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Abstract
Microglia are the resident immune cells in the central nervous system and key players against pathogens and injury. However, persistent microglial activation often exacerbates pathological damage and has been implicated in many neurological diseases. Despite their pivotal physiological and pathophysiological roles, how the survival and death of activated microglia is regulated remains poorly understood. We report here that microglia activated through Toll-like receptors (TLRs) undergo RIP1/RIP3-dependent programmed necrosis (necroptosis) when exposed to the pan caspase inhibitor zVAD-fmk. Although zVAD-fmk and the caspase-8 inhibitor IETD-fmk had no effect on unstimulated primary microglia, they markedly sensitized microglia to TLR1/2,3,4,7/8 ligands or TNF treatment, triggering programmed necrosis that was completely blocked by R1P1 kinase inhibitor necrostatin-1. Interestingly, necroptosis induced by TLR ligands and zVAD was restricted to microglial cells and was not observed in astrocytes, neurons or oligodendrocytes even though they are known to express certain TLRs. Deletion of genes encoding TNF or TNFR1 failed to prevent lipopolysaccharide- and poly(I:C)-induced microglial necroptosis, unveiling a TNF-independent programmed necrosis pathway in TLR3- and TLR4-activated microglia. Microglia from mice lacking functional TRIF were fully protected against TLR3/4 activation and zVAD-fmk-induced necrosis, and genetic deletion of rip3 also prevented microglia necroptosis. Activation of c-jun N-terminal kinase and generation of specific reactive oxygen species were downstream signaling events required for microglial cell death execution. Taken together, this study reveals a robust RIP3-dependent necroptosis signaling pathway in TLR-activated microglia upon caspase blockade and suggests that TLR signaling and programmed cell death pathways are closely linked in microglia, which could contribute to neuropathology and neuroinflammation when dysregulated.
Highlights
Microglia are the immune cells in the CNS and regulate both innate and adaptive immune responses; to date, to our knowledge little information is available on a necroptosis programme in microglia
We demonstrated an operation of two interconnected programmed cell death pathways in Toll-like receptors (TLRs)-activated primary microglia: (1) caspase-dependent apoptosis in overactivated microglia, and (2) RIP3-mediated necroptosis as unmasked by caspase blockade
TLR-receptor agonists as well as TNF were capable of initiating primary microglia necrosis when caspase-8 was suppressed by zVAD-fmk or IETD-fmk, phenomena not observed in other cells of the CNS including primary neurons, astrocytes, and oligodendrocytes
Summary
Accumulating evidence has clearly demonstrated that certain type of necrotic cell death is programmed and could be Multiple lines of evidence have demonstrated that caspase-8, the initiator caspase of the death receptor-induced pathway of apoptosis, and its adaptor protein Fas-associated death domain (FADD) negatively regulate RIP1/RIP3-dependent programmed necrosis by cleaving and inactivating RIP1.21 Suppression of caspase-8 activity with the pan caspase inhibitor zVAD-fmk facilitates TNFa-induced necroptosis in cells expressing RIP3.22 Unlike TNF death receptor-induced necrosis, the details of pathogen-induced necrosis in innate immune cells have just started to emerge. Several studies have shown that when caspase-8 activity is suppressed by IETD-fmk, zVAD-fmk, or the viral caspase inhibitor CrmA, ligation of TLR3 or TLR4 results in programmed necrosis in human macrophages[23] and mouse peritoneal macrophages via RIP3.22,24 whether activated microglia, the innate immune cells in the CNS, undergo programmed necrosis remains unanswered. As necrotic cell death could elicit secondary inflammatory responses, unfolding the molecular mechanisms underlying the demise of activated microglia may hold important clues for future therapeutic intervention of CNS diseases
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