Nox2-dependent Neuroinflammation in An EAE Model of Multiple Sclerosis

Abstract

Background: Multiple sclerosis (MS) is an inflammatory disease of the CNS, characterized by demyelination, focal inflammatory infiltrates and axonal damage. Oxidative stress has been linked to MS pathology. Previous studies have suggested the involvement of NADPH oxidase 2 (Nox2), an enzyme that catalyzes the reduction of oxygen to produce reactive oxygen species, in the MS pathogenesis. The mechanisms of Nox2 activation on MS are unknown. The purpose of this study was to investigate the effect of Nox2 deletion on experimental autoimmune encephalomyelitis (EAE) onset and severity, on astrocyte activation as well as on pro-inflammatory and anti-inflammatory cytokine induction in striatum and motor cortex.

Methodology: Subcutaneous injection of MOG35-55 emulsified with complete Freund's adjuvant was used to evaluate the effect of Nox2 depletion on EAE-induced encephalopathy. Striatum and motor cortices were isolated and evaluated by immunoblotting and RT-PCR.

Results: Nox2 deletion resulted in clinical improvement of the disease and prevented astrocyte activation following EAE induction. Nox2 deletion prevented EAE-induced induction of pro-inflammatory cytokines and stimulated the expression of the anti-inflammatory cytokines IL-4 and IL-10.

Conclusions: Our data suggest that Nox2 is involved on the EAE pathogenesis. IL-4 and IL-10 are likely to be involved on the protective mechanism observed following Nox2 deletion.

Keywords: Multiple sclerosist; NADPH oxidaset; astrocytest; cytokines.

Figure 1

Effect of EAE induction on p47^phox immunoreactivity in striatum (A) and motor cortex (B) of Nox2^+/+ mice. The graph depicts the mean optical density of p47^phox immunostaining. Representative digital images of p47^phox-like immunoreactivity. **p<0.01 (Student’s t-test). N=4. EAE: Experimental autoimmune encephalomyelitis.

Figure 2

Effect of Nox2 deletion on EAE onset and severity. Nox2^+/+ and Nox2^-/- mice were monitored daily up to 20 days for signs of disease following immunization. N=6-9. ***p<0.001 (Bonferroni’ s test).

Figure 3

Effect of EAE on GFAP expression in striatum (A) and motor cortex (B) of Nox2^+/+ and Nox2^-/- mice. Western blots analysis of the GFAP protein levels. The graphs represent mean ratio of GFAP densitometric data in relation to β-actin. *p<0.05 and **p<0.01 vs respective control (Tukey’s test). N=4-6. EAE: Experimental autoimmune encephalomyelitis.

Figure 4

Effect of EAE induction on the mRNA levels of the pro-inflammatory cytokines IL-6, IL1β and MCP-1 in striatum (A) and motor cortex (B) of Nox2^+/+ and Nox2^-/- mice. GAPDH was used as an internal control. **p<0.01 vs respective control, ***p < 0.001 vs respective control (Tukey’s test). N= 5-6. EAE: Experimental autoimmune encephalomyelitis.

Figure 5

Effect of EAE induction on mRNA levels of the anti-inflammatory cytokines IL-4 and IL-10 in striatum (A) and motor cortex (B) of Nox2^+/+ and Nox2^-/- mice. GAPDH was used as an internal control. *p<0.05 vs respective control, **p<0.01 vs respective control (Tukey’s test). N=5-6. EAE: Experimental autoimmune encephalomyelitis.

Figure 6

Effects of EAE induction on IL-6 and IL-10 protein levels in striatum (A) and motor cortex (B) of Nox2^+/+ and Nox2^-/- mice. *p<0.05 and **p<0.01 vs respective control, &p<0.05 vs Nox2^+/+ EAE (Tukey’s test). N=4-5. EAE: Experimental autoimmune encephalomyelitis.