Nasal delivery of Fasudil‐modified immune cells exhibits therapeutic potential in experimental autoimmune encephalomyelitis

Summary Aim Multiple sclerosis (MS) is a relapsing‐remitting inflammatory demyelinating disease that requires long‐term treatment. Although Rho kinase inhibitor Fasudil shows good therapeutic effect in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, certain side effects may limit its clinical use. This study aimed at observing the therapeutic potential of Fasudil‐modified encephalitogenic mononuclear cells (MNCs) via nasal delivery in EAE and exploring possible mechanisms of action. Methods Experimental autoimmune encephalomyelitis was induced with myelin oligodendrocyte glycoprotein 35‐55 in C57BL/6 mice, and encephalitogenic MNCs were treated with Fasudil in vitro. Mice received 3 × 106 cells/10 μL per nasal cavity on day 3 and 11 postimmunization, respectively. Results Fasudil‐modified MNCs reduced clinical severity of EAE, improved demyelination, and decreased inflammatory cells in spinal cords. Immunohistochemical results indicated that CD4+ T cells and CD68+ macrophages were barely detected in Fasudil‐MNCs group. Fasudil‐modified MNCs decreased CD4+IFN‐γ+ and CD4+IL‐17+ T cells, increased CD4+IL‐10+ T cells, restrained M1 markers CD16/32, CCR7, IL‐12, CD8a, enhanced M2 markers CD206, CD200, CD14 in spleen. Fasudil‐modified MNCs inhibited the activation of inflammatory signaling p‐NF‐kB/P38, accompanied by the decrease of COX‐2 and the increase of Arg‐1 in spinal cord, as well as the reduction of IL‐17, TNF‐α, IL‐6 and the elevation of IL‐10 in cultured supernatant of splenocytes. Fasudil‐modified MNCs enhanced the levels of neurotrophic factors BDNF and NT‐3 in spinal cord. Conclusion Our results indicate that intranasal delivery of Fasudil‐modified MNCs have therapeutic potential in EAE, providing a safe and effective cell therapeutic strategy to MS and/or other related disorders.


| INTRODUC TI ON
producing Th17 cells are the main participators in the development of EAE, which is closely related to neuroinflammation and myelin loss. 6,7 Th2 T cells act as immunomodulatory cells and possess the effect of antiinflammatory and myelin protection, which can improve the severity of EAE. 8 Macrophages, an important antigen presenting cells of immune system, participate in the induction and regulation of inflammatory microenvironment. Classically activated macrophages (M1) can activate Th1 and Th17 of T cells and enlarge immunological response.
Alternative activated macrophages (M2) can take part in mediating the differentiation of Th2 cells and CD4 + CD25 + regulatory T cells, which contributes to the myelin regeneration and neuronal survival. [9][10][11] Rho-kinase (ROCK), is expressed both centrally and peripherally where it is involved in fundamental cellular processes, including differentiation, migration, proliferation and survival. 12,13 A series of studies have discovered that many neurodegenerative diseases, such as MS, Alzheimer's disease and Parkinson's disease, have the activation of ROCK signaling pathway that affects the occurrence and development of these diseases. [14][15][16] Therefore, the inhibition of ROCK activity is considered as a potential drug target for the treatment of neurodegenerative diseases. Fasudil (1-[5-isoquinolinesulphonyl]homopiperazine), a selective ROCK inhibitor, has been widely used clinically since 1995 for the treatment of subarachnoid hemorrhage in Japan. Previous investigations from our group and other labs have indicated that Fasudil ameliorates the clinical severity of EAE in different models, accompanied by reduced demyelination and inhibition of neuroinflammation. 17,18 Although Fasudil shows good therapeutic effects in EAE, the following factors restrict its clinical application in MS patients: (a) rapid and obvious vasodilatation; (b) poor oral bioavailability and no oral drug; (c) smaller safety window for long-term use. 19 In recent years, cellular immunotherapy has become a hot spot in the field of neurological disorders and has made great progress, which has the advantages including autologous transplantation, economic security, no ethical problems and tumorigenicity. 20,21 Intranasal administration is a safe and effective delivery in the treatment of CNS diseases. 22 Previous studies show that Fasudil also influence the polarization of T cells and macrophages. 23 In this study, we try to explore the therapeutic effect of encephalitogenic mononuclear cells (MNCs) treated with Fasudil in vitro and possible mechanisms of action in the treatment of EAE by intranasal delivery. MNCs treated with Fasudil in vitro, via direct intranasal delivery, may represent a novel, simple, noninvasive strategy for the treatment of MS.  Cells were then washed three times and re-suspended in medium.

| Induction of EAE and intranasal administration of MNCs
Mice were divided into two groups: Fasudil-MNCs (n = 10) and PBS-MNCs (n = 10). After washing, MNCs from above preparation were resuspended at 6 × 10 6 cells in normal saline (NS) and administrated by bilateral intranasal instillation into mice immunized with MOG  。 For intranasal administration, mice were lightly anesthetized with diethylether, and then received 3 × 10 6 cells/10 μl per nasal cavity on day 3 p.i. and day 11 p.i., respectively. Following cell transplantation, clinical score and body weight were evaluated every other day in a blinded fashion by at least two investigators. Clinical score of EAE was graded according to the following criteria: 0. healthy; 1. limp tail; 2. ataxia and/or paresis of hindlimbs; 3. paralysis of hindlimbs and/ or paresis of forelimbs; 4. tetraparalysis; 5. moribund or death. All animal experiments were repeated three times.

| Histology and immunohistochemistry
On day 28 p.i., mice were perfused with NS and 4% buffered par- (1:1000; Serotec) and anti-microtubule associated protein 2 (MAP2) (1:200; Millipore, Bedford, MA, USA) at 4°C overnight, followed by the corresponding secondary antibodies at room temperature (RT) for 2 hour. As a negative control, additional sections were treated similarly, but the primary antibodies were omitted. The expressions of CD4 + T cells, CD68 + macrophages and MAP2 were determined by Image-Pro Plus software.

| Flow cytometry analysis
On day 28 p.i., mice were sacrificed and spleens were removed under aseptic conditions. MNCs were prepared as above described stained

| Western blot analysis
On day 28 p.i., mice were perfused with NS and spinal cords were homogenized with a microcontent motoroperated tissue homogenizer (Kimble Kontes, Vineland, NJ, USA), using protein extraction kit (Millipore) supplemented with a cocktail of protease inhibitors.
The homogenates were centrifuged at 20 000 g for 20 minutes at 4°C, and the supernatants were collected. Protein extract (20 μg) were separated by SDS-PAGE and electroblotted onto nitrocellulose membrane (Immobilon-P; Millipore). After blocking with 5% nonfat dry milk, the membranes were incubated at 4°C overnight with the following antibodies: antiinducible nitric oxide synthase

| Statistical analysis
GraphPad Prism software (Cabit Information Technology Co., Ltd., Shanghai, China) was used for statistical analysis. The data of clinical mean score was analysed with the Mann-Whitney U test; other data were analysed with Student's t test. A statistically significant difference was assumed at P < 0.05.

| Fasudil-modified MNCs ameliorates severity of EAE via nasal delivery
In the present study, mice were immunized with MOG  peptide to induce EAE model. As shown in Table 1 and Figure 1, the incidence in

| Fasudil-modified MNCs inhibits inflammation and improves demyelination in spinal cords
In the spinal cord of EAE, the inflammatory infiltration CNS and demyelination are major pathological manifestation. As shown in Figure 2A,

| Fasudil-modified MNCs regulate peripheral T cells and macrophages
The activation of the peripheral immune system is the initiating factor in the pathogenesis of EAE, which can infiltrate to CNS and lead to immune attacks and meylin damage. To detect the effect of   Figure 6B, P < 0.05 and P < 0.01, respectively). However, there was no significant difference on the expression of TLR-2 protein in spinal cords between two groups ( Figure 6B).

| Fasudil-modified MNCs induce the expression of neurotrophic factors in spinal cord
It is speculated that the improvement of inflammatory microenvironment can reduce neuron damage. As shown in Figure

| D ISCUSS I ON
Our previous studies found that Fasudil, when injected intraperi-

| CON CLUS ION
In conclusion, our findings provide evidence that nasal delivery of Fasudil-modified MNCs delays the onset and ameliorates the severity in EAE mice, accompanied by the improvement of demyelination.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.