Downstream Toll-like receptor signaling mediates adaptor-specific cytokine expression following focal cerebral ischemia

Abstract

Background: Deletion of some Toll-like receptors (TLRs) affords protection against cerebral ischemia, but disruption of their known major downstream adaptors does not. To determine whether compensation in the production of downstream effectors by one pathway when the other is disrupted can explain these findings, we examined cytokine/chemokine expression and inflammatory infiltrates in wild-type (WT), MyD88(-/-) and TRIF-mutant mice following permanent middle cerebral artery occlusion (pMCAO).

Methods: Cytokine/chemokine expression was measured with a 25-plex bead array in the serum and brains of all three groups of mice at baseline (no surgery/naïve) and at 3 hours and 24 hours following pMCAO. Brain inflammatory and neutrophil infiltrates were examined 24 hours following pMCAO.

Results: IL-6, keratinocyte chemoattractant (KC), granulocyte colony-stimulating factor (G-CSF) and IL-10 were significantly decreased in MyD88(-/-) mice compared to WT mice following pMCAO. Significantly, decreased levels of the neutrophil chemoattractants KC and G-CSF corresponded with a trend toward fewer neutrophils in the brains of MyD88(-/-) mice. IP-10 was significantly decreased when either pathway was disrupted. MIP-1 α was significantly decreased in TRIF-mutant mice, consistent with TRIF-dependent production. MyD88(-/-) mice showed elevations of a number of Th2 cytokines, such as IL-13, at baseline, which became significantly decreased following pMCAO.

Conclusions: Both MyD88 and TRIF mediate pathway-specific cytokine production following focal cerebral ischemia. Our results also suggest a compensatory Th2-type skew at baseline in MyD88(-/-) mice and a paradoxical switch to a Th1 phenotype following focal cerebral ischemia. The MyD88 pathway directs the expression of neutrophil chemoattractants following cerebral ischemia.

Figure 1

MyD88-dependent cytokines following permanent middle cerebral artery occlusion. Significantly decreased levels of IL-6 and neutrophil chemoattractants keratinocyte chemoattractant and granulocyte colony-stimulating factor in MyD88^−/− mice compared to wild type (WT) at the indicated time points in serum (A) and brain (B). ^*p < 0.05, MyD88^−/− compared to WT. N = 9 per group and n = 3 per time point. 0 hours = baseline.

Figure 2

Toll/interleukin 1 receptor domain-containing adaptor-inducing interferon β (TRIF)-dependent cytokines following permanent middle cerebral artery occlusion. Significantly decreased levels of inducible protein 10 (IP-10) and macrophage inflammatory protein 1α (MIP-1α) in TRIF-mutant compared to wild type (WT) at the indicated time points in serum (A) and brain (B) following permanent middle cerebral artery occlusion (pMCAO). IP-10 levels are also significantly decreased in MyD88^−/− mice at 3 hours following permanent middle cerebral artery occlusion. ^*p < 0.05, MyD88^−/− or TRIF compared to WT. N = 9 per group and n = 3 per time point. 0 hours = baseline.

Figure 3

Th2 cytokines at baseline. (A) Abnormal elevations in levels of Th2 cytokines IL-6, IL-10 and IL-13 and (B) Th2-associated chemokines, RANTES (regulated upon activation, normal T-cell expressed, and secreted) and macrophage inflammatory protein 1α (MIP-1α), in MyD88^−/− mice at baseline and significant decreases in levels of these cytokines at the indicated time points following permanent middle cerebral artery occlusion (pMCAO) compared to wild type (WT). Interestingly, Th1-associated monocyte chemoattractant protein 1 significantly increased in the brain of MyD88^−/− mice 24 hours following pMCAO when levels have started to decrease towards baseline in WT mice. *p < 0.05; MyD88^−/− or TRIF-mutant compared to WT. N = 9 per group and n = 3 per time point. 0 hours = baseline.

Figure 4

Leukocyte infiltrate in the brains of (A) wild-type, (B) MyD88^−/−and (C) TRIF-mutant mice 24 hours following permanent middle cerebral artery occlusion. Inset: Graph shows the number of leukocytes per high-powered field (10×) in each group of mice. There were no differences in leukocyte infiltrate in the brains of all three groups of mice at 24 hours following focal ischemia (p = 0.3607). N = 9 mice, and n = 3 mice per wild type, MyD88^−/− and TRIF-mutant.

Figure 5

Neutrophil infiltrate in the brains of (A) wild-type, (B) MyD88^−/−and (C) TRIF-mutant mice at 24 h following permanent middle cerebral artery occlusion. Inset: Graph shows the number of neutrophils per high-powered field (10×) in each group of mice. There was a trend toward fewer neutrophils in the brains of MyD88^−/− mice compared to wild-type (WT) and Toll/interleukin 1 receptor domain-containing adaptor-inducing interferon β (TRIF)-mutant mice at 24 hours following permanent middle cerebral artery occlusion (p = 0.189). N = 9 and n = 3 mice per WT, MyD88^−/− and TRIF-mutant.