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

Abstract

BackgroundKv1.3 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.MethodsWe 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.ResultsLPS-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+CD45low microglia without affecting MHCI expression or trafficking of CD11b+CD45high 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-017-0906-6) contains supplementary material, which is available to authorized users.

The voltage‐gated potassium channel Kv1.3 is required for microglial pro‐inflammatory activation in vivo

“…Although published (Charolidi et al 2015; Nguyen et al 2017; Rangaraju et al 2015; Rangaraju et al 2017; Rus et al 2005) and present data clearly showed that in the brain, Kv1.3 is mainly expressed in activated microglia, it has been described in neurons, most prominently in mitral cells of the olfactory bulb (Fadool et al 2004) and presynaptic terminals of brain stem auditory neurons (Gazula et al 2010), and also in cortical interneurons and in oligodendrocyte progenitor cells but at low levels (Vautier et al 2004; Duque et al 2013), raising the possibility that the above in vivo effects of Kv1.3 knockout may be secondary to Kv1.3 hypomorph in neurons. To address this possibility, we used a chemical biology approach, as in neurons Kv1.3 is typically part of hetereomultimers with Kv1.1, Kv1.2 and Kv1.6 subunits (Helms et al 1997) and therefore has a different pharmacology from the Kv1.3 homotetramers or Kv1.3/Kv1.5 heterotetramers found in T cells, microglia and macrophages (Chandy et al 2004; Feske et al 2015; Wulff et al 2009).…”

“…A first in vivo suggestion of such a role of Kv1.3 came from a recent report showing that systemic inflammation induced by intravenous administration of LPS to adult mice resulted in increased ShK-F6CA (a fluorescein-conjugated Kv1.3-specific peptide inhibitor) labeling of microglia and infiltrating macrophages in the brain (Rangaraju et al 2017). In the current study, we stereotactically injected LPS into the ventricles (Maezawa et al 2006).…”

“…We previously demonstrated that cultured primary microglia activated by lipopolysaccharides (LPS), which induces a classic M1-like activation state, exhibited high Kv1.3 current densities but virtually no activities of the other major microglial K + channel, the inward-rectifier Kir2.1 (Nguyen et al 2017). A systems pharmacology-based study identified functional roles for Kv1.3 in pro-inflammatory microglial activation (Rangaraju et al 2017) while electrophysiological studies by our group demonstrated upregulated Kv1.3 expression in microglia acutely isolated from the infarct areas of mice subjected to experimental stroke (Chen et al 2016). Kv1.3 blockers have further been demonstrated to inhibit microglia mediated neurotoxicity in culture (Fordyce et al 2005) and to protect mice from microglia mediated radiation-induced brain injury (Peng et al 2014).…”

“…Although Kv1.3 appears to be a suitable target for microglia-targeted modulation, a definite in vivo demonstration of the role of Kv1.3 in neuroinflammation is missing, as nearly all prior studies were conducted using BV2 cells, primary microglia, or brain slices in culture (Charolidi et al 2015; Fordyce et al 2005; Khanna et al 2001; Kotecha and Schlichter 1999; Küst et al 1999; Nguyen et al 2017; Rangaraju et al 2017; Schilling and Eder 2003; Schilling and Eder 2011). It is now realized that BV2 cells significantly differ from cultured or in vivo microglia in many aspects (Butovsky et al 2014; Henn et al 2009; Kettenmann et al 2011).…”

Inhibition of the potassium channel Kv1.3 reduces infarction and inflammation in ischemic stroke

“…These findings suggest that Kv1.3 inhibition does not affect phagocytosis in vivo confirming a recent study reporting that Kv1.3 blockers to not impair the ability of isolated brain mononuclear cells to phagocytose polystyrene microspheres. 38…”