Implicating Receptor Activator of NF-κB (RANK)/RANK Ligand Signalling in Microglial Responses to Toll-Like Receptor Stimuli

Abstract

Inflammation in the perinatal brain caused by maternal or intrauterine fetal infection is now well established as an important contributor to the development of perinatal brain injury. Exposure to inflammatory products can impair perinatal brain development and act as a risk factor for neurological dysfunction, cognitive disorders, cerebral palsy, or preterm birth. Pre-exposure to inflammation significantly exacerbates brain injury caused by hypoxic/ischaemic insult. Tumour necrosis factor (TNF) is a family of cytokines largely involved in inflammation signalling. In our previous study, we identified the importance of TNF-related apoptosis-inducing ligand (TRAIL) signalling in the development of perinatal brain injury. We observed a significant increase in the expression levels of a soluble decoy receptor for TRAIL, osteoprotegerin (OPG). Besides TRAIL, OPG is able to bind the receptor activator of the NF-κB (RANK) ligand (RANKL) and inhibit its signalling. The function of the RANK/RANKL/OPG system in the brain has not come under much scrutiny. The aim of this research study was to elucidate the role of RANK, RANKL, and OPG in microglial responses to the proinflammatory stimuli lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (Poly I:C). Here, we show that RANK signalling is important for regulating the activation of the BV2 microglial cell line. We found that LPS treatment causes a significant decrease in the expression of RANK in the BV2 cell line while significantly increasing the expression of OPG, Toll-like receptor (TLR)3, and the adaptor proteins MyD88 and TRIF. We found that pretreatment of BV2 cells with RANKL for 24 h before the LPS or Poly I:C exposure decreases the expression of inflammatory markers such as inducible nitric oxide synthase and cyclooxygenase. This is accompanied by a decreased expression of the TLR adaptor proteins MyD88 and TRIF, which we observed after RANKL treatment. Similar results were obtained in our experiments with primary mouse microglia. Using recently developed CRISPR/Cas9 technology, we generated a BV2 cell line lacking RANK (RANK^-/- BV2). We showed that most effects of RANKL pretreatment were abolished, thereby proving the specificity of this effect. Taken together, these findings suggest that RANK signalling is important for modulating the inflammatory activation of microglial cells to a moderate level, and that RANK attenuates TLR3/TLR4 signalling.

Targeted Gene Editing of Glia Maturation Factor in Microglia: a Novel Alzheimer’s Disease Therapeutic Target

“…It might also be possible that SpCas9 may have relatively inefficient gene editing efficiency as compared to SaCas9. Most recently, Kichev et al have reported on the use of plasmid based approach to achieve gene editing of RANK receptor using Cas9 and HDR plasmids in BV2 cells [63]. Although useful for in vitro experiments, this approach clearly lacks in vivo translational potential.…”

Midkine: The Who, What, Where, and When of a Promising Neurotrophic Therapy for Perinatal Brain Injury

“…Stimuli that induce NFκB activation include reactive oxygen species, proinflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β), and the bacterial endotoxin, lipopolysaccharide (LPS) (102)(103)(104)(105). MK expression can be upregulated during many forms of inflammatory reaction (102)(103)(104)(105). Consistent with this, an elegant study that used a prostate adenocarcinoma cell line showed that induction of MK expression following TNFα exposure occurs in an NFκBdependent manner (7).…”

“…The MK promoter region also possesses a putative nuclear factor kappa light-chain enhancer of activated B cells (NFκB) response element (7,40). Stimuli that induce NFκB activation include reactive oxygen species, proinflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β), and the bacterial endotoxin, lipopolysaccharide (LPS) (102)(103)(104)(105). MK expression can be upregulated during many forms of inflammatory reaction (102)(103)(104)(105).…”

RANK/RANKL/OPG Signaling in the Brain: A Systematic Review of the Literature

“…The RRO axis and osteoclast paradigm is applicable to microglial cells as both osteoclasts and microglia share some developmental and functional properties, including hematopoietic origin, phagocytosis, and immunomodulation (26). Moreover, this can be extended to all myeloid populations within the central nervous system (CNS), including parenchymal microglia in the nerve tissue, perivascular phagocytic cells, meningeal macrophages, and choroid plexus macrophages (27)(28)(29)(30). In contrast to other tissue-resident macrophages which arise from the embryonic yolk sac and fetal liver, microglia originates exclusively from yolk sac-derived hematopoietic progenitors (27).…”