GSK-583

Meningitis occurs when S. pneumonia invade the blood-brain barrier, provoking inflammatory host response and neurological injury. Nucleotide-binding oligomerization domain 2 (NOD2) has been identified to promote microglial activation and autophagy during pneumococcal meningitis, but the mechanism remains unclear. In the present study, we investigated the passway of NOD2-mediated autophagy activation and the role of autophagy in inflammatory damage of murine microglia and mouse meningitis model. We demonstrated that autophagy was activated during S. pneumonia infection, and NOD2-RIP2 signaling was involved in the process. Treatment of microglia with GSK583, the RIP2 kinase inhibitor resulted in reduced autophagy-related protein and p-ULK1, indicating that RIP2 regulated autophagy in a kinase-dependent manner by phosphorylating ULK1. In addition, microglia with ULK1 knockdown exhibited enhanced production of ROS, leading to IL-1β and IL-18 release and cellular pyroptosis. Similar to the in vitro results, NOD2-RIP2 signaling induced autophagy in the brain in a mouse meningitis model. Moreover, ULK1 or RIP2 silencing significantly increased pyroptosis of brain and induced more inflammatory damage of pneumococcal meningitis mice. Taken together, our study demonstrate that NOD2-RIP2 signaling is involved in the activation of autophagy by promoting ULK1 phosphorylation, which alleviates microglial ROS damage and pyroptosis during S. pneumonia infection.

The receptor-interacting serine/threonine-protein kinase-2 (RIPK2, RIP2) is a key player in downstream signaling of nuclear oligomerization domain (NOD)-like receptor (NLR)-mediated innate immune response against bacterial infections. RIPK2 is recruited following activation of the pattern recognition receptors (PRRs) NOD1 and NOD2 by sensing bacterial peptidoglycans leading to activation of NF-κB and MAPK pathways and the production of pro-inflammatory cytokines. Upon NOD1/2 activation, RIPK2 forms complexes in the cytoplasm of human cells, also called RIPosomes. These can be induced by Shigella flexneri or by the inhibition of RIPK2 by small compounds, such as GSK583 and gefitinib.In this chapter, we describe fluorescent light microscopic and Western blot approaches to analyze the cytoplasmic aggregation of RIPK2 upon infection with the invasive, Gram-negative bacterial pathogen Shigella flexneri, or by the treatment with RIPK2 inhibitors. This method is based on HeLa cells stably expressing eGFP-tagged RIPK2 and describes a protocol to induce and visualize RIPosome formation. The described method is useful to study the deposition of RIPK2 in speck-like structures, also in living cells, using live cell imaging and can be adopted for the study of other inhibitory proteins or to further analyze the process of RIPosome structure assembly.