Neuregulin Upregulates Microglial α7 Nicotinic Acetylcholine Receptor Expression in Immortalized Cell Lines: Implications for Regulating Neuroinflammation

Jacobson C (2013) Neuregulin Upregulates Microglial 7 Nicotinic Acetylcholine Receptor Expression in Immortalized Cell Lines: Implications for Regulating Neuroinflammation. PLoS ONE 8(7): e70338. doi:10.1371/journal.pone.0070338 Neuregulin Upregulates Microglial 7 Nicotinic Acetylcholine Receptor Expression in Immortalized Cell Lines: Implications for Regulating Neuroinflammation Malwina Mencel 0 Michelle Nash 0 Christian Jacobson 0 Joseph El Khoury, Massachusetts General Hospital and Harvard Medical School, United States of America 0 1 Department of Biology, University of Waterloo , Waterloo, Ontario , Canada , 2 National School of Medicine, University of Namibia , Pionierspark, Windhoek , Namibia Neuregulin, previously known as ARIA, is a signaling protein involved in cell survival, synaptic plasticity, cell communication and differentiation. Neuregulin has also been described as a potent inducer of acetylcholine receptor transcription in muscle and although both neuregulin and acetylcholine have been individually described to have neuroprotective roles, their relationship in the cholinergic anti-inflammatory pathway of the brain has not been examined. - Funding: MM has been funded by Ontario Graduate Scholarship. Continued funding for MM has been provided by the Department of Biology. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Inflammation is an acute innate immune response to pathogens and functions to eradicate infection by phagocytosis and the recruitment of other effector cells via the rapid inflammatory mediators, by resident tissue macrophages [15]. If this local response becomes over-whelmed by the number of pathogens and/or the amount of pro-inflammatory signals becomes excessive, the response can turn systemic resulting in sepsis, organ failure and even death. Innate deregulation alone can lead to a variety of auto-inflammatory diseases such as arthritis, asthma and Crohns disease [68]. The central nervous system (CNS) is an immunologically privileged site that is inaccessible to cells of the systemic immune system due to the blood brain barrier (BBB). Microglia represent the macrophages of the CNS innate immune system and comprise 10-12% of the total cell population of the brain [9,10]. Microglia are activated under typical pathological conditions and can act as specialized sensors of brain tissue molecules; the synthesis of which correlates with a variety of neurodegenerative diseases, stroke, traumatic injury and tumor invasion [10,11]. For instance, the preliminary stages of Alzheimers disease (AD) progression contain inflammatory components; a result of microglial activation by functional In the periphery, macrophages are able to aid in the inhibition of systemic outcomes via a neuro-immune axis termed the cholinergic anti-inflammatory pathway [14]. Acetylcholine (ACh), secreted by the vagus nerve in response to elevated levels of pro-inflammatory mediators and bacterial products, such as lipopolysaccharide (LPS), binds to homopentameric 7 nicotinic acetylcholine receptors (7nAChRs) expressed by macrophages [1,14]. This prevents further synthesis of pro-inflammatory cytokines, especially TNF-, inhibiting the pro-inflammatory feedback loop [8]. However, this pathway does not affect the synthesis of the homeostatic anti-inflammatory cytokine IL-10 [8]. A similar cholinergic mechanism has also been demonstrated in the CNS by microglia [15]. Finding a pathway that regulates the transcription of 7nAChR in microglia could potentially lead to a mechanism for regulating the inflammatory response in the CNS. The neuregulin (NRG) family of ligands are important in cellcell communication in development and disease and are known to play roles in synaptogenesis and neuronal survival [16]. NRGs all contain an epidermal growth factor (EGF) domain and are extensively alternatively spliced to produce various isoforms [17]. It is through the EGF domain that the various NRG isoforms interact with ErbB family of transmembrane tyrosine kinases receptors [17]. The ErbB family of receptors is comprised of four members: EGFR, ErbB2, ErbB3 and ErbB4. These receptors are expressed in various cell and tissue types with NRG1 binding principally, and with highest affinity, to ErbB3 or ErbB4 [18]. NRG binding to the Erbs induces the formation of functional ErbB homo-and/or heterodimers. Ultimately, these interactions result in receptor phosphotyrosylation and the activation of intracellular signaling cascades. At the neuromuscular junction (NMJ), NRG is involved in the induction of a signal transduction cascade that results in the activation of AChR subunit gene transcription [19]. If NRG is able to regulate AChR in muscle cells, perhaps it is possible that a similar mechanism resulting in regulation of AChR expression exists in microglial cells. Here we investigated the possible effects NRG1 may have on microglia. In particular we were interested in the possible up-regulation of 7nAChR expression and the potential for NRG1 and its receptors, the ErbBs to regulate inflammatory responses. Initial experiments examined ErbB receptor expression in several microglial cell lines. Immunoblots revealed that ErbB4 is the predominat ErbB isoform expressed in microglia and that these receptors were phosphotyrosylated in response to NRG1 treatment. Having established that microglia express functional ErbB4 we turned our attention to the expression of 7nAChRs. In the BV-2 microglial cell line we found a significant increase in 7nAChRs with NRG1 treatment compared to control cultures. To determine if, as in peripheral macrophages, increased 7nAChR expression had any effect on inflammatory responses [1] we then assayed NRG1 treated and LPS induced microglia for changes in TNF- and IL-6 expression with and without ACh. Since TNF- and IL-6 are pro-inflammatory cytokines, a decrease in expression is typically associated with a reduced immune response. Using a commercially available enzyme-linked immunosorbent assay (ELISA) we found that in cells pre-treated with ACh and immunologically challenged with LPS, NRG does appear to induce a decrease in TNF- and IL-6. Collectively, these results suggest a role for NRG working in concert with ACh to decrease inflammation. This has potentially interesting implications in Alzheimers disease and needs to be investigated further. Materials and Methods Cell Lines and Culture The EOC-20 murine microglial cell line (CRL-2469; ATCC) was cultured in Dulbeccos modified Eagles medium (DMEM; Wisent, St. Bruno, QC) with 4mM L-glutamine supplemented with 10% fetal bovine serum (FBS; Wisent), 20% LADMAC conditioned media containing colony stimulating factor (CSF)-1, 100IU/mL penicillin and 100mg/mL streptomycin (Wisent). LADMACs, a murine lymphoblast cell line (CRL-2420; ATCC), were cul (...truncated)