Inhibition of the interaction between microglial adenosine 2A receptor and NLRP3 inflammasome attenuates neuroinflammation posttraumatic brain injury.

AIMS: Adenosine 2A receptor (A2A R) is widely expressed in the brain and plays important roles in neuroinflammation, and the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome is a crucial component of the innate immune system while the regulation of A2A R on it in the central nervous system (CNS) has not been clarified. METHODS: The effects of microglial A2A R on NLRP3 inflammasome assembly and activation were investigated in wild-type, A2A R- or NLRP3-knockout primary microglia with pharmacological treatment. Microglial A2A R or NLRP3 conditional knockout mice were used to interrogate the effects of this regulation on neuroinflammation posttraumatic brain injury (TBI). RESULTS: We found that A2A R directly interacted with NLRP3 and facilitated NLRP3 inflammasome assembly and activation in primary microglia while having no effects on mRNA levels of inflammasome components. Inhibition of the interaction via A2A R agonist or knockout attenuated inflammasome assembly and activation in vitro. In the TBI model, microglial A2A R and NLRP3 were co-expressed at high levels in microglia next to the peri-injured cortex, and abrogating of this interaction by microglial NLRP3 or A2A R conditional knockout attenuated the neurological deficits and neuropathology post-TBI via reducing the NLRP3 inflammasome activation. CONCLUSION: Our results demonstrated that inhibition of the interaction between A2A R and NLRP3 in microglia could mitigate the NLRP3 inflammasome assembly and activation and ameliorate the neuroinflammation post-TBI. It provides new insights into the effects of A2A R on neuroinflammation regulation post-TBI and offers a potential target for the treatment of NLRP3 inflammasome-related CNS diseases.

Ablation of GSDMD Attenuates Neurological Deficits and Neuropathological Alterations After Traumatic Brain Injury.

Pyroptosis plays a significant role in neuroinflammation after traumatic brain injury (TBI). However, the role of pyroptosis executor Gasdermin D (GSDMD) in neurological deficits and neuropathological alterations after TBI have not been elucidated. Our results demonstrated that GSDMD-KO exerted striking neuroprotective effects on motor dysfunction and neuropathological alterations (loss of synaptic proteins, microglia activation, astrogliosis, dendrite injury, and neuron death) at 3 days after TBI. GSDMD-KO inhibited the expression and release of pro-inflammatory cytokine releases (IL-1ß and TNF-α) while promoting those of anti-inflammatory cytokines (IL-10 and TGF-ß1). The temporal pattern of diverse inflammasome signals showed long-lasting elevations of NLRP3, caspase 1, and caspase 1 p20 after TBI, rather than NLRP1, NLRC4 or AIM2, similar to the change in GSDMD postinjury; and NLRP3-KO not only inhibited the expression and cleavage of GSDMD but also attenuated the loss of synaptic proteins and neurological deficits. Notably, RNA sequencing showed both GSDMD-KO and NLRP3-KO reversed the global expression of neuroinflammation- and neuropathology-related genes after TBI. Our findings proved that the inhibition of GSDMD exerts neuroprotective effects after TBI and is mainly driven by the NLRP3 inflammasome. GSDMD serves as a potent therapeutic target for the treatment of TBI.

High glutamate concentration reverses the inhibitory effect of microglial adenosine 2A receptor on NLRP3 inflammasome assembly and activation.

NLRP3 inflammasome plays a crucial role in the innate immune system. Our group previously reported that the microglial adenosine 2A receptor (A2AR) regulates canonical neuroinflammation, which is affected by the glutamate concentration. However, the regulatory effect of A2AR on NLRP3 inflammasome and the effects of glutamate concentration remain unknown. Therefore, we aimed to investigate the regulatory effect of microglial A2AR on NLRP3 inflammasome assembly and activation as well as the effects of glutamate concentration on the inflammasome assembly and activation. Experiments were conducted on magnetically sorted primary microglia from P14 mice. The results showed that pharmacological A2AR activation ameliorated NLRP3 activation under no or low glutamate concentrations, but this effect was reversed by high glutamate concentrations. Moreover, the mRNA levels of NLRP3 inflammasome-related genes were not affected by A2AR activation or the glutamate concentration. We further demonstrated that A2AR activation inhibited the interaction between NLRP3 and caspase 1 under no or low glutamate concentrations while promoting their interaction under high glutamate concentrations. The oligomerization of ASC also showed a similar trend. In conclusion, our findings proved that the high glutamate concentration could reverse the inhibition of A2AR on NLRP3 inflammasome activation by modulating its assembly, which provides new insights into the regulatory effect of A2AR on neuroinflammation under different pathological conditions.