mice were incubated with different concentrations of PamCAG at 37°C for 24 h. LPS (1 µg/ml) was used as a positive control. Total RNA was extracted from stimulated cells and examined for mFPR2 mRNA expression by RT-PCR. (E) Primary mouse microglial cells were stimulated with PamCAG (20 µg/ml) for 24 h and then examined for migration in response to the synthetic mFPR2 agonist peptide MMK-1 (10–4 M). *, Significantly increased cell chemotaxis in response to mFPR2 agonist peptides as compared with medium control (P<0.01).

Activation of NOD2 induces the expression of functional mFPR2 in microglia
NOD2 and TLRs represent two classes of PRRs in the innate immune system. Recently, NOD2 has emerged as one of the central players in the innate immune responses [12 , 16 ]. We found that MDP, a NOD2 ligand, induced mFPR2 expression in N9 and primary microglial cells, which showed increased chemotaxis to mFPR2 agonist peptides (Fig. 2A and 2B ). As MDP is the minimal bioactive PGN motif, we examined the possibility for MDP to activate TLR2. We used mouse (m)TLR2–/– primary microglial cells and found that when stimulated with PamCAG, these cells did not express increased levels of mFPR2 mRNA (data not shown) nor migrated to mFPR2 agonist peptides (Fig. 2C) . In contrast, mTLR2–/– primary microglial cells migrated in response to the mFPR2 agonist peptide after stimulation with LPS (Fig. 2C) or MDP (Fig. 2D) , suggesting that MDP alone induces the expression of functional mFPR2 by activating a receptor other than TLR2.


Figure 2
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  		Figure 2. mFPR2 function in microglia stimulated with NOD2 or TLR2 agonists. N9 microglial cells were incubated with NOD2 agonist MDP (20 µg/ml) at 37°C for 24 h and then were examined for cell chemotaxis to the mFPR2 agonist peptide W-peptide (10–6 M; A). Primary microglia from TLR2+/+ mice were also treated with NOD2 agonist MDP (20 µg/ml) at 37°C for 24 h and then were examined for cell chemotaxis to the mFPR2 agonist peptide MMK-1 (10–4 M; B). Primary microglia from TLR2–/– or TLR2+/+ mice were treated with PamCAG (20 µg/ml) or LPS (0.5 µg/ml) at 37°C for 24 h (C) or with MDP (20 µg/ml) at 37°C for 24 h (D) and then examined for migration in response to the mFPR2 agonist peptide MMK-1 (10–4 M). The results of chemotaxis were expressed as CI. *, Statistically significant (P<0.01) increase in cell migration induced by mFPR2 agonist peptides, as compared with unstimulated cells.

Cooperation between NOD2 and TLR2 in the up-regulation of functional mFPR2 in microglia
We then examined whether activation of NOD2 increases microglial cell responses to TLR2 agonists. We found that the NOD2 agonist MDP was able to prime microglial cell responses to suboptimal concentrations of TLR2 agonists. For instance, the N9 cell line pretreated with MDP (20 µg/ml) followed by PamCAG at 5 µg/ml additively increased mFPR2 mRNA expression as compared with stimulation with MDP or PamCAG alone (Fig. 3A ). A similar effect was observed with another TLR2 agonist Pam3CSK4 as well as PGN (Fig. 3A) . The increase in mFPR2 mRNA was associated with significantly enhanced cell chemotaxis in response to mFPR2 agonist peptides. The cell chemotaxis was more prominent when the cells were treated with NOD2 and TLR2 ligands, as compared with cells treated with NOD2 or TLR2 ligands alone (Fig. 3B and 3C) . Thus, NOD2 agonist MDP primed the microglial cell response to TLR2 ligands by increased expression of functional mFPR2.


Figure 3
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  		Figure 3. Cooperation between NOD2 and TLR2 in the induction of functional mFPR2 in microglial cells. N9 cells were stimulated with MDP (20 µg/ml), PamCAG (CAG; 5 µg/ml), Pam3CSK4 (3CSK4; 0.1 µg/ml), and PGN (5 µg/ml) for 12 h or were pretreated with MDP (20 µg/ml) for 5 h, followed by PamCAG (5 µg/ml), Pam3CSK4 (0.1 µg/ml), and PGN (5 µg/ml) for 7 h. Total RNA was extracted and examined for mFPR2 mRNA expression by RT-PCR (A). RT-PCR products were electrophoresed on an agarose gel and visualized with ethidium bromide staining. The density of RT-PCR product bands was measured by ImageJ (NIH software) and normalized against β-actin. The results are expressed as fold increase of mFRP2 mRNA in NOD2 and TLR2 agonist-treated cells as compared with untreated cells. #, Statistically significant increase in mFPR2 mRNA levels in cells treated with MDP, PamCAG, Pam3CSK4, or PGN as compared with untreated cells (P<0.01). *, Statistically significant (P<0.01) increase in mFPR2 mRNA level in cells treated with MDP in combination with PamCAG, Pam3CSK4, or PGN as compared with cells treated with PamCAG, Pam3CSK4, or PGN alone. (B) N9 cells were stimulated with MDP (20 µg/ml), PamCAG (5 µg/ml), or MDP (20 µg/ml) and PamCAG (5 µg/ml) in combination for 24 h and then were examined for migration in response to Aβ42 (75 µg/ml) or W-peptide (10–6 M). (C) N9 cells were stimulated with MDP (20 µg/ml), Pam3CSK4 (5 µg/ml), or PGN (5 µg/ml) or were pretreated with MDP (20 µg/ml) for 5 h followed by Pam3CSK4 (5 µg/ml) or PGN (5 µg/ml) for 24 h and then were examined for migration in response to W-peptide (10–6 M). The results are expressed as CI. #, Significantly (P<0.05) increased migration of cells treated with PamCAG, Pam3CSK4, or PGN in response to mFPR2 agonist peptide compared with spontaneous cell migration (to medium). *, Significantly (P<0.01) increased migration of microglia treated with a combination of MDP with TLR2 agonists in response to mFPR2 agonist peptides as compared with cells treated with MDP or TLR2 agonists alone (Band).

Activation of NOD2 enhances TLR2 expression on microglial cells
To elucidate the mechanisms of the additive effects shown by MDP and PamCAG on up-regulation of mFPR2 in microglia, we examined the capacity of MDP to regulate the expression level of TLR2. N9 cells constitutively expressed TLR2 on the cell surface, as shown by a high percentage of the TLR2-positive cell pool (Fig. 4 ). Stimulation of the cells with TLR2 or NOD2 ligands had little effect on TLR2 positivity, but rather, each ligand significantly increased the density of TLR2 fluorescence (MFI). There was a marked increase in MFI when the cells were treated with a combination of stimulation of MDP and PamCAG as compared with stimulation with a single ligand (Fig. 4) , suggesting further elevation of TLR2 density on the cell surface. These results indicate that MDP enhanced the level of TLR2 expression on microglia, which may account for its costimulatory effect with PamCAG to promote mFPR2 expression.


Figure 4
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  		Figure 4. NOD2 agonist MDP enhances TLR2 expression in microglial cells. N9 cells cultured in the absence or presence of PamCAG (5 µg/ml), MDP (20 µg/ml) alone, or in combination for 24 h. The cells were then examined for surface expression of TLR2 by flow cytometry. The results are presented as the percentage of positive cells and MFI.

Requirement of NOD2 for MDP to up-regulate TLR2 and to further enhance mFPR2 expression induced by TLR2 ligands
MDP recognition by NOD2 is highly stereospecific for the L-D isomer [17 ]. The core structure required for recognition of NOD2 is N-acetylmuramic acid attached to L-Ala and D-isoGln. Replacement of L-Ala by D-Ala (or D-isoGln by L-isoGln) abolishes the ability of the MDP to stimulate NOD2. We found that the D-D isomer of MDP (MDP control) failed to up-regulate TLR2 (Fig. 5A ) or to further enhance TLR2 ligand-induced mFPR2 expression in murine microglial cells (Fig. 5B and 5C) . The results demonstrate that the activation of NOD2 is restricted to the L-D isomer of MDP.


Figure 5
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  		Figure 5. Requirement of NOD2 recognition for MDP to enhance TLR2 expression and increased microglial response to TLR2 agonist stimulation. N9 cells were cultured in the presence or absence of MDP (20 µg/ml) or D-D MDP (Control MDP; 20 µg/ml) for 24 h at 37°C. The cells were then examined for surface expression of TLR2 by flow cytometry. The results are presented as the percentage of positive cells and MFI (A). (B) N9 cells were treated with PGN (0.5 µg/ml) or pretreated with MDP (20 µg/ml) or control MDP (20 µg/ml) for 5 h followed by PGN (0.5 µg/ml) for 24 h. The cells were then examined for cell migration in response to W-peptide (10–6 M). The results are expressed as CI. *, Statistically significant (P<0.01) increase in migration of cells treated with MDP plus PGN as compared with the cells treated with control MDP plus PGN or PGN alone. (C) N9 cells were treated with PamCAG (5 µg/ml) alone or pretreated with MDP (20 µg/ml) or control MDP (20 µg/ml) followed by PamCAG (5 µg/ml) and then were examined for cell chemotaxis induced by W-peptide. The results are expressed as CI. *, Significantly increased migration of cells treated with MDP plus PamCAG as compared with cells treated with control MDP plus PamCAG or PamCAG alone (P<0.01). (D and E) N9 cells were treated with PGN (0.5 µg/ml), PamCAG (5 µg/ml), or pretreated with MDP (20 µg/ml) or MDP (20 µg/ml) plus IL-10 (100 ng/ml) for 5 h followed by PGN (0.5 µg/ml; D) or PamCAG (5 µg/ml; E) for 24 h. The cells were then examined for migration in response to W-peptide (10–6 M). The results were expressed as CI. *, Significantly (P<0.01) increased migration of cells treated with MDP plus PGN or PamCAG as compared with cells treated with PGN or PamCAG alone or cells treated with MDP plus IL-10 in combination with PGN or PamCAG.

We also tested the effect of IL-10, which has been reported to specifically induce an endogenous NOD2 inhibitor, NOD2-S, in mammalian cells [18 ]. N9 cells pretreated with IL-10 followed with MDP showed reduced expression of functional mFPR2 when further stimulated with TLR2 ligands (Fig. 5D and 5E) , indicating the key role of NOD2 in mediating the priming effect of MDP on microglial responses to TLR2 agonists.

Enhanced activation level of MAPKs and I{kappa}B{alpha} in microglial cells costimulated by NOD2 and TLR2 ligands
We have previously reported the requirement of p38 and ERK1/2 MAPK pathways and I{kappa}B{alpha} activation in the induction of mFPR2 by PGN in microglial cells [4 ]. We confirmed that the capacity of the specific TLR2 agonist PamCAG to up-regulate mFPR2 in microglial cells was indeed dependent on p38, ERK1/2, MAPK, and I{kappa}B{alpha} activation as shown by reduction of the effect of PamCAG by specific inhibitors (Fig. 6A ). The effect of TLR2 ligands on mFPR2 up-regulation in microglia was not dependent on PI3K, as the cells incubated with the specific PI3K inhibitor LY294002 maintained their responses to PamCAG (Fig. 6A) . We also found that Pam3CSK4 is more potent among TLR2 ligands tested in stimulating MAPK and I{kappa}B{alpha} activation (Fig. 6B) . We then further investigated the signaling pathways involved in the cooperative effect of NOD2 and TLR2 on N9 cells. Treatment of the cells with PamCAG alone induced phosphorylation of p38, Akt, and ERK1/2 at 30 min, and activation of I{kappa}B{alpha} was barely detectable at 60 min (Fig. 6C) . When N9 cells were treated with MDP alone, phosphorylation of p38 and Akt was detectable at 5 min, followed by a marked increase at 60 min. MDP also induced the phosphorylation of ERK1/2 at 60 min but without apparent phosphorylation of I{kappa}B{alpha} (Fig. 6D) . In contrast, microglial cells pretreated with MDP followed by PamCAG showed a more rapid phosphorylation of p38 and ERK1/2 (Fig. 6E) . The phosphorylation of I{kappa}B{alpha} was also enhanced significantly at 60 min after microglia were stimulated with MDP and PamCAG (Fig. 6E) . The changes in the levels of total I{kappa}B{alpha} in microglial cells stimulated by MDP and PamCAG alone or in combination were presumably a result of dynamic degradation and de novo synthesis of the molecule (Fig. 6E) . Thus, NOD2 and TLR2 costimulation triggered a more rapid and/or efficient intracellular signaling cascade in microglial cells, which may be responsible for the cooperative effect between NOD2 and TLR2 on mFPR2 up-regulation.


Figure 6
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  		Figure 6. Involvement of MAPKs and I{kappa}B{alpha} in TLR2 agonist-induced mFPR2 expression in microglial cells and in the priming effect of the NOD2 agonist MDP. (A) N9 cells were cultured in the presence or absence of the p38 inhibitor SB202190 (SB; 10 µM), MEK inhibitor PD98059 (PD; 10 µM), I{kappa}B{alpha} inhibitor BAY117082 (BAY; 10 µM), or PI3K inhibitor LY294002 (LY; 20 µM) for 1 h at 37°C before stimulation with PamCAG (20 µg/ml) for 24 h and then were examined for migration in response to the mFPR2 agonist W-peptide (10–6 M). The results are expressed as CI. *, Significantly (P<0.05) reduced migration of the cells treated with SB202190, PD98059, and BAY117082 plus PamCAG as compared with the cells treated with PamCAG alone. (B) N9 cells were cultured in the presence or absence of PamCAG (20 µg/ml), Pam3CSK4 (20 µg/ml), or PGN (20 µg/ml) at 37°C and then were lysed at the indicated time-points. Equal amounts of total proteins were electrophoresed and blotted. Protein bands were detected with antiphospho (P)-p38 and -ERK1/2 and I{kappa}B{alpha}. Total Akt bands were used as loading controls. (C–E) N9 cells were stimulated with PamCAG (5 µg/ml; C) or MDP (20 µg/ml; D), or the cells were pretreated with MDP (20 µg/ml) for 5 h, followed with PamCAG (5 µg/ml; E) for different time periods (min). Whole cell lysates were electrophoresed, and Western immunoblotting was performed using antibodies against phospho-p38, -ERK1/2, -I{kappa}B{alpha}, and -Akt, respectively. The membranes were then stripped and reprobed with antibodies against total p38, ERK, I{kappa}B{alpha}, and Akt.


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DISCUSSION
 
In this study, we showed for the first time that NOD2 and TLR2, two PRRs, cooperate in transmitting signals necessary for induction of the G-protein-coupled receptor mFPR2 in microglial cells. As a consequence, microglial cells exhibited markedly increased chemotaxis in response to exogenous and host-derived mFPR2 agonist peptides.

Microglia are resident mononuclear phagocytes in the CNS and are key mediators of innate immune responses and inflammation in the brain [19 , 20 ]. In fact, activation of microglia is an essential component in the pathogenesis of many CNS diseases, including AD, Parkinson’s disease [21 ], multiple sclerosis, and AIDS dementia [22 ]. Our previous studies showed that murine microglial cells in the nonstimulated state express low levels of mFPR2 [2 3 4 , 6 , 10 ]. When stimulated with LPS, PGN, and CpG, the level of mFPR2 is enhanced, and the cells become more responsive to mFPR2-specific agonists [3 , 4 , 6 ], suggesting that activation of TLR4, TLR2, and TLR9 may promote microglial responses in CNS diseases. Activation of mFPR2 and human FPRL1 in phagocytic leukocytes by their peptide agonists elicits typical proinflammatory responses, including increased cell chemotaxis, phagocytoses, and release of superoxide [23 , 24 ]. Our present study reveals that activation of NOD2 or TLR2 alone increased mFPR2 expression and function in microglial cells, and cells primed with MDP showed enhanced expression of TLR2 and a more efficacious induction of mFPR2 by TLR2 ligands. Thus, TLR2 and NOD2, two classes of PRRs in the innate immune system, are able to cooperate in orchestrating host responses in the CNS. mFPR2 and its human homologue FPRL1 recognize bacterial and host-derived chemotactic agonist peptides, including Aβ42 associated with AD [2 3 4 5 6 , 10 , 24 , 25 ].

It should be noted that our study found that the reversed Aβ42, Aβ42-1 did not activate mFPR2 or human FPRL1. Also, although the 40-aa form of Aβ, Aβ40, has been reported to increase its production in AD, our study showed Aβ40 was a poor agonist of FPRL1 or mFPR2 and thus, may not play a major role in inducing microglial chemotaxis and activation [9 , 25 ].

MDP has been reported to increase TNF-{alpha} production induced by LPS or PGN in a macrophage cell line [26 ], and MDP synergizes with tripalmitoyl-S-glyceryl cysteine in stimulating the production of TNF-{alpha}, IL-1β, and IL-10 in mononuclear cells from healthy donors [27 ]. In rat primary microglial cells, P2X4 receptor was up-regulated after stimulation with Pam3CSK4 or LPS in combination with MDP [28 ]. Synergy between NOD2 and TLRs also has been observed in dendritic cells (DCs) [29 ]. MDP in combination with TLR4-agonistic lipid A, TLR3-agonistic polyinosinic:polycytidylic acid, or TLR9-agonistic CpG DNA synergistically induced IL-12 and IFN-{gamma} production in human DCs to promote Th1 responses [29 ]. MDP priming of mice induced hyper-responsiveness to endotoxin and other bacterial products interacting with TLRs, although MDP by itself showed little activity [30 , 31 ]. Our study demonstrating cooperation between NOD2 and TLR2 in induction of mFPR2 in microglial cells reveals a novel role of these molecules in CNS innate immunology. In addition, we have identified key signal transduction molecules involved in the cooperation of NOD2 and TLR2 in microglial cells.

Mouse microglia stimulated with agonists for TLR4 [3 ], TLR2 [4 ], and TLR9 [6 ] not only exhibited increased chemotactic responses to Aβ42 but also, a markedly enhanced uptake of Aβ42 mediated by mFPR2 [4 5 6 , 10 , 24 ]. Recent studies have shown that TLR activation is essential for microglial cells to acquire the capacity to ingest and process Aβ42, presumably via mFPR2 [32 ]. In this context, the effect of TLRs was almost completely inhibited by pertussis toxin, a deactivator of G-protein-coupled receptors including mFPR2 [32 ]. Other molecules that interact with TLR4 such as Hsp90, Hsp70, and Hsp32 also increased the accumulation of Aβ peptides in microglia [24 , 33 ]. Thus, TLRs in microglia by interacting with host- or pathogen-derived ligands may initiate a protective host response to Aβ42 peptides. Indeed, in mouse AD models, injection of LPS activates microglia and reduces Aβ burden in the hippocampus [34 ]. However, it should be pointed out that other studies also have shown an essential role for microglia to lay down Aβ peptides to form aggregates, and in addition, the Aβ peptide activates mFPR2 or human FPRL1 to promote a proinflammatory response. Therefore, the clearance and deposition of Aβ peptides by microglial cells in the pathogenic process of AD may be dynamic and dependent on multiple factors including the state of microglial activation, the levels of TLRs and mFPR2, as well as Aβ peptide burden. Our study suggests the importance of amplifying the beneficial effects of microglial activation in host defense against noxious agents and to minimize the potential, detrimental impact of an undesirable level of inflammation.


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ACKNOWLEDGEMENTS
 
This project has been funded in part with federal funds from the National Cancer Institute (NCI), NIH, under contract no. NO1-CO-12400. NCI-Frederick is accredited by the American Association for the Accreditation of Laboratory Animal Care International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the "Guide for Care and Use of Laboratory Animals" (National Research Council, 1996, National Academy Press, Washington, DC, USA). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The publisher or recipient acknowledges the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. The authors thank Dr. Joost J. Oppenheim for critically reviewing the manuscript and Ms. Cheryl Fogle and Ms. Cheryl Nolan for secretarial assistance.

Received September 6, 2007; revised January 28, 2008; accepted February 6, 2008.


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