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. 2021 May 7:12:667478.
doi: 10.3389/fimmu.2021.667478. eCollection 2021.

Peripheral Injection of Tim-3 Antibody Attenuates VSV Encephalitis by Enhancing MHC-I Presentation

Ge Li  1 , Lili Tang  1 , Chunmei Hou  1 , Zhiding Wang  1 , Yang Gao  1 , Shuaijie Dou  1 , Rongliang Mo  1 , Ying Hao  1 , Zhenfang Gao  1 , Yuxiang Li  1 , Jie Dong  1 , Jiyan Zhang  1 , Beifen Shen  1 , Renxi Wang  2 , Gencheng Han  1
Affiliations

Peripheral Injection of Tim-3 Antibody Attenuates VSV Encephalitis by Enhancing MHC-I Presentation

Ge Li et al. Front Immunol. .

Abstract

Viral encephalitis is the most common cause of encephalitis. It is responsible for high morbidity rates, permanent neurological sequelae, and even high mortality rates. The host immune response plays a critical role in preventing or clearing invading pathogens, especially when effective antiviral treatment is lacking. However, due to blockade of the blood-brain barrier, it remains unclear how peripheral immune cells contribute to the fight against intracerebral viruses. Here, we report that peripheral injection of an antibody against human Tim-3, an immune checkpoint inhibitor widely expressed on immune cells, markedly attenuated vesicular stomatitis virus (VSV) encephalitis, marked by decreased mortality and improved neuroethology in mice. Peripheral injection of Tim-3 antibody enhanced the recruitment of immune cells to the brain, increased the expression of major histocompatibility complex-I (MHC-I) on macrophages, and as a result, promoted the activation of VSV-specific CD8+ T cells. Depletion of macrophages abolished the peripheral injection-mediated protection against VSV encephalitis. Notably, for the first time, we found a novel post-translational modification of MHC-I by Tim-3, wherein, by enhancing the expression of MARCH9, Tim-3 promoted the proteasome-dependent degradation of MHC-I via K48-linked ubiquitination in macrophages. These results provide insights into the immune response against intracranial infections; thus, manipulating the peripheral immune cells with Tim-3 antibody to fight viruses in the brain may have potential applications for combating viral encephalitis.

Keywords: Tim-3; encephalitis; macrophages; major histocompatibility complex-I; ubiquitination; vesicular stomatitis virus.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Peripheral injection of Tim-3 antibody attenuates VSV encephalitis in mice. (A) C57BL/6 mice were intracranially injected with VSV (1 × 106 pfu/g) on day 0 and intraperitoneally injected with Tim-3 antibody (10 mg/kg, n=15) or isotype control (n=15) on days -2,-1, 1, and 2. Survival rate was analyzed. (B–D) Mice were infected and treated as elaborated in (A). Brain tissues were collected on day 5 post-infection and VSV loads were analyzed by TCID50 assay (B, n=14 in each group). VSV replication were analyzed by real-time polymerase chain reaction (C, n=28 in each group), and (D) Hematoxylin and eosin stain of the brain section. Arrow in a) shows the damaged meningitis which is absent in b). Arrow in c) shows perivascular lymphocyte cuffing which is a well-known indicator of inflammation in the brain. No perivascular lymphocyte cuffing was found in d). The data shown are representative of six samples from each group. Original magnification ×100. (E) Mice underwent the open-field test, and their spontaneous locomotor activity was evaluated by measuring the distance traveled in a defined area for 10 min. (F) Mice underwent the CatWalk analysis assay to evaluate locomotor deficits via measuring the regularity index with a maximum of 10 s permissible to traverse a glass plate. In (B, C, E, F), the data are expressed as mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001.
Figure 2
Figure 2
Peripheral injection of Tim-3 antibody enhances the recruitment of peripheral immune cells. Mice were treated as shown in Figure 1 . On day 5 post-infection, mice were sacrificed, brain tissues were collected from them, and single-cell suspensions were prepared as described in the Material and Methods. The infiltration of CD4+T cells (A), CD8+T cells (B) and peripheral monocyte-derived macrophages (pMDM) cells (CD11b+CD45hi) (C) were examined by flow cytometry analysis. The left panel shows the representative fluorescence-activated cell sorting (FACS) plot, and the right panel are presented as means ± SEM of three independent experiments. **p < 0.01, ***p < 0.001.
Figure 3
Figure 3
Deletion of macrophages abolished Tim-3 antibody-mediated immune protection against VSV encephalitis. Mice were treated as shown in Figure 1 , and untreated mice were used as sham controls. For macrophage depletion, Clodronate Liposomes (CL) were intravenously injected three times on days -2, 0, and 2. On day 5 post-infection, the mice were sacrificed. Brain tissues were collected, and single-cell suspensions were prepared and stained for detecting CD11b and CD45 expression by flow cytometry analysis. (A) shows the representative fluorescence-activated cell sorting (FACS) plot; while, (B) shows the mean ± SEM of three independent experiments. (C) The brain tissues were analyzed for VSV replication by real-time polymerase chain reaction, and the results are expressed as mean ± SEM of three independent experiments. (D) Mice were subjected to the open-field test to evaluate spontaneous locomotor activity by measuring the distance traveled in a defined area for 10 min. Data are expressed as mean ± SEM of three independent experiments. (E) Mice were subjected to the CatWalk analysis to evaluate locomotor deficits by measuring the regularity index, with maximum 10 s to traverse a glass plate. Data are expressed as mean ± SEM of three independent experiments. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4
Figure 4
Peripheral injection of Tim-3 antibody enhances the activation of VSV-specific CD8+ T cells. Mice were treated as described in Figure 1 . On day 5 post-infection, brain tissues were collected. (A) Single-cell suspensions were prepared and stained with antibodies against CD8 and MHC-I-VSV-tetramer. Left panel shows the representative fluorescence-activated cell sorting (FACS) plot and right panel shows the mean percentage ± SEM of three independent experiments. **P < 0.01, ***P < 0.001. (B) The brain tissues were stained with immunofluorescence labels for CD8 (green), MHC-I-VSV-tetramer (red), and DAPI (blue). Data shown are representative images of three independent experiments.
Figure 5
Figure 5
Peripheral injection of Tim-3 antibody enhances the expression of major histocompatibility complex-I (MHC-I). Mice were treated as shown in Figure 1 . On day 5 post-infection, the mice were sacrificed. (A) Peritoneal macrophages were collected and stained with antibodies against F4/80 and MHC-I. The left panel shows the representative fluorescence-activated cell sorting (FACS) plot and right panel shows the mean fluorescence intensity (MFI) ± SEM of three independent experiments. (B, C) Brain tissues were collected, and single-cell suspensions were prepared and stained with anti-CD11b and anti-CD45 antibody. CD11b+CD45hi pMDM (P2) and CD11b+CD45Low microglia (P1) were further gated to analyze the fluorescence intensity of MHC-I using MHC-I antibody. The left panel shows the representative FACS plots and the right panel shows the MFI ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6
Figure 6
Tim-3 promotes proteasome-dependent degradation and ubiquitination of major histocompatibility complex-I (MHC-I). (A) The HEK293T cells were transfected with plasmids encoding Flag-Tim-3 or vector control. Cells were then treated with or without a protein synthesis inhibitor CHX (10 µM) for 6h. Twenty-four hours following transfection, cells were harvested and the level of MHC-I protein was examined by western blot. The upper panel shows the representative image of three independent experiments and the down panel shows the value of the MHC-I/β-tubulin protein ratio which is expressed as mean ± SEM of three independent experiments. (B) The HEK293T cells were transfected with plasmids encoding Flag-Tim-3 or vector control. All cells were treated with CHX (10 µM) in the presence or absence of the proteasome inhibitor MG132 (20 µg/ml) for 6h. Twenty-four hours following transfection, cells were harvested and the level of MHC-I protein was examined by western blot. The upper panel shows the representative image of three independent experiments and the down panel shows the value of the MHC-I/β-tubulin protein ratio which is expressed as mean ± SEM of three independent experiments. (C) Plasmid encoding HA-Tim-3 was transfected into HEK293T cells for 24h, followed by immunoprecipitation with antibody to MHC-I and then Western blot analysis of MHC-I ubiquitination with antibody to HA. The data shown are the representative image for those of three independent experiments. (D&E) The HEK293T cells were transfected with plasmids encoding HA-ubiquitin, Flag-MARCH9 and increasing doses of a plasmid encoding Red-Tim-3 (0.5,1, and 2 µg). The level of MHC-I protein was examined in cells harvested 24h later. D) shows the representative image of three independent experiments and E) shows the value of the MHC-I/β-tubulin protein ratio which is expressed as mean ± SEM of three independent experiments. (F) Plasmids encoding HA-tag-Ubiquitin K48 (K48-lined ubiquitination leads to proteasomal-mediated degradation of the target protein), Flag-MARCH9, and Red-Tim-3 were transfected into HEK293T cells. Cells were treated with MG132 (20 µg/ml) for 6h before harvesting protein lysates, followed by immunoprecipitation with antibody to MHC-I and then Western Blot analysis of K48-linked MHC-I ubiquitination with antibody to HA. These experiments were repeated 3 times with similar results. In A&B, the data shown are expressed as mean ± SEM of three independent experiments. **p<0.01; ***p<0.001.
Figure 7
Figure 7
Schematic diagram of how Tim-3 antibody prevents vesicular stomatitis virus (VSV) encephalitis in mice. Peripheral injection of Tim-3 antibody promoted the activation of peripheral macrophages and CD8+ T cells, which was marked by an increased expression of chemokine receptors including C-C chemokine receptor type 5 (CCR5) and C-X-C chemokine receptor 3 (CXCR3), and increased expression of interferons. Notably, Tim-3 antibody enhanced major histocompatibility complex-I (MHC-I) expression by blocking Tim-3-mediated ubiquitination and degradation of MHC-I. As a consequence of cytokine and chemokine secretion, viral infection of the central nervous system (CNS) resulted in the recruitment of activated macrophages and CD8+ T cells into the brain, where the macrophages present the virus antigen and activate CD8+ T cells to cytotoxic T effector cells. BBB, blood-brain barrier.
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