Abstract 3742: Downstream Toll-like Receptor (TLR) Signaling Modulates The Acute Stress Response To Focal Cerebral Ischemia

Abstract

Background: The Toll-like receptor (TLR) signaling pathway plays an important role in the innate immune response to a number of inflammatory conditions and in the innate immune response to ischemia. However, the downstream TLR signaling events that occur during focal ischemia remain poorly understood. We thereby sought to determine the gene expression profile in the brains of MyD88-/- and TRIF mutant mice following focal cerebral ischemia. Methods: Wild type (WT) mice and mice with disruptions of either MyD88 (MyD88-/-) or TRIF (TRIF mutant) were subjected to permanent Middle Cerebral Artery Occlusion (pMCAO). Total RNA was extracted from brain homogenates from 9 mice per group;3 mice per baseline (without surgery),3 and 24h following focal ischemia. Gene profiling of baseline and ischemic brain cortices was assessed by Affymetrix microarrays and confirmed by Real-time Polymerase Chain Reaction (RT-PCR). Ingenuity Pathway Analysis (IPA) was used for functional analyses of microarray data. Results: A total of 1,833 gene fragments, representing 754 known genes, were significantly differentially regulated following ischemia. IPA functional analyses suggested the association of differentially expressed genes with the acute stress response. RT-PCR confirmed the differential expression of acute stress response genes such as NR4A1 (Nur77), Activity-related Cytoskeleton-associated protein (ARC) and zinc finger transcription factors such as Early Growth Response (EGR) 1 and 2, following focal cerebral ischemia. Expression of these acute stress response elements was increased in the brains of WT mice, but were either unchanged, non-significantly increased or decreased with ischemia, in mice with disruptions of either MyD88 or TRIF. Importantly, these findings were associated with significantly increased expression, at baseline, of these acute stress response elements, such as EGR2, in mice with disruptions of MyD88 (9.7-fold; p=0.027) and TRIF (8.3-fold; p=0.015), compared to baseline WT mice. Interestingly, levels of zinc metallopeptidases such as Insulin-degrading enzyme (IDE), important in clearing of amyloid in the brain, known to be modulated by ischemia, were significantly decreased from baseline following focal ischemia in mice with disruptions of MyD88 or TRIF compared to WT mice. Conclusions: We describe a novel role of the MyD88 and TRIF TLR signaling pathways in modulating the acute stress response to ischemia. These downstream elements may also be involved in regulating the expression of enzymes such as IDE, thereby linking these downstream TLR pathways to the clearance of amyloid in the brain. Finally, the aberrant up-regulation, at baseline, of elements related to the acute stress response in mice with disruptions of MyD88 and TRIF, implicates these downstream TLR pathways in the optimal response to ischemic stress and probably also to other types of cellular stress.