Sigma-1 receptor-regulated efferocytosis by infiltrating circulating macrophages/microglial cells protects against neuronal impairments and promotes functional recovery in cerebral ischemic stroke

Abstract

Background: Efferocytosis of apoptotic neurons by macrophages is essential for the resolution of inflammation and for neuronal protection from secondary damage. It is known that alteration of the Sigma-1 receptor (Sig-1R) is involved in the pathological development of some neurological diseases, including ischemic stroke. The present study aimed to investigate whether and how Sig-1R regulates the phagocytic activity of macrophages/microglia and its significance in neuroprotection and neurological function in stroke. Methods: The roles of Sig-1R in the efferocytosis activity of microglia/macrophages using bone marrow-derived macrophages (BMDMs) or using Sig-1R knockout mice subjected to transient middle artery occlusion (tMCAO)-induced stroke were investigated. The molecular mechanism of Sig-1R in the regulation of efferocytosis was also explored. Adoptive transfer of Sig-1R intact macrophages to recipient Sig-1R knockout mice with tMCAO was developed to observe its effect on apoptotic neuron clearance and stroke outcomes. Results: Depletion of Sig-1R greatly impaired the phagocytic activity of macrophages/microglia, accordingly with worsened brain damage and neurological defects in Sig-1R knockout mice subjected to tMCAO. Adoptive transfer of Sig-1R intact bone marrow-derived macrophages (BMDMs) to Sig-1R knockout mice restored the clearance activity of dead/dying neurons, reduced infarct area and neuroinflammation, and improved long-term functional recovery after cerebral ischemia. Mechanistically, Sig-1R-mediated efferocytosis was dependent on Rac1 activation in macrophages, and a few key sites of Rac1 in its binding pocket responsible for the interaction with Sig-1R were identified. Conclusion: Our data provide the first evidence of the pivotal role of Sig-1R in macrophage/microglia-mediated efferocytosis and elucidate a novel mechanism for the neuroprotection of Sig-1R in ischemic stroke.

Keywords: Efferocytosis; Ischemic stroke; Macrophage/microglia; Rac1; Sigma-1 receptor.

Figure 1

Sig-1R activation promotes efferocytosis and M2 phenotype polarization of macrophages. (A) Bone marrow -derived macrophages (BMDM) pre-treated with PRE-084 (10 μM) or BD1047 (10 μM) followed incubation with dead HT-22 neuronal cells which was prepared as described in Method for 30 min. Dead cells were labeled with CMFDA (Green), macrophages were marked with CD11b (Red). Scale bar: 50 μm. (B) Statistical analysis of phagocytotic index of macrophages. (C) mRNA level of CD206, (D) YM1 and (E) TGF-β was determined 24 h in macrophages after incubation with dead cells. Data are presented as means ± SD, and were analyzed using one-way ANOVA followed by Dunnett's post-hoc tests, n = 3. *P < 0.05; **P < 0.01, ***P < 0.001.

Figure 2

Depletion of Sig-1R abrogates efferocytosis activity of macrophages. Bone marrow derived macrophages (BMDMs) from either wildtype (WT) or Sig-1R knockout (Sig-1R^-/-) mice were incubated with dead cells for 30 min. (A) Flow cytometry analysis of engulfed macrophage or microglia (CMFDA⁺ cells) was conducted as described. Presentative images of wildtype (WT) macrophages (Red line) and Sig-1R^-/- macrophages (blue lines) were exhibited. (B) Phagocytic index of macrophages was calculated as CMFDA⁺ cells among total cells. (C) WT or Sig-1R^-/- BMDMs were incubated with dead cells for 30 min at 1:5 ratio. Dead cells were labeled with CMFDA (Green) and macrophages were marked with CD11b (Red). Scale bar: 50 μm. (D) WT or Sig-1R^-/- primary cortical microglia were incubated with dead cells for 30 min at 1:5 ratio. Dead cells were labeled with CMFDA (Green) and macrophages were marked with Iba1 (Red). Scale bar: 50 μm. Data are presented as means ± SD and were analyzed using student t - test, n = 3. ***P < 0.001.

Figure 3

Depletion of Sig-1R sensitized the brain injure and neurological defects companied with impaired phagocytic activity of macrophage/microglia. WT or Sig-1R^-/- mice were subjected to tMCAO as described. Mice were monitored with neurological scores before sacrificed on day 5 after reperfusion. n = 7 mice / group. (A) Representative image of brain infarct area detected with TTC assay for each treatment. (B) Statistical analysis of infarct volume. (C) Neurological score for animals was assessed according to Material and Methods. (D) Representative images for CD68 (macrophage marker, Red) and NeuN (neuronal marker, Green) which was co-labeled with TUNEL (Blue) in infarct areas. Scale bar: 50μm. High-power 3-D image generated from D (right panel). White arrows indicate microglia / macrophages that engulfed dead / dying neurons (CD68⁺NeuN⁺TUNEL⁺). White arrowheads indicate dead / dying neurons that were not engulfed by microglia / macrophages (CD68^-NeuN⁺TUNEL⁺). Yellow arrows indicate live neurons were engulfed by microglia / macrophages (CD68⁺NeuN⁺TUNEL^-). Yellow arrowhead indicates a TUNEL^- neuron touched by an CD68⁺ cell (CD68⁺NeuN⁺TUNEL^-). Scale bar: 50 μm. (E) The number of total dead / dying neurons in the ischemic striatum of mice. (F) The number of CD68⁺NeuN⁺TUNEL⁺ cells (microglia / macrophages with engulfed dead / dying neurons) in the ischemic striatum of mice. (G) Phagocytic index, as quantified the percentage of dead / dying neurons engulfed by microglia / macrophages ([number of CD68⁺NeuN⁺TUNEL⁺ cells / number of NeuN⁺TUNEL⁺cells] × 100%), in WT and Sig-1R^-/- mice brains. n = 6 mice per group. (H) Percentage of live neurons were engulfed by macrophages / microglia among all engulfed neurons ([number of CD68⁺NeuN⁺TUNEL^- cells / number of CD68⁺NeuN⁺ cells] × 100%) in WT and Sig-1R^-/- mice brains. n = 6 mice per group. Data are presented as means ± SD and were analyzed using student t-test. *P < 0.05; **P < 0.01.

Figure 4

Sig-1R deletion promotes macrophages/microglia activation and its tMCAO. Brains of WT or Sig-1R^-/- mice were subjected to tMCAO as described, and brains were collected on day 5 for further experiments. (A) Representative images of microglia/macrophage marker Iba1 (Red) and inflammatory phenotype marker CD16 (Green) double-staining. Scale bar: 50 μm. (B) Quantification of Iba1⁺CD16⁺ proinflammatory macrophages/microglia in ischemic areas. n = 5 mice per group. (C) Representative images of Iba1 (Red) and CD206 (Green) double staining. Scale bar: 50 μm. (D) Quantification of Iba1⁺ / CD206⁺ macrophages/microglia in ischemic areas. n = 5 mice per group. Data are presented as means ± SD and were analyzed using student t -test. *P < 0.05; ***P < 0.001. (E) Flow cytometry analysis of CD86 expression in CD45⁺CD11b⁺Ly6G^- macrophages in the brain, mean fluorescent intensity (MFI) was calculated (F). (G) Flow cytometry analysis of CD206 level in CD45⁺CD11b⁺Ly6G^- macrophages in the brain, mean fluorescent intensity (MFI) was calculated (H). mRNA expression of proinflammatory cytokines TNF-α (I) and IL-6 (J), resolving cytokines IL-10 (K) and TGF-β (L) were measured with RT-PCR in the ipsilateral hemisphere. n = 5 mice per group. Data are presented as means ± SD and analyzed with one-way ANOVA followed by Dunnett's post-hoc tests. **P < 0.01.

Figure 5

Circulating macrophages attribute to Sig-1R-afforded protection against ischemic stroke. (A) Age- and weight-matched Sig-1R^-/- mice as recipients were subjected tMCAO, BMDMs were prepared as depicted from either WT or Sig-1R^-/- mice. Chimeric mice were constructed by transferring WT or Sig-1R^-/- macrophages through tail vein injection to recipient Sig-1R^-/- mice immediately after reperfusion. (B) Reprehensive images indicating GFP⁺ BMDMs were accumulated at the brain infarct area, scale bar: 50 μm. (C) Reprehensive images depicting transferring BMDMs effectively phagocyte apoptotic neurons (white arrow, GFP⁺CD68⁺NeuN⁺TUNEL⁺), scale bar: 50 μm. (D) Percentage of transferred BMDMs (GFP⁺CD68⁺) among all CD68⁺ microglia/macrophages. (n = 5 mice per group) (E) Percentage of transferred BMDMs contribute to the efferocytosis among all the effectively phagocytes (GFP⁺CD68⁺NeuN⁺TUNEL⁺ / CD68⁺NeuN⁺TUNEL⁺). (n = 5 mice per group). **P < 0.01, one-way ANOVA. (F) Representative image of infarct area of for each treatment on day 5 after tMCAO. (G) The statistical analysis of infarction area and (H) neuronal score in each group was performed on day 5 after tMCAO. n = 7 mice / group. (I) Brains were collected from recipient mice and microglia/macrophage phenotypes were analyzed. Representative images for Iba1 (Green) and CD86 (Red) double staining and Iba1 with Ym1 (Red) double staining. Scale bar: 50 μm. mRNA expression of proinflammatory cytokines TNF-α (J) and IL-6 (K), IL-10 (L) and TGF-β (M) were measured with RT-PCR in the ipsilateral hemisphere. n = 5 mice / group. **P < 0.01, data were analyzed with one-way ANOVA.

Figure 6

Circulating WT macrophages alleviate neuronal deficits after ischemic stroke. Sig-1R knockout mice were used as recipient mice to accepting WT or Sig-1R knockout BMDMs immediately after cerebral ischemia. Neurological function of recipient mice was observed each week. (A) Neurological scores were summarized (n = 6 mice per group). (B) The summary for rotarod test, (C) corner test and (D and E) open-field test were assessed to evaluate the motor and sensory function of recipient mice. n = 12 mice / group. Data are presented as means ± SEM. Two-way ANOVA followed by Dunnett's post-hoc tests were applied. *P < 0.05; **P < 0.01, ***P < 0.001 compared with sham group; #P < 0.05; ##P < 0.01, ###P < 0.001 compared with sham group.

Figure 7

Sig-1R regulate macrophage efferocytosis through Rac1. (A) WT or Sig-1R^-/- BMDMs were incubated with dead cells for 1 h, Rac1 activity was determined. (B) Summary of Rac1 activity in WT and Sig-1R^-/- macrophages. n = 3, data are presented as means ± SD and analyzed with one-way ANOVA followed by Dunnett's post-hoc tests. *P < 0.05 versus WT without dead cells group. ^#P < 0.05 versus WT with dead cells group. (C) Macrophages were pre-treated with Rac1 inhibitor EHop-016 (1 μM) for 30 min followed by Sig-1R agonist PRE084 (10 μM) prior to incubation with dead cells. Dead cells were labeled with CMFDA (Green) and macrophages were marked with CD11b (Red). Scale bar: 20 µM. (D) Phagocytotic index was determined as described. n = 3, data are presented as means ± SD and analyzed with one-way ANOVA followed by Dunnett's post-hoc tests. *P < 0.05, **P < 0.01 versus control group; ###P < 0.001 versus PRE-084-treated group. (E) Effects of Rac1 inhibitor EHop-016 on Sig-1R agonist PRE-084 induced efferocytosis in the mice brain infarct area after cerebral ischemia. Representative Images of CD68 (Red), NeuN (Green), and TUNEL (blue) triple-staining cells in each group. Scale bar: 50 μm. White arrows indicate apoptotic neurons were phagocyted by microglia/macrophages which resented as CD68⁺NeuN⁺TUNEL⁺ cells, yellow arrowheads indicate apoptotic neurons were not effectively phagocyted by microglia/macrophages which presented as CD68^-NeuN⁺TUNEL⁺ cells. (F) Statistical analysis of phagocyte index in each group, which calculated as the percentage of CD68⁺NeuN⁺TUNEL⁺ among all NeuN⁺TUNEL⁺ cells. n = 5, data are presented as means ± SD and analyzed with one-way ANOVA followed by Dunnett's post-hoc tests. *P < 0.05, **P < 0.01 versus vehicle group; ###P < 0.001 versus PRE-084-treated group.

Figure 8

Sig-1R activates and interacts with Rac1 to regulate macrophage efferocytosis. (A) Macrophages were incubated with dead cells for 30 min before processed for immunostaining. Representative images of Rac1 (Red), Sig-1R (Cyan) and CD68 (Green) triple staining were shown from at least three repeated experiments with similar results. Scale bar: 20 μm. (B) Macrophages were pre-treated with BD1047 (10 μM) for 30 min before stimulation with PRE084 (10 μM) for 30 min. The cells were then incubated with dead cells for additional 60 min before collection for further assays. Co-immunoprecipitation (Co-IP) analysis for Rac1 and Sig-1R interaction was shown. (C) HEK293T cells were co-transfected with Myc-Rac1 and Sig-1R plasmids. the interaction between Rac1 and Sig-1R was monitored by co-immunoprecipitation (Co-IP) with anti-Myc beads, followed by SDS-PAGE separation and detected by respective antibody. (D) Single mutation of Rac1 plasmid was prepared as described in Method section, HEK293T cells were transfected with Flag-Sig-1R and wild type Myc-Rac1 or its single mutation, as indicated. After Co-IP with anti-Myc beads, the IP and input samples were separated by SDS-PAGE and probed with indicated antibodies.