Inflammation and the Emerging Role of the Toll-Like Receptor System in Acute Brain Ischemia
Background and Purpose— Systemic administration of cytosine-guanine (CpG) oligodeoxynucleotides provides neuroprotection against subsequent cerebral ischemic injury. We examined the genomic response of leukocytes and brain cells after ischemia in the context of CpG preconditioning.
Methods— RNA was isolated from circulating leukocytes and ischemic cortex 3 and 24 hours after middle cerebral artery occlusion after CpG or saline pretreatment and subjected to microarray analysis. Genes uniquely upregulated in CpG-pretreated mice were examined for overrepresented transcriptional regulatory elements.
Results— CpG preconditioning induced a novel response to middle cerebral artery occlusion within circulating leukocytes that was dominated by natural killer cell-associated genes and the GATA-3 transcriptional regulatory element. Preconditioning also caused a novel brain response to stroke that was dominated by Type I interferon, interferon-associated genes, and transcriptional regulatory elements.
Conclusion— CpG preconditioning invokes novel leukocyte and brain responses to stroke. In this, CpG may be a unique preconditioning agent, coordinating peripheral and brain responses to protect against ischemic injury.
Bacterial nonmethylated cytosine-guanine (CpG) oligodideoxynucleotide motifs alert the body to infection through activation of Toll-like receptor 9 (TLR9). In mice, TLR9 is expressed by B cells, plasmacytoid dendritic cells, macrophages, microglia, and astrocytes. TLR9-activated cells produce the proinflammatory cytokines tumor necrosis factor α, interferon α, and interleukin (IL)-12. These cytokines further activate monocytes, neutrophils, natural killer cells (NK cells), and T cells, facilitating a coordinated inflammatory response to pathogen invasion.
Pre-exposure to CpG reprograms the cellular response to subsequent TLR stimulation. Unlike naïve cells, macrophages pretreated with CpG do not generate tumor necrosis factor α in response to TLR4 stimulation, instead generating IFNβ.1 Furthermore, systemic administration of CpG increases resistance to polymicrobial sepsis.2 Hence, pre-exposure to CpG redirects both cellular and systemic responses to subsequent TLR stimulation.
Systemic administration of CpG also protects the brain from subsequent ischemic damage.3 Such “CpG preconditioning” is time- and dose-dependent and requires tumor necrosis factor α. The precise mechanisms responsible for CpG preconditioning are not well understood but likely involve both direct cellular processes and coordinated systemic responses that minimize ischemic damage.
We hypothesize that CpG preconditioning reprograms the response of the brain and the peripheral immune system to subsequent stroke. We provide evidence for such reprogramming and consider its potential neuroprotective consequences.
Materials and Methods
C57Bl/6 mice (male, 8 to 10 weeks) were obtained from Jackson Laboratories (West Sacramento, Calif). All mice were housed in a facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care International. The animal protocols met National Institutes of Health guidelines with the approval of the Oregon Health and Science University Institutional Animal Care and Use Committee.
CpG oligodeoxynucleotide 1826 (20 to 40 μg; 200 μL; Invivogen; San Diego, Calif) or saline was administered by intraperitoneal injection 72 hours before MCAO.
Mice were anesthetized with isoflurane and ischemia was induced by MCAO as published previously.17 Cerebral blood flow was monitored with laser Doppler flowmetry and temperature was maintained at 37°C. After surgery, mice were kept for 24 hours on a heating pad with access to soft food and water.
Mice were anesthetized and blood was obtained through retro-orbital puncture. Animals were perfused with saline and, under RNase-free conditions, a 1-mm section was removed for infarct area analysis. The ipsilateral cortex region from the frontal 4 mm was snap-frozen. Total RNA was isolated from the blood using the Qiagen PAXgene Blood RNA Kit and from the brain using the Qiagen RNeasy Lipid Mini Kit (Qiagen Inc). RNA from individual animals was hybridized to single arrays.
GeneChip Expression Analyses
Microarray assays were performed in the Affymetrix Microarray Core of the Oregon Health & Sciences University Gene Microarray Shared Resource. RNA samples were labeled using the NuGEN Ovation Biotin RNA Amplification and Labeling System_V1. Quality-tested samples were hybridized to the MOE430 2.0 array and processed with Affymetrix GeneChip Operating Software. Data were normalized using the Robust Multichip Average method. Normalized data were analyzed by multivariant analysis of variance for each gene. Probability values were adjusted for multiple comparisons using the Hochberg and Benjamini method. Significance was determined by P<0.05 and fold change ≥2 for blood analyses and ≥1.5 for brain analyses.
Transcriptional Regulatory Network Analysis
For our reference comparison group, we identified putative TREs in the upstream sequence of transcripts represented on the MOE430 Affymetrix gene chip using TRANSFAC PRO database version 10.4. We then determined the overrepresented TREs in the uniquely upregulated gene cluster compared with the reference group using Promoter Analysis and Interaction Network Toolset version 3.5.
Cytosine-Guanine Preconditioning Induces a Natural Killer Cell-Associated Peripheral Response to Stroke
We evaluated RNA from blood leukocytes 24 hours after middle cerebral artery occlusion (MCAO) using Affymetrix oligonucleotide microarrays. We found 422 genes to be differentially regulated in CpG-pretreated animals relative to saline. We next identified overrepresented transcriptional regulatory elements (TREs) in the genes uniquely increased in CpG-preconditioned animals. In those genes for which the upstream sequence was available for analysis (234), a single TRE, GATA-3, was overrepresented with an adjusted probability value=0.118. A network depiction of interactions between GATA-3 and genes in the CpG-preconditioned cluster is displayed in Figure 1. GATA-3 is linked to 53% of the genes within this upregulated cluster (124 of 234). GATA-3 plays a critical role in the development of natural killer (NK) cells. A literature review identified 24 of the upregulated genes as NK cell-associated: Klra5, Klra7, Klra8, Klra10, Klra18, Klra22, Klrb1a, Klrb1c, Klrb1f, Klrc1, Klrc2, Klre1, Klrg1, Klrk1, Rantes, Cma1, Eomes, Fasl, Gzmb, Il2rb, Ncr1, Ndg1, Prf1, and T-bet. CpG activates NK cells indirectly through IL-12 released from activated dendritic cells. Serum IL-12 levels were significantly increased 24 hours after MCAO in preconditioned animals (data not shown). Together, our data demonstrate that CpG preconditioning induces a novel, systemic NK cell response to stroke.
Cytosine-Guanine Preconditioning Induces a Type I Interferon-Associated Brain Response to Stroke
We evaluated RNA from ischemic cortex 24 hours after MCAO using Affymetrix oligonucleotide microarrays. We found 223 genes to be differentially regulated in CpG-pretreated animals relative to saline. We next identified overrepresented TREs in the genes uniquely upregulated in CpG-preconditioned animals. In those genes for which upstream sequence was available for analysis (136), we identified 4 overrepresented TREs with an adjusted probability value <0.1. Notably, each TRE was Type I IFN-associated (IRF, IRF8, ISRE, 3-hydroxy-3-methylglutaryl-1Y). A network depiction of interactions between the identified TREs and the genes in the CpG-preconditioned cluster is displayed in Figure 2. The IFN-associated TREs are linked to 64% of the genes within this upregulated cluster (88 of 136). A literature review identified 12 of the upregulated genes as Type I IFN-associated: Oas1a, MHC class I (H2-D1, H2-K1, H2-L, H2-Q6), Ifi203, Ifi204, Ifi205, Ifi27, Isg20l1, Lmp7, and Psmb9). Thus, an altered signaling cascade involving Type I IFNs exists in the brain after stroke in CpG-preconditioned mice.
We report the first evidence that CpG preconditioning alters the genomic response to stroke in circulating leukocytes and in the brain. We demonstrate a distinct pattern of NK cell activity in the blood and a clear enhancement of Type I IFN signaling in the brain after MCAO. This pattern of upregulated gene expression underscores a unique response to brain ischemia that may actively protect the brain from injury.
CpG preconditioning induced a novel genomic response in blood leukocytes that was evident 24 hours after stroke. Of those genes uniquely upregulated in preconditioned animals, a majority contained the GATA-3 TRE, which is required for NK cell development. Additionally, 24 of the upregulated genes were NK cell-related and serum IL-12 was increased at this time, supporting the notion of increased NK cell activity.
This unique systemic response may play a role in neuroprotection because NK cells have been shown to limit damaging neuroinflammation in experimental autoimmune encephalomyelitis.4 Interestingly, administration of CpG oligodeoxynucleotides before experimental autoimmune encephalomyelitis induction also reduces disease severity.5 Furthermore, treatment with CpG inhibits inflammatory arthritis in an IL-12- and NK cell-dependent manner.6 Hence, CpG may also initiate a protective NK cell response to cerebral ischemia.
CpG preconditioning induced a novel genomic response in the brain that was evident 24 hours after stroke. Of those genes uniquely upregulated in preconditioned animals, a majority contained one or more Type I IFN-associated TREs. Moreover, 12 of the upregulated genes were associated with Type I IFN signaling, further supporting a role for IFNs after stroke in preconditioned animals.
Microglial, astrocytes, endothelial cells, and neurons all produce the Type I IFN IFNβ. IFNβ can stabilize the blood–brain barrier,7 suppress inflammatory cytokines,8 and protect neurons from cytotoxic microglia.9 Systemic administration of IFNβ reduces infarct damage in several models of ischemic stroke.10,11 Hence, an increase in Type I IFN signaling within the brain has the potential to be neuroprotective.
Our data support a shift toward Type I IFN signaling after stroke in CpG-pretreated animals. How might this shift occur? Mice lacking TLR4 incur significantly less damage from MCAO than wild-type controls,12 indicating a damaging role for this receptor in ischemic injury. Pretreatment with CpG shifts the cellular response to subsequent stimulation of TLR4, leading to a suppression of tumor necrosis factor α and an increase in IFNβ. A similar series of events might occur after CpG preconditioning wherein pretreatment with CpG shifts the response of TLR4 to subsequent stimulation with endogenous ligands such as HSP60, released after stroke, and potentially leads to suppressed cytotoxic tumor necrosis factor α and enhanced neuroprotective IFNβ.
Alternatively, the systemic increase in NK cell activity may explain the Type I IFN shift in the brain. NK cells promote the release of IFNα from plasmacytoid dendritic cells in a CpG- or IL-12-dependent manner.13,14 Hence, pretreatment with CpG may activate dendritic cells to produce IL-12, thereby activating NK cells which, in turn, induce plasmacytoid dendritic cells to produce IFNα.
We have shown that CpG preconditioning reprograms the peripheral and central responses to stroke. The appearance of a novel NK cell and IFN genomic “fingerprints” after ischemia indicates that CpG preconditioning fundamentally changes the body’s inflammatory response to stroke. This is consistent with our previous reports of reprogramming in which ischemic and lipopolysaccharide preconditioning induce novel, protective sets of gene transcripts after stroke.15,16 CpG appears to be a unique preconditioning agent, coordinating both systemic and central immune components to actively protect the body from ischemic injury.
Source of Funding
Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. This work was supported by National Institutes of Health grant R01 NS050567 (M.P.S.-P.).
OHSU, Dr Stenzel-Poore, and Ms Stevens have a financial interest in Neuroprotect, Inc. This potential conflict of interest has been reviewed and managed by OHSU and the Integrity Program Oversight Council. There are no other conflicts to report.
- Received August 15, 2008.
- Accepted August 19, 2008.
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