Frances Rena Bahjat

Proof of Concept: Pharmacological Preconditioning with a Toll-like Receptor Agonist Protects against Cerebrovascular Injury in a Primate Model of Stroke

Cerebral ischemic injury is a significant portion of the burden of disease in developed countries; rates of mortality are high and the costs associated with morbidity are enormous. Recent therapeutic approaches have aimed at mitigating the extent of damage and/or promoting repair once injury has occurred. Often, patients at high risk of ischemic injury can be identified in advance and targeted for antecedent neuroprotective therapy. Agents that stimulate the innate pattern recognition receptor, Toll-like receptor 9, have been shown to induce tolerance (precondition) to ischemic brain injury in a mouse model of stroke. Here, we demonstrate for the first time that pharmacological preconditioning against cerebrovascular ischemic injury is also possible in a nonhuman primate model of stroke in the rhesus macaque. The model of stroke used is a minimally invasive transient vascular occlusion, resulting in brain damage that is primarily localized to the cortex and as such, represents a model with substantial clinical relevance. Finally, K-type (also referred to as B-type) cytosine-guanine-rich DNA oligonucleotides, the class of agents employed in this study, are currently in use in human clinical trials, underscoring the feasibility of this treatment in patients at risk of cerebral ischemia.

Poly-IC preconditioning protects against cerebral and renal ischemia-reperfusion injury

Preconditioning induces ischemic tolerance, which confers robust protection against ischemic damage. We show marked protection with polyinosinic polycytidylic acid (poly-IC) preconditioning in three models of murine ischemia-reperfusion injury. Poly-IC preconditioning induced protection against ischemia modeled in vitro in brain cortical cells and in vivo in models of brain ischemia and renal ischemia. Further, unlike other Toll-like receptor (TLR) ligands, which generally induce significant inflammatory responses, poly-IC elicits only modest systemic inflammation. Results show that poly-IC is a new powerful prophylactic treatment that offers promise as a clinical therapeutic strategy to minimize damage in patient populations at risk of ischemic injury.

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.

Steps to Translate Preconditioning from Basic Research to the Clinic

Efforts to treat cardiovascular and cerebrovascular diseases often focus on the mitigation of ischemia–reperfusion (I/R) injury. Many treatments or “preconditioners” are known to provide substantial protection against I/R injury when administered prior to the event. Brief periods of ischemia itself have been validated as a means to achieve neuroprotection in many experimental disease settings, in multiple organ systems, and in multiple species suggesting a common pathway leading to tolerance. In addition, pharmacological agents that act as potent preconditioners have been described. Experimental induction of neuroprotection using these various preconditioning paradigms has provided a unique window into the brains endogenous protective mechanisms. Moreover, preconditioning agents themselves hold significant promise as clinical-stage therapies for prevention of I/R injury. The aim of this article is to explore several key steps involved in the preclinical validation of preconditioning agents prior to the conduct of clinical studies in humans. Drug development is difficult, expensive, and relies on multifactorial analysis of data from diverse disciplines. Importantly, there is no single path for the preclinical development of a novel therapeutic and no proven strategy to ensure success in clinical translation. Rather, the conduct of a diverse array of robust preclinical studies reduces the risk of clinical failure by varying degrees depending upon the relevance of preclinical models and drug pharmacology to humans. A strong sense of urgency and high tolerance of failure are often required to achieve success in the development of novel treatment paradigms for complex human conditions.

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.

Changes in spontaneous activity assessed by accelerometry correlate with extent of cerebral ischemia-reperfusion injury in the nonhuman primate.

The use of accelerometry to monitor activity in human stroke patients has revealed strong correlations between objective activity measurements and subjective neurological findings. The goal of our study was to assess the applicability of accelerometry-based measurements in experimental animals undergoing surgically induced cerebral ischemia. Using a nonhuman primate cortical stroke model, we demonstrate for the first time that monitoring locomotor activity prior to and following cerebrovascular ischemic injury using an accelerometer is feasible in adult male rhesus macaques and that the measured activity outcomes significantly correlate with severity of brain injury. The use of accelerometry as an unobtrusive, objective preclinical efficacy determinant could complement standard practices involving subjective neurological scoring and magnetic resonance imaging in nonhuman primates. Similar activity monitoring devices to those employed in this study are currently in use in human clinical studies, underscoring the feasibility of this approach for assessing the clinical potential of novel treatments for cerebral ischemia.

Cytosolic Receptor Melanoma Differentiation-Associated Protein 5 Mediates Preconditioning-Induced Neuroprotection Against Cerebral Ischemic Injury.

Background and Purpose— Preconditioning with poly-L-lysine and carboxymethylcellulose (ICLC) provides robust neuroprotection from cerebral ischemia in a mouse stroke model. However, the receptor that mediates neuroprotection is unknown. As a synthetic double-stranded RNA, poly-ICLC may bind endosomal Toll-like receptor 3 or one of the cytosolic retinoic acid–inducible gene-I–like receptor family members, retinoic acid–inducible gene-I, or melanoma differentiation–associated protein 5. Activation of these receptors culminates in type I interferons (IFN-&agr;/&bgr;) induction—a response required for poly-ICLC–induced neuroprotection. In this study, we investigate the receptor required for poly-ICLC–induced neuroprotection. Methods— Toll-like receptor 3, melanoma differentiation–associated protein 5-, and IFN-promoter stimulator 1–deficient mice were treated with poly-ICLC 24 hours before middle cerebral artery occlusion. Infarct volume was measured 24 hours after stroke to identify the receptor signaling pathways involved in protection. IFN-&agr;/&bgr; induction was measured in plasma samples collected 6 hours after poly-ICLC treatment. IFN-&bgr;–deficient mice were used to test the requirement of IFN-&bgr; for poly-ICLC–induced neuroprotection. Mice were treated with recombinant IFN-&agr;-A to test the role of IFN-&agr; as a potential mediator of neuroprotection. Results— Poly-ICLC induction of both neuroprotection and systemic IFN-&agr;/&bgr; requires the cytosolic receptor melanoma differentiation–associated protein 5 and the adapter molecule IFN-promoter stimulator 1, whereas it is independent of Toll-like receptor 3. IFN-&bgr; is not required for poly-ICLC–induced neuroprotection. IFN-&agr; treatment protects against stroke. Conclusions— Poly-ICLC preconditioning is mediated by melanoma differentiation–associated protein 5 and its adaptor molecule IFN-promoter stimulator 1. This is the first evidence that a cytosolic receptor can mediate neuroprotection, providing a new target for the development of therapeutic agents to protect the brain from ischemic injury.

Preconditioning in the Rhesus Macaque Induces a Proteomic Signature Following Cerebral Ischemia that Is Associated with Neuroprotection

Each year, thousands of patients are at risk of cerebral ischemic injury, due to iatrogenic responses to surgical procedures. Prophylactic treatment of these patients as standard care could minimize potential neurological complications. We have shown that protection of brain tissue, in a non-human primate model of cerebral ischemic injury, is possible through pharmacological preconditioning using the immune activator D192935. We postulate that preconditioning with D192935 results in neuroprotective reprogramming that is evident in the brain following experimentally induced cerebral ischemia. We performed quantitative proteomic analysis of cerebral spinal fluid (CSF) collected post-stroke from our previously published efficacy study to determine whether CSF protein profiles correlated with induced protection. Four groups of animals were examined: naïve animals (no treatment or stroke); animals treated with vehicle prior to stroke; D192935 treated and stroked animals, further delineated into two groups, ones that were protected (small infarcts) and those that were not protected (large infarcts). We found that distinct protein clusters defined the protected and non-protected animal groups, with a 16-member cluster of proteins induced exclusively in D192935 protected animals. Seventy percent of the proteins induced in the protected animals have functions that would enhance neuroprotection and tissue repair, including several members associated with M2 macrophages, a macrophage phenotype shown to contribute to neuroprotection and repair during ischemic injury. These studies highlight the translational importance of CSF biomarkers in defining mechanism and monitoring responses to treatment in development of stroke therapeutics.

Toll-Like Receptors in Ischemic Stroke and Other Acute Brain Injuries

Each year a substantial number of Americans suffer from hypoxic injury to the brain due to diminished blood flow and few effective treatments are available. A fruitful area of current investigation involves toll-like receptors (TLRs), which are a family of highly conserved receptors that play a key role in the pathology of brain injury. Studies in animals deficient in specific TLRs as well as genetic data from patients with altered TLR biology suggest that the activation of TLRs exacerbates damage in the setting of ischemia. Paradoxically, the stimulation of TLRs prior to injury is known to induce a state of tolerance to subsequent ischemic injury or “preconditioning”. Such preconditioning results in a profound neuroprotective effect and the mechanisms involved are under intense investigation. Understanding these divergent roles of TLRs in brain injury and neuroprotection offers great promise in the discovery of new therapeutic targets and the mitigation of ischemic brain injury in “at risk” patients through the use of prophylactic TLR stimulation as a therapeutic strategy. This chapter focuses on these two divergent roles of TLRs—one role that promotes and another that prevents ischemic injury in the brain in the context of stroke and other acute brain injuries.

Toll-like receptor agonists as antecedent therapy for ischemic brain injury: Advancing preclinical studies to the nonhuman primate

Antecedent therapy for ischemic brain injury has the potential to protect a large, high-risk patient population from the devastating effects of cerebral ischemia associated with cardiac surgery. Substantial evidence has shown that preconditioning with a modestly damaging stimulus induces powerful endogenous neuroprotection. Pharmacological agents that stimulate toll-like receptors (TLRs) induce robust neuroprotective effects as preconditioning stimuli against cerebral ischemia in mouse and nonhuman primate models of stroke. Here we describe the progress of our preclinical development of TLR agonists as antecedent therapy against cerebral ischemic injury. The objective was to discuss studies that begin with in vitro validation in cell cultures to in vivo efficacy studies using mouse and nonhuman primate models of stroke, with particular emphasis on the TLR9 agonist CpG oligonucleotide. We provide an in-depth discussion of our novel rhesus macaque stroke model and cover the progress we have made in therapeutic testing and evaluation in these animals. These studies represent a logical path for the development of TLR agonists as antecedent therapy for the prevention of the damaging neurological complications resulting from cerebral ischemic injury.