Toll-like receptor 2 deficiency leads to delayed exacerbation of ischemic injury

Abstract

Background: Using a live imaging approach, we have previously shown that microglia activation after stroke is characterized by marked and long-term induction of the Toll-like receptor (TLR) 2 biophotonic signals. However, the role of TLR2 (and potentially other TLRs) beyond the acute innate immune response and as early neuroprotection against ischemic injury is not well understood.

Methods: TLR2-/- mice were subjected to transient middle cerebral artery occlusion followed by different reperfusion times. Analyses assessing microglial activation profile/innate immune response were performed using in situ hybridization, immunohistochemistry analysis, flow cytometry and inflammatory cytokine array. The effects of the TLR2 deficiency on the evolution of ischemic brain injury were analyzed using a cresyl violet staining of brain sections with appropriate lesion size estimation.

Results: Here we report that TLR2 deficiency markedly affects post-stroke immune response resulting in delayed exacerbation of the ischemic injury. The temporal analysis of the microglia/macrophage activation profiles in TLR2-/- mice and age-matched controls revealed reduced microglia/macrophage activation after stroke, reduced capacity of resident microglia to proliferate as well as decreased levels of monocyte chemotactic protein-1 (MCP-1) and consequently lower levels of CD45(high)/CD11b(+) expressing cells as shown by flow cytometry analysis. Importantly, although acute ischemic lesions (24 to 72 h) were smaller in TLR2-/- mice, the observed alterations in innate immune response were more pronounced at later time points (at day 7) after initial stroke, which finally resulted in delayed exacerbation of ischemic lesion leading to larger chronic infarctions as compared with wild-type mice. Moreover, our results revealed that TLR2 deficiency is associated with significant decrease in the levels of neurotrophic/anti-apoptotic factor Insulin-like growth factor-1 (IGF-1), expressed by microglia in the areas both in and around ischemic lesion.

Conclusion: Our results clearly suggest that optimal and timely microglial activation/innate immune response is needed to limit neuronal damage after stroke.

Figure 1

Microglial cells upregulate Toll-like receptor 2 in response to transient ischemic brain injury. (A, B) Photomicrographs of in situ hybridization for Toll-like receptor (TLR) 2 mRNA in wild type (WT) mice 24 h after ischemic injury, indicating marked induction of TLR2 on the ipsilateral side shown in low (A, left) and high (A, right) magnification, in contrast to the contralateral side where no positive S³⁵ signal was observed (B). (C, D, E) Double labeling 3, 7 and 14 days after transient middle cerebral artery occlusion (MCAO) reveals co-localization of TLR2 (green) and Iba1 (red) in WT mice, indicating that TLR2 are expressed almost exclusively by the microglia/macrophage cell population in both the early and late phase of the response to ischemic brain injury. Scale bar: C, 50 μm.

Figure 2

Toll-like receptor 2 deficiency leads to altered microglia/macrophage activation profiles. (A) Schematic representation of the brain section used for the immunohistochemical experiments. Box represents the area magnified in B and D, and in Figure 3A,C,F,G, as well as in Figures 6 and 7. (B) Representative photomicrographs of Iba1 immunoreactivity in control (unlesioned) wild-type (WT) and Toll-like receptor (TLR) 2−/− mice, as well as 3 and 7 days after transient middle cerebral artery occlusion (MCAO), indicate reduced accumulation of Iba1-positive cells in TLR2 deficient mice. (C) Quantification of Iba1 immunoreactivity revealed significantly decreased immunoreactivity in TLR2−/− mice compared with the WT group of mice 3 and 7 days after transient MCAO. (D) Photomicrographs of CD68 immunoreactivity in unlesioned (control) WT and TLR2−/− mice 3 and 7 days after transient ischemic lesion. (E) Quantitative analysis of CD68 immunoreactivity in both groups reveals significant reduction of the signal intensity 7 days after MCAO in the TLR2−/− group. Values (C, E) indicate mean ± SEM (n = 4; **P <0.001 ***P <0.0001). Scale bars: B, D: 100 μm.

Figure 3

Decreased levels of MCP-1 and CD45^high/CD11b⁺expressing cells in Toll-like receptor 2 deficient mice. (A-C) Expression analysis of inflammatory cytokines IL-1β, IL-6 and TNF-α on protein levels 1 and 4 days after transient middle cerebral artery occlusion (MCAO) revealed no significant difference between wild-type (WT) and Toll-like receptor (TLR) 2−/− groups. (D) Significant reduction of MCP-1 levels in the TLR2−/− group compared with their WT controls 1 day after transient MCAO. (E) Topographic representation of isolated brain mononuclear cells from ischemic hemispheres of control, WT and TLR2−/− mice 3 and 7 days after stroke, analyzed using two-color flow cytometry. Cells were analyzed for CD45 and CD11b, thus allowing us to distinguish two different cell populations: CD45^high/CD11b⁺ (that is, the macrophage-like) population (red); and CD45^low/CD11b⁺ (microglial) population (green). (F) Flow cytometric analysis showed a significant decrease in the number of CD45^high/CD11b⁺ cells in TLR2−/− mice compared with WT mice at both 3 and 7 days after stroke. (G) Quantitative analysis of CD45^low/CD11b⁺ cells (microglia) indicates a significant reduction in cell numbers in TLR2−/− mice as compared with WT 7 days after stroke, while 3 days after stroke the difference is not significant, but there is a tendency toward reduced cell numbers in TLR2−/− mice. Values (A-D) are expressed as mean ± SEM (n = 4, *P <0.05). Data (F, G) are expressed as percentage of CD45⁺ events ± SEM (n = 4; *P <0.05).

Figure 4

Toll-like receptor 2 deficient mice display reduced proliferation of resident microglial cells. (A) Photomicrographs of BrdU immunoreactivity in wild-type (WT) and Toll-like receptor (TLR) 2−/− mice 3 days after transient middle cerebral artery occlusion (MCAO) and their unlesioned controls, respectively. (B) Quantification of BrdU immunoreactivity indicates reduced cell proliferative capacity in TLR2−/− mice compared with WT mice 3 days after transient MCAO. (C) Representative photomicrographs of double immunostaining on WT brain sections 3 days after transient MCAO show almost complete co-localization of BrdU (green) positive cells with Iba1 (red; white arrows). (D) Flow cytometric analysis of the CD45^low/CD11b⁺ (microglial) population proliferation in WT and TLR2−/− mice 3 days after MCAO compared with their contralateral (unlesioned) hemisphere controls. (E) Graph presents quantification of microglial proliferation measured by flow cytometric analysis. Data indicate reduced microglial proliferation in TLR2−/− mice compared with WT mice 3 days after MCAO. (F) Double immunofluorescent labeling reveals co-localization of BrdU (green) and Mac-2 (red; white arrows) in WT brain sections 3 days after transient MCAO. (G) Representative photomicrographs of Mac-2 immunofluorescence in WT and TLR2−/− mice observed 3 days after transient MCAO. (H) Quantification of Mac-2 immunoreactivity 3 days after transient MCAO revealed reduced signals in TLR2−/− mice compared with WT mice. Data (B, H) are indicated as mean ± SEM (n = 4; *P <0.05). Data (E) are expressed as percentage of CD45⁺ events ± SEM (n = 4; *P <0.05). Scale bars: A, G, 100 μm; C, F, 50 μm.

Figure 5

Toll-like receptor 2 deficiency delays evolution of ischemic brain lesion. (A) Representative low-magnification photomicrographs of cresyl violet stained brain sections of wild type (WT) and Toll-like receptor (TLR) 2−/− mice 3, 7 and 14 days after transient middle cerebral artery occlusion (MCAO). The ischemic area is emphasized by a black dashed line. (B) Significantly reduced direct stroke area in TLR2−/− mice 3 days after transient MCAO compared with WT mice. This was followed by marked lesion exacerbation in TLR2−/− mice 7 days after injury, while lesions in WT mice showed a reduction in size. The evolution of direct stroke area stagnated 14 days after injury, indicating significantly larger final lesion size in TLR2−/− mice compared with the WT group. (C) A smaller indirect stroke area in TLR2−/− mice compared with WT mice 3 days after transient MCAO. In contrast, 7 days after injury, indirect stroke area in the TLR2−/− group was larger compared with WT mice, and the same relationship was observed 14 days after ischemic injury. Values are expressed as mean ± SEM (n = 10, *P <0.05, **P <0.001).

Figure 6

Delayed exacerbation of neuronal apoptosis in Toll-like receptor 2 deficient mice. (A) Photomicrographs of cleaved caspase-3 immunoreactivity in wild-type (WT) and Toll-like receptor (TLR) 2 −/− mice 3,7 and 14 days after transient middle cerebral artery occlusion (MCAO). (B) Quantification of labeled cells showed a higher number of apoptotic cells in WT compared with TLR2−/− mice 3 days after injury, while 7 days after injury the number of cleaved caspase-3-positive cells was significantly higher in the TLR2−/− group compared with the WT group of mice. The number of cleaved caspase-3-positive cells was higher in TLR2−/− mice 14 days after transient MCAO, but not significantly because of high variation within the group. (C) Double immunofluorescence analysis reveals a high proportion of cleaved caspase-3 (green) positive cells correlating with the neuronal marker NeuN (red, co-localization marked with white arrows). (D) No co-localization was observed for GFAP (red) and cleaved caspase-3 (green). (E) Only a few cleaved caspase-3-positive cells (green) showed co-localization with Iba1 (red, co-localization marked with white arrow heads), while the vast majority were not co-localizing. Data (B) are expressed as mean ± SEM (n = 4, *P <0.05, **P <0.001). Scale bars: A, C, D, E, 25 μm.

Figure 7

Reduced levels of Insulin-like growth factor 1 in brains of Toll-like receptor 2 deficient mice after stroke. (A) Photomicrographs of (IGF)-1 immunoreactivity in control (unlesioned) and lesioned brain hemispheres at 3 and 7 days after stroke, showing reduced levels of IGF-1 immunoreactivity in Toll-like receptor (TLR) 2−/− mice. (B) Quantification of signal intensity indicates a reduced level of IGF-1 present in the brains of TLR2−/− mice following transient ischemia compared with wild-type (WT) mice. (C, D) Double immunofluorescence labeling reveals that IGF-1 expression was present in microglia/macrophages within and around the infarcted region in both 3 and 7 days after ischemia. Data (B) are indicated as mean ± SEM (n = 4; *P <0.05, **P <0.001). Scale bars: A, 100 μm; C, D: 50 μm.