Philberta Y. Leung

Systemic lipopolysaccharide protects the brain from ischemic injury by reprogramming the response of the brain to stroke: a critical role for IRF3.

Lipopolysaccharide (LPS) preconditioning provides neuroprotection against subsequent cerebral ischemic injury through activation of its receptor, Toll-like receptor 4 (TLR4). Paradoxically, TLR activation by endogenous ligands after ischemia worsens stroke damage. Here, we define a novel, protective role for TLRs after ischemia in the context of LPS preconditioning. Microarray analysis of brains collected 24 h after stroke revealed a unique set of upregulated genes in LPS-pretreated animals. Promoter analysis of the unique gene set identified an overrepresentation of type I interferon (IFN)-associated transcriptional regulatory elements. This finding suggested the presence of type I IFNs or interferon regulatory factors (IRFs), which upregulate interferon-stimulated genes. Upregulation of IFNβ was confirmed by real-time reverse transcription-PCR. Direct administration of IFNβ intracerebroventricularly at the time of stroke was sufficient for neuroprotection. TLR4 can induce both IFNβ and interferon-stimulated genes through its adapter molecule Toll/interleukin receptor domain-containing adaptor-inducing IFNβ (TRIF) and the IRF3 transcription factor. We show in oxygen glucose deprivation of cortical neurons, an in vitro model of stroke, that activation of TRIF after stroke reduces neuronal death. Furthermore, mice lacking IRF3 were not protected by LPS preconditioning in our in vivo model. Our studies constitute the first demonstration of the neuroprotective capacity of TRIF/IRF3 signaling and suggest that interferon-stimulated genes, whether induced by IFNβ or by enhanced TLR signaling to IRF3, are a potent means of protecting the brain against ischemic damage.

Factor XII inhibition reduces thrombus formation in a primate thrombosis model

The plasma zymogens factor XII (fXII) and factor XI (fXI) contribute to thrombosis in a variety of mouse models. These proteins serve a limited role in hemostasis, suggesting that antithrombotic therapies targeting them may be associated with low bleeding risks. Although there is substantial epidemiologic evidence supporting a role for fXI in human thrombosis, the situation is not as clear for fXII. We generated monoclonal antibodies (9A2 and 15H8) against the human fXII heavy chain that interfere with fXII conversion to the protease factor XIIa (fXIIa). The anti-fXII antibodies were tested in models in which anti-fXI antibodies are known to have antithrombotic effects. Both anti-fXII antibodies reduced fibrin formation in human blood perfused through collagen-coated tubes. fXII-deficient mice are resistant to ferric chloride-induced arterial thrombosis, and this resistance can be reversed by infusion of human fXII. 9A2 partially blocks, and 15H8 completely blocks, the prothrombotic effect of fXII in this model. 15H8 prolonged the activated partial thromboplastin time of baboon and human plasmas. 15H8 reduced fibrin formation in collagen-coated vascular grafts inserted into arteriovenous shunts in baboons, and reduced fibrin and platelet accumulation downstream of the graft. These findings support a role for fXII in thrombus formation in primates.

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.

Inhibition of factor XI activation attenuates inflammation and coagulopathy while improving the survival of mouse polymicrobial sepsis

Severe bacterial sepsis often leads to a systemic procoagulant and proinflammatory condition that can manifest as disseminated intravascular coagulation, septic shock, and multiple organ failure. Because activation of the contact proteases factor XII (FXII), prekallikrein, and factor XI (FXI) can trigger coagulation and inflammatory responses, the contact factors have been considered potential targets for the treatment of sepsis. However, the pathogenic role of contact activation in severe infections has not been well defined. We therefore investigated whether an anticoagulant antibody (14E11) that selectively inhibits prothrombotic FXI activation by activated FXII (FXIIa) modifies the course of bowel perforation-induced peritoneal sepsis in mice. Early anticoagulation with 14E11 suppressed systemic thrombin- antithrombin complex formation, IL-6, and TNF-α levels, and reduced platelet consumption in the circulation and deposition in the blood vessels. Treatment with 14E11 within 12 hours after bowel perforation significantly improved survival compared with vehicle treatment, and the saturating dose did not increase tail bleeding. These data suggest that severe polymicrobial abdominal infection induces prothrombotic FXI activation, to the detriment of the host. Systemic anticoagulation by inhibiting FXI activation or FXIIa procoagulant activity during sepsis may therefore limit the development of disseminated intravascular coagulation without increasing bleeding risks.

Toll-Like Receptor 7 Preconditioning Induces Robust Neuroprotection Against Stroke by a Novel Type I Interferon-Mediated Mechanism

Background and Purpose— Systemic administration of Toll-like receptor (TLR) 4 and TLR9 agonists before cerebral ischemia have been shown to reduce ischemic injury by reprogramming the response of the brain to stroke. Our goal was to explore the mechanism of TLR-induced neuroprotection by determining whether a TLR7 agonist also protects against stroke injury. Methods— C57Bl/6, TNF−/−, interferon (IFN) regulatory factor 7−/−, or type I IFN receptor (IFNAR)−/− mice were subcutaneously administered the TLR7 agonist Gardiquimod (GDQ) 72 hours before middle cerebral artery occlusion. Infarct volume and functional outcome were determined after reperfusion. Plasma cytokine responses and induction of mRNA for IFN-related genes in the brain were measured. IFNAR−/− mice also were treated with the TLR4 agonist (lipopolysaccharide) or the TLR9 agonist before middle cerebral artery occlusion and infarct volumes measured. Results— The results show that GDQ reduces infarct volume as well as functional deficits in mice. GDQ pretreatment provided robust neuroprotection in TNF−/− mice, indicating that TNF was not essential. GDQ induced a significant increase in plasma IFN&agr; levels and both IRF7−/− and IFNAR−/− mice failed to be protected, implicating a role for IFN signaling in TLR7-mediated protection. Conclusions— Our studies provide the first evidence that TLR7 preconditioning can mediate neuroprotection against ischemic injury. Moreover, we show that the mechanism of protection is unique from other TLR preconditioning ligands in that it is independent of TNF and dependent on IFNAR.

It's all in the family: multiple Toll-like receptors offer promise as novel therapeutic targets for stroke neuroprotection

Ischemic tolerance is a biological process that can be utilized to unlock the brains own endogenous protection mechanisms and, as such, holds true promise for patients at risk of ischemic injury. Experimentally, preconditioning with various Toll-like receptor (TLR) agonists has now been demonstrated to successfully attenuate ischemic damage, partly through genomic reprogramming of the bodys response to stroke. This treatment diminishes the inflammatory response to stroke and at the same time enhances the production of anti-inflammatory cytokines and neuroprotective mediators. This review discusses recent discoveries about the role of TLRs in preconditioning and ischemic tolerance.

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.

A New Paradigm in Protecting Ischemic Brain: Preserving the Neurovascular Unit Before Reperfusion

Of the ~795,000 strokes that occur each year in the USA, ~695,000 are ischemic strokes (IS) where a clot occludes a major cerebral artery. About half of these IS patients present with so-called penumbra, defined as a hypoperfused tissue immediately surrounding the ischemic core that is severely deprived of oxygen and at risk for deterioration. Collateral vessels can provide sufficient oxygen and nutrients to temporarily maintain neuronal structure in the penumbra but not enough to support function. Thus, the at-risk tissue has the potential for functional recovery if blood flow is restored, but will irreversibly infarct if recanalization is not achieved, resulting in neurological deterioration. Additionally, though collateral circulation can transiently maintain penumbra viability, injury mechanisms such as excitotoxicity and ATP depletion will have already been initiated. Thus, it is imperative to administer therapies that can alleviate ischemia-induced cell death, restore energy metabolism, and halt pathogenic cascades as soon as possible after occlusion in order to protect the at-risk tissue until reperfusion therapies can be employed. Excitingly, the recent breakthroughs in acute IS reperfusion therapy have opened new opportunities for such adjunct neuroprotective treatments. This chapter provides a description of the penumbra tissue, followed by a brief overview of the emerging standard of care for acute IS based on the recent positive clinical trials using IV tPA and mechanical thrombectomy devices. We will then describe the promising use of adjunctive therapies to enhance the benefits of recanalization therapies. In particular, we will discuss the concept of oxygen therapy and oxygen carriers as a valid approach for “combination therapy” to protect the penumbra until reperfusion. Finally, we will discuss the future challenges of clinical trials in acute IS patients and highlight the need for new trial designs to test the potential benefit of combination therapies.

Abstract 4686: Omx a hypoxia modulator reverses the immunosuppressive glioblastoma microenvironment by stimulating T cell infiltration and activation that results in increased number of long-term survivors

Oxygen is one of the key modulators of tumor microenvironment whereby low oxygen or hypoxia is associated with resistance to chemo- and radio- therapies and poor patient outcomes. Hypoxia favors an immunosuppressive tumor microenvironment by promoting Treg recruitment and activation and suppressing T cell and NK cell proliferation and effector function and pro-inflammatory cytokine secretion. Therefore, reversing tumor hypoxia could create an immunopermissive microenvironment and improve the efficacy of several immunotherapies. Omniox has developed an oxygen carrier OMX that can specifically deliver oxygen to hypoxic tumor regions without affecting oxygenation of tissues within physiologic oxygen levels. Due to its biochemical features, OMX is well tolerated in small (rats and mice) and large (sheep and dogs) animals. Following intravenous administration, OMX extravasates through leaky tumor vasculature and accumulates within immunocompetent rodent orthotopic glioblastoma models as well as spontaneous canine brain tumors. Consequently, OMX decreases hypoxia levels in the tumor tissue measured directly using oxygen sensor probes and indirectly with exogenous hypoxia markers using ELISA, immunohistochemistry and flow cytometry methods. Here we evaluated OMX’ activity in reversing the immunosupressive tumor microenvironment using a combination of immunohistochemistry, flow cytometry and Luminex methods. Moreover, we investigated the efficacy of OMX in improving mouse survival and effectiveness of checkpoint inhibitors (CPI). Similar to previously published findings, we demonstrated that T lymphocytes are mostly excluded from hypoxic tumor areas in the GL261 model. A single OMX treatment in GL261 tumor-bearing mice reduces tumor hypoxia, enhances T cell localization in previously hypoxic tumor areas, and increases CD8 accumulation by ~4-fold. Specifically, OMX treatment increased the activated cytotoxic T lymphocytes (CTLs) fraction by ~2 fold and reduced the immunosuppressive Treg fraction by 2-fold, resulting in a 3-fold increase of Teff/Treg ratio, which indicates a switch from an immunosupressive to an immunopermissive microenvironment. When combined with CPI, OMX reverses the immunosuppressive tumor microenvironment by increasing CD8 T cell infiltration, proliferation and cytotoxic activity, and modulating IFNg and IFNg-inducible cytokines that may polarize T cells towards a Th1 phenotype. Furthermore, treatment of late-stage GL261 tumor-bearing mice with the combination of OMX-CPI increases mouse survival by 80%. By delivering oxygen specifically to the hypoxic tumor microenvironment, OMX may restore anti-cancer immune responses in glioblastoma patients and synergize with radiotherapy and immunotherapy to enhance tumor control and improve patient outcomes. Citation Format: Natacha Le Moan, Philberta Leung, Sarah Ng, Tina Davis, Carol Liang, Jonathan W. Winger, Stephen P. Cary, Nicolas Butowski, Ana Krtolica. Omx a hypoxia modulator reverses the immunosuppressive glioblastoma microenvironment by stimulating T cell infiltration and activation that results in increased number of long-term survivors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4686. doi:10.1158/1538-7445.AM2017-4686