Insensitivity of astrocytes to interleukin 10 signaling following peripheral immune challenge results in prolonged microglial activation in the aged brain

Abstract

Immune-activated microglia from aged mice produce exaggerated levels of cytokines. Despite high levels of microglial interleukin (IL)-10 in the aged brain, neuroinflammation was prolonged and associated with depressive-like deficits. Because astrocytes respond to IL-10 and, in turn, attenuate microglial activation, we investigated if astrocyte-mediated resolution of microglial activation was impaired with age. Here, aged astrocytes had a dysfunctional profile with higher glial fibrillary acidic protein, lower glutamate transporter expression, and significant cytoskeletal re-arrangement. Moreover, aged astrocytes had reduced expression of growth factors and IL-10 receptor-1 (IL-10R1). After in vivo lipopolysaccharide immune challenge, aged astrocytes had a molecular signature associated with reduced responsiveness to IL-10. This IL-10 insensitivity of aged astrocytes resulted in a failure to induce IL-10R1 and transforming growth factor β and resolve microglial activation. In addition, adult astrocytes reduced microglial activation when co-cultured ex vivo, whereas aged astrocytes did not. Consistent with the aging studies, IL-10R^KO astrocytes did not augment transforming growth factor β after immune challenge and failed to resolve microglial activation. Collectively, a major cytokine-regulatory loop between activated microglia and astrocytes is impaired in the aged brain.

Introduction

The bi-directional communication between the immune system and brain is altered with age (Corona et al., 2012, Jurgens and Johnson, 2010). This neuroimmune communication is relevant because it is essential for mounting the appropriate immunologic, physiological, and behavioral responses to pathogens. This communication results in the propagation of cytokines and chemokines by microglia (Dantzer et al., 2008, Henry et al., 2009, Nguyen et al., 2002, Norden et al., 2014) and the induction of sickness behavior (Dantzer, 2001, Kelley et al., 2003). Altered neuroimmune communication in aging is significant because older individuals have an increased risk of infection with concomitant neurobehavioral deficits (Jackson et al., 2004, Rockwood et al., 1999), including depression (Koenig et al., 1988, Penninx et al., 1999) and cognitive impairment (Ahmed et al., 2014, Dunn et al., 2005, Iwashyna et al., 2010). Thus, immune challenges in the elderly can be triggers for ongoing inflammatory processes that negatively affect mood and cognition.

The mechanism by which peripheral infection in the elderly causes neuropsychiatric complications is unknown, but it may be related to impaired regulation of microglia. For instance, immune challenge (lipopolysaccharide [LPS] or Escherichia coli) in aged rodents results in amplified glia-mediated neuroinflammation associated with neuropsychiatric complications (Barrientos et al., 2009, Barrientos et al., 2010, Godbout et al., 2005, Godbout et al., 2008, Wynne et al., 2010). Several studies show that exaggerated microglial responses are related to impairments in regulatory systems making it difficult to resolve microglial activation (Jurgens and Johnson, 2010, Norden and Godbout, 2013). For example, microglia produce exaggerated levels of both pro- and anti-inflammatory cytokines, including interleukin (IL)-10 (Henry et al., 2009, Sierra et al., 2007). This simultaneous induction of IL-1β and IL-10 is likely related to enhanced NF-κB activity because it drives the transcription of both pro- and anti-inflammatory mediators (Cao et al., 2006). Despite higher levels of IL-10 in the aged brain, microglial activation and sickness behavior were prolonged, and depression and acute cognitive impairments developed (Chen et al., 2008, Godbout et al., 2008, Huang et al., 2008, Wynne et al., 2010). Thus, anti-inflammatory responses to IL-10 may be impaired in the aged brain.

Astrocytes are potentially key to this IL-10 puzzle because they respond to IL-10 and inhibit microglial activation in a transforming growth factor (TGF) β–dependent manner (Norden et al., 2014). The IL-10 receptor has 2 necessary components for functional signaling: ligand binding domain (IL-10R1) and signaling domain (IL-10R2; Moore et al., 2001). Although IL-10R2 is expressed constitutively by most cells, IL-10R1 expression is cell specific and low under homeostatic conditions (Moore et al., 2001). IL-10 binding to IL-10R results in Jak1 and Tyk2 phosphorylation and subsequent STAT3 activation. STAT3 induces suppressor of cytokine signaling 3 (SOCS3), which inhibits IL-1β, IL-6, and tumor necrosis factor (TNF)α. STAT3 also enhances antiapoptotic and cell-cycle progression genes including Myc, cyclin-D1, and cyclin-dependent kinase inhibitors (CDKNs; Donnelly et al., 1999, Lin et al., 2005, O'Farrell et al., 2000). Furthermore, IL-10 has anti-inflammatory and neuroprotective roles within the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) (Cua et al., 2001), spinal cord injury (Bethea et al., 1999, Ishii et al., 2013), stroke (Frenkel et al., 2005), and cytokine-induced sickness behavior (Bluthe et al., 1999, Lynch et al., 2004, Richwine et al., 2009).

The induction of TGFβ by astrocytes after IL-10 (Norden et al., 2014) is important because TGFβ has neuroprotective and anti-inflammatory effects in the CNS (Qian et al., 2008). In microglia, increased pro-inflammatory mediators and cytokine expression initiated by LPS challenge was attenuated by TGFβ (Butovsky et al., 2014, Herrera-Molina and von Bernhardi, 2005, Norden et al., 2014, Tichauer et al., 2014). Therefore, TGFβ produced by activated astrocytes can inhibit microglial activation.

Based on these findings, we surmise that key regulatory communication between microglia and astrocytes is impaired with age. Thus, the purpose of this study was to investigate the degree to which IL-10 and TGFβ cytokine interactions between microglia and astrocytes were impaired in the aged brain following an immune challenge. Here, we provide novel evidence that aged astrocytes are less sensitive to IL-10, that this IL-10 insensitivity is related to IL-10R1, and that IL-10 insensitivity occurs concomitantly with decreased TGFβ feedback on active microglia.