Effects Of LOX-1 And Extracellular HSP60 On Microglia-mediated Neuroinflammation And Nerve Injury

PARTI Study on the role of LOX-1in microglial activation, neuroinflammation and neurotoxicityInflammation in the central neuronal system (CNS) is a double-edged sword. Microglial overactivation and uncontrolled neuroinflammation participates in many CNS disorders. Stimuli that promote microglial overactivation are diverse. Besides the exogenous pathogen-associated molecular patterns (PAMPs), the endogenous damage-associated molecular patterns (DAMPs) released by damaged/dying neurons were also reported to alarm the immune system. In this study, we used medium conditioned by necrotic differentiated-PC12cells to confirm that damaged neurons can release soluble DAMPs to efficiently promote microglial activation, stimulate microglial expression of pro-inflammatory cytokines (IL-1β, IL-6and TNF-a), increase microglial production of NO and ROS, and propagate the neurotoxicity.Lectin-like oxidized low-density lipoprotein receptor-1(LOX-1) belongs to the C-type lectin-like receptor family and the scavenger receptor family. Significant evidence points to the deleterious role of LOX-1in the progression of endothelial inflammation and cardiovascular disorders. In the CNS however, the functions of LOX-1are still not understood. In this study, we explored the potential role of LOX-1in microglial activation and neuroinflammation. We observed that LOX-1expression is induced upon LPS-stimulated toxic microglial activation, and discovered that LOX-1is necessary in microglia for sensing soluble DAMP(s) in the conditioned medium of injured neurons. LOX-1knockdown reduces soluble DAMP-stimulated microglial generation of pro-inflammatory mediators (IL-1β, TNF-a, NO and ROS), and attenuates the subsequent microglial neurotoxicity.These results indicate that microglial LOX-1detects and responds to the injured-neuron released soluble DAMPs to promote the vicious cycle of neuroinflammation and neurotoxicity. PART II Extracellular HSP60acts on microglial LOX-1to drive the vicious cycle of neuroinflammation and neurotoxicityChronic neurodegeneration is in part caused by a vicious cycle of persistent microglial activation and progressive neuronal cell loss. However, the driving force behind this cycle remains poorly understood. HSP60is a highly conserved and ubiquitously expressed molecular chaperone. In addition to the essential intracellular chaperone activities for maintenance of cellular homeostasis, HSP60can also appear in the extracellular milieu where it elicits a potent proinflammatory response in the peripheral immune system. However, the contribution of extracellular HSP60to neuroinflammation is poorly understood. In this study, we demonstrated that damaged neurons can release soluble heat shock protein60(HSP60). Employing a unique eukaryotic HSP60-overexpression method, we further demonstrated that extracellular HSP60can act as soluble DAMP in the central immune system (CNS), promote microglial activation, boost the production of pro-inflammatory factors (IL-1β, NO and ROS) in microglia and propagate neuronal damage.Since lectin-like oxidized low-density lipoprotein receptor-1(LOX-1) has previously been identified as a novel receptor for HSP60, we hypothesize that LOX-1through binding to extracellular HSP60promotes microglia-mediated neuroinflammation. To explore this hypothesis, we knocked down LOX-1expression by RNA interference technology, and discovered that LOX-1knockdown significantly reduces extracellular HSP60-stimulated microglial generation of pro-inflammatory mediators (IL-1β, TNF-a, NO and ROS), and attenuates the subsequent HSP60-mediated microglial neurotoxicity.Thus, our results indicate that extracellular HSP60, which is released by injured neuronal cells, acts on microglial LOX-1to drive the vicious cycle of neuroinflammation and neurotoxicity.