The TRIF-dependent signaling pathway is not required for acute cerebral ischemia/reperfusion injury in mice

Abstract

TIR domain-containing adaptor protein (TRIF) is an adaptor protein in Toll-like receptor (TLR) signaling pathways. Activation of TRIF leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB). While studies have shown that TLRs are implicated in cerebral ischemia/reperfusion (I/R) injury and in neuroprotection against ischemia afforded by preconditioning, little is known about TRIF’s role in the pathological process following cerebral I/R. The present study investigated the role that TRIF may play in acute cerebral I/R injury. In a mouse model of cerebral I/R induced by transient middle cerebral artery occlusion, we examined the activation of NF-κB and IRF3 signaling in ischemic cerebral tissue using ELISA and Western blots. Neurological function and cerebral infarct size were also evaluated 24 h after cerebral I/R. NF-κB activity and phosphorylation of the inhibitor of kappa B (IκBα) increased in ischemic brains, but IRF3, inhibitor of κB kinase complex-ε (IKKε), and TANK-binding kinase1 (TBK1) were not activated after cerebral I/R in wild-type (WT) mice. Interestingly, TRIF deficit did not inhibit NF-κB activity or p-IκBα induced by cerebral I/R. Moreover, although cerebral I/R induced neurological and functional impairments and brain infarction in WT mice, the deficits were not improved and brain infarct size was not reduced in TRIF knockout mice compared to WT mice. Our results demonstrate that the TRIF-dependent signaling pathway is not required for the activation of NF-κB signaling and brain injury after acute cerebral I/R.

Introduction

Cerebral ischemia is the third leading cause of death and remains the leading cause of long-term disability in the United States. Current evidence suggests that the inflammatory responses to injury initiated by the innate immune system contribute to the pathogenesis of cerebral ischemia/reperfusion (I/R) [1], [2]. Many reports indicate that Toll-like receptors (TLRs) are implicated in the pathological processes of cerebral I/R injury, as well as in neuroprotection against ischemia afforded by preconditioning [3], [4], [5], [6], [7].

TLRs are a family of signaling molecules which play a critical role in mediating immunity and inflammation [8], [9], [10]. At present, at least ten human TLRs have been identified [11], [12] and eleven TLRs have been found in mice [13]. TLR signaling is mediated by cytosolic adaptor molecules that bind to the intracellular domain of TLRs. One of the intracellular adaptors, myeloid differentiation primary-response protein-88 (MyD88), recruits serial downstream kinases and then phosphorylates the inhibitor of κB (IκB), resulting in nuclear translocation of NF-κB and the transcription of genes associated with innate immune and inflammation (Fig. 1). This MyD88-dependent pathway is shared by all the TLRs with the exception of TLR3 [14]. TLR signaling is also mediated through another adaptor, TIR domain-containing adaptor protein (TRIF), which activates TANK-binding kinase1 (TBK1) and inhibitor of κB kinase complex-ε (IKKε), leads to the activation of IFN regulatory factor 3 (IRF3), and thereby activates interferon-β (IFN-β) and IFN-β-inducible genes (Fig. 1). This TRIF-dependent pathway is unique to TLR3 and TLR4 signaling [15], [16], [17]. Thus, TLR4 is the only TLR that mediates both MyD88-dependent and TRIF-dependent pathways. Of all the TLRs, TLR4 has been the most extensively investigated and implicated in ischemic brain injury. Transcription and expression of TLR4 increased and TLR4-mediated NF-κB signaling was activated in ischemic brain, which induced in situ inflammatory responses and contributed to brain damage after cerebral I/R [4], [5]. Modulation of TLR4 inhibited the activation of NF-κB, decreased inflammatory responses and attenuated brain damage induced by cerebral I/R [4], [5], [7]. In contrast, Marsh et al. reported that activation of TLR4 by a specific ligand, lipopolysaccharide (LPS), provides neuroprotection against subsequent cerebral ischemic injury which can be attributed to the activation of TLR4-mediated TRIF/IRF3 signaling, and activation of TRIF reduces neuronal death [18]. In this experiment we hoped to determine whether TRIF-dependent signaling plays a role in the acute brain damage that accompanies cerebral I/R.