Disruption of downstream MyD88 or TRIF Toll-like receptor signaling does not protect against cerebral ischemia

Abstract

Toll-like receptor (TLR) signaling plays an important role in cerebral ischemia, but downstream signaling events, which can be organ-specific, are incompletely understood. We thereby investigated involvement of the MyD88-dependent (MyD88) and MyD88-independent (TRIF) TLR signaling pathways in 2 in vitro and 2 in vivo models of cerebral ischemia. For in vitro studies, we used a model of oxygen-glucose deprivation (OGD) followed by flow cytometric analysis to determine:1) viability of PC12 cells following knock-down with MyD88 siRNA compared to negative control siRNA and 2) viability, apoptosis and necrosis of cortical neurons from MyD88 null (-/-), TRIF mutant, and wild type (WT) mice. In addition, in vivo, 1) We examined CA1 neuronal survival 7 days after global forebrain ischemia and 2) infarct volumes 24h after Middle Cerebral Artery Occlusion (MCAO) in all 3 types of mice. OGD: 1) There were no differences in either percent viability of PC12 cells transfected with MyD88 compared to negative control siRNA or 2) in percent viability, apoptosis and necrosis of cortical neurons from MyD88-/-,TRIF mutant and WT mice. Global ischemia: neuronal survival was similar in all 3 groups of mice. Finally, MCAO: infarct volumes were not statistically different among all 3 groups of mice: MyD88 -/-, 23.9 ± 9.9 mm3, TRIF mutant, 26.7 ± 5.8mm3 and WT, 17.9 ± 8.4mm3. These findings show that disruption of MyD88 or TRIF signaling does not confer protection in brain ischemia and suggests the possibility of additional or alternate downstream adaptors during TLR signaling in cerebral ischemia.

A New Role for Downstream Toll-like Receptor Signaling in Mediating Immediate Early Gene Expression during Focal Cerebral Ischemia

“…However, our previous studies showed similar expression of MyD88-mediated cytokines in TRIF-mutant and WT mice, 11 but mice with disruption of TRIF were not protected against focal cerebral ischemia. 12 We postulate that the baseline cytokine changes, reported in our previous study, 11 were limited to the MyD88 versus WT comparison, in part, because immediate early genes, such as EGR1, which was increased at baseline in MyD88 À / À and TRIF-mutant mice, compared to WT mice, in our current study, are known to affect the expression of MyD88-specific cytokines, such as IL-6. 28,29 Taken together, these previous results of a cytokine profile similar to WT mice, and the current additional role for TRIF in ischemia-mediated immediate early gene expression, suggest that both optimal cytokine and immediate early gene expression might be essential for the optimal acute phase response to focal cerebral ischemia.…”

“…26 However, disruption of MyD88 signaling does not protect against focal cerebral ischemia, in in vitro and in vivo models. 12 Surprisingly, this lack of protection occurs in spite of decreased levels of proinflammatory cytokines in the serum and brains of MyD88 À / À mice following focal cerebral ischemia. 11 To date, a molecular basis for these confounding results has not been known.…”

Novel therapeutic strategies targeting innate immune responses and early inflammation after stroke

“…However, in an experiment using TLR2-or TLR4-deficient mice with wild-type BM, no improvement was observed in ischemic brain injury (Yang et al 2011;Shichita et al 2012). Moreover, mice lacking MyD88, the adaptor protein required for almost all TLR signaling cascades (other than TLR3), showed either no improvement or exaggeration of ischemic brain injury (Famakin et al 2011). These results indicate that the function of TLRs is dependent of the cell types in the brain and the timing of their activation.…”

The role of Toll-like receptors (TLRs) in stroke

“…However, Famakin et al illustrated that the elimination of neither MyD88 adaptor nor TRIF does protect against ischemia. They performed the experiments in OGD cell culture and PC12 cell viability as two in vitro models and two in vivo models of permanent middle cerebral artery occlusion and global cerebral ischemia (Famakin et al, 2011). The investigation did not point to a significant difference among groups.…”