Key role of CD36 in Toll-like receptor 2 signaling in cerebral ischemia

Abstract

Background and purpose: Toll-like receptors (TLRs) and the scavenger receptor CD36 are key molecular sensors for the innate immune response to invading pathogens. However, these receptors may also recognize endogenous "danger signals" generated during brain injury, such as cerebral ischemia, and trigger a maladaptive inflammatory reaction. Indeed, CD36 and TLR2 and 4 are involved in the inflammation and related tissue damage caused by brain ischemia. Because CD36 may act as a coreceptor for TLR2 heterodimers (TLR2/1 or TLR2/6), we tested whether such interaction plays a role in ischemic brain injury.

Methods: The TLR activators FSL-1 (TLR2/6), Pam3 (TLR2/1), or lipopolysaccharide (TLR4) were injected intracerebroventricularly into wild-type or CD36-null mice, and inflammatory gene expression was assessed in the brain. The effect of TLR activators on the infarct produced by transient middle cerebral artery occlusion was also studied.

Results: The inflammatory response induced by TLR2/1 activation, but not TLR2/6 or TLR4 activation, was suppressed in CD36-null mice. Similarly, TLR2/1 activation failed to increase infarct volume in CD36-null mice, whereas TLR2/6 or TLR4 activation exacerbated postischemic inflammation and increased infarct volume. In contrast, the systemic inflammatory response evoked by TLR2/6 activation, but not by TLR2/1 activation, was suppressed in CD36-null mice.

Conclusions: In the brain, TLR2/1 signaling requires CD36. The cooperative signaling of TLR2/1 and CD36 is a critical factor in the inflammatory response and tissue damage evoked by cerebral ischemia. Thus, suppression of CD36-TLR2/1 signaling could be a valuable approach to minimize postischemic inflammation and the attendant brain injury.

Figure 1

In monocytic cells, LPS binds to TLR4, Pam3 to TLR2/1, and FSL-1 to TLR2/6. Such binding triggers proinflammatory gene expression through nuclear factor (NF)-κB activation.

Figure 2

Pam3 does not increase proinflammatory gene mRNAs in CD36-null mice. Vehicle or TLR ligands were administered intracerebroventricularly to nonischemic wild-type (WT), CD36−/−, or TLR2−/− mice. Sham-operated mice served as controls. Increases relative to sham-treated mice are shown. *P<.05 from WT; #P<0.05 from vehicle; ANOVA and Newman-Keuls test; n=4/group. ICAM-1 indicates intercellular adhesion molecule-1; ELAM-1, endothelial leukocyte adhesion molecule-1; IL-6, interleukin-6; and MCP-1, monocyte chemotactic protein-1.

Figure 3

Pam3 does not increase infarct volume and motor deficits in CD36- or TLR2-null mice, but is effective in COX-2– null mice. Motor deficits, expressed as latency to fall during the hanging wire test in wild-type (WT, E) and CD36−/− (F) mice, are also shown. *P<0.01 from vehicle; n=6–10/group except for COX-2–null mice (for vehicle and FSL-1, n=5/group; for Pam3, n=4/group).

Figure 4

Pam3 does not increase neutrophil infiltration or microglial activation in CD36-null mice 72 hours after MCA occlusion. Effect of intracerebroventricular administration of Pam3 or FSL-1 on the astroglial marker glial fibrillary acid protein (GFAP; a, d, g, j), the microglial/macrophage marker F4/80 (b, e, h, k), and the neutrophil marker MPO (c, f, i, l) in CD36+/+ (a, b, c) and CD36-null mice (d–l) after intracerebroventricular administration of FSL-1 (g, h, i) or Pam3 (j, k, l). Images in A were taken at the medial border zone of the cortical infarct. Number of MPO-positive cells (B) or F4/80-positive microglia/macrophages (C) throughout the rostrocaudal extent of the infarct are also shown. *P<0.05 from CD36+/+ mice; #P<0.05 from CD36-null mice treated with FSL-1; n=6/group.

Figure 5

Intraperitoneal administration of FSL-1 does not increase plasma TNF-α concentrations in CD36-null mice. ND indicates not detected; n=4 or 5/group.