Neuronal Release of Cytokine IL-3 Triggered by Mechanosensitive Autostimulation of the P2X7 Receptor Is Neuroprotective

No idea how you would mechanically stretch brain neurons and we'll never find out with no followup from anyone in stroke. We need neuroprotection, a boring/unknown term for neuronal cascade of death.

Neuronal Release of Cytokine IL-3 Triggered by Mechanosensitive Autostimulation of the P2X7 Receptor Is Neuroprotective

Jason C. Lim¹, Wennan Lu¹, Jonathan M. Beckel² and Claire H. Mitchell^1,3,4*

• ¹Department of Anatomy and Cell Biology, University of Pennsylvania, Philadelphia, PA, USA
• ²Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA
• ³Department of Physiology, University of Pennsylvania, Philadelphia, PA, USA
• ⁴Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA, USA

Mechanical strain due to increased pressure or swelling activates inflammatory responses in many neural systems. As cytokines and chemokine messengers lead to both pro-inflammatory and neuroprotective actions, understanding the signaling patterns triggered by mechanical stress may help improve overall outcomes. While cytokine signaling in neural systems is often associated with glial cells like astrocytes and microglia, the contribution of neurons themselves to the cytokine response is underappreciated and has bearing on any balanced response. Mechanical stretch of isolated neurons was previously shown to trigger ATP release through pannexin hemichannels and autostimulation of P2X7 receptors (P2X7Rs) on the neural membrane. Given that P2X7Rs are linked to cytokine activation in other cells, this study investigates the link between neuronal stretch and cytokine release through a P2X7-dependent pathway. Cytokine assays showed application of a 4% strain to isolated rat retinal ganglion cells (RGCs) released multiple cytokines. The P2X7R agonist BzATP also released multiple cytokines; Interleukin 3 (IL-3), TNF-α, CXCL9, VEGF, L-selectin, IL-4, GM-CSF, IL-10, IL-1Rα, MIP and CCL20 were released by both stimuli, with the release of IL-3 greatest with either stimuli. Stretch-dependent IL-3 release was confirmed with ELISA and blocked by P2X7R antagonists A438079 and Brilliant Blue G (BBG), implicating autostimulation of the P2X7R in stretch-dependent IL-3 release. Neuronal IL-3 release triggered by BzATP required extracellular calcium. The IL-3Rα receptor was expressed on RGCs but not astrocytes, and both IL-3Rα and IL-3 itself were predominantly expressed in the retinal ganglion cell layer of adult retinal sections, implying autostimulation of receptors by released IL-3. While the number of surviving ganglion cells decreased with time in culture, the addition of IL-3 protected against this loss of neurons. Expression of mRNA for IL-3 and IL-3Rα increased in rat retinas stretched with moderate intraocular pressure (IOP) elevation; BBG blocked the rise in IL-3, implicating a role for the P2X7R in transcriptional regulation in vivo. In summary, mechanical stretch triggers release of cytokines from neurons that can convey neuroprotection. The enhancement of these signals in vivo implicates P2X7R-mediated IL-3 signaling as an endogenous pathway that could minimize damage following neuronal exposure to chronic mechanical strain.

Introduction

Mechanical strain can trigger neuronal loss, and inflammatory signals are increasingly recognized as contributing to the response. Cytokines and chemokines have been implicated in traumatic brain injury (TBI), tumor-related swelling, increased intraocular pressure (IOP) and other pathological conditions linked to mechanical strain (Ziebell and Morganti-Kossmann, 2010; Freedman and Iserovich, 2013; Wei et al., 2014; Gyoneva and Ransohoff, 2015). Given that cytokines and chemokines can initiate both pro-inflammatory and protective responses, understanding the pathways linking mechanical strain to cytokine signaling has broad relevance for neural trauma.

Although cytokines are traditionally associated with release from cell types of the inflammatory system, they are now recognized as general signaling molecules (Iwasaki and Medzhitov, 2010; Lacy and Stow, 2011; Arango Duque and Descoteaux, 2014). Within neural tissues, microglial cells are a major source of cytokines (Hanisch, 2002), but other glial cells like astrocytes can also signal with cytokines in health and disease (Domanska et al., 2011; Kan et al., 2012; Choi et al., 2014). Neurons are typically examined as a target for cytokine signaling, with various cytokine receptors expressed on neural membranes (Bajetto et al., 2002; Gougeon et al., 2013). However, neurons are also a source of releasable cytokines; while this has been known for some time, the signaling pathways that link neural stimulation to cytokine release remain unclear (Freidin et al., 1992; Yamamoto et al., 2011).

Purinergic signaling pathways provide a likely route to connect mechanical strain with neuronal cytokine release. Throughout the body, mechanical strain leads to ATP release, with pannexin hemichannels implicated as a conduit for mechanosentitive ATP release in many cell types (Bao et al., 2004; Beckel et al., 2015; Furlow et al., 2015). Isolated neurons also respond to mechanical strain with the release of ATP through pannexin hemichannels (Xia et al., 2012). This released ATP autostimulates P2X7 receptors (P2X7Rs) on the neural membrane to elevate cytoplasmic calcium levels. Given that cytoplasmic calcium can contribute to the vesicular release of cytokines (Stow et al., 2009; Stanley and Lacy, 2010), and the stimulation of P2X7Rs is associated with cytokine release from multiple cell types (Pizzirani et al., 2007; Mingam et al., 2008; Clark et al., 2010), this study asked whether neurons could release cytokines in response to mechanical strain, whether this involved P2X7Rs, and probed the consequences of cytokine release for neuronal survival.