ABSTRACT: MicroRNAs (miRs) contribute to both neuronal and immune cell fate, but their involvement in inter-tissue communication remained unexplored. The brain, via vagal secretion of acetylcholine (ACh), suppresses peripheral inflammation by intercepting cytokine production; therefore, we predicted that microRNAs targeting acetylcholinesterase (AChE) can attenuate inflammation. Here, we report that inflammatory stimuli induce leukocyte over-expression of the AChE-targeting miR-132. Injected LNA-modified anti-miR-132 oligonucleotide depleted miR-132 levels while elevating AChE in mouse circulation and tissues. In transfected cells, a mutated 3âUTR miR-132 binding site increased AChE mRNA expression, whereas cells infected with a lentivirus expressing pre-miR-132 showed suppressed AChE. Transgenic mice over-expressing 3'UTR-null AChE showed excessive inflammatory mediators and impaired cholinergic anti-inflammatory regulation, in spite of substantial miR-132 up-regulation in brain and bone marrow. Our findings identify the AChE mRNA-targeting miR-132 as a functional regulator of the brain-to-body resolution of inflammation, opening new avenues for study and therapeutic manipulations of the neuro-immune dialogue. Pathogen-negative leukopacks (Buffy coat) obtained from the Blood Bank at the Hadassah Medical Center, Ein Kerem were transferred into 50 ml conical tubes (~10 ml/tube) and diluted 1:2 with Dulbecco's phosphate buffered saline (PBS, Biological Industries, Beit Haemek, Israel). The diluted blood was gently layered on top of 20 ml of lymphocyte separation medium (LSM)/Ficoll (MP Biomedicals, Solon, OH) without mixing the layers. Following centrifugation (500g, 30 min, 20°C, without brake), three layers were obtained (plasma & platelets, lymphocytes, erythrocytes & granulocytes). The upper phase was drawn with a Pasteur pipette until reaching 2 mm above the interphase cloud, and then 5 to 10 ml interphase lymphocytes were drawn gently with a 1ml pipette, washed once with 45 ml PBS, and centrifuged (400g , 10 min, room temp.). Pellets from all tubes were resuspended in a total of 40 ml PBS. An aliquot of cells was counted in a hemacytometer using 0.5% Trypan Blue Solution (Biological Industries) to determine cell number and viability. Cells were then centrifuged (200g, 5 min, room temp) and resuspended at a concentration of ~ 5 X 106 cells/ml in RPMI-1640 (Sigma, St. Louis, MO) supplemented with 2.5% heat-inactivated (56°C, 30 min) human serum (Sigma). 10 ml of cell suspension were plated in 75 ml flasks and incubated (2 hrs, 37°C, 5% CO¬2). Non-adherent cells were then removed and the adherent monocytes washed 3 times with warm PBS. Cells were incubated for 2hr in 10 ml per dish of RPMI-1640 supplemented with LPS-free 10% foetal bovine serum (FBS), 2 mM L-Glutamine and 0.5% penicillin/streptomycin (GIBCO, Carlsbad, CA) and washed 3 times with warm PBS and 10 ml of complete RPMI supplemented with 10 U/ml granulocyte-monocyte colony stimulating factor (GM-CSF, Sigma, G5035) added every 2-3 days. By ~day 7 monocytes were differentiated to macrophages. Escherichia coli LPS (Sigma) was added to the growth medium to a final concentration of 1μg/ml, which was found optimal to induce innate immune reaction. For comparison, 1 μM of the TLR9 ligand CpG 2006 (type B; Microsynth GmbH) were added for 24 hr. Cells were washed 2-3 times with cold PBS, then incubated in 5 ml of cold filtered PBS + EDTA 2 mM (pH = 7.2) for 10-15 min on ice. Flasks were then tapped firmly to loosen cell attachment, and cells were gently scraped and collected by centrifugation (4°C, 800 rpm, 10 min). Short RNAs isolated from LPS-treated, CpG-treated and control cells were labeled with CyDyes 3 and 5 (each with a dye-swap), and hybridized with slides with spotted miRvana probe set (Ambion) as described for each sample.
