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, 63 (10), 1797-808

Alkylindole-sensitive Receptors Modulate Microglial Cell Migration and Proliferation

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Alkylindole-sensitive Receptors Modulate Microglial Cell Migration and Proliferation

Susan Fung et al. Glia.

Abstract

Ligands targeting G protein-coupled receptors (GPCR) expressed by microglia have been shown to regulate distinct components of their activation process, including cell proliferation, migration and differentiation into M1 or M2 phenotypes. Cannabinoids, including the active component of the Cannabis plant, tetrahydrocannabinol (THC), and the synthetic alkylindole (AI) compound, WIN55212-2 (WIN-2), activate two molecularly identified GPCRs: CB1 and CB2 . Previous studies reported that WIN-2 activates an additional unknown GPCR that is not activated by plant-derived cannabinoids, and evidence indicates that microglia express these receptors. Detailed studies on the role of AI-sensitive receptors in microglial cell activation were difficult as no selective pharmacological tools were available. Here, three newly-developed AI analogues allowed us to determine if microglia express AI-sensitive receptors and if so, study how they regulate the microglial cell activation process. We found that mouse microglia in primary culture express functional AI-sensitive receptors as measured by radioligand binding and changes in intracellular cAMP levels, and that these receptors control both basal and ATP-stimulated migration. AI analogues inhibit cell proliferation stimulated by macrophage-colony stimulating factor (M-CSF) without affecting basal cell proliferation. Remarkably, AI analogues do not control the expression of effector proteins characteristic of M1 or M2 phenotypes; yet activating microglia with M1 and M2 cytokines reduces the microglial response to AI analogues. Our results suggest that microglia express functional AI-sensitive receptors that control select components of their activation process. Agonists of these novel targets might represent a novel class of therapeutics to influence the microglial cell activation process.

Keywords: GPCR; cannabinoid; cell migration and cell proliferation; microglia.

Figures

Figure 1
Figure 1
Microglial AI-sensitive receptors couple to Gs proteins. (A-C) Chemical structures of ST-11 (A), ST-48 (B), and ST-47 (C). (D) WIN55212-2 (WIN), ST-11 and ST-48 dose-dependent competition of [3H]WIN55212-2 binding in microglial membranes. (E) The inactive compound ST-47 (1 μM) and cannabinoid ligands, SR2 (300 nM) and O-2050 (100 nM) do not compete for [3H]WIN55212-2 binding in microglial membranes. (F) ST-11 and ST-48 dose-dependently increase intracellular cAMP levels in primary cultured microglia (**P < 0.01 and ***P < 0.001 compared with vehicle-treated cells). (G) PTX (1 μg/ml) does not block the ST-11 (300 nM) or ST-48 (1 μM) stimulated increase of cAMP levels (*P < 0.05, **P < 0.01, and ***P < 0.001 compared with vehicle-treated cells). Each data point represents at least 3 experiments, each performed in triplicate.
Figure 2
Figure 2
AI-sensitive receptors inhibit basal and stimulated microglial cell migration. (A) Representative images of fluorescent microglia that have migrated through 10 μm pores. Background fluorescence was subtracted from all conditions tested and is represented by the blank condition. This filter shows ST-11 treatment (300 nM) in the presence or absence of ATP (300 μM). (B) ATP (300 μM) stimulates primary microglia migration 2.9 fold over basal migration (***P < 0.001 compared with vehicle-treated cells. (C) ST-11 dose-dependently inhibits ATP-stimulated migration (*P < 0.05, ***P < 0.001 compared with vehicle-treated cells). (D) ST-11 does not cause cellular toxicity to primary microglia during a 3-hr period (***P < 0.001 compared with vehicle-treated cells). Each data point represents at least 3 experiments, each performed in triplicate.
Figure 3
Figure 3
AI-sensitive receptors inhibit microglial cell proliferation stimulated by M-CSF. (A) ST-11 does not affect basal cell proliferation but dose-dependently inhibits cell proliferation stimulated M-CSF (1% L929 condition media) (*P < 0.05 compared with L929 response). (B) 1% L929 added to cell culture media significantly increases cell proliferation (***P < 0.001 compared with cells treated with 0.1% DMSO vehicle). (C) ST-11 does not affect microglial cell viability over a 72 hr period. Each data point represents at least 4 experiments, each performed in triplicate.
Figure 4
Figure 4
AI-sensitive receptors do not modulate the induction of microglial M1 phenotype. (A) Nitrite accumulation measured based of a calibration curve. (B) TNFα/IFNγ significantly increases nitrite levels (**P < 0.01 compared with vehicle-treated cells). Both the basal and TNFα/IFNγ-stimulated increase in nitrate levels are within the linear range of the calibration curve. Nitrite production measured under vehicle or TNFα/IFNγ-stimulated conditions shows no change when treated with ST-11 (300 nM). (C) TNFα/IFNγ treatment significantly increases IP-10 levels (**P < 0.01 compared with vehicle treated cells). IP-10 production measured under basal (vehicle-treated) or when stimulated with TNFα/IFNγ shows no change when treated with ST-11 (1 μM). Each data point represents at least 3 experiments, each performed in triplicate.
Figure 5
Figure 5
AI-sensitive receptors do not modulate the induction of microglial M2 phenotype. (A, B) Representative ct curves of Ym1 (A) and FIZZ1 (B) markers measured by qRT-PCR following treatment with vehicle or IL-4 (10 ng/mL). (C) The mRNA expression levels of Ym1 and FIZZ1 in microglia show no change when treated with ST-11 (300 nM). Each data point represents at least 3 experiments, each performed in triplicate.
Figure 6
Figure 6
M1 and M2 phenotypes differentially regulate the functionality of AI-sensitive receptors. (A, B) Both TNFα/IFNγ (A) and IL-4 (B) do not affect basal cAMP levels. ST-11 (300 nM) induced a significant increase in cAMP levels compared with vehicle in treated cells (P < 0.05 compared with vehicle levels in cytokine-treated cells), but did not induce a significant response in IL-4 treated cells. Both cytokine treatments potentiate the 10 μM isoproterenol-stimulated increase in cAMP levels (+++P < 0.001 compared with isoproterenol response in non-treated cells). (C, D) Both TNFα/IFNγ (C) and IL-4 (D) significantly reduce basal migration (***P<0.001 compared with vehicle level in non-treated cells). ST-11 (300 nM) further reduced migration in both TNFα/IFNγ- and IL-4-treated cells (#P<0.05, ###P<0.001 compared with ST-11 response in non-treated cells). ST-11 further reduced basal migration in TNFα/INFγ-treated cells (%P<0.05 compared to vehicle levels in cytokine-treated cells) but not in IL-4 treated cells. TNFα/IFNγ treatment did not significantly affect the ATP (300 μM) stimulated migration when compared to the ATP response in non-treated cells, whereas IL-4 treatment significantly reduced the ATP response (++P<0.001 compared with ATP response in non-treated cells). (E, F) Both TNFα/IFNγ (E) and IL-4 (F) significantly reduced basal microglial cell proliferation (***P<0.001 compared with vehicle level in non-treated cells). ST-11 (300 nM) did not further reduce proliferation in these cells. Both cytokine treatments blocked the ability of L929 (1%) conditioned media to increase proliferation (+++ P < 0.001 compared with L929 response in non-treated cells). All data points represent at least 3 experiments, each performed in triplicate.

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