Cysteinyl Leukotriene CysLT₂Receptor Mediates Ischemic Neuronal Injury Through Activating Microglia

BackgroundsCysteinyl leukotrienes (CysLTs), namely leukotriene C4(LTC4), LTD4and LTE4, are arachidonic acid-derived lipid mediators. CysLTs act on least two G protein-coupled receptors, CysLT1R and CysLT2R, and play important regulatory roles. In the periphery, the CysLT1R is involved in various inflammatory diseases such as bronchial asthma and allergic rhinitis, and the CysLT2R mediates increase of vascular permeability and aggravates myocardial ischemia/reperfusion injury. In the central nervous system (CNS), the production of CysLTs increases after ischemic injury in rat brain, primary neurons and astrocytes. Increased CysLTs induce various CNS responses through activating their receptors. It has been reported that the expression of CysLT1R and CysLT2R was up-regulated in the brain after focal cerebral ischemia. These up-regulated receptors were distributed in injured neurons in the acute phase (-24h), and activated microglia and proliferating astrocytes in the late phases (3-28days). These findings suggest that CysLT1R and CysLT2R may mediate acute ischemic neuronal injury and the sequential microgliosis and astrocytosis in vivo.In cellular level, neuron-like PC12cells transfected with CysLT1R and CysLT2R showed distinct sensitivities to ischemic injury, namely CysLT1R transfection lowered, but CysLT2R transfection increased, the sensitivity to ischemia-like injury induced by oxygen-glucose deprivation (OGD). However, whether and how these receptors regulate ischemic neuronal injury is necessary to be investigated. In primary neurons, OGD induced injury, but the agonist LTD4of CysLT receptors did not induce ischemia-like injury, which cannot explain the in vivo findings. Therefore, it is possible that CysLTs may regulate ischemic neuronal injury via interactions between neurons and surrounding cells, such as astrocyte and microglia. We have found that CysLT1R and CysLT2R play distinct roles in regulation of astrocytes; however, their roles in regulation of microglial activation need investigation.Pharmacologically, the CysLT1R antagonists pranlukast and montelukast have protective effects on focal or global cerebral ischemia in rats and mice. They attenuated neuronal injury, blood-brain barrier disruption, inflammatory responses, chronic brain injury and the relevant glial scar formation. However, montelukast had no effect on OGD-induced reduction in neuron viability, or merely moderate effect on neuron morphological changes after OGD. On the other hand, because of lack of selective CysLT2R antagonists for a long time, their effects on ischemic neuronal injury are unknown. Recently, N-methyl leukotriene C4(NMLTC4) has been reported as a potent selective agonist for the CysLT2receptor, and Bay CysLT2and HAMI3379as selective CysLT2R antagonists, which provide useful tools for CysLT2R investigation. We have reported that intracerebroventricular injection of HAMI3379protects rats from acute brain injury after focal cerebral ischemia, but its effects on in vitro ischemic neuronal injury need investigation.AimsIn the present study, we investigated the detailed regulatory roles of CysLTiR and CysLT2R in OGD-induced ischemic neuronal injury, including the direct action on neurons and the indirect action through activating microglia. We performed cellular experiments to answer the following questions:(1) In primary rat cortical neurons, whether CysLT receptor subtypes directly mediate OGD-induced injury?(2) In mixed cultures of rat cortical cells, whether CysLT receptor subtypes mediate OGD-induced cell injury?(3) In neuron-glial transwell co-cultures, whether CysLT receptor subtypes mediate OGD-induced neuronal injury through interactions with astrocyte or microglia?(4) In primary rat microglia, whether CysLT receptor subtypes mediate OGD-induced microglial activation (phagocytosis and cytokine release) and thereby induce neuronal injury? The effects of microglial conditioned medium on neuronal injury were also included.MethodsIn primary rat cortical neuron, the mixed cultures of rat cortical cells, neuron-glial transwell co-cultures and rat primary microglia, ischemic injury was induced by OGD. The agonist LTD4or NMLTC4at various concentrations was applied as stimuli to induce neuronal injury. The effects of the CysLT1R antagonist montelukast and the CysLT2R antagonist HAMI3379were observed as well. The gene expression of CysLTiR was silenced by a targeted siRNA and that of CysLT2R was silenced by a targeted lentivirus shRNA. Thus, OGD-induced neuronal injury and microglia activation were investigated by application of the agonists, antagonists and RNA interference of CysLT1R and CysLT2R.ResultsPart I Roles of CysLT1R and CysLT2R in OGD-induced injury in primary rat cortical neuronsAt first, we determined whether CysLTs directly affect primary neurons via activating their receptors. The results showed that OGD time-dependently induced ischemic neuronal injury, while the non-selective agonist LTD4and the selective CysLT2R agonist NMLTC4did not induce neuronal injury. The CysLT1R antagonist montelukast attenuated OGD/R (OGD1h, recovery24h)-induced injury, including attenuation of neuronal viability reduction, LDH release and necrosis. However, the selective CysLT2R antagonist HAMI3379did not show these protective effects. In addition, inhibition of CysLT1R expression by CysLT1R siRNA and CysLT2R expression by CysLT2R shRNA had been confirmed in the neurons. Neither CysLT1R siRNA nor CysLT2R shRNA inhibited OGD/R-induced neuronal injury.These findings indicated that CysLTs and their receptors might not directly mediate neuronal injury; the effects of montelukast might result from CysLT1R-independent mechanisms..Part Ⅱ Roles of CysLT1R and CysLT2R in OGD-induced injury in the mixed cultures of cortical cellsNext, we determined whether CysLT receptor subtypes regulate OGD-induced injury in a relatively intact cellular environment, namely in the mixed cultures of cortical cells. We found that OGD/R (OGD1h, recovery24h), LTD4or NMLTC4induced cell viability reduction, LDH release increase, cell necrosis and apoptosis, and morphological changes of microglial activation. CysLT2shRNA and HAMI3379inhibited OGD/R-, LTD4-or NMLTC4-induced responses. Montelukast, not CysLT1R siRNA, attenuated the changes induced by OGD/R and LTD4.Therefore, CysLT2R might mediate neuronal injury and microglial activation in the mixed cultures of cortical cells, suggesting a close relationship between microglial activation and neuronal injury. CysLT1R might not mediate these responses, while montelukast partially inhibited neuronal injury and microglial activation through CysLT1R-independent mechanisms.Part Ⅲ Roles of CysLT1R and CysLT2R in OGD-induced neuronal injury in neuron-glial co-cultures Then, to further confirm the relation between microglial activation and neuronal injury, we observed the changes in neuron-glial transwell co-cultures. In neuron-astrocyte co-cultures, OGD/R (OGD1h, recovery24h), LTD4and NMLTC4induced mild neuronal injury, i.e. reduction in neuron number and induction of necrosis. None of the CysLT receptor antagonists and RNA interference affected these changes in neuron-astrocyte co-cultures. However, in neuron-microglial co-cultures, OGD/R, LTD4or NMLTC4induced more profound neuronal injury. CysLT2R shRNA and HAMI3379significantly decreased neuronal injury in neuron-microglial co-cultures. In neuron-microglial co-cultures, montelukast attenuated neuronal injury induced by OGD/R or LTD4, and CysLT1R siRNA had mild inhibition on LTD4-induced injury as well.These findings indicated that CysLT2R might mediate the neuronal injury mediated by microglial activation, which might be the primary way for regulating ischemic neuronal injury. CysLT1R might partially regulate microglial activation, and montelukast might inhibit microglia-mediated neuronal injury through both CysLT1R-dependent and independent mechanisms. However, whether and how CysLT receptors regulate neuronal injury mediated by astrocytes remains to be investigated.Part Ⅳ Roles of CysLT1R and CysLT2R in OGD-induced microglial activation, and effects of microglial conditioned medium on neuronal injuryFinally, to reveal the regulatory roles of CysLT receptors in microglial action, we observed the changes in primary cultures of rat microglia. Immunofluoresence examination showed that CysLT1R and CysLT2R were weakly expressed in microglia under normal conditions; and their expression was significantly up-regulated after OGD/R (1-h OGD and24-h recovery). OGD/R, LTD4or NMLTC4induced microglial activation, i.e. enhancing phagocytosis and increasing cytokine (TNF-a and IL-1β) release. HAMI3379and CysLT2shRNA inhibited all the responses induced by the three stimuli. Montelukast decreased the responses induced by OGD/R and LTD4, while CysLT1R siRNA partially inhibited cytokine release. On the other hand, the conditioned medium from microglia pre-treated with OGD/R, LTD4or NMLTC4induced neuronal necrosis, and CysLT2shRNA and HAMI3379inhibited the induced necrosis. Montelukast showed an inhibitory trend but no significant effect on OGD/R-and LTD4-induced necrosis, while CysLT1R siRNA had no effect.These findings demonstrated that the CysLT2R, as the primary regulatory factor, might mediate microglial activation (enhancing phagocytosis and cytokine release), and thereby induce neuronal injury. CysLT1R might partially regulate cytokine release from microglia, and montelukast might inhibit neuronal injury through CysLT1R-dependent and independent mechanisms.Conclusions:1. In primary rat cortical neurons, CysLTs and their receptors do not directly mediate OGD/R-induced ischemic injury.2. In the mixed cultures of rat cortical cells, CysLT1R, but not CysLT1R, mediates OGD/R-induced neuronal injury and microglial activation. In neuron-glial co-cultures, CysLT2R as the primary factor mediates OGD/R-induced neuronal injury via activating microglia, while CysLT1R partially regulates cytokine release from microglia; but the roles of CysLT receptors in astrocyte-mediated neuronal injury need investigation.3. CysLT2R mediates OGD/R-induced microglial activation, and thereby induces neuronal injury via enhancing microglial phagocytosis and cytokine release. CysLT1R partially mediates neuronal injury via regulating cytokine release from microglia.4. HAMI3379blocks CysLT2R-mediated responses, but montelukast exerts both CysLT1R-independent and dependent effects. Both of them may be potentially valuable for inhibition of inflammation after ischemic stroke.