Keri B. Vartanian

Multiple Preconditioning Paradigms Converge on Interferon Regulatory Factor-Dependent Signaling to Promote Tolerance to Ischemic Brain Injury

Ischemic tolerance can be induced by numerous preconditioning stimuli, including various Toll-like receptor (TLR) ligands. We have shown previously that systemic administration of the TLR4 ligand LPS or the TLR9 ligand unmethylated CpG oligodeoxynucleotide before transient brain ischemia in mice confers substantial protection against ischemic damage. To elucidate the molecular mechanisms of preconditioning, we compared brain genomic profiles in response to preconditioning with these TLR ligands and with preconditioning via exposure to brief ischemia. We found that exposure to the TLR ligands and brief ischemia induced genomic changes in the brain characteristic of a TLR pathway-mediated response. Interestingly, all three preconditioning stimuli resulted in a reprogrammed response to stroke injury that converged on a shared subset of 13 genes not evident in the genomic profile from brains that were subjected to stroke without prior preconditioning. Analysis of the promoter region of these shared genes showed sequences required for interferon regulatory factor (IRF)-mediated transcription. The importance of this IRF gene network was tested using mice deficient in IRF3 or IRF7. Our data show that both transcription factors are required for TLR-mediated preconditioning and neuroprotection. These studies are the first to discover a convergent mechanism of neuroprotection induced by preconditioning—one that potentially results in reprogramming of the TLR-mediated response to stroke and requires the presence of IRF3 and IRF7.

LPS preconditioning redirects TLR signaling following stroke: TRIF-IRF3 plays a seminal role in mediating tolerance to ischemic injury

BackgroundToll-like receptor 4 (TLR4) is activated in response to cerebral ischemia leading to substantial brain damage. In contrast, mild activation of TLR4 by preconditioning with low dose exposure to lipopolysaccharide (LPS) prior to cerebral ischemia dramatically improves outcome by reprogramming the signaling response to injury. This suggests that TLR4 signaling can be altered to induce an endogenously neuroprotective phenotype. However, the TLR4 signaling events involved in this neuroprotective response are poorly understood. Here we define several molecular mediators of the primary signaling cascades induced by LPS preconditioning that give rise to the reprogrammed response to cerebral ischemia and confer the neuroprotective phenotype.MethodsC57BL6 mice were preconditioned with low dose LPS prior to transient middle cerebral artery occlusion (MCAO). Cortical tissue and blood were collected following MCAO. Microarray and qtPCR were performed to analyze gene expression associated with TLR4 signaling. EMSA and DNA binding ELISA were used to evaluate NFκB and IRF3 activity. Protein expression was determined using Western blot or ELISA. MyD88-/- and TRIF-/- mice were utilized to evaluate signaling in LPS preconditioning-induced neuroprotection.ResultsGene expression analyses revealed that LPS preconditioning resulted in a marked upregulation of anti-inflammatory/type I IFN-associated genes following ischemia while pro-inflammatory genes induced following ischemia were present but not differentially modulated by LPS. Interestingly, although expression of pro-inflammatory genes was observed, there was decreased activity of NFκB p65 and increased presence of NFκB inhibitors, including Ship1, Tollip, and p105, in LPS-preconditioned mice following stroke. In contrast, IRF3 activity was enhanced in LPS-preconditioned mice following stroke. TRIF and MyD88 deficient mice revealed that neuroprotection induced by LPS depends on TLR4 signaling via TRIF, which activates IRF3, but does not depend on MyD88 signaling.ConclusionOur results characterize several critical mediators of the TLR4 signaling events associated with neuroprotection. LPS preconditioning redirects TLR4 signaling in response to stroke through suppression of NFκB activity, enhanced IRF3 activity, and increased anti-inflammatory/type I IFN gene expression. Interestingly, this protective phenotype does not require the suppression of pro-inflammatory mediators. Furthermore, our results highlight a critical role for TRIF-IRF3 signaling as the governing mechanism in the neuroprotective response to stroke.

Endothelial cell cytoskeletal alignment independent of fluid shear stress on micropatterned surfaces

Endothelial cells (ECs) in athero-protective regions are elongated with actin and microtubule fibers aligned parallel to the direction of blood flow. Fluid shear stress (FSS) affects EC shape and functions, but little is known about shape-dependent EC properties that are independent of FSS. To evaluate these properties, ECs were elongated on micropatterned (MP) 25mum wide collagen-coated lanes (MPECs) and characterized by cell shape index, actin and microtubule alignment, and polarization of the microtubule-organizing center (MTOC). ECs on non-patterned surfaces were also exposed to FSS. MPEC elongation was microtubule-dependent (and actin-independent); shape indices and cytoskeletal alignment were comparable to FSS-elongated ECs. Cytoskeletal alignment was lost when MPECs were exposed to perpendicular FSS, but not parallel FSS. MTOC polarization was FSS-dependent. Thus, by isolating EC elongation and cytoskeletal alignment from FSS, micropatterning creates a platform for studying EC shape-related cellular functions that are independent of FSS.

Toll-Like Receptor Tolerance as a Mechanism for Neuroprotection

It has been discovered recently that Toll-like receptors (TLRs) are key mediators of tissue injury in response to stroke. This revelation has identified a new target critical to understanding the underlying mechanisms of stroke injury and potential therapies. Much of the interest in TLRs centers around their ability to self-regulate—a process commonly referred to as “tolerance,” wherein prior exposure to low-level TLR activation induces protection against a subsequent challenge that would otherwise cause damage. This endogenous process has been exploited in the setting of stroke. Recent studies show that TLR pathways can be reprogrammed via prior exposure to TLR ligands, leading to decreased infarct size and improved neurological outcomes in response to ischemic injury. Efforts to understand the molecular mechanisms of TLR reprogramming have led to the identification of multiple routes of TLR regulation including inhibitors that target signaling mediators, microRNAs that suppress genes posttranscriptionally, and epigenetic changes in chromatin remodeling that affect global gene regulation. In this review, we discuss the role of TLRs in mediating injury due to stroke, evidence for TLR-preconditioning-induced TLR reprogramming in response to stroke, and possible mechanisms of TLR-induced neuroprotection.

Poly‐ICLC preconditioning protects the blood–brain barrier against ischemic injury in vitro through type I interferon signaling

Preconditioning with a low dose of harmful stimulus prior to injury induces tolerance to a subsequent ischemic challenge resulting in neuroprotection against stroke. Experimental models of preconditioning primarily focus on neurons as the cellular target of cerebral protection, while less attention has been paid to the cerebrovascular compartment, whose role in the pathogenesis of ischemic brain injury is crucial. We have shown that preconditioning with polyinosinic polycytidylic acid (poly‐ICLC) protects against cerebral ischemic damage. To delineate the mechanism of poly‐ICLC protection, we investigated whether poly‐ICLC preconditioning preserves the function of the blood–brain barrier (BBB) in response to ischemic injury. Using an in vitro BBB model, we found that poly‐ICLC treatment prior to exposure to oxygen‐glucose deprivation maintained the paracellular and transcellular transport across the endothelium and attenuated the drop in transendothelial electric resistance. We found that poly‐ICLC treatment induced interferon (IFN) β mRNA expression in astrocytes and microglia and that type I IFN signaling in brain microvascular endothelial cells was required for protection. Importantly, this implicates a potential mechanism underlying neuroprotection in our in vivo experimental stroke model, where type I IFN signaling is required for poly‐ICLC‐induced neuroprotection against ischemic injury. In conclusion, we are the first to show that preconditioning with poly‐ICLC attenuates ischemia‐induced BBB dysfunction. This mechanism is likely an important feature of poly‐ICLC‐mediated neuroprotection and highlights the therapeutic potential of targeting BBB signaling pathways to protect the brain against stroke.

Cytoskeletal structure regulates endothelial cell immunogenicity independent of fluid shear stress

The cardiovascular disease atherosclerosis is directly linked to the functions of endothelial cells (ECs), which are affected by fluid shear stress (FSS). High, unidirectional FSS causes EC elongation with aligned cytoskeletal components and nonimmunogenic EC functions that protect against atherosclerosis. In contrast, low, oscillatory FSS is associated with cobblestone-shaped ECs with randomly oriented cytoskeletons and proinflammatory EC functions that promote atherosclerosis. Whether EC shape plays a role in EC immunogenic functions, independent of FSS, has not been previously determined. The goal of this study was to determine the effect of EC elongation and cytoskeletal alignment on the expression of inflammatory genes and functions. With the use of micropatterned lanes, EC elongation and cytoskeletal alignment were achieved in the absence of FSS. EC gene expression of key inflammation markers determined that the elongation and cytoskeletal alignment of micropattern-elongated ECs (MPECs) alone significantly downregulated VCAM-1 while having no effect on E-selectin and ICAM-1. The positive control of FSS-elongated ECs promoted E-selectin and VCAM-1 downregulation and upregulation of ICAM-1. Functionally, monocytic U937 cells formed weaker interactions on the surface of MPECs compared with cobblestone ECs. Interestingly, MPEC expression of the known FSS-dependent transcription factor krüppel-like factor 2 (KLF2), which promotes a nonimmunogenic EC phenotype, was significantly upregulated in MPECs compared with cobblestone ECs. Cytoskeletal regulation of KLF2 expression was shown to be dependent on microtubules. Therefore, the cellular elongation and cytoskeletal alignment of MPECs regulated immunogenic gene expression and functions and may act synergistically with FSS to create an EC surface with reduced inflammatory capability.

Distinct extracellular matrix microenvironments of progenitor and carotid endothelial cells.

Endothelial cells (ECs) produce and maintain the local extracellular matrix (ECM), a critical function that contributes to EC and blood vessel health. This function is also crucial to vascular tissue engineering, where endothelialization of vascular constructs require a cell source that readily produces and maintains ECM. In this study, baboon endothelial progenitor cell (EPC) deposition of ECM (laminin, collagen IV, and fibronectin) was characterized and compared to mature carotid ECs, evaluated in both elongated and cobblestone morphologies typically found in vivo. Microfluidic micropatterning was used to create 15-microm wide adhesive lanes with 45-microm spacing to reproduce the elongated EC morphology without the influence of external forces. Both EPCs and ECs elongated on micropatterned lanes had aligned actin cytoskeleton and readily deposited ECM. EPCs deposited and remodeled the ECM to a greater extent than ECs. Since a readily produced ECM can improve graft patency, EPCs are an advantageous cell source for endothelializing vascular constructs. Furthermore, EC deposition of ECM was dependent on cell morphology, where elongated ECs deposited more collagen IV and less fibronectin compared to matched cobblestone controls. Thus micropatterned surfaces controlled EC shape and ECM deposition, which ultimately has implications for the design of tissue-engineered vascular constructs.

Reprogramming the Response to Stroke by Preconditioning

Stroke causes neuronal injury and death because of deprivation of oxygen and nutrients that are essential for cell survival. In addition, inflammatory mediators are released that trigger a cascade of responses that exacerbate injury. Although these injurious pathways are well defined, it has been a major challenge to identify ways to mitigate these pathways to reduce ischemic damage. One approach to the management of stroke injury under development in the research laboratory involves tapping into powerful endogenous mechanisms of protection through a process known as preconditioning. Preconditioning is a well-defined phenomenon whereby a small dose of an otherwise-harmful stimulus confers tolerance to a subsequent injurious event. Preconditioning stimuli that provide significant protection against ischemic brain injury include exposure to brief ischemia, small seizures, immune activation, exposure to hypo- and hyperthermia, and inhalation of volatile anesthetics.1 Although distinct, these preconditioning stimuli initiate a cascade of endogenous neuroprotective pathways that produce tolerance to ischemic injury.2–7 Preconditioning with the immune activators Toll-like receptor (TLR) ligands has shown exceptional efficacy in the induction of ischemic tolerance. Systemic administration of ligands for TLR2, TLR4, TLR7, or TLR9 before focal cerebral ischemia profoundly reduces ischemic injury in rodent models of stroke.8–12 TLR preconditioning has also been shown to be effective in a neonatal ischemia model, demonstrating significant cerebral protection against hypothermic circulatory arrest in neonatal pigs.13 In addition, the TLR9 ligand has shown significant efficacy in a clinically relevant nonhuman primate model of experimental stroke.14 Many TLR ligands have been approved for clinical use in other indications,15 making them ideal candidates for translation of pharmacological preconditioning from the laboratory to the clinic. Preconditioning is being developed as a prophylactic treatment for cerebral ischemia and is being investigated at …

TLR9 bone marrow chimeric mice define a role for cerebral TNF in neuroprotection induced by CpG preconditioning

Systemic preconditioning with the TLR9 ligand CpG induces neuroprotection against brain ischemic injury through a tumor necrosis factor (TNF)-dependent mechanism. It is unclear how systemic administration of CpG engages the brain to induce the protective phenotype. To address this, we created TLR9-deficient reciprocal bone marrow chimeric mice lacking TLR9 on either hematopoietic cells or radiation-resistant cells of nonhematopoietic origin. We report that wild-type mice reconstituted with TLR9-deficient hematopoietic cells failed to show neuroprotection after systemic CpG preconditioning. Further, while hematopoietic expression of TLR9 is required for CpG-induced neuroprotection it is not sufficient to restore protection to TLR9-deficient mice that are reconstituted with hematopoietic cells bearing TLR9. To determine whether the absence of protection was associated with TNF, we examined TNF levels in the systemic circulation and the brain. We found that although TNF is required for CpG preconditioning, systemic TNF levels did not correlate with the protective phenotype. However, induction of cerebral TNF mRNA required expression of TLR9 on both hematopoietic and nonhematopoietic cells and correlated with neuroprotection. In accordance with these results, we show the therapeutic potential of intranasal CpG preconditioning, which induces brain TNF mRNA and robust neuroprotection with no concomitant increase in systemic levels of TNF.

CpG preconditioning regulates miRNA expression that modulates genomic reprogramming associated with neuroprotection against ischemic injury

Cytosine-phosphate-guanine (CpG) preconditioning reprograms the genomic response to stroke to protect the brain against ischemic injury. The mechanisms underlying genomic reprogramming are incompletely understood. MicroRNAs (miRNAs) regulate gene expression; however, their role in modulating gene responses produced by CpG preconditioning is unknown. We evaluated brain miRNA expression in response to CpG preconditioning before and after stroke using microarray. Importantly, we have data from previous gene microarrays under the same conditions, which allowed integration of miRNA and gene expression data to specifically identify regulated miRNA gene targets. CpG preconditioning did not significantly alter miRNA expression before stroke, indicating that miRNA regulation is not critical for the initiation of preconditioning-induced neuroprotection. However, after stroke, differentially regulated miRNAs between CpG- and saline-treated animals associated with the upregulation of several neuroprotective genes, implicating these miRNAs in genomic reprogramming that increases neuroprotection. Statistical analysis revealed that the miRNA targets were enriched in the gene population regulated in the setting of stroke, implying that miRNAs likely orchestrate this gene expression. These data suggest that miRNAs regulate endogenous responses to stroke and that manipulation of these miRNAs may have the potential to acutely activate novel neuroprotective processes that reduce damage.