Inhibition of STAT3- and MAPK-dependent PGE2 synthesis ameliorates phagocytosis of fibrillar β-amyloid peptide (1-42) via EP2 receptor in EMF-stimulated N9 microglial cells

Abstract

Background: Prostaglandin E₂ (PGE₂)-involved neuroinflammatory processes are prevalent in several neurological conditions and diseases. Amyloid burden is correlated with the activation of E-prostanoid (EP) 2 receptors by PGE₂ in Alzheimer's disease. We previously demonstrated that electromagnetic field (EMF) exposure can induce pro-inflammatory responses and the depression of phagocytosis in microglial cells, but the signaling pathways involved in phagocytosis of fibrillar β-amyloid (fAβ) in microglial cells exposed to EMF are poorly understood. Given the important role of PGE₂ in neural physiopathological processes, we investigated the PGE₂-related signaling mechanism in the immunomodulatory phagocytosis of EMF-stimulated N9 microglial cells (N9 cells).

Methods: N9 cells were exposed to EMF with or without pretreatment with the selective inhibitors of cyclooxygenase-2 (COX-2), Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinases (MAPKs) and antagonists of PG receptors EP1-4. The production of endogenous PGE₂ was quantified by enzyme immunoassays. The phagocytic ability of N9 cells was evaluated based on the fluorescence intensity of the engulfed fluorescent-labeled fibrillar β-amyloid peptide (1-42) (fAβ₄₂) measured using a flow cytometer and a fluorescence microscope. The effects of pharmacological agents on EMF-activated microglia were investigated based on the expressions of JAK2, STAT3, p38/ERK/JNK MAPKs, COX-2, microsomal prostaglandin E synthase-1 (mPGES-1), and EP2 using real-time PCR and/or western blotting.

Results: EMF exposure significantly increased the production of PGE₂ and decreased the phagocytosis of fluorescent-labeled fAβ₄₂ by N9 cells. The selective inhibitors of COX-2, JAK2, STAT3, and MAPKs clearly depressed PGE₂ release and ameliorated microglial phagocytosis after EMF exposure. Pharmacological agents suppressed the phosphorylation of JAK2-STAT3 and MAPKs, leading to the amelioration of the phagocytic ability of EMF-stimulated N9 cells. Antagonist studies of EP1-4 receptors showed that EMF depressed the phagocytosis of fAβ₄₂ through the PGE₂ system, which is linked to EP2 receptors.

Conclusions: This study indicates that EMF exposure could induce phagocytic depression via JAK2-STAT3- and MAPK-dependent PGE₂-EP2 receptor signaling pathways in microglia. Therefore, pharmacological inhibition of PGE₂ synthesis and EP2 receptors may be a potential therapeutic strategy to combat the neurobiological deterioration that follows EMF exposure.

Keywords: EMF; Microglia; PGE2; Phagocytosis; Synthesis.

Fig. 1

Flow cytometry and confocal microscopy analyses of the effect of EMF exposure on phagocytic capacity in N9 cells. N9 cells were exposed to 2.45-GHz EMF for 20 min. Untreated cultures were used as sham-exposed controls. N9 cells were subjected to a 1-h process of phagocytosis of fAβ₄₂ with a strong fluorescent signal label HiLyte™ Fluor 647 (647-fAβ₄₂) at the indicated time points after EMF exposure. a Continuous engulfment of 647-fAβ₄₂ within 3 h in N9 cells. b Normalized average fluorescence intensity of 647-fAβ₄₂ ingested per group estimated using a flow cytometer and a confocal microscope. *P < 0.05 vs the sham-exposed control group. c Microscopy images of 647-fAβ₄₂ phagocytosis in N9 cells 3 and 12 h after EMF exposure. Scale bar = 20 μm

Fig. 2

Improvement in phagocytic ability for EMF-exposed N9 cells with the addition of celecoxib. N9 cells were pretreated with or without celecoxib (1, 5, and 25 μM) for 30 min and then exposed to 2.45-GHz EMF (+) or sham exposed (−) for 20 min. Then, cells were subjected to a 1-h process of phagocytosis of 647-fAβ₄₂ at the indicated time points after EMF exposure. a Enzyme immunoassay of PGE₂ production in N9 cells pretreated with or without celecoxib 3 and 12 h after EMF exposure. b Normalized average fluorescence intensity of 647-fAβ₄₂ ingested per group 12 h after EMF exposure estimated using a flow cytometer and a confocal microscope. For a and b, *P < 0.05 vs the sham-exposed control group; ^# P < 0.05 vs the EMF-exposed group. c Microscopy images of 647-fAβ₄₂ phagocytosis in N9 cells pretreated with or without celecoxib 12 h after EMF exposure. Scale bar = 20 μm

Fig. 3

EMF exposure induces phosphorylation of JAK2, STAT3, and MAPKs in N9 cells. N9 cells were pretreated with or without JAK2 inhibitor AG490 (25 μM), STAT3 inhibitor S3I-201 (30 μM), p38 inhibitor SB203580 (10 μM), mitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase (ERK) (MEK1/2-ERK1/2) inhibitor PD98059 (30 μM), c-Jun N-terminal kinase (JNK) inhibitor SP600125 (5 μM), for 30 min and then exposed to 2.45-GHz EMF (+) or sham exposed (−) for 20 min. The phosphorylation and expression of JAK2 (a), STAT3(b), p38(c), ERK1/2(d), and JNK(e) were determined, and the corresponding densitometric analyses were represented. *P < 0.05 vs the sham-exposed control group; ^# P < 0.05 vs the EMF-exposed group

Fig. 4

Inhibition of JAK2, STAT3, and MAPKs ameliorated the phagocytosis of 647-fAβ₄₂ and abrogated the induction of TNF-α and NO in EMF-stimulated N9 cells. N9 cells were pretreated with or without celecoxib (25 μM), AG490 (25 μM), S3I-201 (30 μM), SB203580 (10 μM), PD98059 (30 μM), and SP600125 (5 μM), for 30 min and then exposed to 2.45-GHz EMF or sham exposed for 20 min. Then, cells were subjected to a 1-h process of phagocytosis of 647-fAβ₄₂ 12 h after EMF exposure. a Normalized average fluorescence intensity of 647-fAβ₄₂ ingested per group 12 h after EMF exposure estimated using a flow cytometer. Enzyme immunoassay of PGE₂ (b) and TNF-α (c) production and Griess determination of nitrite (d) in N9 cells pretreated with or without the mentioned pharmacologic compounds of interest 12 h after EMF exposure. *P < 0.05 vs the sham-exposed control group; ^# P < 0.05 vs the EMF-exposed group

Fig. 5

Involvement of JAK2, STAT3, and MAPKs in the regulation of the expression of COX-2 and mPGES-1 in EMF-stimulated N9 cells. N9 cells were pretreated with or without celecoxib (25 μM), AG490 (25 μM), S3I-201 (30 μM), SB203580 (10 μM), PD98059 (30 μM), and SP600125 (5 μM) for 30 min and then exposed to 2.45-GHz EMF or sham exposed for 20 min. Relative mRNA (a) and protein (b) levels of COX-2 and mPGES-1 in N9 cells pretreated with or without the mentioned pharmacologic compounds of interest 12 h after EMF exposure. *P < 0.05 vs the sham-exposed control group; ^# P < 0.05 vs the EMF-exposed group

Fig. 6

Involvement of EP2 activity in the restoration of impaired phagocytosis of 647-fAβ₄₂ in EMF-stimulated N9 cells. N9 cells were pretreated with or without PG receptor EP1 antagonist GW848687X (5 μM), EP2 antagonist AH6809 (10 μM), EP3 antagonist L-798106 (10 μM), and EP4 antagonist GW627368X (10 μM). a Normalized average fluorescence intensity of 647-fAβ₄₂ ingested per group 12 h after EMF exposure estimated using a flow cytometer and a confocal microscope. *P < 0.05 vs the sham-exposed control group; ^# P < 0.05 vs the EMF-exposed group. b Microscopy images of 647-fAβ₄₂ phagocytosis in N9 cells pretreated with or without AH6809 12 h after EMF exposure. Scale bar = 20 μm

Fig. 7

Involvement of COX-2, JAK2, STAT3, and MAPKs in the regulation of the expression of EP2 in EMF-stimulated N9 cells. N9 cells were pretreated with or without celecoxib (25 μM), AG490 (25 μM), S3I-201 (30 μM), SB203580 (10 μM), PD98059 (30 μM), SP600125 (5 μM), and AH6809 (10 μM) for 30 min and then exposed to 2.45-GHz EMF or sham exposed for 20 min. Relative mRNA (a) and protein (b) levels of EP2 in N9 cells pretreated with or without the mentioned pharmacologic compounds of interest 12 h after EMF exposure. *P < 0.05 vs the sham-exposed control group; ^# P < 0.05 vs the EMF-exposed group

Fig. 8

Schematic diagram illustrating the proposed immunomodulatory phagocytosis of fAβ₄₂ via PGE₂-related signaling mechanism in EMF-stimulated N9 microglial cells. External electromagnetic emission as a physical stimulation directly triggers an initial activation of microglia. Activation of JAK2-STAT3 and MAPKs signaling occurs in parallel with microglial activation, leading to PGE₂ synthesis via COX-2-mPGES-1 system. Finally, PGE₂ decreased microglial phagocytosis through EP2 receptor. Preventing phosphorylation of JAK2-STAT3 and MAPKs, inhibiting COX-2 activity, or abolishing EP2 activity efficiently ameliorated microglial phagocytosis during EMF stimulation