A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation

• Published in: Journal of neuroinflammation Vol. 14; no. 1; p. 128
• Main Authors: Rangaraju, Srikant, Raza, Syed Ali, Pennati, Andrea, Deng, Qiudong, Dammer, Eric B, Duong, Duc, Pennington, Michael W, Tansey, Malu G, Lah, James J, Betarbet, Ranjita, Seyfried, Nicholas T, Levey, Allan I
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 26.06.2017; BioMed Central; BMC
• Subjects: Analysis; Animals; Antigen presentation; Brain; CD11b antigen; CD8 antigen; Cell growth; Cell Line; Cell proliferation; Cell surface; Cells, Cultured; Central nervous system; Cytokines; Data processing; Disease; Dose-Response Relationship, Drug; Experiments; Female; Flow cytometry; Health aspects; Homeostasis; Immunoblotting; Immunomodulation; Kv1.3; Kv1.3 Potassium Channel - biosynthesis; Kv1.3 Potassium Channel - immunology; Leukocyte migration; Leukocytes (mononuclear); Lipopolysaccharides; Lipopolysaccharides - pharmacology; Lymphocytes; Lymphocytes T; Macrophages; Macrophages - drug effects; Macrophages - immunology; Macrophages - metabolism; Mice; Mice, Inbred C57BL; Microglia; Microglia - drug effects; Microglia - immunology; Microglia - metabolism; Nervous system diseases; Neuroimmunomodulation; Neuroinflammation; Neurological diseases; Peritoneum; Phagocytes; Phagocytosis; Potassium channels; Potassium channels (voltage-gated); Proteomics; Proteomics - methods; Reactive oxygen species; Rodents; Software; Studies; T cells; Transcription; Neuroinflammation; Potassium channels; Kv1.3; Proteomics; Microglia
• ISSN: 1742-2094; 1742-2094
• DOI: 10.1186/s12974-017-0906-6

Kv1.3 potassium channels regulate microglial functions and are overexpressed in neuroinflammatory diseases. Kv1.3 blockade may selectively inhibit pro-inflammatory microglia in neurological diseases but the molecular and cellular mechanisms regulated by Kv1.3 channels are poorly defined. We performed immunoblotting and flow cytometry to confirm Kv1.3 channel upregulation in lipopolysaccharide (LPS)-activated BV2 microglia and in brain mononuclear phagocytes freshly isolated from LPS-treated mice. Quantitative proteomics was performed on BV2 microglia treated with control, LPS, ShK-223 (highly selective Kv1.3 blocker), and LPS+ShK-223. Gene ontology (GO) analyses of Kv1.3-dependent LPS-regulated proteins were performed, and the most representative proteins and GO terms were validated. Effects of Kv1.3-blockade on LPS-activated BV2 microglia were studied in migration, focal adhesion formation, reactive oxygen species production, and phagocytosis assays. In vivo validation of protein changes and predicted molecular pathways were performed in a model of systemic LPS-induced neuroinflammation, employing antigen presentation and T cell proliferation assays. Informed by pathway analyses of proteomic data, additional mechanistic experiments were performed to identify early Kv1.3-dependent signaling and transcriptional events. LPS-upregulated cell surface Kv1.3 channels in BV2 microglia and in microglia and CNS-infiltrating macrophages isolated from LPS-treated mice. Of 144 proteins differentially regulated by LPS (of 3141 proteins), 21 proteins showed rectification by ShK-223. Enriched cellular processes included MHCI-mediated antigen presentation (TAP1, EHD1), cell motility, and focal adhesion formation. In vitro, ShK-223 decreased LPS-induced focal adhesion formation, reversed LPS-induced inhibition of migration, and inhibited LPS-induced upregulation of EHD1, a protein involved in MHCI trafficking. In vivo, intra-peritoneal ShK-223 inhibited LPS-induced MHCI expression by CD11b CD45 microglia without affecting MHCI expression or trafficking of CD11b CD45 macrophages. ShK-223 inhibited LPS-induced MHCI-restricted antigen presentation to ovalbumin-specific CD8 T cells both in vitro and in vivo. Kv1.3 co-localized with the LPS receptor complex and regulated LPS-induced early serine (S727) STAT1 phosphorylation. We have unraveled novel molecular and functional roles for Kv1.3 channels in pro-inflammatory microglial activation, including a Kv1.3 channel-regulated pathway that facilitates MHCI expression and MHCI-dependent antigen presentation by microglia to CD8 T cells. We also provide evidence for neuro-immunomodulation by systemically administered ShK peptides. Our results further strengthen the therapeutic candidacy of microglial Kv1.3 channels in neurologic diseases.