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. 2018 Mar 28;92(8):e02029-17.
doi: 10.1128/JVI.02029-17. Print 2018 Apr 15.

MALT1 Controls Attenuated Rabies Virus by Inducing Early Inflammation and T Cell Activation in the Brain

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Free PMC article

MALT1 Controls Attenuated Rabies Virus by Inducing Early Inflammation and T Cell Activation in the Brain

E Kip et al. J Virol. .
Free PMC article

Abstract

MALT1 is involved in the activation of immune responses, as well as in the proliferation and survival of certain cancer cells. MALT1 acts as a scaffold protein for NF-κB signaling and a cysteine protease that cleaves substrates, further promoting the expression of immunoregulatory genes. Deregulated MALT1 activity has been associated with autoimmunity and cancer, implicating MALT1 as a new therapeutic target. Although MALT1 deficiency has been shown to protect against experimental autoimmune encephalomyelitis, nothing is known about the impact of MALT1 on virus infection in the central nervous system. Here, we studied infection with an attenuated rabies virus, Evelyn-Rotnycki-Abelseth (ERA) virus, and observed increased susceptibility with ERA virus in MALT1-/- mice. Indeed, after intranasal infection with ERA virus, wild-type mice developed mild transient clinical signs with recovery at 35 days postinoculation (dpi). Interestingly, MALT1-/- mice developed severe disease requiring euthanasia at around 17 dpi. A decreased induction of inflammatory gene expression and cell infiltration and activation was observed in MALT1-/- mice at 10 dpi compared to MALT1+/+ infected mice. At 17 dpi, however, the level of inflammatory cell activation was comparable to that observed in MALT1+/+ mice. Moreover, MALT1-/- mice failed to produce virus-neutralizing antibodies. Similar results were obtained with specific inactivation of MALT1 in T cells. Finally, treatment of wild-type mice with mepazine, a MALT1 protease inhibitor, also led to mortality upon ERA virus infection. These data emphasize the importance of early inflammation and activation of T cells through MALT1 for controlling the virulence of an attenuated rabies virus in the brain.IMPORTANCE Rabies virus is a neurotropic virus which can infect any mammal. Annually, 59,000 people die from rabies. Effective therapy is lacking and hampered by gaps in the understanding of virus pathogenicity. MALT1 is an intracellular protein involved in innate and adaptive immunity and is an interesting therapeutic target because MALT1-deregulated activity has been associated with autoimmunity and cancers. The role of MALT1 in viral infection is, however, largely unknown. Here, we study the impact of MALT1 on virus infection in the brain, using the attenuated ERA rabies virus in different models of MALT1-deficient mice. We reveal the importance of MALT1-mediated inflammation and T cell activation to control ERA virus, providing new insights in the biology of MALT1 and rabies virus infection.

Keywords: ERA; MALT1; immunity; neuroinflammation; rabies virus.

Figures

FIG 1
FIG 1
MALT1 is critical to control the pathogenicity of ERA virus. MALT1−/− (n = 10) and MALT1+/+ littermates (n = 10) were infected intranasally with ERA virus. Clinical symptoms (A) and survival rates (B) were assessed. All MALT1−/− mice developed severe disease and had to be euthanized. MALT1+/+ mice developed only mild symptoms. The results shown are representative of two independent experiments.
FIG 2
FIG 2
Virus spread in the brains of MALT1−/− and MALT1+/+ mice after intranasal inoculation. (A) Schematic overview of the experiment. Mice were inoculated intranasally with ERA virus and sacrificed at 10, 17, and 35 dpi. (B) Profile of viral RNA load in total brain determined by RT-qPCR. (C and D) Profile of viral RNA load in different parts of the brain (**, P ≤ 0.01). (E) Immunofluorescence staining for viral nucleocapsid in the brain tissue. At 10 dpi, green fluorescent spots indicate the abundant spread of virus in the brain of MALT1−/− and MALT1+/+ mice. At 17 dpi, only small amounts of viral antigens were still visible in MALT1+/+ mice, whereas viral antigens were still abundant in the brains of MALT1−/− mice. These results are representative of three mice per time point and per genotype. Scale bars, 20 μm; magnification, ×40.
FIG 3
FIG 3
Defective expression of antiviral and inflammatory genes in the brains of ERA virus-infected MALT1−/− mice. Quantitative RT-qPCR measurements of the indicated mRNA expression levels in brains of MALT1+/+ (n = 7) and MALT1−/− littermate mice (n = 7) at 10 and 17 dpi are shown. The results are represented as the fold increases compared to noninfected MALT1+/+ and MALT1−/− littermate mice, respectively. Differences in the fold increase at the same time point were determined by two-way ANOVA and Sidak's multiple-comparison test, and statistical differences between MALT1+/+ and MALT1−/− mice are denoted by asterisks. Asterisks (****, ***, **, and *) represent P values of <0.0001, 0.001, 0.01, and 0.05, respectively.
FIG 4
FIG 4
Reduced infiltration and activation of inflammatory cells in the brains of MALT1−/− mice at 10 dpi with ERA virus. Immunohistochemical analysis of CNS sections from ERA virus-infected MALT1+/+ and MALT1−/− mice at 10 and 17 dpi. PBS-inoculated MALT1+/+ mice were used as controls. Sections of the cerebellum and hippocampus are shown. Brain sections were immunostained for Iba-1 (microglial cells), CD3 (T cells), Mac-3 (macrophages), B220 (B cells), and GFAP (astrocytes). PBS-injected mice showed abundant inactive ramified microglial cells and astrocytes, but no B cell, macrophage, or T cell infiltration. At 10 dpi, ERA virus-infected MALT1+/+ mice showed activation of microglial and astroglial cells, infiltration of T lymphocytes and macrophages in the parenchyma, and infiltration of B cells around the blood vessels and choroid plexus. In MALT1−/− mice, T lymphocyte and macrophage infiltration, as well as microglial activation, were reduced at 10 dpi. Moreover, B cells could not be observed around the blood vessels or the choroid plexus in MALT1−/− mice. At 17 dpi, pronounced microglial activation and T lymphocyte infiltration were observed in both MALT1+/+ and MALT1−/− mice. Macrophages were no longer visible. Astrogliosis increased further but was more pronounced in MALT1+/+ mice than in MALT1−/− mice. Scale bars represent 20 μm (magnification, ×20), 50 μm (magnification, ×10), or 100 μm (magnification, ×4). Data are representative of two mice per condition.
FIG 5
FIG 5
Flow cytometric analysis of immune cell activation and infiltration in the brains of ERA virus-infected mice. Immune cells were isolated from the brains of naive mice and infected mice (MALT1+/+ and MALT1−/−) at 10 dpi. (A) Absolute numbers of leukocytes present in the brain were first determined, and the total numbers of each cell type were determined by the percentage of marker expression on the total number of leukocytes. A significant decrease in total leukocytes was observed in ERA virus-infected MALT1−/− mice compared to ERA virus-infected MALT1+/+ mice. (B) CD49b and CD3 markers were used to distinguish NK cells (CD49b+ CD3), NKT cells (CD49b+ CD3+), and T cells (CD49b CD3+). CD8 and CD4 markers were also used. A significant decrease in NK cells, NKT cells, and CD8+ T cells was observed in ERA virus-infected MALT1−/− mice compared to ERA virus-infected MALT1+/+ mice. (C) CD45 and CD11b markers were used to distinguish T cells (CD3+ CD11b CD45high), microglial cells (CD11b+ CD45int), and MMDCs (monocytes, macrophages, and DCs; CD11b+ CD45high). CD45int cells were selected to analyze microglial activation by using CD11b and CD86, a costimulatory molecule expressed on activated antigen-presenting cells. CD45high cells were selected to analyze monocyte, macrophage, and DC activation. Activation of microglial cells and MMDCs was determined by CD86 expression represented as the MFI. A significant decrease of CD86 MFI was observed in ERA virus-infected MALT1−/− mice compared to ERA virus-infected MALT1+/+ mice, which corresponds to the dot plots. The dot plots are representative of five mice per condition. Statistical differences between MALT1+/+ and MALT1−/− mice were determined using a Student t test . Asterisks (***, **, and *) represent P values of <0.001, 0.01 and 0.05, respectively.
FIG 6
FIG 6
Reduced granzyme B, IL-17, and IFN-γ production in T cells from ERA virus-infected MALT1−/− mice. Immune cells were isolated from the brains of naive mice and infected mice (MALT1+/+ and MALT1−/−) at 10 dpi and stained for the intracellular markers IFN-γ, IL-17, and granzyme B. T cells were gated for their phenotypic marker CD3. (A and B) FACS analysis revealed less production of IL-17, granzyme B, and IFN-γ in the T cells of ERA virus-infected MALT1−/− mice compared to ERA virus-infected MALT1+/+ mice. The dot plots are representative of five mice per condition. (C) A significant decrease in T cells expressing IFN-γ, granzyme B, and IL-17 was observed in infected MALT1−/− mice compared to infected MALT1+/+ mice. Statistical differences between MALT1+/+ and MALT1−/− mice were determined by using a Student t test. Asterisks (***, **, and *) represent P values of <0.001, 0.01 and 0.05, respectively.
FIG 7
FIG 7
Defective humoral immune response in MALT1−/− mice. In MALT1+/+ mice, antibody production was first detected in 1 out 7 mice at 10 dpi. At 17 dpi, neutralizing antibodies were detected in all MALT1+/+ mice (n = 7), and antibody levels increased further at 35 dpi (n = 7). Neutralizing antibodies were not detected in MALT1−/− mice (n = 7) at 10 dpi and in 5 of 7 mice at 17 dpi. The two remaining MALT1−/− mice had low levels of antibodies just above the cutoff at 17 dpi.
FIG 8
FIG 8
Transfer of neutralizing antibodies does not rescue MALT1−/− mice. (A) Schematic overview of the experimental setup. MALT1+/+ mice were inoculated intranasally with the ERA virus and sacrificed at 35 dpi. Immune sera were collected, pooled, heat inactivated, and titrated prior to i.p. transfer to MALT1−/− mice at 10 dpi (500 μl, 4.8 IU/ml). (B) Survival of infected mice. All MALT1−/− mice developed severe disease and had to be euthanized, despite transfer of immune serum.
FIG 9
FIG 9
Impact of specific MALT1 deficiency in T cells, myeloid cells, or cells from neuroectodermal origin on ERA virus infection. (A) Schematic overview of conditional MALT1−/− mouse generation. Mice expressing the CRE recombinase gene under the influence of the CD4, LysM, or Nestin promoter were crossed with MALT1FL/FL mice to generate conditional mice lacking MALT1 in T cells, myeloid cells, or cells from a neuroectodermal origin, respectively. Conditional mice and their wild-type littermates were genotyped and selected for each experiment. (B) Survival of infected mice. Mice were infected intranasally with ERA virus and monitored for disease development and survival. Mice lacking MALT1 in cells from neuroectodermal origin (Nestin-Cretg/+ MALT1FL/FL) and myeloid cells (LysM-Cretg/+ MALT1FL/FL) developed only mild disease, comparable to their wild-type littermates. Mice lacking MALT1 in T cells (CD4-Cretg/+ MALT1FL/FL) presented the same phenotype as the full MALT1−/− and developed severe disease requiring euthanasia at 15 dpi. (C) Virus neutralizing antibodies in serum. Except for one, all CD4-Cretg/+ MALT1FL/FL mice failed to mount protective levels of neutralizing antibodies. (D) Viral RNA in total brain. All T cell-specific MALT1−/− mice (CD4-Cretg/+ MALT1FL/FL) presented a high viral load at 15 dpi, comparable to full MALT1−/− mice.
FIG 10
FIG 10
Treatment with mepazine in ERA virus-infected MALT1+/+ mice: impact on survival, viral load, and neutralizing antibody production. (A) Schematic overview of the experimental setup. MALT1+/+ mice were treated daily with mepazine (n = 7) or a control solution (0.9% NaCl) (n = 7) starting at day −2 before virus inoculation until the end of the experiment. Two days after the first treatment, mice were inoculated intranasally with ERA virus and monitored daily for signs of disease. (B) Survival curves. Four of seven mepazine-treated mice developed severe disease and had to be euthanized. Control mice and the remaining three mepazine-treated mice survived the infection. (C) Profile of viral RNA in total brains of mepazine-treated MALT1+/+ mice (n = 7) and control mice (n = 7) at sacrifice determined by RT-qPCR. Mepazine-treated mice with severe disease had higher viral loads. (D) Humoral immune response in mepazine-treated mice and control mice. Mepazine-treated mice with severe disease had no neutralizing antibodies. Control mice and the three surviving mepazine-treated mice had developed protective levels of neutralizing antibodies. The results were obtained from one experiment.
FIG 11
FIG 11
Hypothetical model of the impact of MALT1 inactivation on ERA virus-induced immune responses in the brain. In MALT1+/+ mice, virus-infected neuronal cells produce CXCL10 (as shown in other studies), leading to the activation of microglia and recruitment of CXCR3-expressing T cells and NK cells to the brain parenchyma. Activated microglia and astrocytes start to produce proinflammatory cytokines, iNOS and CXCL10, which amplifies the recruitment and activation of several immune cell types. Activated macrophages migrate to the cervical lymph nodes, where they serve as antigen-presenting cells for naive T cells and trigger the differentiation to effector T cells, mainly CD4+ Th1 and Th17 subsets and cytotoxic CD8+ T cells. Only activated T cells can infiltrate the brain. IFN-γ-producing CD8+ T cells are the main T cells recruited to the site of infection. Th1 cells and CD8+ T cells can further activate macrophages by IFN-γ production, and antigen-presenting cells can reactivate the T cells that have entered the brain. CD8+ T cells also produce granzyme B, mediating their cytotoxic activity and virus clearance. Th1 cells provide help for B cell activation and immunoglobulin production outside the brain, whereas Th17 cells produce IL-17, contributing to the activation of microglia and astrocytes, as well as enhancing blood-brain barrier permeability. The impact of MALT1 deficiency is represented by orange arrows. MALT1 deficiency in T cells reduces their activation and differentiation by antigen-presenting cells in the periphery, resulting in less effector T cells entering the brain. This not only results in less IFN-γ and IL-17 production but also lowers T cell help to activate B cells and CD8+ cytotoxic T cells, leading to defective production of virus neutralizing antibodies and reduced killing of virus-infected cells. As a consequence, viral load in the brain increases, exacerbating disease development.

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