Ischemic stroke is a leading cause of death and disability around the world, with an estimated annual cost of $721 billion, as of 2022 (Feigin et al., 2022). An ischemic stroke occurs when blood flow to the brain is restricted due to the blockage of a cerebral artery. This results in the rapid death of neurons and other brain cells downstream of the cerebral artery as these cells succumb to the hypoxic and energy-deprived conditions. This, in turn, promotes a highly neuroinflammatory state within the brain parenchyma as glial cells, like microglia, respond to the damage caused by stroke injury by secreting cytokines, chemokines, and blood-brain barrier (BBB)-compromising matrix metalloproteinases (MMPs) (Candelario-Jalil et al., 2022). Peripheral immune cells are then drawn to the site of injury via chemotactic signals, aided by the breakdown of the BBB, where they further contribute to the neuroinflammatory state by releasing additional inflammatory factors (Iadecola et al., 2020). In this way, neuroinflammation contributes greatly to secondary brain injury after stroke. Modulation of neuroinflammatory pathways represents an attractive target to lessen the deleterious progression of stroke injury.

Various cell types utilize pattern recognition receptors (PRRs) to respond to molecular patterns and facilitate a rapid response to potentially dangerous stimuli (Li and Wu, 2021). In the context of stroke, PRRs like toll-like receptors (TLRs) and nucleotide oligomerization domain (NOD)-like receptors (NLRs) are used by immune cells and neurons to respond to damage-associated molecular patterns (DAMPs) released from dead/dying cells and promote NF-κB and MAPK pro-inflammatory pathway activation (Kumar, 2019; Santoni et al., 2015). Overactivation of these pathways promotes a highly neuroinflammatory environment with the consequence of direct damage to otherwise healthy neurons.

Receptor-interacting protein kinase 2 (RIPK2) is well characterized for being the obligate kinase for NOD1 and NOD2 NLR signaling. NOD1 and NOD2 are PRRs that are traditionally known for recognizing pathogen-associated molecular patterns (PAMPs) of bacterial origin. However, more recent evidence suggests that they may have a lesser-defined role in the recognition of DAMPs (Pei et al., 2021). Upon NOD1/NOD2 activation, RIPK2 autophosphorylates, initiating a signaling cascade that results in both NF-κB and MAPK pro-inflammatory pathway activation. Besides their role in the propagation of inflammatory signaling, NOD1/2 and RIPK2 have been shown to be involved in the cascade of mitochondrial endoplasmic reticulum (ER) stress (Keestra-Gounder et al., 2016), and overactivation of ER stress can shunt cells toward a more pro-apoptotic pathway (Han et al., 2021).

In addition to its association with and propagation of NOD1/2 signaling, RIPK2 has alternative roles that implicate it in the progression of stroke injury. For instance, RIPK2 was shown to associate with caspase-1 and contribute to the direct cell death of neurons under hypoxic conditions (Zhang et al., 2003). RIPK2 has also been implicated in the autophagy pathway, and its inhibition was shown to decrease the phosphorylation of the autophagy-initiating protein ULK1 in a mouse meningitis model (Gao et al., 2019; Wang et al., 2022). Excessive activation of the autophagy pathway in the brain after stroke is associated with worse outcomes in animal models of the disease (Mo et al., 2020; Shi et al., 2021). Previous work from our group found that global Ripk2 knockout mice and mice where Ripk2 is specifically deleted from microglia experienced dramatically reduced post-stroke deficits compared to their respective control animals (Larochelle et al., 2023).

By inhibiting RIPK2's autophosphorylation event, RIPK2's signaling activity can be ablated (Tigno-Aranjuez et al., 2010). Inhibition of RIPK2 has proven effective in a variety of inflammatory disease models; in particular, inflammatory bowel disease (Hollenbach et al., 2004), Crohn's disease and ulcerative colitis (Haile et al., 2016; Hollenbach et al., 2005), multiple sclerosis (Nachbur et al., 2015), arthritis (Rosenzweig et al., 2011), and intracerebral hemorrhage (Wang et al., 2020). Multiple inhibitors for RIPK2 have been developed, and currently, there is a focused effort to create novel inhibitors for RIPK2 with enhanced potency and selectivity, as well as other molecules that block RIPK2 activity, such as the development of RIPK2-specific proteolysis-targeting chimeras (PROTACs) (Bondeson et al., 2015; Mares et al., 2020; Miah et al., 2021). Herein, we utilize an inhibitor of RIPK2 that was proven to be highly efficacious at doses ranging from 1 to 15 mg/kg of body weight in mice, while also avoiding typical off-target effects that were common in previous iterations of RIPK2 inhibitors (Salla et al., 2018).

In our present study, we subjected adult male mice to 45 min of transient middle cerebral artery occlusion (tMCAO) and administered doses of either RIPK2 inhibitor or vehicle control upon reperfusion. We hypothesized that inhibition of RIPK2 will improve animal outcomes after ischemic stroke primarily by dampening neuroinflammatory processes, thus preserving functional brain tissue from the sequelae of stroke injury. We assessed the effects of RIPK2 inhibition on infarct volume and post-stroke behavioral deficits, dampening neuroinflammation, and preservation of the BBB in the acute phase of stroke injury. Finally, to delve into molecular mechanisms underlying the effects of RIPK2 inhibition in stroke, we performed bulk RNAseq in the ischemic cerebral cortex of vehicle-administered and inhibitor-pretreated animals to investigate how RIPK2 blockade impacts stroke-induced transcriptional programs during the early stages of ischemic brain injury.