Research ReportDisruption of downstream MyD88 or TRIF Toll-like receptor signaling does not protect against cerebral ischemia
Research highlights
► MyD88 knock-down does not affect PC12 cell viability in vitro. ► Disruption of MyD88 or TRIF has no effect on cortical neuron viability in vitro. ► CA1 neuronal survival is unaffected by MyD88 or TRIF disruption in global ischemia. ► No differences in infarct sizes in WT, MyD88 and TRIF mutant mice after pMCAO.
Introduction
Stroke remains the third leading cause of death and the leading cause of long-term serious disability in the United States and new approaches for treatment are desperately needed. One such approach involves understanding the role of pathways leading to inflammation such as the Toll-like receptor (TLR) signaling pathway in controlling the brain's response to ischemia.
Toll-like receptors are evolutionarily conserved pattern-recognition receptors important for initiation of innate immune responses to pathogen-associated molecular patterns (PAMPs). However, the “danger model” of immunity suggests that “danger signals” that activate the innate immune response to infection are not restricted to PAMPs such as lipopolysaccharide (LPS;TLR4 ligand) (Matzinger, 2002a, Matzinger, 2002b), but can include endogenous danger-associated molecular patterns (DAMPs) released during ischemic stress or during the subsequent restoration of blood flow (reperfusion) (Medzhitov, 2001).
Evidence supporting the involvement of the TLR signaling pathway in the innate immune response to brain ischemia includes elevated TLR4 message levels in blood cells of patients with acute cerebral ischemia compared to controls (Yang et al., 2008) and protection against cerebral ischemia in mice with Toll-like receptor deletions/mutations (Caso et al., 2007, Ziegler et al., 2007, Tang et al., 2007). While these studies focus on upstream TLR signaling events, very little is known about the molecules that propagate TLR signals downstream during cerebral ischemia. Understanding these downstream signaling events is important as it may lead to the identification of specific points along the pathway that can be modulated during cerebral ischemia. Also, there is evidence that downstream TLR signaling pathways may be organ-specific. For instance in cardiac ischemia, disruption of myeloid differentiation factor 88 (MyD88) signaling is protective (Hua et al., 2005). In hepatic ischemia–reperfusion, however, downstream TLR signaling seems to occur via the Toll/IL-1 Receptor-containing adapter inducing IFN-β (TRIF) as opposed to the MyD88 pathway (Tsung et al., 2006). In addition, in renal ischemia, downstream TLR 2 signaling seems to be mediated via a MyD88-independent pathway, as mice with deletions of TLR2 are better protected from renal ischemia compared to mice with disruptions of the currently known TLR2 downstream adaptor, MyD88 (Shigeoka et al., 2007).
Currently, downstream TLR signaling is known to occur via two major pathways, the MyD88-dependent pathway and the MyD88-independent pathway or TRIF pathway (Kumar et al., 2009).
In light of the fact that the Toll-like receptors previously shown to be important in cerebral ischemia both use the MyD88 and TRIF adaptors for downstream signaling, we sought to systematically determine whether one, both, or neither, of these two known TLR adaptors, MyD88 and TRIF, were important or preferentially used in downstream TLR signaling during cerebral ischemia. We used two in vitro and two in vivo models of cerebral ischemia in our study.
Section snippets
Confirmation of MyD88−/− and TRIF mutant genotypes
Polymerase Chain Reaction (PCR): PCR of tail DNA, from all 3 types of mice, using MyD88 primers, to confirm the genotype of MyD88−/− mice compared to TRIF mutant and WT mice, yielded the expected DNA size fragments (data not shown).
MyD88 knock-down does not affect PC12 cell viability in vitro
PC12 cells transfected
Discussion
Our results showed that neither deletion of MyD88 nor disruption of TRIF had any clear effect on 1) cell viability in in vitro ischemia, 2) CA1 neuron survival in global forebrain ischemia or 3) on infarct size in a model of permanent focal ischemia in contrast to the previously reported roles of MyD88 and TRIF in downstream TLR signaling in cardiac and hepatic ischemia respectively (Hua et al., 2005, Tsung et al., 2006). However, there was an unexpected trend toward larger infarct volumes in
Animals
We used 12- to 16-week-old, male, WT C57BL6/J and TRIF mutant mice (Jackson Laboratories) and MyD88 knock-out mice (breeder pairs from Institute for Systems Biology, Seattle, WA). Genotypes of both MyD88−/− and TRIF mutant mice were confirmed by Polymerase Chain Reaction (PCR) and DNA sequencing. WT C57BL6/J mice served as controls since both TRIF mutant and MyD88 knock-out mice were on a C57BL6/J background. All mice were housed and bred together in the institutional animal facility. Mice had
Disclosures
We have no conflict of interest and nothing to disclose.
Acknowledgments
Dr. Maria Spatz for editorial and scientific suggestions and Dr. Sungyoung Auh for help with the statistical analysis. This research was supported by the Intramural Research Program of the National Institutes of Health, National institute of Neurological Disorders and Stroke.
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MyD88 contributes to neuroinflammatory responses induced by cerebral ischemia/reperfusion in mice
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2015, Respiratory Physiology and NeurobiologyCitation Excerpt :Some indicate that MyD88 contributes to ischemic injury and both MYD88 and TRIF involved in development of postoperative cognitive dysfunction (Gao et al., 2009; Wang et al., 2013). While other reports indicate that knocking out of MyD88 or TRIF does not confer protection in ischemic brain damage (Famakin et al., 2011; Hua et al., 2009b; Yang et al., 2011). The molecular mechanisms underlying these effects are not clear.
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2015, Brain, Behavior, and ImmunityCitation Excerpt :Higher susceptibility to neuronal death was already reported in MyD88-deficient mice following NO-induced excitoxicity (Simard and Rivest, 2007). Two different studies using mouse model of stroke reported no differences between WT, MyD88−/− and TRIF−/− mice (Famakin et al., 2011; Yang et al., 2008), but Downes et al. recently reported a protective role for MyD88 signaling in hematopoietic cells after middle cerebral artery occlusion (Downes et al., 2013). Our results using the KA model of excitotoxicity differ from these results as we found that MyD88 signaling in CNS resident cells mediates the protective effects.
Chloroquine pretreatment inhibits toll-like receptor 3 signaling after stroke
2013, Neuroscience LettersCitation Excerpt :Because of the limitation of effective therapeutics, it is also a leading cause of serious, long-term disability [19]. Although the cause of ischemic brain injury is multifactorial, increasing experimental evidence suggests an important role for the innate immune system in initiating the inflammatory cascade leading to detrimental changes and poor outcome after stroke [7,13]. Therefore, investigation of post-ischemic inflammation is warranted.
Identification of TLR downstream pathways in stroke patients
2013, Clinical BiochemistryCitation Excerpt :A recent study with models of focal, global, or in vitro ischemia in mice found no significant differences in infarct size or neuron survival in those with deletions or mutations of the downstream TLR signaling adaptor molecules, MyD88 and Toll/interleukin 1 receptor TRIF β. Instead, the infarct sizes showed trends toward being larger in MyD88 −/− and TRIF-mutant mice compared to wild-type (WT) mice following focal ischemia [26]. This raised a question about the potential benefit of targeting TLR pathways for ischemic stroke.