Abstract
BackgroundMicroglia are critical mediators of neuroimmune pathology across multiple neurologic disorders. Microglia can be persistently activated or “primed” by Toll-like receptor (TLR) activation, ethanol, stress, and other insults. Thus, strategies to prevent or reverse microglial priming may be beneficial for conditions that involve progressively increasing microglial activation. Microglial depletion with repopulation is emerging as a potential therapy to normalize chronic immune activation. Primary organotypic hippocampal slice culture (OHSC) allows for the study of neuroimmune activation as well as microglial depletion and repopulation without involvement of peripheral immune activation. OHSC undergoes functional maturation and retains cytoarchitecture similar to in vivo.MethodsOHSC underwent microglial depletion with the CSF1R antagonist PLX3397 with or without repopulation after removal of PLX3397. Immune, trophic, and synaptic gene changes in response to agonists of TLRs 2, 3, 4, 7, and 9 as well as ethanol were assessed in the settings of microglial depletion and repopulation. Gi-DREADD inhibition of microglia was used to confirm select findings seen with depletion. The ability of microglial repopulation to prevent progressive proinflammatory gene induction by chronic ethanol was also investigated.ResultsMicroglia were depleted (> 90%) by PLX3397 in OHSC. Microglial depletion blunted proinflammatory responses to several TLR agonists as well as ethanol, which was mimicked by Gi-DREADD inhibition of OHSC microglia. Removal of PLX3397 was followed by complete repopulation of microglia. OHSCs with repopulated microglia showed increased baseline expression of anti-inflammatory cytokines (e.g., IL-10), microglial inhibitory signals (e.g., CX3CL1), and growth factors (e.g., BDNF). This was associated with blunted induction (~ 50%) of TNFα and IL-1β in response to agonists to TLR4 and TLR7. Further, chronic cycled ethanol from 4 days in vitro (DIV) to 16DIV caused immediate 2-fold inductions of TNFα and IL-1β that grew to ~4-fold of age-matched control slices by 40DIV. This persistent inflammatory gene expression was completely reversed by microglial depletion and repopulation after chronic ethanol.ConclusionsMicroglia in OHSCs mediate proinflammatory responses to TLR agonists and ethanol. Microglial repopulation promoted an anti-inflammatory, trophic neuroenvironment and normalized proinflammatory gene expression. This supports the possibility of microglial depletion with repopulation as a strategy to reverse chronic neuroimmune activation.
Highlights
Microglia are critical mediators of neuroimmune pathology across multiple neurologic disorders
Microglial depletion by PLX3397 in brain slice culture blunts Toll-like receptor (TLR) immune responses Emerging studies have suggested that microglial depletion by multiple methods has beneficial effects on pathology [17, 18]
organotypic hippocampal slice culture (OHSC) maintain the cellular structure of the hippocampus ex vivo [29], and we confirmed by RT-PCR that OHSC retain expression of Sall1, a critical microglial transcriptional regulator lost in some microglial culture preparations [56]
Summary
Microglia are critical mediators of neuroimmune pathology across multiple neurologic disorders. Microglial priming or sensitization to proinflammatory signaling has been associated with the progression of neurodegenerative diseases as well as normal aging [1,2,3], with “memory”-like adaptation of innate immune microglial signaling [4]. Microglial priming of proinflammatory signaling is increasingly being linked to depression, drug and alcohol dependence, brain stress responses, and neurological diseases [3, 8,9,10,11]. For these reasons, the inhibition of microglia has been explored as a therapeutic approach for multiple neurologic disorders. Microglial depletion is a novel approach that can be used to provide insight into microglial biology and may have therapeutic potential in many brain diseases that involve neuroimmune dysfunction [17, 18]
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