Article Figures & Data
- Figure 1
C-C motif chemokine ligand 17 (CCL17) expression was enhanced after acute subarachnoid haemorrhage (SAH) in rats. (A) Western blot analysis of CCL17 expression after SAH (n=3/group). (B) Western blot analysis of CCL17 expression at the base, cortex, and hippocampus regions at 24 hours post-SAH (n=3). (C) CCL17 (green) co-localised with NeuN (red) in basal ganglia, temporal cortex and hippocampus region in sham and 24 hours after SAH. *p < 0.05; ***p <0.001. Nuclei were stained with DAPI (blue), magnification: 200×, scale bar=20 µm, n=3. DAPI, 4',6-diamidino-2-phenylindole.
- Figure 2
Administration of recombinant C-C motif chemokine ligand 17 (rCCL17) reduced neuronal apoptosis and brain water content, and improved short-term neurological function after subarachnoid haemorrhage (SAH). (A) TUNEL staining in the ipsilateral cortex and FJC staining in the hippocampal area at 24 hours after SAH. Magnification: 200×, scale bar=20 µm, n=3. Modified Garcia test (B), beam balance score (C) and brain water content (D) at 24 or 72 hours after SAH revealed that rCCL17 treatment (60 μg/kg) improved short-term neurological function compared with vehicle or low dosage groups (n=6). *p < 0.05; **p < 0.01. DG, dentate gyrus; i.n., intranasal; LH, left hemisphere; RH, right hemisphere; Cb, cerebellum; BS, brain stem.
- Figure 3
Time course and co-location of endogenous C-C motif chemokine receptor 4 (CCR4) expression after subarachnoid haemorrhage (SAH). (A) Western blot analysis of CCR4 expression after SAH in rats (n=3). (B) Co-localisation of CCR4 (green) with microglia (Iba-1, red), astrocytes (GFAP, red) and neurons (NeuN, red) in the ipsilateral cortex at 24 hours after SAH in rats. Magnification: 200×, scale bar=20 µm, n=3. Nuclei were stained with DAPI (blue), scale bar=20 µm. (C) Western blot analysis of CCR4 expression in primary astrocyte, neuron and microglia cultures after treatment with haemoglobin for 24 hours (n=3/group). GFAP, glial fibrillary acidic protein.
- Figure 4
Altered morphology and M2-like polarisation of microglia with recombinant C-C motif chemokine ligand 17 (rCCL17) treatment after subarachnoid haemorrhage (SAH) in rats. (A) Altered morphology of microglia in the ipsilateral cortex with a concentration gradient treatment of rCCL17 at 24 hours after SAH. Right panel: representative cell subjected to image analysis combined with three-dimensional representation of the determined skeleton. Magnification: 200×, scale bar=20 µm, n=3. (B) Quantification of branches, process length and end points per single cell. (C) Relative messenger RNA expression of M2-like associated genes (CD301, MRC1 and ARG1) in brain tissue post-SAH (n=4). **p < 0.01, ***p < 0.001.
- Figure 5
C-C motif chemokine receptor 4 (CCR4)/mammalian target of rapamycin complex 2 (mTORC2) mediates C-C motif chemokine ligand 17 (CCL17)-induced M2-like polarisation in primary microglia culture. (A) Representative M2-like microglia marker gene expression of microglia stimulated with a concentration gradient of recombinant CCL17 (rCCL17), n=6. (B) Altered morphology of microglia staining with CD206 (green) and Iba-1 (red) after stimulation with a concentration gradient of rCCL17. Magnification: 200×, scale bar=20 µm. (C) Western blot analysis showed effects of rCCL17 (100 ng/mL) treatment on STAT6 and AKT(ser473) phosphorylation in microglia, n=3/group. (D) Representative immunoblots showing effects of rCCL17 (100 ng/mL) treatment on mTORC2 downstream targets and ERK activation in microglia. (E) Relative M2-like microglia marker gene expression, n=3/group. (F) Examination of mTORC2 activity through in vitro kinase assay in rat primary microglia stimulated with rCCL17 or rCCL17 combined with AZD2098. ***p < 0.001.
- Figure 6
The effect of recombinant C-C motif chemokine ligand 17 (rCCL17)/C-C motif chemokine receptor 4 (CCR4)/mammalian target of rapamycin complex 2 (mTORC2) axis was reversed by administration of CCR4 inhibitor and mTORC2 inhibitor after subarachnoid haemorrhage (SAH) in rats. (A) TUNEL staining in the ipsilateral cortex and FJC staining in the hippocampal area at 24 hours after SAH. Magnification: 200×, scale bar=20 µm, n=3. Modified Garcia test (B), beam balance score (C) and brain water content at 24 or 72 hours post-SAH showed that the activation of CCR4/mTORC2 axis with rCCL17 treatment was abolished by CCR4 inhibitor, AZD2098 and mTORC2 inhibitor, JR-AB2-011, n=5. (D) Western blot analysis of phosphorylation of AKT473 (E), PKC (F) and ERK (G) were performed. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 7
Recombinant C-C motif chemokine ligand 17 (rCCL17) induced M2-like polarisation of microglia was reversed by inhibition of C-C motif chemokine receptor 4 (CCR4)/mammalian target of rapamycin complex 2 (mTORC2) axis after subarachnoid haemorrhage (SAH) in rats. (A) Altered morphology of microglia in the ipsilateral cortex from Sham or SAH rats model with treatment of rCCL17 and administration of CCR4 inhibitor, AZD2098 and mTORC2 inhibitor, JR-AB2-011, at 24 hours post-SAH. Right panel: representative cell subjected to image analysis combined with three-dimensional representation of the determined skeleton. Magnification: 200×, scale bar=20 µm, n=3. (B) Total number of branches, process length and end points per single cell were calculated. (C) Relative messenger RNA expression of M2-like associated genes (CD301, MRC1 and ARG1) in brain tissue at 24 hours post-SAH. All t-tests were two-tailed, n=4/group. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 8
Recombinant C-C motif chemokine ligand 17 (rCCL17) induced M2-like polarisation of microglia was reversed by the inhibition of mammalian target of rapamycin complex 2 (mTORC2) signal pathway. Sprague-Dawley rats were intracerebroventricularly pretreated with AAV-CD68-Control or AAV-CD68-shRictor before surgery to target and knockdown the Rictor expression in microglia. (A) Representative images of microglia in the ipsilateral cortex from Sham or subarachnoid haemorrhage (SAH) rats model with treatment of rCCL17 at 24 hours post-SAH. Right panel: representative cell subjected to image analysis combined with three-dimensional representation of the determined skeleton. Magnification: 200×, scale bar=20 µm, n=3. (B) Total number of branches, process length and end points per single cell were calculated. (C) Relative messenger RNA expression of M2-like associated genes (CD301, MRC1 and ARG1) in brain tissue at 24 hours post-SAH, n=4/group. ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 9
Activation of C-C motif chemokine receptor 4 (CCR4)/mammalian target of rapamycin complex 2 (mTORC2) axis in microglia mediated the neuroprotective function of recombinant C-C motif chemokine ligand 17 (rCCL17). Sprague-Dawley rats were intracerebroventricularly pretreated with AAV-CD68-Control or AAV-CD68-shRictor before surgery to target and knockdown the Rictor expression in microglia. (A) Representative image of the TUNEL in the ipsilateral cortex and FJC staining in the hippocampal area at 24 hours postsubarachnoid haemorrhage (SAH). Magnification: 200×, scale bar=20 µm, n=3. Modified Garcia test (B), beam balance score (C) and brain water content at 24 or 72 hours after SAH showed that the neuroprotective effect of rCCL17 was mediated through the activation of CCR4/mTORC2 axis in microglia, n=5/group. (D) Western blot analysis of the quantification of Rictor (E), phosphorylation of AKT473 (F), PKC (G) and ERK (H) were performed. ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 10
Schematic model showing the mechanism of C-C motif chemokine ligand 17 (CCL17)-mediated crosstalk between neuron and microglia attenuate neurological injury after subarachnoid haemorrhage (SAH). Overproduction of CCL17 presents an essential role in neuroprotection and immune regulation. Following SAH onset, CCL17 secreted by neuron promotes microglia towards M2-like phenotype polarisation via the C-C motif chemokine receptor 4 (CCR4)/mammalian target of rapamycin complex 2 (mTORC2) axis. In return, the M2-like microglia present neuroprotective functions and attenuate neurological injury.
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