Increased expression of T cell immunoglobulin and mucin domain 3 aggravates brain inflammation via regulation of the function of microglia/macrophages after intracerebral hemorrhage in mice

Abstract

Background: Microglia/macrophages are known to play important roles in initiating brain inflammation after spontaneous intracerebral hemorrhage (ICH). T cell immunoglobulin and mucin domain-3 (Tim-3) have been proven to play a critical part in several inflammatory diseases through regulation of both adaptive and innate immune responses. Tim-3 can be expressed by microglia/macrophages and regulates their function in the innate immune response. However, the effect of Tim-3 on inflammatory responses following ICH is unclear.

Methods: In this study, we investigated Tim-3 expression, the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and brain water content in peri-hematomal brain tissue at 12 hours and at 1, 3, 5, and 7 days post-ICH in wild type (WT) ICH and Tim-3-/- ICH mice. The numbers of Tim-3 positive cells,astrocytes, neutrophils and microglia/macrophages were detected using immunofluorescence staining. Cytokines were measured by ELISA. Double immunofluorescence labeling was performed to identify the cellular source of Tim-3 expression. Mouse neurological deficit scores were assessed through animal behavior.

Results: Expression of Tim-3 increased early in mouse peri-hematomal brain tissue after autologous blood injection, peaked at day 1, and was positively correlated with the concentrations of TNF-α, IL-1β, and brain water content. Tim-3 was predominantly expressed in microglia/macrophages. Compared with WT mice, Tim-3-/- mice had reduced ICH-induced brain inflammation with decreased TNF-α and IL-1β, cerebral edema and neurological deficit scores. Moreover, Tim-/- inhibited activation of microglia/macrophages. The number of activated microglia/macrophages in Tim-3-/- ICH mice was much lower than that in WT ICH mice.

Conclusions: Our findings demonstrate that Tim-3 plays an important role in brain inflammation after ICH, and may be a potential treatment target.

Figure 1

Expression of Tim-3 positive cells and Tim-3 mRNA in peri-hematomal brain tissues at time-tested points following intracerebral hemorrhage (ICH). A) and B) Immunofluorescence staining showed that the number of Tim-3 positive cells increased at 12 hours in the wild type (WT) ICH group mice (n = 6), with the maximum at Day 1, **P <0.01 versus the WT sham group (mean ± SD). Scale bar = 20 μm. C) Real-time RT-PCR indicated that the expression of Tim-3 mRNA increased at all time-tested points in the ICH group mice (n = 6) and peaked at Day 1. **P <0.01 versus WT sham group (mean ± SD). ‘Relative fold’ meant the level of mRNA of Tim-3 relative to that of beta-actin.

Figure 2

The expression of Tim-3 in different brain cells at day 1 after intracerebral hemorrhage (ICH). Double-immunofluorescence staining displayed that: A) A few of Tim-3 positive cells could express in GFAP⁺ cells (astrocytes). B) A few of Tim-3 positive cells could express in MPO⁺ cells (neutrophils), but more than were expressed in GFAP⁺ cells (astrocytes). The arrows indicate co-expressed cells of Tim-3 and MPO. C) Almost all CD11b⁺ cells (microglia/macrophages) were Tim-3 positive cells at day 1 after ICH. The arrows indicate co-expressed cells of Tim-3 and CD11b. Scale bar = 20 μm.

Figure 3

The change of IL-1β, TNF-a,brain water content and neurological deficit scores (NDS) after intracerebral hemorrhage (ICH). A) indicates the change of the concentration of IL-1β in the peri-hematomal brain tissues at 12 hours andat 1, 3, 5 and 7 days post-ICH. B) shows the change of the concentration of TNF-a. C) displays the change of brain water content. D) shows the NDS between the Tim-3^−/− ICH group and the wild type (WT) ICH group. The WT ICH group (n = 6) versus the WT sham group (n = 6): **P <0.01; The Tim-3^−/− ICH group (n = 6) versus the Tim-3^−/− sham group (n = 6): &&P < 0.01; The Tim-3^−/− ICH group (n = 6) versus the WT ICH group (n = 6): ^#P < 0.05, ^##P < 0.01.

Figure 4

The correlation between the expression of Tim-3 and IL-1β, TNF-a, brain water content. A), B) and C) indicate that the expression of Tim-3 was positively correlated with IL-1β (r = 0.618, P <0.001), TNF-a (r = 0.610, P <0.001) and brain water content (r = 0.566, P = 0.001).

Figure 5

The change of the number of astrocytes, neutrophils and microglia/macrophages after intracerebral hemorrhage (ICH).A) indicates the change in the number of astrocytes in the peri-hematomal brain tissues at 12 hours and at 1, 3, 5 and 7 days post-ICH. B) shows the change in the number of neutrophils. C) displays the change in the number of microglia/macrophages in the wild type (WT) ICH group (n = 6) versus the WT sham group (n = 6): **P <0.01; The Tim-3^−/− ICH group (n = 4) versus the Tim-3^−/− sham group (n = 4): &&P <0.01; the Tim-3^−/− ICH group (n = 4) versus the WT ICH group (n = 6): ^##P <0.01.