Regulatory T cells ameliorate intracerebral hemorrhage-induced inflammatory injury by modulating microglia/macrophage polarization through the IL-10/GSK3β/PTEN axis

Abstract

Inflammation mediated by the peripheral infiltration of inflammatory cells plays an important role in intracerebral hemorrhage (ICH) induced secondary injury. Previous studies have indicated that regulatory T lymphocytes (Tregs) might reduce ICH-induced inflammation, but the precise mechanisms that contribute to ICH-induced inflammatory injury remain unclear. Our results show that the number of Tregs in the brain increases after ICH. Inducing Tregs deletion using a CD25 antibody or Foxp3DTR-mice increased neurological deficient scores (NDS), the level of inflammatory factors, hematoma volumes, and neuronal degeneration. Meanwhile, boosting Tregs using a CD28 super-agonist antibody reduced the inflammatory injury. Furthermore, Tregs depletion shifted microglia/macrophage polarization toward the M1 phenotype while boosting Tregs shifted this transition toward the M2 phenotype. In vitro, a transwell co-culture model of microglia and Tregs indicated that Tregs changed the polarization of microglia, decreased the expression of MHC-II, IL-6, and TNF-α and increased CD206 expression. IL-10 originating from Tregs mediated the microglia polarization by increasing the expression of Glycogen Synthase Kinase 3 beta (GSK3β), which phosphorylates and inactivates Phosphatase and Tensin homologue (PTEN) in microglia, TGF-β did not participate in this conversion. Thus, Tregs ameliorated ICH-induced inflammatory injury by modulating microglia/macrophage polarization toward the M2 phenotype through the IL-10/GSK3β/PTEN axis.

Keywords: Intracerebral hemorrhage; T-cells; inflammation; macrophages; microglia.

Figures

Figure 1.

The infiltration of Tregs into the CNS after ICH. (a) Representative FACS plots showing that CD45hiCD3+CD4+ cells were defined as brain-invading T helper cells and further analyzed to identify Tregs. FSC-A indicates the forward scatter channel area, and SSC-A indicates side scatter channel area. (b) Temporal changes in brain-invading CD4+T cells and Tregs after ICH, and Sham-operated mice that were analyzed on day 4 post-hemorrhage. (c) Representative FACS plots showing that CD4+Foxp3+ cells are considered Tregs in spleen. (d) The proportions of Foxp3+ cells in CD4+ cells in the brain and the spleen were separately analyzed at 1, 4, 7 and 14 d after ICH (4 individual experiments with four pooled animals per group in each experiment; *p < 0.05 versus the sham group in brain, #p < 0.05 versus the sham group in spleen).

Figure 2.

Foxp3DTR mice exhibited increased inflammatory injury after ICH. (a) The NDS of WT and Foxp3DTR mice at 1, 3, 5, and 7 d after ICH (*p < 0.05 versus the WT-ICH group at the corresponding time points, n = 12 for each time point). (b) Brain water content at 4 d after hemorrhage or sham operation. (c) Serial coronal sections of mouse brain tissues at 4 d after ICH. (d) Brain sections were used to measure the hematoma volume. (e) Brain homogenate was used to measure the hemoglobin level (**p < 0.01, *p < 0.05 versus the WT-ICH group, n = 4). (f) Representative FJB-positive cells in peri-hematomal tissue of WT and Foxp3DTR groups (bars=20 µm). (g) FJB-positive cells were quantified and compared (**p < 0.01 versus WT-ICH, n = 6). (h) Global changes in M1-related inflammatory factors. (i) Global changes in M2-related inflammatory factors at 4 d after hemorrhage or sham operation were analyzed using RT-PCR (*p < 0.05, **p < 0.01 versus the WT-ICH group, n = 4). Cereb, cerebellum; Cont BG, contralateral basal ganglia; Cont CX, contralateral cortex; Ipsi BG, ipsilateral basal ganglia; Ipsi CX, ipsilateral cortex.

Figure 3.

CD25 antibodies increased inflammatory injury after ICH. (a) The NDSs in the IgG and AntiCD25 groups at 1, 3, 5, and 7 d after ICH (*p < 0.05 versus the IgG group at the corresponding time points, n = 12 for each time point). (b) Brain water content at 4 d after hemorrhage or sham operation. (c) Serial coronal sections of mouse brain tissues at 4 d after ICH. (d) Brain sections were used to measure the hematoma volume. (e) Brain homogenate was used to measure the hemoglobin level (**p < 0.01, *p < 0.05 versus the WT-ICH group, n = 4). (f) Representative FJB-positive cells in the AntiCD25 and IgG groups (bars=20 µm). (g) FJB-positive cells were quantified and compared (**p < 0.01 versus IgG, n = 6). (h) Global changes in M1-related inflammatory factors. (i) Global changes in M2-related inflammatory factors at 4 d after hemorrhage or sham operation were analyzed using RT-PCR (*p < 0.05, **p < 0.01 versus the IgG group, n = 4).

Figure 4.

CD28-SA ameliorated inflammatory injury after ICH. (a) The NDSs in the IgG and CD28-SA groups at 1, 3, 5, and 7 d after ICH (*p < 0.05 versus the IgG group at the corresponding time points, n = 12 for each time point). (b) Brain water content at 4 d after hemorrhage or sham operation. (c) Serial coronal sections of mouse brain tissues at 4 d after ICH. (d) Brain sections were used to measure the hematoma volume. (e) Brain homogenate was used to measure the hemoglobin level (**p < 0.01, *p < 0.05 versus the WT-ICH group, n = 4). (f) Representative FJB-positive cells in the CD28-SA and IgG groups (bars=20 µm). (g) FJB-positive cells were quantified and compared (**p < 0.01 versus IgG, n = 6). (h) Global changes in M1-related inflammatory factors. (i) Global changes in M2-related inflammatory factors at 4 d after hemorrhage or sham operation were analyzed using RT-PCR (*p < 0.05, **p < 0.01 versus the IgG group, n = 4).

Figure 5.

IL-10 originating from Tregs influenced microglia polarization. In vivo experiments. (a) Representative FACS plots for the sham-operated, WT-ICH, Foxp3DTR-ICH, and CD28SA-ICH groups showing that microglia/macrophage were gated by CD45+CD11b+ from brain inflammatory cells and that the expression levels of MHC-II and CD206 were determined by analyzing mean fluorescence intensity (MFI). (b) The absolute number of microglia/macrophages was calculated using flow cytometry, and the MFI of MHC-II and CD206 on these cells were analyzed (*p < 0.05, **p < 0.01 versus the sham group, four individual experiments with three pooled animals per group are shown for each experiment). The in vitro co-culture model further indicated the function of Tregs. (c) TNF-α and IL-6 were detected using RT-PCR in microglia (d) The numbers of microglia and MFI of CD206 and MHC-II on microglia. (e) The MFI was calculated for CD206 and MHC-II (##p < 0.01 versus the vehicle; *p < 0.05, **p < 0.01 versus the microglia + Hb group, statistics were from four separate experiments).

Figure 6.

GSK3β/PTEN axis mediated the IL-10 induced microglia polarization. (a) A representative Western-blot shows the expression of GSK3b, P-GSK3b, PTEN, and P-PTEN in microglia 40 h after co-culture. (b) The expression of GSK3β was calculated. (c) Representative Western-blot shows the expression of P-PTEN and P-GSK3β in Lentiviral shRNA-infected microglia. (d) The expression of P-PTEN was calculated (##p < 0.01 versus the vehicle; *p < 0.05, **p < 0.01 versus the microglia + Hb group; statistics were from four separate experiments with four wells per trail).