Negative regulation of glial Tim-3 inhibits the secretion of inflammatory factors and modulates microglia to antiinflammatory phenotype after experimental intracerebral hemorrhage in rats

Abstract

Aims: To investigate the critical role of Tim-3 in the polarization of microglia in intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI).

Methods: An in vivo ICH model was established by autologous whole blood injection into the right basal ganglia in rats. The primary cultured microglia were treated with oxygen-hemoglobin (OxyHb) to mimic ICH in vitro. In this experiment, specific siRNA for Tim-3 and recombinant human TIM-3 were exploited both in vivo and in vitro.

Results: Tim-3 was increased in the brain after ICH, which mainly distributed in microglia, but not neurons and astrocytes. However, the blockade of Tim-3 by siRNA markedly reduced secretion of inflammatory factors, neuronal degeneration, neuronal cell death, and brain edema. Meanwhile, downregulation of Tim-3 promoted the transformation of microglia phenotype from M1 to M2 after ICH. Furthermore, upregulation of Tim-3 can increase the interaction between Tim-3 and Galectin-9 (Gal-9) and activate Toll-like receptor 4 (TLR-4) pathway after ICH. Increasing the expression of Tim-3 may be related to the activation of HIF-1α.

Conclusion: Tim-3 may be an important link between neuroinflammation and microglia polarization through Tim-3/Gal-9 and TLR-4 signaling pathways which induced SBI after ICH.

Keywords: HIF-1α; Tim-3; inflammation; intracerebral hemorrhage; microglia.

Figure 1

Intracerebral hemorrhage (ICH) model and levels of Tim‐3 in brain tissues which were mainly located in microglia and the levels of inflammatory factors after ICH. A, Whole brain and the largest coronal section of hematoma. B, C, Western blot analysis showed the protein levels of Tim‐3 at various time points in brain tissues after ICH. D, E, ELISA assay was used to detect the brain tissues of IL‐1β and IL‐17. F, Double immunofluorescence analysis was performed with Tim‐3 (green) and astrocytic marker GFAP, microglia marker CD11b, or neurotic maker NeuN (red) in brain sections. Nuclei were fluorescently labeled with DAPI (blue). Scale bar=50 μm. G, The relative Tim‐3 fluorescent intensity in different brain cells was shown. Data are mean ± SD. Except for (G), the rest of the graph, *P < 0.05 vs sham group; **P < 0.01 vs sham group; ***P < 0.001 vs sham group, n = 6; ^† P < 0.05 vs 12 h ICH group; ^†† P < 0.01 vs 12 h ICH group, n = 6; ^‡ P < 0.05 vs ICH (1 d) group; ^‡‡ P < 0.01 vs ICH (1 d) group, n = 6; (G), ***P < 0.001 vs astrocyte group, NS, no significant differences, ^††† P < 0.001 vs microglia group, n = 6

Figure 2

Effects of rhTIM‐3 and Tim‐3 siRNA treatments on the protein level of Tim‐3 in the brain tissues of IL‐1β and IL‐17 under ICH conditions. A and B, Western blot analysis showed the protein level of Tim‐3 in brain tissues in various groups. C and D, ELISA assay was used to detect the brain tissues content of IL‐1β and IL‐17 at 1 d after ICH. Data are mean ± SD. NS: no significant differences; *P < 0.05; **P < 0.01; ***P < 0.001, n = 6. E, Neurological behavior scores. F, Adhesive removal test. G, Foot‐fault test. Data are mean ± SD. NS: no significant differences; *P < 0.05; **P < 0.01; ***P < 0.001, n = 6. rhTIM‐3, recombinant human TIM‐3

Figure 3

Effects of Tim‐3 siRNA on neuronal degeneration and cell death, and brain water content under ICH conditions. A, Fluoro‐Jade B (FJB) staining (green) shows neuronal degeneration in the cerebral cortex. Scale bar = 100 μm. B, Degeneration of neuronal cells in brain tissues is shown. C, Percentage of apoptotic neurons is shown. D, Double immunofluorescence for NeuN (red) and TUNEL (green) counterstained with DAPI (blue) was performed. Arrows point to NeuN/TUNEL‐positive cells. Scale bar = 100 μm. E, Bar graphs showing the effects of rhTIM‐3 and Tim‐3 siRNA on brain water content. Cont: contralateral; Ipsi: ipsilateral; CX: cortex; BG: basal ganglia; Cerebel; cerebellum. Data are mean ± SD. NS: no significant differences; *P < 0.05; **P < 0.01, n = 6. rhTIM‐3, recombinant human TIM‐3

Figure 4

Effects of Tim‐3 on ICH‐induced microglia polarization and changes of phenotype. Sections were stained for CD16/CD11b (pro‐inflammatory microglia marker) or CD206/CD11b (antiinflammatory microglia marker). Representative images were shown in (A) and (C) and percentage of CD16‐positive cells or CD206‐positive cells was shown in (B) and (D). Scale bar = 50 μm. Data are mean ± SD. NS: no significant differences; *P < 0.05; **P < 0.01; ***P < 0.001, n = 6. E and G, The immunoblots showed TNF‐α, IL‐1β, iNOS, arginase1, IL‐4, and IL‐10 produced by microglia under indicated treatment. F and H, The quantitative analyses of TNF‐α, IL‐1β, iNOS, arginase1, IL‐4, and IL‐10 in the immunoblots. Data are mean ± SD. NS: no significant differences; *P < 0.05; **P < 0.01; ***P < 0.001, n = 6

Figure 5

Effects of Tim‐3 on Gal‐9/Tim‐3 and Gal‐9/TLR‐4 interactions after ICH. A and B, Gal‐9/Tim‐3 interaction and Gal‐9/TLR‐4 interactions in brain tissues were determined using immunoprecipitation (IP). C and D, Quantitative analysis was performed. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, n = 6. E, Double immunofluorescence for Tim‐3 or TLR‐4 (red) and Gal‐9 (green) counterstained with DAPI (blue) was performed. Arrows indicated the overlap of Gal‐9 and Tim‐3 or TLR‐4 around the cell edge. Scale bar = 10 μm

Figure 6

The expression level of HIF‐1α protein in cytoplasm and nucleus is closely related to the regulation of Tim‐3 after ICH. A‐D, Western blot analysis and quantification of the protein level of HIF‐1α in the cytoplasm and the nucleus. Data are mean ± SD. NS: no significant differences. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, n = 6. Hypothesis of potential mechanisms of Tim‐3 in inflammatory signaling pathway under ICH condition. Green colored arrows indicate the ICH‐induced Tim‐3 actions, and red colored arrows indicate the effects of rhTIM‐3 and Tim‐3 siRNA. HIF‐1α transports from the cytoplasm to the nucleus, Tim‐3 expression increased after ICH. A large number of Tim‐3 interact with Gal‐9, and the results are activated Tim‐3/Gal‐9 signaling pathway which promotes the production of inflammatory factors. In addition, a large number of TLR‐4 exposure, activation of TLR‐4 signaling pathway which promotes microglia to the pro‐inflammatory state. Two inflammatory pathways are activated, leading to SBI after ICH. rhTIM‐3, recombinant human TIM‐3