Abstract
Given the modulatory role of neuropeptide Y (NPY) in the immune system, we investigated the effect of NPY on the production of NO and IL-1β in microglia. Upon LPS stimulation, NPY treatment inhibited NO production as well as the expression of inducible nitric-oxide synthase (iNOS). Pharmacological studies with a selective Y(1) receptor agonist and selective antagonists for Y(1), Y(2), and Y(5) receptors demonstrated that inhibition of NO production and iNOS expression was mediated exclusively through Y(1) receptor activation. Microglial cells stimulated with LPS and ATP responded with a massive release of IL-1β, as measured by ELISA. NPY inhibited this effect, suggesting that it can strongly impair the release of IL-1β. Furthermore, we observed that IL-1β stimulation induced NO production and that the use of a selective IL-1 receptor antagonist prevented NO production upon LPS stimulation. Moreover, NPY acting through Y(1) receptor inhibited LPS-stimulated release of IL-1β, inhibiting NO synthesis. IL-1β activation of NF-κB was inhibited by NPY treatment, as observed by confocal microscopy and Western blotting analysis of nuclear translocation of NF-κB p65 subunit, leading to the decrease of NO synthesis. Our results showed that upon LPS challenge, microglial cells release IL-1β, promoting the production of NO through a NF-κB-dependent pathway. Also, NPY was able to strongly inhibit NO synthesis through Y(1) receptor activation, which prevents IL-1β release and thus inhibits nuclear translocation of NF-κB. The role of NPY in key inflammatory events may contribute to unravel novel gateways to modulate inflammation associated with brain pathology.
Highlights
Brain inflammation is characterized primarily by microglia activation (1)
We revealed a novel signaling pathway through which neuropeptide Y (NPY) inhibits nitric oxide (NO) production induced by IL-1; Y1 receptor (Y1R) signaling prevents NF-B activation triggered by IL-1, in an inflammatory context occurring in a microglial cell line
The N9 microglial cell line did not abundantly express NPY in control conditions, there was a significant increase of NPY cDNA when cells were treated with 100 ng/ml LPS (p Ͻ 0.001, n ϭ 3) (Fig. 1A)
Summary
Cell Line Culture—The murine N9 microglia cell line (a kind gift from Prof. Claudia Verderio, CNR Institute of Neuroscience, Cellular and Molecular Pharmacology, Milan, Italy) was grown in RPMI medium supplemented with 30 mM glucose (Sigma), 100 units/ml penicillin, and 100 g/ml streptomycin (Invitrogen). Membranes were blocked in Tris-buffered saline containing 5% low fat milk and 0.1% Tween 20 (Sigma) for 1 h at room temperature and incubated overnight at 4 °C with the primary antibody solution diluted in 1% TBS-Tween, 0.5% low fat milk. Cells were left at room temperature for 5 min in lysis buffer (137 mM NaCl, 20 mM Tris-HCl, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 g/ml aprotinin, 1 g/ml leupeptin, 0.5 mM sodium orthovanadate (all from Sigma), pH 8.0). 100 l/well detection antibody diluted in 1ϫ assay diluent was added, and the plate was sealed and incubated at room temperature for 1 h. Every experimental condition was tested in three sets of independent experiments, unless stated otherwise, and performed in duplicates
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