Genetic inhibition of RIPK3 ameliorates functional outcome in controlled cortical impact independent of necroptosis

Abstract

Traumatic brain injury (TBI) is a leading cause of death and disability with no specific effective therapy, in part because disease driving mechanisms remain to be elucidated. Receptor interacting protein kinases (RIPKs) are serine/threonine kinases that assemble multi-molecular complexes that induce apoptosis, necroptosis, inflammasome and nuclear factor kappa B activation. Prior studies using pharmacological inhibitors implicated necroptosis in the pathogenesis of TBI and stroke, but these studies cannot be used to conclusively demonstrate a role for necroptosis because of the possibility of off target effects. Using a model of cerebral contusion and RIPK3 and mixed lineage kinase like knockout (MLKL−/−) mice, we found evidence for activation of RIPK3 and MLKL and assembly of a RIPK1-RIPK3-MLKL necrosome complex in pericontusional brain tissue. Phosphorylated forms of RIPK3 and MLKL were detected in endothelium, CD11b + immune cells, and neurons, and RIPK3 was upregulated and activated in three-dimensional human endothelial cell cultures subjected to CCI. RIPK3−/− and MLKL−/− mice had reduced blood-brain barrier damage at 24 h (p < 0.05), but no differences in neuronal death (6 h, p = ns in CA1, CA3 and DG), brain edema (24 h, p = ns), or lesion size (4 weeks, p = ns) after CCI. RIPK3−/−, but not MLKL−/− mice, were protected against postinjury motor and cognitive deficits at 1–4 weeks (RIPK3−/− vs WT: p < 0.05 for group in wire grip, Morris water maze hidden platform trials, p < 0.05 for novel object recognition test, p < 0.01 for rotarod test). RIPK3−/− mice had reduced infiltrating leukocytes (p < 0.05 vs WT in CD11b + cells, microglia and macrophages), HMGB1 release and interleukin-1 beta activation at 24–48 h (p < 0.01) after CCI. Our data indicate that RIPK3 contributes to functional outcome after cerebral contusion by mechanisms involving inflammation but independent of necroptosis.

Mitochondria dysregulation contributes to secondary neurodegeneration progression post-contusion injury in human 3D in vitro triculture brain tissue model

“…4, 6; Supplementary Fig. 13,21,[23][24]. Additionally, we observed some differences in the inflammatory markers' secretion and the response to P110 treatment between tricultures with iPSC microglia and HMC3 cells, indicating the importance of the genetic contribution to the severity of injury progression and potential drug treatments.…”

“…Next, we looked at the activation of the necroptotic pathway, a known mechanism of neuronal death post-controlled cortical impact injury in rodents (21). We have confirmed necroptotic death execution in Nc and NAMc (the groups that showed neuronal network degeneration) through phosphorylation of mixed lineage kinase (pMLKL), known as necroptotic executor (Supplementary Fig.…”

“…21). Culture conditions, such as viability, neuronal network density, glial cell-specific markers, were optimized for the survival of all three cell types when combined in the tricultures (Supplementary Fig [1][2][3]21). Confocal image analysis, gene and protein expression studies of tri-cultures demonstrated upregulation of quintessential cell-type specific transcription factors (NeuN for mature neurons, Sox9 for astrocytes, and PU.1 for microglia) concomitant with an increase in density of beta-3-tubulin (Tuj1) positive neuronal networks (Fig.…”

Enterococcus faecalis -Induced Macrophage Necroptosis Promotes Refractory Apical Periodontitis

“…Interestingly, in addition to its well-established roles in necroptosis, RIPK3 can also serve as a regulator of inflammation and cell death, independent of necroptotic function. Recent studies have shown that RIPK3 promotes the activation of NLRP3 inflammasomes and contributes to inflammation in an MLKL-independent manner ( 29 , 30 ). The combined application of MLKL -deficient mice in future studies is recommended to provide more robust evidence of the involvement of necroptosis in RAP progression.…”

RIPK3 modulates sarcoma through immune checkpoint HAVCR2

“…However, this function was not found to be dependent on the expression level of RIPK3 in the cells, indicating that RIPK3 might not act through classical inflammatory pathways ( 36 ). RIPK3 has also shown inflammation-modulating effects in certain types of immune cells, such as monocytes and macrophages ( 37 ); however neither of these cell types were assayed in sarcomas conducted by Shekhar et al ( 36 ).…”