J Neuroimmunol. 2015 Oct 15;287:43-53. doi: 10.1016/j.jneuroim.2015.08.005.
Experimental autoimmune encephalomyelitis and age-related correlations of NADPH oxidase, MMP-9, and cell adhesion molecules: The increased disease severity and blood-brain barrier permeability in middle-aged mice.
Ji-Eun Seoa,c, Mahbub Hasana,c, Joon-Seung Hana,d, Min-Jung Kangb, Byung-Hwa Jungb, Seung-Ki Kwoke, Ho-Youn Kime and Oh-Seung Kwona,c*
aToxicology Laboratory, Doping Control Center; bMolecular Recognition Research Center, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea
cDepartment of Biological Chemistry, Korea University of Science and Technology, Daejeon, 34113, Republic of Korea
dDepartment of Pharmaceutical Biochemistry, College of Pharmacy, Kyunghee University, Seoul, 02447, Republic of Korea
eRheumatism Research Center, Catholic Research Institute of Medical Science, Catholic University of Korea, Seoul, 06591, Republic of Korea
The aim of the present study was to investigate effect of two different ages (6 weeks [6 W] vs. 6 months [6 M]) on blood-brain barrier (BBB) disruption in EAE and evaluate the expression and correlations of NADPH oxidase, MMP-9, ICAM-1, and VCAM-1 following increased age and EAE induction. Higher disease severity was observed in 6 M-EAE than 6 W-EAE. The four factors were significantly elevated and correlated in 6 M-EAE. BBB permeability increased with statistically significant interaction between age and EAE effects. We suggest strong correlations between NADPH oxidase and the other factors play important roles in increased BBB disruption and EAE susceptibility in middle-aged mice.
KEYWORDS: Adhesion molecules; Age; Blood–brain barrier; Experimental autoimmune encephalomyelitis; MMP-9; NADPH oxidase
Multiple sclerosis (MS) is demyelinating autoimmune disease of the central nervous system (CNS). Aging is associated with declines of immune functions, and it can increase severity of autoimmune disease . Although MS typically begins between the ages of 20 and 40 years, the disease is more quickly developed showing severe clinical symptoms at older age compared with younger age [2, 3]. Therefore, aging is assumed to play an important role in MS disease. To elucidate the relationship between multiple sclerosis and age-related changes, experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, was induced in different ages of mice (6 weeks, 6W vs. 6 months, 6M) by immunization of myelin oligodendrocyte glycoprotein (MOG)35–55 emulsion with corresponding controls (same treatment without MOG35-55).
We found that EAE severity was markedly higher in 6 M-EAE than 6 W-EAE with the significant decrease of body weight and increase of clinical score after EAE induction (Figure 1). BBB disruption was increased in 6M-EAE compared with other groups with the elevated levels of NADPH oxidase, MMP-9 activities, ICAM-1, VCAM-1, and inflammatory cytokines (IFN-γ, IL-6, IL-10 and IL-17A) in tissues (brain, spinal cord, thymus, and spleen) and kinetic plasma samples (days 0, 11, 22, 32, 41, 53, and 60). Significant changes in Vmax and Km values of NADPH oxidase and MMP-9 enzymes in response to increased age and EAE effects resulted in the increases of these enzyme activities. In the kinetic study of in vivo plasma and brain samples, the significant correlation between NADPH oxidase activity, MMP-9 activity, ICAM-1 and VCAM-1 (4 factors) were observed in EAE groups. In particular, 6 M-EAE showed strong correlations between NADPH oxidase and the other factors and between MMP-9 and cell adhesion molecules.
Figure 1. Clinical presentation of EAE mice. Maximum clinical scores (CS) were apparently 3 in 6 W-EAE (two hind-limb weakness) and 4 in 6 M-EAE (severe hind limb paralysis).
We summarized possible mechanisms of EAE with aging (Figure 2). This study demonstrates the increased age influences the expressions of BBB-related factors leading to the increases of disruption of BBB and EAE susceptibility. BBB permeability was elevated in response to the effects of both increased age and EAE induction with a significant interaction between age and EAE effects. The enhanced BBB permeability with aging might increase the facilitation for infiltrating leukocytes into the CNS. Subsequently, the higher auto-reactive T cells in CNS could lead demyelination with higher pro-inflammatory immune responses in 6 M group compared with 6 W group.
Figure 2. Overview of EAE mechanism with aging. The increased NADPH oxidase activity with aging contributes to expression of MMP-9, ICAM-1, and VCAM-1, and the interactions of the factors that elevate BBB disruption and infiltration of immune cells. Consequently, the severe demyelination with aging enhances EAE vulnerability (Black, EAE effects; Red, Aging effects).
Additionally, we emphasize the role of NADPH oxidase on the EAE pathogenesis with age effect. While positive correlations between MMP-9 and cell adhesion molecules, and between ICAM-1 and VCAM-1 were observed in CON group, strong correlations between NADPH oxidase activity and other factors were only found in EAE group. Besides, the factors were more strongly correlated in 6M-EAE. NADPH oxidase-dependent ROS appears to influence the expression of MMP-9, ICAM-1, and VCAM-1 and plays a critical role in their response to pro-inflammatory mediators. Our other study showed that NADPH oxidase activity had direct influences on MMP-9 activity, ICAM-1 and VCAM-1, but not vice versa, and there are interchangeably bidirectional reactions between MMP-9 activity and adhesion molecules in the endothelial cells . As a result, we suggest the increased NADPH oxidase activity with increased age effect strengthened the relationship between MMP-9, ICAM-1 and VCAM-1 that contributed to BBB permeability. Consequently, the enhanced correlation of the 4 factors in the middle aged-mice leads to BBB disruption that helps leukocytes more easily migrate across the BBB and increases EAE vulnerability.
The importance of this study:
Although the NADPH oxidase, MMP-9, or adhesion molecules are reportedly known to be affected pathogenesis of EAE, a few studies of the relationship with these biomarkers in EAE disease models have been found. Our results showed the changes of these biomarkers in tissues and kinetic plasma samples for 60 days in 4 groups with EAE- and age-related correlations of the factors. Thus, this study is meaningful in not only EAE effect but also aging effect. Eventually, NADPH oxidase activity plays important roles in controlling MMP-9 activity, ICAM-1 and VCAM-1, showing positive correlations between each of the factors in middle-aged EAE mice. This finding can be applied to the discovery of biomarkers related to the aged-immunity and EAE progress. Furthermore, NADPH oxidase inhibitor can be considered a potential therapeutic candidate for multiple sclerosis therapy.
Acknowledgments: This study was supported by both Public Welfare & Safety Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant number 2011-0020952) and Korea Institute of Science and Technology (2V03600).
- Busse PJ, Mathur SK: Age-related changes in immune function: effect on airway inflammation. J Allergy Clin Immunol 2010, 126(4):690-699; quiz 700-691.
- Bermel RA, Rae-Grant AD, Fox RJ: Diagnosing multiple sclerosis at a later age: more than just progressive myelopathy. Mult Scler 2010, 16(11):1335-1340.
- Tullman MJ: Overview of the epidemiology, diagnosis, and disease progression associated with multiple sclerosis. Am J Manag Care 2013, 19(2 Suppl):S15-20.
- Seo JE, Hasan M, Rahaman KA, Kang MJ, Jung BH, Kwon OS: A leading role for NADPH oxidase in an in-vitro study of experimental autoimmune encephalomyelitis. Mol Immunol 2016, 72:19-27.
Contact: Oh-Seung Kwon, Ph.D.
Principal Research Scientist, Toxicology Laboratory, Doping Control Center, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of Korea. Tel.: +82 2 958 5184; Fax: +82 2 958 6677; E-mail address: firstname.lastname@example.org (O.S. Kwon)