Abstract
Microglia rapidly respond to CNS injury and disease and can assume a spectrum of activation states. While changes in gene expression and production of inflammatory mediators have been extensively described after classical (LPS-induced) and alternative (IL4-induced) microglial activation, less is known about acquired de-activation in response to IL10. It is important to understand how microglial activation states affect their migration and invasion; crucial functions after injury and in the developing CNS. We reported that LPS-treated rat microglia migrate very poorly, while IL4-treated cells migrate and invade much better. Having discovered that the lamellum of migrating microglia contains a large ring of podosomes – microscopic structures that are thought to mediate adhesion, migration and invasion – we hypothesized that IL4 and IL10 would differentially affect podosome expression, gene induction, migration and invasion. Further, based on the enrichment of the KCa2.3/SK3 Ca2+-activated potassium channel in microglial podosomes, we predicted that it regulates migration and invasion. We found both similarities and differences in gene induction by IL4 and IL10 and, while both cytokines increased migration and invasion, only IL10 affected podosome expression. KCa2.3 currents were recorded in microglia under all three activation conditions and KCNN3 (KCa2.3) expression was similar. Surprisingly then, of three KCa2.3 inhibitors (apamin, tamapin, NS8593), only NS8593 abrogated the increased migration and invasion of IL4 and IL10-treated microglia (and invasion of unstimulated microglia). This discrepancy was explained by the observed block of TRPM7 currents in microglia by NS8593, which occurred under all three activation conditions. A similar inhibition of both migration and invasion was seen with a TRPM7 inhibitor (AA-861) that does not block KCa2.3 channels. Thus, we conclude that TRPM7 (not KCa2.3) contributes to the enhanced ability of microglia to migrate and invade when in anti-inflammatory states. This will be an important consideration in developing TRPM7 inhibitors for treating CNS injury.
Highlights
Microglial cells become activated within the CNS after acute injury and with disease, but it is increasingly clear that they exist in a spectrum of activation states and are not proor anti-inflammatory [1,2,3]
The findings described below indicate that IL4 and IL10 induce different expression patterns in rat microglia despite both being considered anti-inflammatory cytokines
Very few studies have directly compared IL4 and IL10 treatment, and the most extensive gene expression comparison suggests that IL10 outcomes resemble LPS in murine microglia [12]
Summary
Microglial cells become activated within the CNS after acute injury and with disease, but it is increasingly clear that they exist in a spectrum of activation states and are not proor anti-inflammatory [1,2,3]. Mechanistic, in vitro studies generally exploit several stimuli to evoke discrete activation states. ‘Classical’ activation increases pro-inflammatory mediators that can exacerbate injury, and this state is commonly evoked by lipopolysaccharide (LPS) in vitro [1,4]. ‘Alternative’ activation is typically evoked by interleukin (IL)-4, ‘acquired deactivation’ by IL10, and both states are thought to help resolve acute inflammation by decreasing pro-inflammatory mediators (e.g., cytokines, reactive oxygen and nitrogen species) (reviewed in [1,5,6,7]). A recent comparative study of murine microglia showed that IL10 evoked gene expression changes more closely resembling LPS- than IL4treated cells [12]. It is important to compare the effects of IL4 and IL10 on specific microglial functions
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