Transforming growth factor-β-activated kinase 1 (TAK1) is upregulated after cerebral ischemia and contributes to an aggravation of brain injury. TAK1 acts as a key regulator of NF-ΚB and the MAP kinases JNK and p38 and modulates post-ischemic neuroinflammation and apoptosis. Microglia are the main TAK1-expressing immunocompetent cells of the brain. However, little is known about the function and regulation of microglial TAK1 after cerebral ischemia. Tamoxifen-dependent conditional depletion of TAK1 in microglial cells was induced in Cx3cr1creER-Tak1fl/fl mice. The creER-negative Tak1fl/fl mice and vehicle-treated (corn oil) mice served as control groups. A transient intraluminal middle cerebral artery occlusion of 30 min followed by 6 h and 72 h of reperfusion was performed in male mice. Oxygen-glucose-deprivation (OGD) was performed with primary cortical glial cell cultures to examine the effect of microglial-specific and general (5Z-7-Oxozeaenol) TAK1 inhibition after different reperfusion times (1 h, 6 h, and 72 h). Cx3cr1creER-Tak1fl/fl mice showed reduced infarct sizes and improved neurological outcomes compared to the control group. The mRNA and protein levels of pro-inflammatory Il1b/IL-1β and Tnf/TNF-α in the peri-infarct zones of microglial-specific TAK1-depleted mice were significantly reduced. Furthermore, TAK1 depletion in vitro led to reduced cell death rates after OGD. Moreover, hypoxia-mediated activation of TAK1 and its downstream signalling proteins, JNK and p38, were dampened by microglial TAK1 depletion. In contrast, 5Z-7-Oxozeaenol-induced pharmacological inhibition of TAK1 completely diminished MAPK-signalling including the kinases JNK and p38 in all cells. Microglial TAK1 depletion abrogates post-ischemic neuroinflammation and apoptosis in the acute phase, hence might be considered as a potential target in the treatment of cerebral hypoxia. Key messages TAK1 is activated after cerebral ischemia and induces MAP kinases p38 and JNK. Activated TAK1 increases apoptosis rate and the level pro-inflammatory cytokines IL-1β and TNF-α. Microglial cells seem to be the main source of TAK1-mediated post-ischemic neuroinflammation. Microglial-specific TAK1-depletion mediates sustainable neuroprotective effects, which might be superior to global TAK1 inhibition.

Inflammasomes are known to contribute to brain damage after acute ischemic stroke (AIS). TAK1 is predominantly expressed in microglial cells and can regulate the NLRP3 inflammasome, but its impact on other inflammasomes including NLRC4 and AIM2 after AIS remains elusive. EPO has been shown to reduce NLRP3 protein levels in different disease models. Whether EPO-mediated neuroprotection after AIS is conveyed via an EPO/TAK1/inflammasome axis in microglia remains to be clarified. Subjecting mice deficient for TAK1 in microglia/macrophages (Mi/MΦ) to AIS revealed a significant reduction in infarct sizes and neurological impairments compared to the corresponding controls. Post-ischemic increased activation of TAK1, NLRP3, NLRC4, and AIM2 inflammasomes including their associated downstream cascades were markedly reduced upon deletion of Mi/MΦ TAK1. EPO administration improved clinical outcomes and dampened stroke-induced activation of TAK1 and inflammasome cascades, which was not evident after the deletion of Mi/MΦ TAK1. Pharmacological inhibition of NLRP3 in microglial BV-2 cells did not influence post-OGD IL-1β levels, but increased NLRC4 and AIM2 protein levels, suggesting compensatory activities among inflammasomes. Overall, we provide evidence that Mi/MΦ TAK1 regulates the expression and activation of the NLRP3, NLRC4, AIM2 inflammasomes. Furthermore, EPO mitigated stroke-induced activation of TAK1 and inflammasomes, indicating that EPO conveyed neuroprotection might be mediated via an EPO/TAK1/inflammasome axis. Graphical Abstract

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Recombinant human erythropoietin (rhEPO) has been shown to exert anti-apoptotic and anti-inflammatory effects after cerebral ischemia. Inflammatory cytokines interleukin-1β and -18 (IL-1β and IL-18) are crucial mediators of apoptosis and are maturated by multiprotein complexes termed inflammasomes. Microglia are the first responders to post-ischemic brain damage and are a main source of inflammasomes. However, the impact of rhEPO on microglial activation and the subsequent induction of inflammasomes after ischemia remains elusive. To address this, we subjected human microglial clone 3 (HMC-3) cells to various durations of oxygen-glucose-deprivation/reperfusion (OGD/R) to assess the impact of rhEPO on cell viability, metabolic activity, oxidative stress, phagocytosis, migration, as well as on the regulation and activation of the NLRP1, NLRP3, NLRC4, and AIM2 inflammasomes. Administration of rhEPO mitigated OGD/R-induced oxidative stress and cell death. Additionally, it enhanced metabolic activity, migration and phagocytosis of HMC-3. Moreover, rhEPO attenuated post-ischemic activation and regulation of the NLRP1, NLRP3, NLRC4, and AIM2 inflammasomes as well as their downstream effectors CASPASE1 and IL-1β. Pharmacological inhibition of NLRP3 via MCC950 had no effect on the activation of CASPASE1 and maturation of IL-1β after OGD/R, but increased protein levels of NLRP1, NLRC4, and AIM2, suggesting compensatory activities among inflammasomes. We provide evidence that EPO-conveyed anti-inflammatory actions might be mediated via the regulation of the inflammasomes.

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