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• W2013852511 abstract "MicroRNAs (miRNAs) contribute to both neuronal and immune cell fate, but their involvement in intertissue 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 induced leukocyte overexpression of the AChE-targeting miR-132. Injected locked nucleic acid (LNA)-modified anti-miR-132 oligonucleotide depleted miR-132 amounts while elevating AChE in mouse circulation and tissues. In transfected cells, a mutated 3′UTR miR-132 binding site increased AChE mRNA expression, whereas cells infected with a lentivirus expressing pre-miR-132 showed suppressed AChE. Transgenic mice overexpressing 3′UTR null AChE showed excessive inflammatory mediators and impaired cholinergic anti-inflammatory regulation, in spite of substantial miR-132 upregulation 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 avenues for study and therapeutic manipulations of the neuro-immune dialog. MicroRNAs (miRNAs) contribute to both neuronal and immune cell fate, but their involvement in intertissue 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 induced leukocyte overexpression of the AChE-targeting miR-132. Injected locked nucleic acid (LNA)-modified anti-miR-132 oligonucleotide depleted miR-132 amounts while elevating AChE in mouse circulation and tissues. In transfected cells, a mutated 3′UTR miR-132 binding site increased AChE mRNA expression, whereas cells infected with a lentivirus expressing pre-miR-132 showed suppressed AChE. Transgenic mice overexpressing 3′UTR null AChE showed excessive inflammatory mediators and impaired cholinergic anti-inflammatory regulation, in spite of substantial miR-132 upregulation 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 avenues for study and therapeutic manipulations of the neuro-immune dialog. MicroRNAs (miRNAs) are evolutionarily conserved short noncoding RNAs that regulate multiple molecular pathways in specific cell or tissue types (Ambros, 2004Ambros V. The functions of animal microRNAs.Nature. 2004; 431: 350-355Crossref PubMed Scopus (8378) Google Scholar, Landgraf et al., 2007Landgraf P. Rusu M. Sheridan R. Sewer A. Iovino N. Aravin A. Pfeffer S. Rice A. Kamphorst A.O. Landthaler M. et al.A mammalian microRNA expression atlas based on small RNA library sequencing.Cell. 2007; 129: 1401-1414Abstract Full Text Full Text PDF PubMed Scopus (2795) Google Scholar, Lim et al., 2003Lim L.P. Glasner M.E. Yekta S. Burge C.B. Bartel D.P. Vertebrate microRNA genes.Science. 2003; 299: 1540Crossref PubMed Scopus (973) Google Scholar); however, their relevance for systemic interactions such as brain-body signaling is yet unknown. miRNAs often bind their mRNA targets based on sequence complementarity in specific locations on the 3′ untranslated region (UTR) of the mRNA, termed miRNA response elements (MREs). This leads to translational repression and/or degradation of the mRNA. Each miRNA potentially binds multiple mRNAs (Krek et al., 2005Krek A. Grun D. Poy M.N. Wolf R. Rosenberg L. Epstein E.J. MacMenamin P. da Piedade I. Gunsalus K.C. Stoffel M. Rajewsky N. Combinatorial microRNA target predictions.Nat. Genet. 2005; 37: 495-500Crossref PubMed Scopus (3702) Google Scholar). miRNA expression varies with cell type, tissue, and developmental stage (Baek et al., 2008Baek D. Villen J. Shin C. Camargo F.D. Gygi S.P. Bartel D.P. The impact of microRNAs on protein output.Nature. 2008; 455: 64-71Crossref PubMed Scopus (2780) Google Scholar) and spans both neural and immune cells (Liang et al., 2007Liang Y. Ridzon D. Wong L. Chen C. Characterization of microRNA expression profiles in normal human tissues.BMC Genomics. 2007; 8: 166Crossref PubMed Scopus (823) Google Scholar, Plasterk, 2006Plasterk R.H. Micro RNAs in animal development.Cell. 2006; 124: 877-881Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Specifically, bacterial endotoxin- or lipopolysaccharide (LPS)-exposed human monocytes overproduce several miRNAs, e.g., miR-146a, 155, and 132, whereas cytokines such as TNF upregulate miR-155 and 125b (Tili et al., 2007Tili E. Michaille J.J. Cimino A. Costinean S. Dumitru C.D. Adair B. Fabbri M. Alder H. Liu C.G. Calin G.A. Croce C.M. Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation and their possible roles in regulating the response to endotoxin shock.J. Immunol. 2007; 179: 5082-5089PubMed Google Scholar) and immune regulators like IFN-β reduce miR-122 as part of an antiviral protection response (Pedersen et al., 2007Pedersen I.M. Cheng G. Wieland S. Volinia S. Croce C.M. Chisari F.V. David M. Interferon modulation of cellular microRNAs as an antiviral mechanism.Nature. 2007; 449: 919-922Crossref PubMed Scopus (716) Google Scholar). The NF-κB-dependent miR-146a targets the tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) (Taganov et al., 2006Taganov K.D. Boldin M.P. Chang K.J. Baltimore D. NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses.Proc. Natl. Acad. Sci. USA. 2006; 103: 12481-12486Crossref PubMed Scopus (3205) Google Scholar). miR-155 targets the interleukin-1 signaling pathway in monocytes (Ceppi et al., 2009Ceppi M. Pereira P.M. Dunand-Sauthier I. Barras E. Reith W. Santos M.A. Pierre P. MicroRNA-155 modulates the interleukin-1 signaling pathway in activated human monocyte-derived dendritic cells.Proc. Natl. Acad. Sci. USA. 2009; 106: 2735-2740Crossref PubMed Scopus (556) Google Scholar) as well as angiotensin II type I receptor in fibroblasts (Martin et al., 2006Martin M.M. Lee E.J. Buckenberger J.A. Schmittgen T.D. Elton T.S. MicroRNA-155 regulates human angiotensin II type 1 receptor expression in fibroblasts.J. Biol. Chem. 2006; 281: 18277-18284Crossref PubMed Scopus (207) Google Scholar) and regulates T cell differentiation and antibody response through the JNK kinase pathway, which activates the AP-1 complex (Rodriguez et al., 2007Rodriguez A. Vigorito E. Clare S. Warren M.V. Couttet P. Soond D.R. van Dongen S. Grocock R.J. Das P.P. Miska E.A. et al.Requirement of bic/microRNA-155 for normal immune function.Science. 2007; 316: 608-611Crossref PubMed Scopus (1505) Google Scholar). miR-155 null mice are immune deficient (Thai et al., 2007Thai T.-H. Calado D.P. Casola S. Ansel K.M. Xiao C. Xue Y. Murphy A. Frendewey D. Valenzuela D. Kutok J.L. et al.Regulation of the germinal center response by microRNA-155.Science. 2007; 316: 604-608Crossref PubMed Scopus (1189) Google Scholar), suggesting that LPS-induced miRs regulate immunity. Our study focused on the inflammation-associated role(s) of miR-132, which has demonstrated function and high expression in the brain (Cheng et al., 2007Cheng H.Y. Papp J.W. Varlamova O. Dziema H. Russell B. Curfman J.P. Nakazawa T. Shimizu K. Okamura H. Impey S. Obrietan K. microRNA modulation of circadian-clock period and entrainment.Neuron. 2007; 54: 813-829Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). We predicted miR involvement in brain-body communication such as the neuro-endocrine modulation of inflammation (De Kloet et al., 1998De Kloet E.R. Vreugdenhil E. Oitzl M.S. Joels M. Brain corticosteroid receptor balance in health and disease.Endocr. Rev. 1998; 19: 269-301Crossref PubMed Scopus (2107) Google Scholar, McEwen, 2007McEwen B.S. Physiology and neurobiology of stress and adaptation: Central role of the brain.Physiol. Rev. 2007; 87: 873-904Crossref PubMed Scopus (2558) Google Scholar), whereby innate immunity is regulated by acetylcholine (ACh) to optimize the resolution of inflammation (Sternberg, 2006Sternberg E.M. Neural regulation of innate immunity: A coordinated nonspecific host response to pathogens.Nat. Rev. Immunol. 2006; 6: 318-328Crossref PubMed Scopus (733) Google Scholar). Specifically, afferent fibers of the vagus nerve signal the presence of circulating proinflammatory cytokines to the brain (Watkins and Maier, 1999Watkins L.R. Maier S.F. Implications of immune-to-brain communication for sickness and pain.Proc. Natl. Acad. Sci. USA. 1999; 96: 7710-7713Crossref PubMed Scopus (200) Google Scholar). Reciprocally, efferent vagus nerve fibers release ACh, which binds α7 nicotinic receptors on macrophages, intercepts the nuclear translocation of NFκB, and inhibits the production of proinflammatory mediators (de Jonge et al., 2005de Jonge W.J. van der Zanden E.P. The F.O. Bijlsma M.F. van Westerloo D.J. Bennink R.J. Berthoud H.R. Uematsu S. Akira S. van den Wigngaard R.M. Boeckxstaens G.E. Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway.Nat. Immunol. 2005; 6: 844-851Crossref PubMed Scopus (704) Google Scholar, Metz and Tracey, 2005Metz C.N. Tracey K.J. It takes nerve to dampen inflammation.Nat. Immunol. 2005; 6: 756-757Crossref PubMed Scopus (75) Google Scholar, Watkins and Maier, 1999Watkins L.R. Maier S.F. Implications of immune-to-brain communication for sickness and pain.Proc. Natl. Acad. Sci. USA. 1999; 96: 7710-7713Crossref PubMed Scopus (200) Google Scholar). Activation of this “cholinergic reflex” has been shown to alleviate inflammatory disease, including endotoxemia (Pavlov et al., 2007Pavlov V.A. Ochani M. Yang L.H. Gallowitsch-Puerta M. Ochani K. Lin X. Levi J. Parrish W.R. Rosas-Ballina M. Czura C.J. et al.Selective alpha7-nicotinic acetylcholine receptor agonist GTS-21 improves survival in murine endotoxemia and severe sepsis.Crit. Care Med. 2007; 35: 1139-1144Crossref PubMed Scopus (260) Google Scholar), sepsis (van Westerloo et al., 2005van Westerloo D.J. Giebelen I.A. Florquin S. Daalhuisen J. Bruno M.J. de Vos A.F. Tracey K.J. van der Poll T. The cholinergic anti-inflammatory pathway regulates the host response during septic peritonitis.J. Infect. Dis. 2005; 191: 2138-2148Crossref PubMed Scopus (249) Google Scholar), colitis (Pullan et al., 1994Pullan R.D. Rhodes J. Ganesh S. Mani V. Morris J.S. Williams G.T. Newcombe R.G. Russell M.A. Feyerabend C. Thomas G.A. et al.Transdermal nicotine for active ulcerative colitis.N. Engl. J. Med. 1994; 330: 811-815Crossref PubMed Scopus (428) Google Scholar), pancreatitis (van Westerloo et al., 2006van Westerloo D.J. Giebelen I.A. Florquin S. Bruno M.J. Larosa G.J. Ulloa L. Tracey K.J. van der Poll T. The vagus nerve and nicotinic receptors modulate experimental pancreatitis severity in mice.Gastroenterology. 2006; 130: 1822-1830Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), ischemia reperfusion (Altavilla et al., 2006Altavilla D. Guarini S. Bitto A. Mioni C. Giuliani D. Bigiani A. Squadrito G. Minutoli L. Venuti F.S. Messineo F. et al.Activation of the cholinergic anti-inflammatory pathway reduces NF-kappab activation, blunts TNF-alpha production, and protects againts splanchic artery occlusion shock.Shock. 2006; 25: 500-506Crossref PubMed Scopus (81) Google Scholar), and acute lung injury (Su et al., 2007Su X. Lee J.W. Matthay Z.A. Mednick G. Uchida T. Fang X. Gupta N. Matthay M.A. Activation of the alpha7 nAChR reduces acid-induced acute lung injury in mice and rats.Am. J. Respir. Cell Mol. Biol. 2007; 37: 186-192Crossref PubMed Scopus (129) Google Scholar). At the tissue level, synaptophysin-positive nerve endings interact with spleen macrophages, and surgical ablation of the splenic nerve indicates that these interactions are required for the cholinergic anti-inflammatory control of endotoxemia (Rosas-Ballina et al., 2008Rosas-Ballina M. Ochani M. Parrish W.R. Ochani K. Harris Y.T. Huston J.M. Chavan S. Tracey K.J. Splenic nerve is required for cholinergic antiinflammatory pathway control of TNF in endotoxemia.Proc. Natl. Acad. Sci. USA. 2008; 105: 11008-11013Crossref PubMed Scopus (412) Google Scholar). Thus, brain-body communication through ACh is intimately involved in the regulation of inflammation. ACh is hydrolyzed by acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) (Soreq and Seidman, 2001Soreq H. Seidman S. Acetylcholinesterase—new roles for an old actor.Nat. Rev. Neurosci. 2001; 2: 294-302Crossref PubMed Scopus (961) Google Scholar); therefore, high amounts of these enzymes could potentially negate the cholinergic anti-inflammatory signal (Ofek et al., 2007Ofek K. Krabbe K.S. Evron T. Debecco M. Nielsen A.R. Brunnsgaad H. Yirmiya R. Soreq H. Pedersen B.K. Cholinergic status modulations in human volunteers under acute inflammation.J. Mol. Med. 2007; 85: 1239-1251Crossref PubMed Scopus (85) Google Scholar). Supporting this notion, AChE inhibition restricts inflammation in both the peripheral and the central nervous system (CNS) (Pollak et al., 2005Pollak Y. Gilboa A. Ben-Menachem O. Ben-Hur T. Soreq H. Yirmiya R. Acetylcholinesterase inhibitors reduce brain and blood interleukin-1beta production.Ann. Neurol. 2005; 57: 741-745Crossref PubMed Scopus (89) Google Scholar, Pavlov et al., 2009Pavlov V.A. Parrish W.R. Rosas-Ballina M. Ochani M. Puerta M. Ochani K. Chavan S. Al-Abed Y. Tracey K.J. Brain acetylcholinesterase activity controls systemic cytokine levels through the cholinergic anti-inflammatory pathway.Brain Behav. Immun. 2009; 23: 41-45Crossref PubMed Scopus (290) Google Scholar). We therefore predicted that microRNAs targeting AChE can attenuate inflammation. In murine splenocytes, AChE (but not BChE; Figure S1A available online) mRNA, protein, and activity were all substantially reduced 24 hr after LPS (endotoxin, a Toll-like receptor TLR4 ligand) treatment (Figures 1A–1C). The post-LPS downregulation of AChE could be reproduced in vivo, by intraperitoneal (i.p.) injection of LPS that upregulated interleukin (IL)-6 and IL-1β while reducing serum AChE activity (Figures 1D–1F), suggesting that systemic reduction of AChE activity is an integral part of the postinflammatory response. To find miRNAs that could potentially downregulate cholinesterases, we used PicTar (Krek et al., 2005Krek A. Grun D. Poy M.N. Wolf R. Rosenberg L. Epstein E.J. MacMenamin P. da Piedade I. Gunsalus K.C. Stoffel M. Rajewsky N. Combinatorial microRNA target predictions.Nat. Genet. 2005; 37: 495-500Crossref PubMed Scopus (3702) Google Scholar), miRanda (John et al., 2004John B. Enright A.J. Aravin A. Tuschl T. Sander C. Marks D.S. Human MicroRNA targets.PLoS Biol. 2004; 2: e363Crossref PubMed Scopus (2695) Google Scholar), and an in-house target prediction algorithm. To test whether the in silico-identified miRNAs respond to inflammation, we used an in-house spotted microarray. No miRNAs were predicted to target both AChE and BChE, and miRNAs induced by immune challenges did not include sequences that could target BChE (Figure S1B). In contrast, two miRNAs complementary to AChE's 3′ UTR were induced by LPS: miR-132 and miR-182∗ (processed from the same precursor molecule as miR-182; sequences and alignments in Figures S1C and S1D), compatible with the immune modulation of AChE activity in nucleated blood cells (Pick et al., 2006Pick M. Perry C. Lapidot T. Guimaraes-Sternberg C. Naparstek E. Deutsch V. Soreq H. Stress-induced cholinergic signaling promotes inflammation-associated thrombopoiesis.Blood. 2006; 107: 3397-3406Crossref PubMed Scopus (49) Google Scholar). miR-132 and miR-182∗ are intergenically encoded and dissimilar. Binding sites for miR-132 and 182∗ at bases 961 and 704 of AChE 3′ UTR, respectively (Figure S1C), showed high conservation across species, supporting their physiological relevance. Spotted array analysis showed that miR-132, among others, was consistently upregulated in primary human macrophages by LPS (Figure 1G) as well as by additional immunogens, e.g., the TLR9 ligand CpG oligonucleotide (ODN) 2006, which is known to react to LPS (Figures S1E and S1F; Tables S2 and S3 of array results; Hartmann et al., 1999Hartmann G. Weiner G.J. Krieg A.M. CpG DNA: A potent signal for growth, activation, and maturation of human dendritic cells.Proc. Natl. Acad. Sci. USA. 1999; 96: 9305-9310Crossref PubMed Scopus (540) Google Scholar). QRT-PCR validation confirmed that LPS exposure elevates miR-132 and 182∗ but not other hematopoietic miRs (e.g., miR-181a), in a dose-dependent manner (Figure 1H; Figure S1G). This increase, which occurred 12–24 hr after the onset of inflammation, was paralleled by a reduction in AChE protein (Figures 1A–1C; Figure S1H) that could enable the attenuation of inflammation by ACh in a physiological system. Supporting this notion, AChE reduction was observed both in the bone marrow (BM) and spleen of LPS-injected FVB-N mice (Figures 1A–1C and 1F) and in human- and mouse-derived cell lines (Figure S2). Both AChE-targeting miRNAs increased in an LPS dose-dependent manner, peaking at 1 μg/ml LPS (Figure S1G) and at 24 hr postexposure in cultured macrophages, and LPS-exposed mice reached double the expression seen in bone marrow (BM) and splenocytes of control mice at the same time point (Figure 1I). Taken together, these results indicate inherent involvement of changes in miR-132 and miR-182∗ in the inflammatory reaction to LPS exposure. To manipulate miR-132 amounts in vivo, we intravenously injected FVB-N mice with 3 daily 3.3 mg/kg doses of a phosphorothioated, LNA-modified oligonucleotide (Elmen et al., 2008Elmen J. Lindow M. Schutz S. Lawrence M. Petri A. Obad S. Lindholm M. Hedtjarn M. Hansen H.F. Berger U. et al.LNA-mediated microRNA silencing in non-human primates.Nature. 2008; 452: 896-899Crossref PubMed Scopus (1343) Google Scholar) complementary to mature miR-132 (anti-132) or a scrambled sequence control oligonucleotide (scr) (Figure 2A). 24 hr after the last injection, anti-132-treated mice showed dramatic reductions in miR-132 but not miR-182∗ expression in both BM and spleen (Figure 2B), accompanied by increases in AChE but not BChE (data not shown) activity in the small intestine, serum, and bone marrow (Figure 2C). Likewise, AChE protein expression increased in anti-132-treated mice (Figure 2D). In individual mice, relative spleen miR-132 expression (in anti-132-, scr-, and mock-treated groups) showed an inverse correlation when plotted against serum AChE activities of the same mice (Figure 2E), suggesting that miR-132 is a major systemic regulator of AChE. These findings further demonstrate that the anti-132 oligonucleotide can serve to manipulate the systemic expression of AChE. In the RAW 264.7 murine macrophage-derived cell line, LPS exposure induced robust increases of nitric oxide (NO), IL-6, and TNF-α within 24 hr, reflecting an inflammatory response. This was accompanied by robust decreases in AChE activity (Figure S2B) and a marked elevation in miR-132 and miR-182∗ expression, whereas AChE mRNA expression was essentially unchanged in this test (Figures S2D–S2F). By 24 hr after exposure, both human-derived U937 cells and mouse-derived RAW 264.7 cells showed reduced AChE activity (Figure S2C), similar to that achieved by treatment with 10 μM of the selective AChE inhibitor, BW 284 C51 (Figure S2D). Inversely, ACh blocked the LPS-induced increase in NO as efficiently as dexamethasone (Figure S2E). Also, untreated cells showed NF-κB labeling in the cytoplasm, whereas LPS exposure shifted NF-κB labeling to the nucleus, but coexposure to both LPS and ACh inhibited this translocation (data not shown), demonstrating the cholinergic anti-inflammatory reflex. The “Cister” algorithm (Frith et al., 2001Frith M.C. Hansen U. Weng Z. Detection of cis-element clusters in higher eukaryotic DNA.Bioinformatics. 2001; 17: 878-889Crossref PubMed Scopus (191) Google Scholar) identified binding motifs for inflammation-associated transcription factors in the 5 kb genomic regions upstream of the miR-132 and miR-182∗ precursors (Figures S3A and S3B), suggesting that the promoters of both of these AChE-targeting miRs bind AP-1, involved in cellular proliferation, transformation, and death (Shaulian and Karin, 2002Shaulian E. Karin M. AP-1 as a regulator of cell life and death.Nat. Cell Biol. 2002; 4: E131-E136Crossref PubMed Scopus (2074) Google Scholar). AP-1 is composed of members of the Jun and Fos family of transcription factors, and c-Jun plays essential roles in the immune responses of pattern recognition receptors (Akira, 2006Akira S. TLR signaling.Curr. Top. Microbiol. Immunol. 2006; 311: 1-16PubMed Google Scholar). For miR-132, the Cister algorithm also identified the calcium-response element CRE. Its binding protein, CREB, responds to cholinergic signals via the α7 nicotinic ACh receptor (Bitner et al., 2007Bitner R.S. Bunnelle W.H. Anderson D.J. Briggs C.A. Buccafusco J. Curzon P. Decker M.W. Frost J.M. Gronlien J.H. Gubbins E. et al.Broad-spectrum efficacy across cognitive domains by alpha7 nicotinic acetylcholine receptor agonism correlates with activation of ERK1/2 and CREB phosphorylation pathways.J. Neurosci. 2007; 27: 10578-10587Crossref PubMed Scopus (168) Google Scholar) as a late event in inflammatory reactions (Kang et al., 2007Kang Y.J. Kim S.O. Shimada S. Otsuka M. Seit-Nebi A. Kwon B.S. Watts T.H. Han J. Cell surface 4-1BBL mediates sequential signaling pathways ‘downstream’ of TLR and is required for sustained TNF production in macrophages.Nat. Immunol. 2007; 8: 601-609Crossref PubMed Scopus (80) Google Scholar). Thus, miRNAs targeting AChE mRNA, like AChE itself, emerged as likely mediators of inflammatory reactions. To more directly test for regulation of AChE by miR-132, we transfected CHO cells with AChE expression vectors encoding either a native UTR or UTR with mutated miR-132 MRE. CHO cells express miR-132 at about 20 fmoles/mg (104 molecules per ng RNA), higher than the basal expression in mouse splenocytes yet lower than that in the mouse cortex (Figure 3A). In contrast, CHO cells have almost no endogenous AChE (Figure 3B). After transfection, cells expressing the 3′UTR-mutated construct produced higher AChE activities than those expressing the native 3′UTR control, validating the importance of the miR-132 MRE in regulation of AChE (Figure 3B). To further test the regulation of AChE by miR-132, we overexpresed miR-132 by infecting BM-derived macrophages, extracted from FVB-N mice, with either a pre-miR-132 expression vector or a control vector containing a scrambled sequence (Figure 3C). 72 hr after infection, AChE activity of BM cells infected by the miR-132 vector was downregulated by 60% (Figure 3D) relative to cells infected with scrambled vector. Together with the above mutation studies, these findings demonstrate that creating miR-132 loss of function by ablating the miR-132 binding site elevates, whereas implementing miR-132 gain of function by enhancing miR-132 expression through lentiviral infection suppresses, AChE expression. Transgenic mice expressing the TgR construct (Figure 4A; Gilboa-Geffen et al., 2007Gilboa-Geffen A. Lacoste P.P. Soreq L. Cizeron-Clairac G. Le Panse R. Truffault F. Shaked I. Soreq H. Berrih-Aknin S. The thymic theme of acetylcholinesterase splice variants in myasthenia gravis.Blood. 2007; 109: 4383-4391Crossref PubMed Scopus (31) Google Scholar, Sternfeld et al., 2000Sternfeld M. Shoham S. Klein O. Flores-Flores C. Evron T. Idelson G.H. Kitsberg D. Patrick J.W. Soreq H. Excess “readthrough” acetylcholinesterase attenuates but the “synaptic” variant intensifies neurodeterioration correlates.Proc. Natl. Acad. Sci. USA. 2000; 97: 8647-8652Crossref PubMed Scopus (112) Google Scholar) served to challenge in vivo the importance of AChE regulation by miRs in the context of the anti-inflammatory reflex. Because of the absence of the functionally relevant MRE, we predicted that AChE activity in TgR mice should not be suppressed by miR-132, even when it is elevated after inflammatory insults, and that ACh should therefore fail to attenuate innate immunity reactions, e.g., the response to LPS (Scheme, Figure 4B). Supporting this hypothesis, intestinal explants from TgR mice, in addition to showing elevated AChE activity (Figure 4C) and AChE protein expression (Figure 4D), displayed higher basal amounts of circulation cytokines (IL-1β and Il-6 and IL-10) compared to FVB-N counterparts. The cytokine overproduction in the TgR intestine could be rebalanced with i.p. injection of the ACh receptor agonist nicotine (Figures 4E–4G), demonstrating that the cholinergic suppression of inflammation was not impaired in these mice because of missing ACh receptors and/or downstream signaling. AChE protein in TgR intestine reached similar amounts as those in anti-132-treated mice (Figure 4D), consistent with the role of miR-132 as a major physiological regulator of ACh signaling. Mice notably respond to LPS administration with dynamic changes in their body temperature. First, they develop hyperthermia that is thereafter followed by hypothermia (due to peripheral vasodilatation) (Splawinski et al., 1977Splawinski J.A. Zacny E. Gorka Z. Fever in rats after intravenous E. coli endotoxin administration.Pflugers Arch. 1977; 368: 125-128Crossref PubMed Scopus (19) Google Scholar). To assess the impact of 3′UTR null AChE overexpression on this robust physiological response, rectal temperatures were measured in FVB-N and TgR mice after i.p. injection of LPS. TgR mice notably differed from FVB-N wild-type mice both in their lower basal body temperatures and in their LPS fever response. Whereas FVB-N mice display transient hyperthermia, TgR mice respond to LPS by initial hypothermia followed by prolonged hyperthermia (Figure 4H). TgR mice further showed higher expression of miR-132 than wild-type mice, in the periphery, BM, and the brain, especially in the pyrogenic cytokine-responding hypothalamus (Figure 4I; Blatteis, 2007Blatteis C.M. The onset of fever: New insights into its mechanism.Prog. Brain Res. 2007; 162: 3-14Crossref PubMed Scopus (121) Google Scholar), suggesting feedback regulation between miR-132 and AChE expression that spans both body and relevant regions in the brain. In agreement with the effects in intestinal tissue, BM-derived macrophages from both wild-type and TgR mice overproduced IL6, IL12, and TNF-α when challenged with LPS ex vivo; however, coadministration of LPS and ACh prevented this increase in FVB-N but not TgR-derived macrophages (Figures 4J–4L), demonstrating that excessive ACh hydrolysis impairs the cholinergic anti-inflammatory response. TgR mice further presented excessive leukocyte recruitment into the peritoneum after thioglycolate injection, displayed by higher numbers of Mac-1- (CD11b, a complement receptor typical of activated macrophages) expressing cells than control mice (Figures S3C and S3D), suggesting systemically deregulated immunity in these mice. Our findings demonstrate that AChE-targeting miR-132 can attenuate inflammation by reducing AChE amounts, thus enhancing the brain's ability to govern inflammation via cholinergic signaling. We found that excessive inflammation upregulates miR-132. In miR-132-expressing cultured cells, ectopic AChE expression was elevated by introducing a point mutation in the native miR-132 MRE. Infection with lentivirus expressing pre-miR-132 suppressed AChE in cultured BM cells. Depleting miR-132 in vivo in the BM and spleen increased peripheral AChE activity in the intestine, serum, and bone marrow. TgR mice, overexpressing MRE null AChE, lost the ability to attenuate inflammation by cholinergic signaling, although their excessive inflammatory response was quenchable by nicotine, a cholinergic agonist not hydrolyzed by AChE. Despite expressing miR-132 at high amounts, TgR MRE null mice lacked the ability to benefit from the regulation of AChE expression by miRNAs. The nervous system was shown to profoundly regulate the innate immune system. Direct innervation of lymphatic organs (Pavlov et al., 2007Pavlov V.A. Ochani M. Yang L.H. Gallowitsch-Puerta M. Ochani K. Lin X. Levi J. Parrish W.R. Rosas-Ballina M. Czura C.J. et al.Selective alpha7-nicotinic acetylcholine receptor agonist GTS-21 improves survival in murine endotoxemia and severe sepsis.Crit. Care Med. 2007; 35: 1139-1144Crossref PubMed Scopus (260) Google Scholar) or inflamed tissues (Tracey, 2007Tracey K.J. Physiology and immunology of the cholinergic antiinflammatory pathway.J. Clin. Invest. 2007; 117: 289-296Crossref PubMed Scopus (1037) Google Scholar) restricts the inflammatory burden. Neurotransmitters and neuropeptides bind to specific receptors expressed by leukocytes, inhibiting production of proinflammatory mediators (Sternberg, 2006Sternberg E.M. Neural regulation of innate immunity: A coordinated nonspecific host response to pathogens.Nat. Rev. Immunol. 2006; 6: 318-328Crossref PubMed Scopus (733) Google Scholar). Neuronal and leukocyte miRNAs can thus provide a fine-tuning tool for these sensitive neuro-immune checkpoints. We focused our current study on miR-132, well characterized by others in brain neurons, because of its robust induction in leukocytes after LPS. miR-132 is upregulated by the cAMP-response element binding protein (CREB) (Vo et al., 2005Vo N. Klein M.E. Varlamova O. Keller D.M. Yamamoto T. Goodman R.H. Impey S. A cAMP-response element binding protein-induced microRNA regulates neuronal morphogenesis.Proc. Natl. Acad. Sci. USA. 2005; 102: 16426-16431Crossref PubMed Scopus (662) Google Scholar) and downregulated by the RE1 silencing transcription factor (REST) (Conaco e" @default.
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• W2013852511 date "2009-12-01" @default.
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• W2013852511 title "MicroRNA-132 Potentiates Cholinergic Anti-Inflammatory Signaling by Targeting Acetylcholinesterase" @default.
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• W2013852511 doi "https://doi.org/10.1016/j.immuni.2009.09.019" @default.
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