NEMO (IKKγ) citrullination in microglia promotes neuroinflammation in the post-ischemic brain

Peptidylarginine deiminase (PAD) catalyzes the post-translational conversion of positively charged arginine residues into neutral citrulline. This modification, known as citrullination, is implicated in various pathological conditions, including those affecting the central nervous system. This study investigated the pro-inflammatory role of PAD in microglia using a middle cerebral artery occlusion (MCAO) animal model of ischemic stroke. Pharmacological inhibition of PAD via BB-Cl-amidine (BBCA; a pan-PAD inhibitor) exerted robust anti-inflammatory and neuroprotective effects in the post-ischemic brain in a broad therapeutic window. A significant accumulation of citrullinated proteins was detected in activated microglia following ischemia, which was suppressed by BBCA. Notably, we demonstrate that citrullination of NEMO (IKKγ), a key regulator that activates the NF-κB signaling pathway, was significantly induced in the post-ischemic brain, facilitating NEMO-IKKα/IKKβ assembly. Importantly, BBCA inhibited NEMO citrullination and disrupted subsequent IKK assembly, highlighting the critical role of PAD in this process. Targeted knockdown experiments using siRNA in BV2 microglial cells revealed that both PAD2 and PAD4 play crucial role in NEMO citrullination and NF-κB-mediated pro-inflammatory response. Furthermore, inhibiting NEMO citrullination with a NEMO-binding domain peptide (NBDp) or co-treatment with NBDp and BBCA, further supports a crucial role for PAD-mediated NEMO citrullination in post-ischemic neuroinflammation. Collectively, these results suggest that PAD2 and PAD4 drive pro-inflammatory processes following cerebral ischemia by upregulating NEMO citrullination and subsequent IKK complex formation.