Fasudil mediates cell therapy of EAE by immunomodulating encephalomyelitic T cells and macrophages.

Although Fasudil has shown therapeutic potential in EAE mice, the mechanism of action are still not fully understood. Here, we examined the immunomodulatory effect of Fasudil on encephalitogenic mononuclear cells (MNCs), and tested the therapeutic potential of Fasudil-treated MNCs in active EAE. Fasudil inhibited expression of CCL20 on T cells and migration of T cells, decreased CD4(+) IFN-γ(+) and CD4(+) IL-17(+) T cells, but increased CD4(+) IL-10(+) and CD4(+) TGF-ß(+) T cells. Fasudil reduced expression of CD16/32 and IL-12, while elevating expression of CD206, CD23, and IL-10. Fasudil also decreased levels of iNOS/NO, enhanced levels of Arg-1, and inhibited the TLR-4/NF-κB signaling and TNF-α, shifting M1 macrophage to M2 phenotype. These modulatory effects of Fasudil on T cells and macrophages were not altered by adding autoantigen MOG35-55 to the culture, i.e., autoantigen-independent. Further, we observed that, in vitro, Fasudil inhibited the capacity of encephalitogenic MNCs to adoptively transfer EAE and reduced TLR-4/p-NF-κB/p65 and inflammatory cytokines in spinal cords. Importantly, Fasudil-treated encephalitogenic MNCs exhibited therapeutic potential when injected into actively induced EAE mice. Together, our results not only provide evidence that Fasudil mediates the polarization of macrophages and the regulation of T cells, but also reveal a novel strategy for cell therapy in MS.

[Immunoregulative effect of Fasudil on encephalomyelitic T cells in experimental autoimmune encephalomyelitis mice].

OBJECTIVE: To explore the therapeutic effect of Fasudil-modified splenic mononuclear cells (MNCs) in experimental autoimmune encephalomyelitis (EAE) and the possible mechanisms.
 METHODS: C57BL/6 female mice were immunized with myelin oligodendrocyte glycoprotein peptide 35-55 to establish active immunity EAE model. Splenic MNCs were isolated on the 9th day after immunization and treated with or without Fasudil for 72 h in vitro. These cells were collected for analysis of the variance of T cell subtypes, the level of cytokines and the activity of Rho kinase (ROCK). MNCs (5×107 cells) were resuspended in 500 µL of phosphate buffer solution (PBS) and transferred into EAE model (intraperitoneal injection), which was divided into a PBS-MNCs group and a Fasudil-MNCs group. Changes of body weight and clinical symptom scores were observed.
 RESULTS: Splenic encephalitogenic MNCs from EAE mice on the 9th day after immunization could establish passive transfer EAE model. But Fasudil-treated MNCs did not trigger EAE development. Compared with the PBS-MNCs group, the loss of body weight was less in the Fasudil-MNCs group. The in vitro experiment indicated that Fasudil could suppress the activity of ROCK on MNCs (P<0.01), decrease the percentage of CD4+ T cells with the expression of interferon-γ (IFN-γ) and interleukin-17 (IL-17) (IFN-γ: P<0.01; IL-17: P<0.05), while increase the secretion of CD4+ T cells with the expression of transforming growth factor-ß (TGF-ß) and IL-10 (all P<0.001) . Furthermore, Fasudil could inhibit the release of IL-17 (P<0.001) and enhance the level of IL-10 (P<0.05).
 CONCLUSION: Fasudil-modified cell therapy affects the occurrence and development of EAE by inhibiting the inflammatory reaction of helper T cell 1 (Th1) and Th17 while enhancing the immunoregulative effect of Th2.

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

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.

[Studies on the correlation of 489 cases of bronchial asthma complicated with allergic rhinitis in Datong].

OBJECTIVE: To discuss the disease incidence of bronchial asthma (BA) complicated with allergic rhinitis (AR) and the correlation of their age, classification, concomitant symptom. METHOD: Four hundred-nine cases of AR were identified by means of random sampling, physical examination, laboratory test and questionnaire. According to the diagnostic criteria of BA, they were divided into two groups: group I ( control group) including 298 cases suffering from only AR, group II ( experimental group) 191 cases of BA concomittent with AR. RESULT: 1) This study showed that BA concomittent with AR account for 39% of all cases; 2) Incidence of aspirin triad syndrome (asthma, rhinopolyps and aspirin intolerance) was significantly different in two groups (P<0.01); 3) As for age group distribution, there was significant difference between groups in age segment 10-19 and 40-49 (P<0.05); 4) Disease classification: there were significant differences in the incidence of moderate to severe intermittent AR, mild continuous AR, and moderate to severe continuous AR between two groups (P<0.05); 5) Other major concomitant diseases and symptoms distribution; the concomittant occurrence of allergic pharyngitis, sinusitis, conjunctivitis, secretory tympanitis, hypertrophic rhinitis, dermatosis, discomfort of gastrointestinal tract, and headache of unknown origin were significantly different between two groups (P<0.05). The difference above showed that the occurrence in experimental group was higher than that in control group. CONCLUSION: the incidence of BA complicated with AR were relatively high in Datong, with a peak incidence at age 10 to 19 and 40 to 49. The common types of disease were moderate to severe intermittent AR, mild continuous AR, and moderate to severe continuous AR. The number of patients with BA complicated with AR were growing.