NOD1/RIP2 signalling enhances the microglia-driven inflammatory response and undergoes crosstalk with inflammatory cytokines to exacerbate brain damage following intracerebral haemorrhage in mice.

BACKGROUND: Secondary brain damage caused by the innate immune response and subsequent proinflammatory factor production is a major factor contributing to the high mortality of intracerebral haemorrhage (ICH). Nucleotide-binding oligomerization domain 1 (NOD1)/receptor-interacting protein 2 (RIP2) signalling has been reported to participate in the innate immune response and inflammatory response. Therefore, we investigated the role of NOD1/RIP2 signalling in mice with collagenase-induced ICH and in cultured primary microglia challenged with hemin. METHODS: Adult male C57BL/6 mice were subjected to collagenase for induction of ICH model in vivo. Cultured primary microglia and BV2 microglial cells (microglial cell line) challenged with hemin aimed to simulate the ICH model in vitro. We first defined the expression of NOD1 and RIP2 in vivo and in vitro using an ICH model by western blotting. The effect of NOD1/RIP2 signalling on ICH-induced brain injury volume, neurological deficits, brain oedema, and microglial activation were assessed following intraventricular injection of either ML130 (a NOD1 inhibitor) or GSK583 (a RIP2 inhibitor). In addition, levels of JNK/P38 MAPK, IκBα, and inflammatory factors, including tumour necrosis factor-α (TNF-α), interleukin (IL)-1ß, and inducible nitric oxide synthase (iNOS) expression, were analysed in ICH-challenged brain and hemin-exposed cultured primary microglia by western blotting. Finally, we investigated whether the inflammatory factors could undergo crosstalk with NOD1 and RIP2. RESULTS: The levels of NOD1 and its adaptor RIP2 were significantly elevated in the brains of mice in response to ICH and in cultured primary microglia, BV2 cells challenged with hemin. Administration of either a NOD1 or RIP2 inhibitor in mice with ICH prevented microglial activation and neuroinflammation, followed by alleviation of ICH-induced brain damage. Interestingly, the inflammatory factors interleukin (IL)-1ß and tumour necrosis factor-α (TNF-α), which were enhanced by NOD1/RIP2 signalling, were found to contribute to the NOD1 and RIP2 upregulation in our study. CONCLUSION: NOD1/RIP2 signalling played an important role in the regulation of the inflammatory response during ICH. In addition, a vicious feedback cycle was observed between NOD1/RIP2 and IL-1ß/TNF-α, which could to some extent result in sustained brain damage during ICH. Hence, our study highlights NOD1/RIP2 signalling as a potential therapeutic target to protect the brain against secondary brain damage during ICH.

Dectin-1/Syk signaling triggers neuroinflammation after ischemic stroke in mice.

BACKGROUND: Dendritic cell-associated C-type lectin-1 (Dectin-1) receptor has been reported to be involved in neuroinflammation in Alzheimer's disease and traumatic brain injury. The present study was designed to investigate the role of Dectin-1 and its downstream target spleen tyrosine kinase (Syk) in early brain injury after ischemic stroke using a focal cortex ischemic stroke model. METHODS: Adult male C57BL/6 J mice were subjected to a cerebral focal ischemia model of ischemic stroke. The neurological score, adhesive removal test, and foot-fault test were evaluated on days 1, 3, 5, and 7 after ischemic stroke. Dectin-1, Syk, phosphorylated (p)-Syk, tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS) expression was analyzed via western blotting in ischemic brain tissue after ischemic stroke and in BV2 microglial cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) injury in vitro. The brain infarct volume and Iba1-positive cells were evaluated using Nissl's and immunofluorescence staining, respectively. The Dectin-1 antagonist laminarin (LAM) and a selective inhibitor of Syk phosphorylation (piceatannol; PIC) were used for the intervention. RESULTS: Dectin-1, Syk, and p-Syk expression was significantly enhanced on days 3, 5, and 7 and peaked on day 3 after ischemic stroke. The Dectin-1 antagonist LAM or Syk inhibitor PIC decreased the number of Iba1-positive cells and TNF-α and iNOS expression, decreased the brain infarct volume, and improved neurological functions on day 3 after ischemic stroke. In addition, the in vitro data revealed that Dectin-1, Syk, and p-Syk expression was increased following the 3-h OGD and 0, 3, and 6 h of reperfusion in BV2 microglial cells. LAM and PIC also decreased TNF-α and iNOS expression 3 h after OGD/R induction. CONCLUSION: Dectin-1/Syk signaling plays a crucial role in inflammatory activation after ischemic stroke, and further investigation of Dectin-1/Syk signaling in stroke is warranted.

Suppressors of cytokine signalling (SOCS)-1 inhibits neuroinflammation by regulating ROS and TLR4 in BV2 cells.

OBJECTIVE: The suppressors of cytokine signaling (SOCS) proteins are physiological suppressors of cytokine signaling which have been identified as a negative feedback loop to weaken cytokine signaling. However, the underlying molecular mechanisms is unknown. This study was to investigate the role of SOCS1 in the oxygen-glucose deprivation and reoxygenation (OGDR) or LPS-induced inflammation in microglia cell line BV-2 cells. MATERIALS AND METHODS: BV-2 microglial cells were used to construct inflammation model. A SOCS1 over-expression plasmid was constructed, and the SOCS1-deficient cells were generated by utilizing the CRISPR/CAS9 system. BV-2 microglial cells were pretreated with over-expression plasmid or SOCS1 CRISPR plasmid before OGDR and LPS stimulation. The effect of SOCS1 on proinflammatory cytokines, toll-like receptor 4 (TLR4), and reactive oxygen species (ROS) were evaluated. RESULTS: We found that SOCS1 increased in OGDR or LPS-treated BV-2 microglial cells in vitro. SOCS1 over-expression significantly reduced the production of proinflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and IL-6, and CRISPR/CAS9-mediated SOCS1 knockout reversed this effect. Also we determined that SOCS1 over-expression reduced the level of reactive oxygen species (ROS) while the absence of SOCS1 increased the production of ROS after OGDR or LPS-stimulated inflammation. Furthermore, we found that OGDR and LPS induced the expression of toll-like receptor 4 (TLR4) in BV2 cells. Nevertheless, SOCS1 over-expression attenuated the expression of TLR4, while knockdown of SOCS1 upregulated TLR4. CONCLUSIONS: Our study indicated that SOCS1 played a protective role under inflammatory conditions in OGDR or LPS treated BV-2 cells through regulating ROS and TLR4. These data demonstrated that SOCS1 served as a potential therapeutic target to alleviate inflammation after ischemic stroke.