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Clinical Trial
. 2014 Jun 15;192(12):5984-92.
doi: 10.4049/jimmunol.1400054. Epub 2014 May 7.

CD36-mediated hematoma absorption following intracerebral hemorrhage: negative regulation by TLR4 signaling

Huang Fang  1 , Jing Chen  1 , Sen Lin  2 , PengFei Wang  1 , YanChun Wang  1 , XiaoYi Xiong  1 , QingWu Yang  3
Affiliations
Clinical Trial

CD36-mediated hematoma absorption following intracerebral hemorrhage: negative regulation by TLR4 signaling

Huang Fang et al. J Immunol. .

Abstract

Promoting hematoma absorption is a novel therapeutic strategy for intracerebral hemorrhage (ICH); however, the mechanism of hematoma absorption is unclear. The present study explored the function and potential mechanism of CD36 in hematoma absorption using in vitro and in vivo ICH models. Hematoma absorption in CD36-deficient ICH patients was examined. Compared with patients with normal CD36 expression, CD36-deficient ICH patients had slower hematoma adsorption and aggravated neurologic deficits. CD36 expression in perihematomal tissues in wild-type mice following ICH was increased, whereas the hematoma absorption in CD36(-/-) mice was decreased. CD36(-/-) mice also showed aggravated neurologic deficits and increased TNF-α and IL-1β expression levels. The phagocytic capacity of CD36(-/-) microglia for RBCs was also decreased. Additionally, the CD36 expression in the perihematoma area after ICH in TLR4(-/-) and MyD88(-/-) mice was significantly increased, and hematoma absorption was significantly promoted, which was significantly inhibited by an anti-CD36 Ab. In vitro, TNF-α and IL-1β significantly inhibited the microglia expression of CD36 and reduced the microglia phagocytosis of RBCs. Finally, the TLR4 inhibitor TAK-242 upregulated CD36 expression in microglia, promoted hematoma absorption, increased catalase expression, and decreased the H2O2 content. These results suggested that CD36 mediated hematoma absorption after ICH, and TLR4 signaling inhibited CD36 expression to slow hematoma absorption. TLR4 inhibition could promote hematoma absorption and significantly improve neurologic deficits following ICH.

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Figures

FIGURE 1.
FIGURE 1.
Reduced hematoma absorption and aggravated neurologic deficits in CD36-deficient patients with ICH. (A) Typical cranial CT images of CD36-normal (n = 11) and CD36-deficient (n = 11) ICH patients. Original magnification ×10. (B) Comparison of hematoma volumes in the CD36-deficient and CD36-normal groups at admission and 7 d after the onset of ICH. *p < 0.05 versus the CD36-normal group. (C) Decreased absorption rate in the CD36-deficient patients relative to the normal group. **p < 0.01 versus the CD36-normal group. (D) NIHSS scores and (E) mRS scores for ICH patients. *p < 0.05 versus the CD36-normal group, **p < 0.01 versus the CD36-normal group.
FIGURE 2.
FIGURE 2.
Enhanced CD36 expression in perihematomas following ICH. (A) Detection of CD36 mRNA expression in mice subjected to ICH using real-time quantitative RT-PCR. The data are expressed as fold increases relative to naive animals. **p < 0.01 versus sham, n = 6. (B) Detection of CD36 protein expression in mice subjected to ICH using Western blot. **p < 0.01 versus sham, n = 6. (C) Detection of CD36 expression in astrocytes, neurons, and microglia in the perihematomal tissues of mice using flow cytometry at 3 d after ICH. The left panel is the representative flow cytometry plot and the lower right panel is the percentage of CD36+ cells in neurons, astrocytes, and microglia. Microglia were marked by CD45intCD11b+, whereas neurons were identified as β-III tubulin+, and astrocytes were identified as GFAP+. **p < 0.01 versus sham, ##p < 0.01 versus astrocytes or neurons, n = 3. (D) Detection of CD36 expression in human perihematomal tissues of ICH patients using fluorescence immunohistochemistry; CD36 expression was labeled with a CD36 Ab (red), astrocytes, neurons, and microglia were labeled with GFAP (green), Neun (green), and Iba-1 (green), respectively. The merged images of the overlay of CD36 together with astrocytes, neurons, and microglia were shown as yellow, and the nuclei were stained with DAPI (blue). The arrows indicate positive cells. Scale bars, 80 μm.
FIGURE 3.
FIGURE 3.
CD36-mediated hematoma absorption in mice with ICH. (A) Serial coronal sections of mouse brain tissues 5 d after the onset of ICH. Original magnification ×1.5. (B) Comparison of hematoma volumes between WT and CD36−/− mice. The data in the left and right panels are the Image-Pro Plus and hemoglobin detection results, respectively. **p < 0.01 versus the WT group, n = 6. (C) The NDS of WT and CD36−/− mice at 1, 3, 5, and 7 d after the onset of ICH. *p < 0.05 versus the WT group at the corresponding time points, n = 6. (D) Brain water content and (E) mRNA expression of inflammatory factors 3 d after the onset of ICH. The data in (E) were assessed by real-time quantitative RT-PCR and were expressed as fold increases relative to naive animals. *p < 0.05 versus the WT group; **p < 0.01 versus the WT group, n = 6. Cereb, cerebellum; Cont BG, contralateral basal ganglia; Cont CX, contralateral cortex; Ipsi BG, ipsilateral basal ganglia; Ipsi CX, ipsilateral cortex.
FIGURE 4.
FIGURE 4.
CD36-mediated engulfment of RBCs by microglia. (A) Observation of the phagocytosis of RBCs (CFSE-labeled, green) by microglia (Alexa Fluor 594–labeled, red) using confocal microscopy. The nuclei were stained with Hoechst. Serial sections along the z-axis were acquired and compiled as images. The x–z-axis is below, and the y–z-axis is on the right. The arrows indicate the RBCs engulfed by microglia. Scale bars, 40 μm. Detection of changes in the phagocytic ability of microglia using flow cytometry is shown. Microglia were stained with anti-mouse CD11b-PE and RBCs were stained with CFSE. (B) Representative flow cytometry plot (left panel) and the percentage of PE+CFSE+ microglia that had engulfed RBCs (right panel). (C) Average fluorescence intensity of CFSE in the PE+CFSE+ microglia. (D) The number of nonphagocytosed RBCs in the supernatant. **p < 0.01 versus the WT microglia group, n = 6 for all graphs.
FIGURE 5.
FIGURE 5.
TLR4 signaling downregulates CD36 expression and increases hematoma absorption. (A) Detection of CD36 expression in perihematomal tissues 3 d after the onset of ICH using Western blot. **p < 0.01 versus the WT group, n = 6. (B) Detection of microglia CD36 expression using flow cytometry. **p < 0.01 versus the WT group, n = 6. (C) Five days after the onset of ICH, the hematoma volume was smaller in MyD88−/− and TLR4−/− mice than in WT mice. Measurement of hematoma volume using Image-Pro Plus (left panel) and hemoglobin detection (right panel) are shown. **p < 0.01 versus the WT group, n = 6. Detection of changes in the phagocytic capacity of microglia using flow cytometry is depicted. Microglia were stained with anti-mouse CD11b-PE, and RBCs were stained with CFSE. (D) Representative flow cytometry plot (left panel) and the percentage of PE+CFSE+ microglia that had engulfed RBCs (right panel); (E) average fluorescence intensity of CFSE in PE+CFSE+ microglia; (F) number of nonphagocytosed RBCs in the supernatant. **p < 0.01 versus the WT microglia group, ##p < 0.01 versus the groups with anti-CD36 Abs, n = 6 for all flow cytometry graphs.
FIGURE 6.
FIGURE 6.
Effects of TNF-α, IL-1β, and IL-10 on CD36 expression and phagocytosis in microglia. (A) Detection of changes in microglial CD36 protein levels using Western blot. *p < 0.05 versus the vehicle group, **p < 0.01 versus the vehicle group, n = 6. (B) Detection of microglial CD36 expression using flow cytometry. **p < 0.01 versus the vehicle group, n = 6. Detection of changes in the phagocytic ability of microglia using flow cytometry. Microglia were stained with anti-mouse CD11b-PE, and RBCs were stained with CFSE. (C) Representative flow cytometry plot (left panel) and the percentage of PE+CFSE+ microglia that had engulfed RBCs (right panel). **p < 0.01 versus the vehicle group, n = 6. (D) Average fluorescence intensity of CFSE in PE+CFSE+ microglia. **p < 0.01 versus the vehicle group, n = 6. (E) Number of nonphagocytosed RBCs in the supernatant. *p < 0.05 versus the vehicle group, **p < 0.01 versus the vehicle group, n = 6.
FIGURE 7.
FIGURE 7.
TAK-242 (TAK) increases CD36 expression and promotes hematoma absorption. (A) Detection of CD36 protein expression using Western blot. **p < 0.01 versus the vehicle group, #p < 0.05 versus the TAK group, ##p < 0.01 versus the TAK group, n = 6. (B) Detection of microglia CD36 expression using flow cytometry. **p < 0.01 versus the vehicle group, ##p < 0.01 versus the TAK group, n = 6. Detection of changes in the phagocytic capacity of microglia using flow cytometry. Microglia were stained with anti-mouse CD11b-PE, and RBCs were stained with CFSE. (C) A representative flow cytometry plot (left panel) displaying the percentage of PE+CFSE+ microglia (right panel) that had engulfed RBCs. **p < 0.01 versus the vehicle group, ##p < 0.01 versus the TAK group, n = 6. (D) The average fluorescence intensity of CFSE in PE+CFSE+ microglia. **p < 0.01 versus the vehicle group, ##p < 0.01 versus the TAK group, n = 6. (E) The number of nonphagocytosed RBCs in the supernatant. **p < 0.01 versus the vehicle group, ##p < 0.01 versus the TAK group, n = 6. (F) Detection of microglial catalase expression using real-time quantitative RT-PCR. The data are expressed as fold increases relative to the vehicle group. **p < 0.01, n = 6. (G) The changes in H2O2 content in the supernatant were measured. The results are presented as fold changes relative to vehicle group. **p < 0.01, n = 6. (H) Changes in hematoma volume in mice at 5 d after the onset of ICH. Measurement of hematoma volume using Image-Pro Plus (left panel) and detection of hemoglobin (right panel). **p < 0.01 versus the ICH plus vehicle group, #p < 0.05 versus the ICH plus TAK plus TNF-α group, n = 6.
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