Toll-like receptor 9: a new target of ischemic preconditioning in the brain

Abstract

Preconditioning with lipopolysaccharide (LPS), a toll-like receptor 4 (TLR4) ligand, provides neuroprotection against subsequent cerebral ischemic brain injury, through a tumor necrosis factor (TNF)alpha-dependent process. Here, we report the first evidence that another TLR, TLR9, can induce neuroprotection. We show that the TLR9 ligand CpG oligodeoxynucleotide (ODN) can serve as a potent preconditioning stimulus and provide protection against ischemic brain injury. Our studies show that systemic administration of CpG ODN 1826 in advance of brain ischemia (middle cerebral artery occlusion (MCAO)) reduces ischemic damage up to 60% in a dose- and time-dependent manner. We also offer evidence that CpG ODN preconditioning can provide direct protection to cells of the central nervous system, as we have found marked neuroprotection in modeled ischemia in vitro. Finally, we show that CpG preconditioning significantly increases serum TNFalpha levels before MCAO and that TNFalpha is required for subsequent reduction in damage, as mice lacking TNFalpha are not protected against ischemic injury by CpG preconditioning. Our studies show that preconditioning with a TLR9 ligand induces neuroprotection against ischemic injury through a mechanism that shares common elements with LPS preconditioning via TLR4.

Figure 1. CpG protects primary mixed cortical cultures from OGD-induced cell death through TLR9

A) Mixed cortical cultures were stimulated with increasing doses of CpG 1826 (0.5–5 ug/ml) 24 hr prior to 3 hr OGD. Cell death was assessed 24 hr following OGD by propidium iodide (PI) staining. For all experiments, values are mean ± SEM, **p<0.01, ***p<0.001 versus media-treated OGD controls, n=5 individually repeated experiments. CpG 1826 treatment alone at the highest dose (5ug/ml) did not result in increased cell death over media alone (grey bar). B) Mixed cortical cultures were stimulated with either CpG or the TLR4 agonist LPS (1ug/ml) in the presence or absence of the TLR9 antagonist ODN 2088 (2 or 5 ug/ml) 24 hr prior to 3 hr OGD. Cell death was assessed 24 hr following OGD by PI staining. Values are mean ± SEM, *p<0.05 vs media-treated OGD controls, #p<0.05 vs CpG-treated OGD; n=2–4 individually repeated experiments, except for LPS +5ug/ml ODN 2088 which represents a single experiment.

Figure 2. Preconditioning with CpG reduces infarct size in a mouse model of focal ischemia

C57BL/6 (males, 6–10/dose) received various doses of CpG 1826 (5–40ug; i.p.) 72 hr prior to ischemic challenge (60 min MCAO). Infarct volume was determined 24 hrs following MCAO by TTC staining. Values are group means ± SEM; *p<0.05 comparison to saline controls by one way ANOVA followed by Bonferroni’s multiple comparison test.

Figure 3. Time window of CpG preconditioning

C57Bl/6 mice received an injection of saline (4 mice/time point) or CpG (20ug; i.p.) 1, 3, 7 or 14 days (6 mice/time point) prior to 60 min MCAO. Infarct volume was determined 24 hr following MCAO by TTC staining. No statistical difference was observed between the saline groups, thus they were combined for analysis. Values are group means ± SEM; ***p<0.001 comparison to saline controls by one way ANOVA followed by Bonferroni’s multiple comparison test.

Figure 4. Serum TNF-α levels significantly increased 1-hour post CpG treatment

Mice (n=4/time point) were administered CpG 1826 (20 ug; i.p.) and blood was collected at 1, 3, 24 or 72 hrs post injection. Blood was allowed to clot for 2 hr at room temperature and the serum collected. TNFα levels (pg/ml of blood) were measured with a TNFα ELISA (R&D Systems). Values are group means ± SEM; ***p < 0.001 by two way ANOVA followed by Bonferroni’s multiple comparison test. LPS (5 ug; i.p.) treated mice were included in the same experiment for comparison.

Figure 5. TNF induction is required for CpG preconditioning

Control (C57Bl/6; TNFα^+/+) or TNFα knockout (B6.129S-Tnf^tm1Gkl/J; TNFα^−/−) mice were treated with CpG 1826 (20 ug; i.p.) at 72 hr prior to 40 min MCAO and infarcts were assessed 24 hr post MCAO. Values are mean + SEM, *p<0.05 versus saline treatment, n = 4–6 mice/group.