key: cord-0007275-z5lv81bi
authors: Hirano, Yuta; Kobayashi, Kunitoshi; Tomiki, Hiroki; Inaba, Yuhji; Ichikawa, Motoki; Kim, Byung S.; Koh, Chang-Sung
title: The role of α4 integrin in Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease: an infectious animal model for multiple sclerosis (MS)
date: 2016-11-01
journal: Int Immunol
DOI: 10.1093/intimm/dxw045
sha: 08912859bba139ba760b709510984b9415e5af03
doc_id: 7275
cord_uid: z5lv81bi

Natalizumab, which is an antibody against α4 integrin, has been used for the treatment of multiple sclerosis (MS). In the present study, we investigated both the role of α4 integrin and the therapeutic effect of HCA3551, a newly synthesized orally active small-molecule α4 integrin antagonist, in the development of TMEV-induced demyelinating disease (TMEV-IDD). The mRNA levels of α4 integrins were significantly up-regulated in the CNS of mice with TMEV-IDD as compared with naïve mice (*p<0.05). HCA3551 treatment in the effector phase significantly suppressed both the clinical and histological development of TMEV-IDD. The number of infiltrating mononuclear inflammatory cells in the CNS was significantly decreased in the mice treated with HCA3551 (**p<0.01). The labeling indices for CD68 antigen and the absolute cell numbers of TNF-α-producing CD4(+) T cells and IFN-γ-producing CD8(+) T cells were significantly decreased in the CNS of mice treated with HCA3551 (*p<0.05). HCA3551 treatment in the effector phase might inhibit the binding of α4 integrin to VCAM-1, thereby decreasing the number of MNCs in the CNS.

Multiple sclerosis (MS) is an immune-mediated chronic demyelinating disease that is associated with inflammation in the white matter of the human central nervous system (CNS).

Histologically, MS is characterized by CNS lesions displaying inflammation, demyelination and axonal damage. Although its etiology remains unclear, MS is generally considered to be an autoimmune disease mediated by T helper (Th) 1 and Th 17 cells (1, 2) . The morphology of the acute lesion and the susceptibility to MS that is apparently conferred through certain MHC class II-restricted antigens suggest that autoreactive CD4 + T cells play a crucial role in the disease process.

Moreover, CD8 + T cells might participate in the pathogenesis of MS based on animal models of this disease (3, 4) . Epidemiological evidence suggests that one or more infectious agents might be involved in the initial tissue damage, leading to autoimmunity. Evidence indicates that infection with the Epstein-Barr virus plays a major role in the pathogenesis of MS, although precise role for this virus remains incompletely understood (5, 6) . Several virus-induced and autoimmune models have been used to examine the underlying mechanisms of this disease (7) (8) (9) (10) . In particular, Theiler's murine encephalomyelitis virus (TMEV)-IDD provides an excellent infectious model for several reasons (9, 11 ).

Theiler's murine encephalomyelitis virus (TMEV) is a positive sense single-stranded RNA (ssRNA) virus of the Picornaviridae family (12) . The infection of TMEV in CNS induces immune-mediated demyelinating disease in susceptible mouse strains. TMEV-induced demyelinating disease (TMEV-IDD) serves as a relevant infectious animal model for human MS, because this disease displays histopathological, genetic and clinical similarities to human MS. In addition, the development and progression of TMEV-IDD well correlated with the level of Th 1 responses specific for viral epitopes (13) (14) (15) .

The α4 integrins impact hematopoiesis, leukocyte trafficking in immune surveillance and inflammation, and leukocyte activation and survival (16) . α4 integrin, which is expressed on the surface of leukocytes, pairs with one of two β subunits, : β1 and β7 (17) . α4β1 integrin primarily binds to vascular cell adhesion molecule-1 (VCAM-1) and the CS1 fragment of fibronectin (18) .

Specifically, the interaction of α4β1/VCAM-1 plays a major role in the recruitment of mononuclear leukocytes to inflammatory sites in vivo (19) . Anti-α4 integrin antibody significantly suppressed the development of paralysis and the infiltration of leukocytes into the CNS in rat experimental autoimmune encephalomyelitis (EAE) (20). Anti-α4 integrin antibody suppressed the clinical score and pathological features of EAE in the guinea pigs by inhibiting leukocyte infiltration into the CNS (21) . In addition, magnetic resonance imaging revealed that an antibody against α4 integrin significantly decreased the percentage of pixels as a result of the leakage of contrast material and CNS abnormalities associated with cerebral edema and inflammation compared with control animals of guinea pig EAE (22) . In a placebo-controlled trial involving 213 patients with relapsing-remitting or relapsing secondary progressive MS, treatment with natalizumab, an antibody against α4 integrin, showed a marked suppression of the mean number of new lesions and relapses compared with the placebo-group (23) . However, progressive multifocal leukoencephalopathy (PML) has been reported

in MS and Crohn's disease patients who are treated with natalizumab (24) (25) (26) . In the present study, we investigated the role of α4 integrin and the effects of orally active small molecules α4 integrin antagonists in the development of TMEV-IDD, which is an infectious animal model for MS.

Here, we demonstrated that the mRNA levels of α4 integrins were significantly up-regulated in the development and treatment of TMEV-IDD with the newly synthesized orally active small molecule α4 integrin antagonist, HCA3551, which markedly suppressed the development of TMEV-IDD.

Female SJL/J mice, at 6 weeks old, were purchased from Charles River Laboratories, Inc.

(Ibaraki, Japan) and, housed and maintained for in an approved facility, in accordance with the Shinshu University Guide for Laboratory. The animals were housed in aluminum cages containing pine chips and fed food and water ad libitum. The protocol for animal experiments was approved through the Animal Care Committee of Shinshu University.

The BeAn strain of TMEV was expanded in baby hamster kidney (BHK) cells in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 7% donor calf serum (Life Technologies, Gaithersburg, MD, USA). Cell lysates with known plaque forming units (PFU) were used as viral stocks for animal experiments. Partially purified virus was prepared following centrifugation through 30% sucrose as previously described and used for in vitro assays (27). The viral titer was determined using a standard plaque assay on BHK cells.

The mice were intracerebrally (i.c.) infected with 30 μl (0.6710 6 PFU) of the BeAn strain of TMEV on day 0, and clinically observed and scored from day 0 to the end of the experiments. The mice were examined daily for clinical neurological signs, scored and recorded using the following grading scale: grade 0 = no clinical signs; grade 1 = mild waddling gait; grade 2 = moderate waddling gait and hindlimb paresis; grade 3 = severe hindlimb paralysis; grade 4 = severe hindlimb paralysis and loss of righting reflex; and grade 5 = moribund or death. Two independent investigators, who were blinded to the treatment of each animal, assessed the clinical score. The cumulative clinical score was calculated for the individual mice in each group.

Six-week-old female SJL/J mice were separated into groups (Table 1) . TMEV was injected (i.c.) into SJL/J mice i.c. on day 0. To block α4 integrins, we used HCA3551 (Ajinomoto Pharmaceuticals Co., Ltd, Tokyo, Japan) an orally active small molecule antagonist that inhibits the binding of α4β1 integrin to VCAM-1, but not αLβ2 integrin to ICAM-1. The IC50 values were 8.2

and >5,000 nM, respectively. In addition, the inhibitory activity of HCA3551 was not reduced after adding 50% human serum (IC; 11nM), although the inhibitory activity of the α4 integrin antagonist, afirategrast (GlaxoSmithKline Co., UK) was dramatically reduced (28). HCA3551 was dissolved in 0.5% hydroxypropyl methyl cellulose (methylcellulose) (Sigma-Aldrich, St Louis, MO, USA) as vehicle. The BeAn strain of TMEV induces a clinically undetectable level of early-phase disease, whereas the DA strain of TMEV induces significant polio-like symptoms (29). We used the BeAn strain of TMEV in previous studies, specifically a recent batch of the BeAn strain, which causes early onset TMEV-IDD. The onset of TMEV-IDD occurred at approximately 14 days post-infection using this batch of the BeAn strain of TMEV (30). Therefore, the mice were treated with 100 mg/kg HCA3551 twice a day via oral gavage from 11 to 40 days post infection (dpi) or at 41 dpi in the effector phase (group B and E, respectively) and from -3 to 14 dpi in the induction phase (group D).

In a preliminary study, to evaluate the efficacy of HCA3551, HCA3551 was dissolved in 0.5% methylcellulose and orally administered at doses of 30, 100, and 300 mg/kg BID twice a day to female SCID mice to generate a mouse model of colitis through the adoptive transfer of CD4+ T cells from diseased IL-10 -/mice at Ajinomoto pharmaceuticals. HCA3551 suppressed the severity of the mouse model of colitis in a dose-dependent manner. The inhibition of the severity of mouse colitis was statically significant in experimental groups treated with HCA3551 at doses of 30, 100 and 300 mg/kg. However, there was no significant difference between the 100 and 300 mg/kg doses (data not shown). Therefore, we assessed the effects of the 100 mg/kg dosage of HCA3551. Control groups (group A and C) were treated with 0.5% methylcellulose/H 2 O as a vehicle during the same Table 2 . The primers for β-actin (Mm01205647_g1) were obtained from Applied Biosystems. Amplification was conducted in a total volume of 10 μl for 40 cycles of 15 s at 95°C, 30 s at 60°C and 30 s at 72°C for α4 integrin and β-actin, and for 40 cycles of 1 s at 95°C and 20 s at 60°C for TMEV and β-actin. The mRNA levels were determined after normalizing the RNA concentration against β-actin.

Infiltrating mononuclear cells (MNCs) in the CNS (brain and spinal cord) were isolated Percoll-NaCl solution diluted with culture medium and overlaid onto 2.5 ml of 70% Percoll-NaCl solution diluted with culture medium. The gradient was centrifuged at 2,000g for 20 min and the MNCs were harvested from the 37%-70% interface, followed by washing and counting. 

The mice were sacrificed for histological examination at 40 dpi (group A and B). Subsequently, the spinal cords were removed and fixed as previously described (32) . Spinal cord sections from paraffin-embedded tissues were prepared at thickeness of 3-or 6-μm. The sections were stained with hematoxylin-eosin (H&E) or Klüver-Barerra's (KB) staining. Immunohistochemical staining was performed using indirect immunoperoxidase techniques. The following primary antibodies were used: rabbit polyclonal anti-CD3 antibody (Abcam, Tokyo, Japan), rat monoclonal anti-mouse CD45R/B220 antibody (BD Biosciences) and rabbit polyclonal anti-CD68 antibody (Abcam). The Furthermore, we adopted labeling indices (LIs) to more quantitatively show the positive cells. The

The LIs for CD3, a T cells marker, CD45R, a B cells marker, and CD68, a macrophages markers, were examined in the same serial sections and photographed using a light microscope at a magnification of ×200 in three randomly selected fields. Using the photographs, we examined at 

Comparisons were performed with the Mann-Whitney U-test using JMP software (SAS institute Inc., Tokyo, Japan). The differences were considered statistically significant when the two-tailed p-value was <0.05.

To investigate the mRNA levels of α4 integrins during TMEV infection, we compared the mRNA levels of α4 integrins in the CNS (brain and spinal cord) between naïve SJL/J and TMEV-IDD mice using real-time RT-PCR. The α4 integrins mRNA levels were significantly increased in the CNS (brain and spinal cord) of mice with TMEV-IDD at 40 dpi (n=7) compared with naïve SJL/J mice (n=6) (*p<0.05) (1.0 and 2.5, respectively) ( Fig. 1) .

To determine the effects of HCA3551, the orally active small molecule α4 integrin antagonist, on the development of TMEV-IDD, we treated mice with HCA3551 at the induction phase (from -3 to 14 dpi) or the effector phase (from 11 to 40 or 41 dpi) of the disease. The experimental design and the results of the effects of HCA3551 on the clinical course are summarized in Table 1 and Fig. 2, respectively, including two independent experiments. TMEV-infected control animals (groups A and C treated with 0.5% methylcellulose as a vehicle at the effector phase) showed the typical course of TMEV-IDD. There were no significant differences in the clinical signs between control groups C and group D, which was treated with HCA3551 at the induction phase. However, the clinical signs of groups B and E, which were treated with HCA3551 at the effector phase, were significantly 

The representative mice were blindly selected from groups A and B (Table 1) Percoll, from the CNS of TMEV-infected mice treated with HCA3551 or methylcellulose (n=17 and n=14, respectively). The isolated cells were calculated. The mean number of MNCs isolated from the CNS of the mice treated with methylcellulose was 1.89±0.1510 6 . In contrast, the mean number of MNCs isolated from the CNS of HCA3551-treated mice in the effector phase was significantly decreased to 1.03±0.1810 6 (**p<0.01) (Fig. 3. 3A) . These results suggest that oral treatment with HCA3551, an antagonist against α4 integrin, suppressed disease development by inhibiting cellular infiltration to the CNS of TMEV-infected mice.

To examine the effect of HCA3551 in the effector phase on the number of pro-inflammatory cytokine-producing cells, we assessed the intracellular levels of IFN-γ-, TNF-α-, IL-4-, IL-10-and IL-17A-producing MNCs in the CNS at 40 dpi using flow cytometry after stimulation with VP2 [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] or VP3 159-166 peptides. The number of TNF-α-producing CD4 + T cells and IFN-γ-producing CD8 + T cells in the CNS was significantly decreased in mice treated with HCA3551 compared with the control mice (*p<0.05 and **p<0.01, respectively) (Fig. 4B) . Notably, CD4 + T cells producing TNF-α and VP3 159-166 -specific CD8 + T cells were particularly reduced among T cells in the CNS of HCA3551-treated mice, as these T cells have been associated with the pathogenesis of TMEV-IDD (34, 35) .

To determine the effect of the orally active small molecule α4 integrin antagonist, HCA3551, on TMEV replication in the CNS, the viral mRNA levels in the CNS at 40 dpi were assessed by real-time RT-PCR (Fig. 5) . There was no significant difference in the TMEV mRNA levels between methylcellulose-treated control mice and mice treated with the orally active small molecule α4 integrin antagonist. These results suggest that the administration of orally active small molecule α4 integrin antagonist, HCA3551, during the effector phase does not affect TMEV replication. These results suggest that the inhibition of α4 integrin function might also affect the migration of protective T cells involved in the clearance of TMEV persistence in the CNS, in addition to the pathogenic T cells.

The importance of α4 integrin in EAE is well established (20,36,37), but there are no reports on the importance and the role of α4 integrin in TMEV-IDD. Therefore, in the present study, we investigated the role of α4 integrin and the effects of the orally active small molecule α4 integrin antagonist, HCA3551, in the development of TMEV-IDD. HCA3551 is an orally active small molecule α4 integrin antagonist with an IC 50 value for VCAM-1 cell adhesion of 8.2 nM. Previous studies have suggested that T cells in inflamed CNS express high levels of active α4β1 integrin, and that the general expression of α4 integrin is increased on the surface of circulating monocytes in EAE (36, 38) . Moreover, compared with remission, the expression of α4 integrin was significantly up-regulated during relapse in relapsing-remitting MS patients (39) . In the present study, we demonstrated that the mRNA levels of α4 integrins were significantly increased in the CNS (brain and spinal cord) of mice with TMEV-IDD at 40 dpi compared with naïve SJL/J mice (*p<0.05) (Fig.   1 ). α4 integrin is generally expressed on the surface of T cells, macrophages and circulating monocytes. Infiltrating cells to the CNS in TMEV-IDD are primarily macrophages, T cells and monocytes. Therefore, the up-regulated α4 integrin mRNA of primarily reflects infiltrated macrophages, T cells and monocytes. These findings indicate that α4 integrin might be closely associated with the development of TMEV-IDD.

Anti-α4 integrin antibodies or small molecule α4 integrin antagonists have been demonstrated as effective for the treatment of EAE (20- 22, 38) . However, to our knowledge, the effects of α4 integrin and its antagonist on the development of TMEV-IDD, which one of only a few infectious animal models of MS, have not yet been studied. The results of present study revealed that HCA3551 treatment significantly suppressed the development of TMEV-IDD (Fig. 2) . Furthermore, HCA3551 treatment dramatically decreased demyelination and the infiltration of MNCs into the CNS, consistent with a decrease in the number of TNF-α-producing CD4 + T cells and IFN-γ-producing CD8 + T cells (**p<0.01 and *p<0.05, respectively) (Fig. 4B ). This study is the first to demonstrate the efficacy of orally active small molecule α4 integrin antagonists in TMEV-IDD.

However HCA3551 is not effective in TMEV-IDD when administrated during the induction phase.

After TMEV inoculation (i.c.), initial viremia followed by persistent low-level CNS infection is observed (40) . TMEV infects neurons, glial cells and macrophages in the spinal cord (41) . This stage has been referred to as the 'induction phase'. During this phase, infiltration of macrophage and T cells into the CNS is low, and α4 integrin may not be important in this phase, thereby HCA3551 does not suppress TMEV-IDD when in the induction phase.

To further clarify the effects of the treatment of orally active small molecule α4 integrin antagonist, HCA3551, in TMEV-IDD, we measured the number of infiltrating cells, and the LIs for CD3, CD45R and CD68 antigen and assessed Th1, Th2 and Th17 responses in the CNS. The VCAM-1/α4β1 integrin ligand-receptor pair plays major roles in the recruitment of mononuclear leukocytes to inflammatory sites in vivo (19) . We showed that the administration of HCA3551 significantly suppressed the number of CNS inflammatory MNCs compared with administration of control vehicle methylcellulose (**p<0.01) (Fig. 3. 3A) , suggesting that HCA3551 inhibits the infiltration of MNCs into the CNS through the inhibition of α4 integrin binding to VCAM-1.

Furthermore, the LIs for CD3, CD45R and CD68 antigen were significantly decreased in the CNS of mice treated with HCA3551 compared with mice treated with methylcellulose (*p<0.05) (Fig. 3. 2A-2C). The numbers of TNF-α-producing CD4 + T cells and IFN-γ-producing CD8 + T cells in the CNS were significantly decreased and the numbers of IL-17A-producing CD4 + T cells in the CNS were decreased in mice treated with HCA3551 compared with control mice treated with methylcellulose (Fig. 4B ). Previous studies have suggested that Th17 cells express lower amounts of α4 integrin than Th1 cells and Th1 cells preferentially infiltrate the spinal cord via an α4 integrin-mediated mechanism, whereas the entry of Th17 cells into the brain parenchyma occurs in the absence of α4 integrins but is dependent on αLβ2 expression (42) . Therefore, the differential expression of integrin molecules might result in the significant decrease in TNF-α-producing CD4 + T cells and IFN-γ-producing CD8 + T cells, but unaltered level of IL-17-producing CD4 + T cells compared with control mice. Thus, the significant suppression of TMEV-IDD was accompanied with the reduction of the LIs for the CD68 antigen, macrophages markers, and the reduced numbers of TNF-α-producing CD4 + cells and IFN-γ-producing CD8 + cells in the CNS of mice treated with HCA3551, because as these cell types have been implicated to play pathogenic roles in the disease development (13, 34, 35) . These results suggest that treatment with HCA3551 decreased the number of MNCs infiltrated into the CNS of TMEV-infected mice, theby suppressed the development of TMEV-IDD with decreasing the number of MNCs infiltrated into the CNS of TMEV-infected mice.

We examined whether the suppressive effect of HCA3551 treatment on TMEV-IDD might

reflect the elimination of viral persistence. We assessed the levels of replication in the CNS of HCA3551-treated mice or methylcellulose-treated mice using real-time RT-PCR. The results showed that there were no significant differences in the mRNA levels of TMEV between methylcellulose-treated control mice and HCA3551-treated mice (Fig. 5) . Thus, it seems that HCA3551 treatment reduced the development of TMEV-IDD without interfering viral persistence.

Consistently, viral levels might not necessarily be associated with the development of demyelinating disease in this model (43, 44) .

The results of the present study suggested that α4 integrin might play a critical role in the development of TMEV-IDD. Treatment with HCA3551 might ameliorate TMEV-IDD through the inhibition of α4 integrin binding to VCAM-1 accompanied a decrease in the number of MNCs in the CNS. HCA3551 also has a potential risk for PML. However, the pharmacological half-life of HCA3551 is 12 hours, which is much shorter than that of natalizumab (approximately 11 days) (28).

The potential advantage is that the early removal of this drug from the body after the onset of PML could lead to more favorable outcomes, but this is a hypothesis which needs further validation (45) . 

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Immunology of multiple sclerosis

CD4(+) and CD8(+) T cells make discrete contributions to demyelination and neurologic disease in a viral model of multiple sclerosis

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The essential role of Epstein-Barr virus in the pathogenesis of multiple sclerosis

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Theiler's Murine Encephalomyelitis Virus (TMEV)-Induced Demyelination: A Model for Human Multiple Sclerosis

Murine coronavirus infection: a paradigm for virus-induced demyelinating disease

Encephalomyelitis of Mice : I. Characteristics and Pathogenesis of the Virus

Analysis of the complete nucleotide sequence of the picornavirus Theiler's murine encephalomyelitis virus indicates that it is closely related to cardioviruses

Pathogenesis of virus-induced immune-mediated demyelination

Class II-restricted T cell responses in Theiler's murine encephalomyelitis virus-induced demyelinating disease. V. Mapping of a dominant immunopathologic VP2 T cell epitope in susceptible SJL/J mice

A predominant viral epitope recognized by T cells from the periphery and demyelinating lesions of SJL/J mice infected with Theiler's virus is located within VP1(233-244)

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Epitope-specific CD8+ T cells play a differential pathogenic role in the development of a viral disease model for multiple sclerosis

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Th17 lymphocytes traffic to the central nervous system independently of alpha4 integrin expression during EAE

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Representative FACS histograms of infiltrating CD4 + or CD8 + T cells into the CNS were shown. Numbers in the FACS histograms represent % of IFN-γ-, TNF-α-, IL-4-, IL-10-or IL-17A-producing CD4 + T cells among total CD4 + T cells and % of IFN-γ-producing CD8 + T cells among total CD8 + T cells. (B) The total numbers of cytokine-producing CD4 + or CD8 + T cells in the CNS are shown. The numbers of TNF-α-producing CD4 + and IFN-γ-producing CD8 + T cells were significantly decreased in mice

Relative expression levels of TMEV-mRNA in the CNS of each group at 40 dpi. The transcriptional levels of TMEV in the CNS were measured using real-time RT-PCR. The data represent the expression levels normalized to β-actin. There was no significant difference in the mRNA level of TMEV between methylcellulose-treated control mice and HCA3551-treated mice

This work was financially supported in part through Health and Labor Sciences Research Grants for research on intractable disease from the Ministry of Health, Labor and Welfare of Japan, and grants 20 / 38 from the Terumo Lifescience Foundation. HCA3551 was kindly provided from Ajinomoto Pharmaceuticals Co., Ltd.Conflicts of interest statement: the authors declared no conflicts of interest.