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

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-&amp;#x03BA;B (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&lt;sup&gt;-/-&lt;/sup&gt; 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.</jats:p

Lipopolysaccharide-induced alteration of mitochondrial morphology induces a metabolic shift in microglia modulating the inflammatory response in vitro and in vivo

Accumulating evidence suggests that changes in the metabolic signature of microglia underlie their response to inflammation. We sought to increase our knowledge of how pro‐inflammatory stimuli induce metabolic changes. Primary microglia exposed to lipopolysaccharide (LPS)‐expressed excessive fission leading to more fragmented mitochondria than tubular mitochondria. LPS‐mediated Toll‐like receptor 4 (TLR4) activation also resulted in metabolic reprogramming from oxidative phosphorylation to glycolysis. Blockade of mitochondrial fission by Mdivi‐1, a putative mitochondrial division inhibitor led to the reversal of the metabolic shift. Mdivi‐1 treatment also normalized the changes caused by LPS exposure, namely an increase in mitochondrial reactive oxygen species production and mitochondrial membrane potential as well as accumulation of key metabolic intermediate of TCA cycle succinate. Moreover, Mdivi‐1 treatment substantially reduced LPS induced cytokine and chemokine production. Finally, we showed that Mdivi‐1 treatment attenuated expression of genes related to cytotoxic, repair, and immunomodulatory microglia phenotypes in an in vivo neuroinflammation paradigm. Collectively, our data show that the activation of microglia to a classically pro‐inflammatory state is associated with a switch to glycolysis that is mediated by mitochondrial fission, a process which may be a pharmacological target for immunomodulation

How to reprogram microglia toward beneficial functions

Microglia, brain cells of nonneural origin, orchestrate the inflammatory response to diverse insults, including hypoxia/ischemia or maternal/fetal infection in the perinatal brain. Experimental studies have demonstrated the capacity of microglia to recognize pathogens or damaged cells activating a cytotoxic response that can exacerbate brain damage. However, microglia display an enormous plasticity in their responses to injury and may also promote resolution stages of inflammation and tissue regeneration. Despite the critical role of microglia in brain pathologies, the cellular mechanisms that govern the diverse phenotypes of microglia are just beginning to be defined. Here we review emerging strategies to drive microglia toward beneficial functions, selectively reporting the studies which provide insights into molecular mechanisms underlying the phenotypic switch. A variety of approaches have been proposed which rely on microglia treatment with pharmacological agents, cytokines, lipid messengers, or microRNAs, as well on nutritional approaches or therapies with immunomodulatory cells. Analysis of the molecular mechanisms relevant for microglia reprogramming toward pro-regenerative functions points to a central role of energy metabolism in shaping microglial functions. Manipulation of metabolic pathways may thus provide new therapeutic opportunities to prevent the deleterious effects of inflammatory microglia and to control excessive inflammation in brain disorders

Expression of GPR17 receptor in a murine model of perinatal brain neuroinflammation and its possible interaction with Wnt pathway

Oligodendrocyte precursor cells (OPCs) are generated in specific germinal regions and progressively maturate to myelinating cells. Oligodendrocytes (OLs) differentiation is regulated by a complex interplay of intrinsic, epigenetic and extrinsic factors, including Wnt and the G protein-coupled receptor referred to as GPR17 (Mitew et al., 2014). This receptor responds to both extracellular nucleotides (UDP, UDP-glucose) and cysteinyl-leukotrienes (Ciana et al., 2006), endogenous signaling molecules involved in inflammatory response and in the repair of brain lesions. GPR17 is highly expressed in OPCs during the transition to immature OLs, but it is down-regulated in mature cells. Accordingly, GPR17-expressing OPCs are already present in mice at birth, increase over time, reach a peak at P10, before the peak of myelination, and then decline in the adult brain (Boda et al., 2011). Of note, in cultured OPCs, early GPR17 silencing has been shown to profoundly affect their ability to generate mature OLs (Fumagalli et al., 2011, 2015). Myelination defects characterize many brain disorders, including perinatal brain injury caused by systemic inflammation (Favrais et al., 2011), which is a leading cause of preterm birth. It has already been suggested that an imbalance in the Wnt/\u3b2-catenin/TCF4 pathway could be involved in the maturation arrest of OLs that is observed in premature infants (Yuen et al., 2014). No data are currently available on GPR17 in perinatal brain injury and on its possible interaction with Wnt pathway. Based on these premises, the aim of this work was to assess if the maturational blockade of OLs due to mild systemic perinatal inflammation, induced by intraperitoneal injections of interleukin-1\u3b2 (IL- 1\u3b2), is accompanied by defects in GPR17 expression and whether the Wnt pathway is involved in the regulation of GPR17. Data showed that in newborn mice exposed to IL-1\u3b2, which induces a blockade of oligodendrocyte maturation, GPR17 expression is not affected at early time point (P5), but it is downregulated at P10, when its expression should be maximal. Moreover, in vitro studies revealed that the maturation blockade of the oligodendroglial cell line Oli-Neu, after treatment with a Wnt Agonist II, is accompanied by a severe inhibition of GPR17 expression. In conclusion, our data have shown that myelination defects observed in perinatal brain injury are associated with defects in GPR17 expression; further studies are needed to characterize the molecular link between Wnt pathway and GPR17 receptor

Heat shock factor 2 is a stress‐responsive mediator of neuronal migration defects in models of fetal alcohol syndrome

Fetal alcohol spectrum disorder (FASD) is a frequent cause of mental retardation. However, the molecular mechanisms underlying brain development defects induced by maternal alcohol consumption during pregnancy are unclear. We used normal andHsf2‐deficient mice and cell systems to uncover a pivotal role for heat shock factor 2 (HSF2) in radial neuronal migration defects in the cortex, a hallmark of fetal alcohol exposure. Upon fetal alcohol exposure, HSF2 is essential for the triggering of HSF1 activation, which is accompanied by distinctive post‐translational modifications, and HSF2 steers the formation of atypical alcohol‐specific HSF1–HSF2 heterocomplexes. This perturbs the in vivo binding of HSF2 to heat shock elements (HSEs) in genes that control neuronal migration in normal conditions, such as p35 or the MAPs(microtubule‐associated proteins, such as Dclk1 and Dcx), and alters their expression. In the absence of HSF2, migration defects as well as alterations in gene expression are reduced. Thus, HSF2, as a sensor for alcohol stress in the fetal brain, acts as a mediator of the neuronal migration defects associated with FASD

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling and cell death in the immature central nervous system after hypoxia-ischemia and inflammation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family. The interaction of TRAIL with death receptor 4 (DR4) and DR5 can trigger apoptotic cell death. The aim of this study was to investigate the role of TRAIL signaling in neonatal hypoxia-ischemia (HI). Using a neonatal mouse model of HI, mRNA, and protein expression of TRAIL, DR5 and the TRAIL decoy receptors osteoprotegerin (OPG), mDcTRAILR1, and mDcTRAILR2 were determined. In vitro, mRNA expression of these genes was measured in primary neurons and oligodendrocyte progenitor cells (OPCs) after inflammatory cytokine (TNF-α/IFN-γ) treatment and/or oxygen and glucose deprivation (OGD). The toxicity of these various paradigms was also measured. The expression of TRAIL, DR5, OPG, and mDcTRAILR2 was significantly increased after HI. In vitro, inflammatory cytokines and OGD treatment significantly induced mRNAs for TRAIL, DR5, OPG, and mDcTRAILR2 in primary neurons and of TRAIL and OPG in OPCs. TRAIL protein was expressed primarily in microglia and astroglia, whereas DR5 co-localized with neurons and OPCs in vivo. OGD enhanced TNF-α/IFN-γ toxicity in both neuronal and OPC cultures. Recombinant TRAIL exerted toxicity alone or in combination with OGD and TNF-α/IFN-γ in primary neurons but not in OPC cultures. The marked increases in the expression of TRAIL and its receptors after cytokine exposure and OGD in primary neurons and OPCs were similar to those found in our animal model of neonatal HI. The toxicity of TRAIL in primary neurons suggests that TRAIL signaling participates in neonatal brain injury after inflammation and HI

Therapies for neonatal encephalopathy: Targeting the latent, secondary and tertiary phases of evolving brain injury

In term and near-term neonates with neonatal encephalopathy, therapeutic hypothermia protocols are well established. The current focus is on how to improve outcomes further and the challenge is to find safe and complementary therapies that confer additional protection, regeneration or repair in addition to cooling. Following hypoxia-ischemia, brain injury evolves over three main phases (latent, secondary and tertiary), each with a different brain energy, perfusion, neurochemical and inflammatory milieu. While therapeutic hypothermia has targeted the latent and secondary phase, we now need therapies that cover the continuum of brain injury that spans hours, days, weeks and months after the initial event. Most agents have several therapeutic actions but can be broadly classified under a predominant action (e.g., free radical scavenging, anti-apoptotic, anti-inflammatory, neuroregeneration, and vascular effects). Promising early/secondary phase therapies include Allopurinol, Azithromycin, Exendin-4, Magnesium, Melatonin, Noble gases and Sildenafil. Tertiary phase agents include Erythropoietin, Stem cells and others. We review a selection of promising therapeutic agents on the translational pipeline and suggest a framework for neuroprotection and neurorestoration that targets the evolving injury

Neuroinflammatory markers at school age in preterm born children with neurodevelopmental impairments

Background: Immune system activation in the neonatal period is associated with white matter injury in preterm infants. In animal studies, neonatal priming of the immune system leads to chronic activation of i.e. microglia cells and altered neuroinflammatory responses potentially years after preterm birth. This may contribute further to brain injury and neurodevelopmental impairment. It is unknown to what extend this also occurs in human. Aim: To identify neuro-inflammatory markers at school age that relate to motor, cognitive and behavioral impairments in preterm born children in a pilot case-control study. Methods: We included n = 20 preterm born children (GA < 28 weeks) in this study, of which n = 10 with motor, cognitive and behavorial impairments and n = 10 preterm born controls next to n = 30 healthy adult controls. In the preterm children, at 8–9 years, 39 inflammatory markers were assessed by Luminex assay in blood serum samples. Firstly, the preterm concentrations of these markers were compared to n = 30 adult controls. Then a univariate analysis was performed to determine differences in values between preterm children with and without impairment at school age. Finally, a principal component analysis and hierarchical clustering was performed to identify protein profiles in preterm born children that relate to impairment at school age. Results: Inflammatory proteins in preterm children at school age differed from values of adult controls. Within the group of preterm children, we found significantly higher levels of GM-CSF in preterms with impairment (p < 0.01) and a trend towards significance for Gal1 and TRAIL (p = 0.06 and p = 0.06 respectively) when compared to preterms without impairment. In addition, differences in clustering of proteins between preterm children was observed, however this variance was not explained by presence of neurodevelopmental impairments. Conclusion: The inflammatory profile at school age in preterm children is different from that of adult controls. The immune modulating cytokines GM-CSF, Gal1 and TRAIL were higher in preterm children with impairment than control preterm children, suggesting that immune responses are altered in these children. No specific cluster of inflammatory markers could be identified. Results indicate that even at school age, neuroinflammatory pathways are activated in preterm born children with neurodevelopmental impairments

Inhaled 45-50% argon augments hypothermic brain protection in a piglet model of perinatal asphyxia

Cooling to 33.5 °C in babies with neonatal encephalopathy significantly reduces death and disability, however additional therapies are needed to maximize brain protection. Following hypoxia–ischemia we assessed whether inhaled 45–50% Argon from 2–26 h augmented hypothermia neuroprotection in a neonatal piglet model, using MRS and aEEG, which predict outcome in babies with neonatal encephalopathy, and immunohistochemistry. Following cerebral hypoxia–ischemia, 20 Newborn male Large White piglets < 40 h were randomized to: (i) Cooling (33 °C) from 2–26 h (n = 10); or (ii) Cooling and inhaled 45–50% Argon (Cooling + Argon) from 2–26 h (n = 8). Whole-brain phosphorus-31 and regional proton MRS were acquired at baseline, 24 and 48 h after hypoxia–ischemia. EEG was monitored. At 48 h after hypoxia–ischemia, cell death (TUNEL) was evaluated over 7 brain regions. There were no differences in body weight, duration of hypoxia–ischemia or insult severity; throughout the study there were no differences in heart rate, arterial blood pressure, blood biochemistry and inotrope support. Two piglets in the Cooling + Argon group were excluded. Comparing Cooling + Argon with Cooling there was preservation of whole-brain MRS ATP and PCr/Pi at 48 h after hypoxia–ischemia (p < 0.001 for both) and lower 1H MRS lactate/N acetyl aspartate in white (p = 0.03 and 0.04) but not gray matter at 24 and 48 h. EEG background recovery was faster (p < 0.01) with Cooling + Argon. An overall difference between average cell-death of Cooling versus Cooling + Argon was observed (p < 0.01); estimated cells per mm2 were 23.9 points lower (95% C.I. 7.3–40.5) for the Cooling + Argon versus Cooling. Inhaled 45–50% Argon from 2–26 h augmented hypothermic protection at 48 h after hypoxia–ischemia shown by improved brain energy metabolism on MRS, faster EEG recovery and reduced cell death on TUNEL. Argon may provide a cheap and practical therapy to augment cooling for neonatal encephalopathy

Functional roles of extracellular vesicles derived from microglia with diverse activation states

Microglia respond to all types of CNS injury and acquire different activated phenotypes, participating not only in mechanisms of injury but also in tissue repair (1). However, the mode(s) of action of these cells in fostering or inhibiting CNS repair is still largely unclear. Here, we investigated the action of extracellular vesicles (EVs) released by microglia with diverse activation states (2) on Oligodendrocyte Precursor Cells (OPCs) and hippocampal neurons. Fluorescence analysis of OPCs exposed to EVs together with the proliferative marker EdU showed that EVs produced by pro-inflammatory cells limit OPC proliferation, while EVs released by pro-regenerative microglia tend to increase it. Stronger proliferative action was observed in vivo upon delivery of EVs derived from pro-regenerative microglia to mice with focal myelin lesions, with a significant increase in the density of proliferating NG2+ cells at the lesion site. Immunocytochemistry and western-blot analysis of markers of mature oligodendrocytes revealed that EVs derived from both inflammatory and pro- regenerative microglia, but not from unstimulated cells, promote OPC maturation in vitro, with EVs released by pro-regenerative microglia displaying higher differentiation activity and significantly fostering myelin deposition in an in vitro system of OPCs co-cultured with DRG neurons. Globally these results show that through EVs, pro-regenerative microglia and, at lesser extent, pro-inflammatory and resting cells may exert a beneficial action on OPCs, promoting their differentiation and myelin formation. Conversely, a clear detrimental action of EVs derived from inflammatory microglia was observed in cultured hippocampal neurons, highlighting a previously unrecognized role of microglia-derived EVs in inflammation-induced synaptic alteration. Indeed, immunofluorescence and western blotting analysis for synaptic markers showed that EVs secreted from inflammatory but not pro-regenerative microglia decrease the density of dendritic spines and cause destabilization of excitatory synapses. Molecular mechanisms underlying such synaptic alterations will be discussed