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.


Introduction
Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS), characterized by demyelination, focal inflammatory infiltrates and axonal damage [1,2]. Several brain areas are affected, including but not limited to the cerebellum, brainstem, optic nerves and spinal cord [2].
Studies revealed atrophy of the temporal and frontal cortical areas, as well as demyelination in the thalamus, basal ganglia, hypothalamus, hippocampus, cerebellum and neocortex [3].
Experimental autoimmune encephalomyelitis (EAE) is the most commonly used experimental model for MS induction [4]. EAE can be induced in rodents by immunization with myelin constituents, such as myelin basic protein, myelin oligodendrocyte glycoprotein (MOG) and proteolipid protein [5]. A subcutaneous injection of the antigen in complete Freund's adjuvant (CFA) followed by two intraperitoneal injections of pertussis toxin (on the day of immunization and two days later) induces activation of myelin-specific T lymphocytes in the periphery and its migration into the CNS where T cells are reactivated by antigenpresenting cells leading to a subsequent inflammatory cascade and, eventually, demyelination and axonal degeneration [6].
Following EAE induction, activated macrophages and microglial cells produce multiple mediators of tissue damage, including proteases, nitric oxide (NO) and reactive oxygen species (ROS), resulting in neurodegeneration [7]. ROS generated by macrophages appear to be involved in demyelination and axonal damage induced by EAE [8]. ROS derived from mononuclear cells mediate oxidation of total DNA as well as mitochondrial DNA in patients with MS [9,10]. Moreover, evidence of lipid peroxidation has been described in exhaled breath samples of MS patients [11].
Additional studies have shown decreased levels of antioxidant enzymes in blood and cerebrospinal fluid of MS patients [12,13]. NADPH oxidases (Noxes) are a family of neuropathological changes associated with EAE in MOG35-55-induced EAE model [15].
Moreover, an in vitro study demonstrated that the treatment with NADPH oxidase inhibitors (diphenyleneiodonium or apocynin) prevented myelin phagocytosis by peritoneal macrophages [8]. Furthermore, Nox2 mRNA expression was found to be upregulated in spinal cord in both relapsing-remitting and chronic models of EAE [16]. In MS patients, Nox2 was shown to be upregulated in activated microglia found in demyelinating and chronic MS lesions in brain tissue [17]. In a different study, it has been shown that microglial cells express

Western blotting
Mice were sacrificed and the regions of  ΔCt method for quantification [22]. Primer sequences are indicated in Table 1.

Cytokine ELISAs
IL-4, IL-6 and IL-10 levels in brain tissue were measured using specific ELISA kits according to the manufacturer's instructions (R&D Systems).

Statistical analysis
Data are expressed as the mean ± SEM. For individual comparisons, statistical analysis was performed using unpaired Student's t-test.
Statistical analysis for EAE-induced changes in Nox2 +/+ and Nox2 -/mice were performed by one-way analysis of variance (ANOVA), followed by pairwise comparisons (Tukey's HSD test).
For the clinical score evaluation, a two-way ANOVA followed by a Bonferroni post hoc test was used to assess significance between Nox2 +/+ and Nox2 -/groups. In all cases, p ≤ 0.05 was considered to be statistically significant.
Statistical analyses of data were generated using GraphPad Prism, version 3.02 (GraphPad Software Inc., San Diego, CA, USA).

Effect of EAE on p47phox expression
In both structures analyzed p47 phox , a Nox2

Effect of Nox2 deletion on EAE onset and severity
As shown in Figure

Nox2 deletion abrogates GFAP protein expression induced by EAE
Western blotting was used to quantitatively evaluate GFAP protein expression in the striatum and motor cortex following EAE induction. In both striatum and motor cortex, an increase in GFAP protein expression was observed in Nox2 +/+ mice (p<0.05 and p<0.01, respectively), which was abrogated in Nox2 -/mice ( Figure 3).

Nox2 mediates proinflammatory cytokine induction following EAE
To

Effect of Nox2 deletion on the induction of anti inflammatory cytokines following EAE
In order to determine the effect of Nox2  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.
10 protein levels were found increased in the striatum of Nox2 -/mice after EAE but not in the motor cortex (Figure 6, p<0.05 vs Nox2 +/+ EAE).

Discussion
In the present study, we present evidence to support our hypothesis that Nox2 plays an important role on the neuroinflammation induced by EAE. Our findings suggest that The human Nox2 gene is associated with MS and its expression is correlated with disease severity [23]. As noted earlier, p47 phox is among the most important subunits regulating Nox2 activity. We found a substantial increase in p47 phox immunoreactivity in both structures analyzed following EAE induction, suggesting the activation of Nox2 isoform. Of note, since Nox1 is also activated by forming a complex with p47 phox in a similar manner to Nox2 [24], we cannot rule out the possibility that p47 phox may also influence Nox1-specific signaling. Corroborating our results, Nox2 mRNA expression was found upregulated in spinal cord [25] and brain tissue [26] after EAE induction. Moreover, gene expression of the subunits p47 phox , p67 phox , and gp91 phox , were all found increased after MOG-induced EAE.
The Nuclear factor-erythroid 2-related factor 2 (Nrf2), a transcription factor that regulates genetic expression of many protective antioxidant enzymes, can be responsible for this neuroinflammatory response, since all these subunits were also significantly increased in Nrf2 knockout mice compared to the WT mice following EAE induction [27].
Nox2 has been implicated in several neuropathological conditions [26,28]. We have recently shown that Nox2 deletion protects mice against cognitive impairment induced by streptozotocin [28]. In the present study, Nox2 deletion significantly decreased clinical signs of EAE induced by the treatment with MOG35-55.
In agreement with our data, Nox2 knockdown improved clinical scores, prevented body weight loss and oxidative stress-induced nitrotyrosine formation in a mouse model of EAE [29]. It has been demonstrated that Nox2 alters the pattern of proteolytic digestion. forming a glial scar [31]. MS patients exhibited increased expression of GFAP in MS lesions [32] and in the cerebrospinal fluid [33]. Similarly, up-regulation of GFAP has been described in the spinal cord of mice in the chronic phase of EAE induced by MOG   [33]. In our study we observed that EAE induction increased GFAP expression in striatum and motor cortex. Nox2 conditions, such as Parkinson's disease [21], sepsis induced encephalopathy [34] and Alzheimer's disease [28]. It has been previously demonstrated that the expression of IL-6 is increased in macrophages and astrocytes in MS [35]. Moreover, its concentration was found elevated in serum and CSF [36]. IL-6 blockade by the treatment with an anti-IL-6 receptor monoclonal antibody inhibited the development of EAE in mice [37].
Corroborating our data, elevated levels of IL-6 have been described in CSF [38] and in brain structures in experimental models of MS [39]. IL-1β has been shown to be released by monocytes, microglia, astrocytes and brain endothelial cells and it is involved in inflammatory reactions within the CNS. Similarly to IL-6, enhanced levels of IL-1β have been described to be increased in MS lesions [40]. Of note, the correlation between IL-6, IL-1β and Nox2 was demonstrated in a study involving traumatic brain injury (TBI). TBI significantly increased mRNA levels of IL-6 and IL-1β in in the cortex, which was attenuated upon Nox2 knockdown [41]. Nox2 deletion has also been shown to inhibit streptozotocin-induced IL1β production in the hippocampus [28].
MCP-1, a monocyte chemoattractant, is produced by several immune and nonimmune cells [42]. In MS, this chemokine has been demonstrated to be released from astrocytes and macrophages and it seems to play a role in the demyelination of the CNS [43]. Increased expression of MCP-1 has been detected in astrocytes in the spinal cord following EAE induction [44]. Diphenyliodonium, a non-  release induced by LPS treatment in mice [45].
IL-10, a cytokine with potent antiinflammatory and immunosuppressive activities, has been shown to be associated with both EAE [46] and MS remission [47].
In addition, several research groups have demonstrated that IFN-β treatment stimulates IL-10 production in MS, suggesting that IL-10 is involved in neuroprotection afforded by the treatment [48][49][50][51]. On the basis of these reports, we hypothesized that the Nox2 knockdown would increase IL-10 induction, which is in fact what we observed. IL-10 was found upregulated in the striatum of Nox2 -/mice at the protein and mRNA level, suggesting a role for IL-10 on the protective mechanism observed following Nox2 deletion. Similarly, Nox2 knockdown induced increased IL-10 mRNA expression in the cortex after TBI induction [52].
IL-4, another potent anti-inflammatory cytokine, is able to regulate the immune response, the production of proinflammatory cytokines and expression of major histocompatibility complex class II molecules [53]. The exogenous administration of IL-4 attenuates the disease severity after EAE induction [54]. Moreover, IL-4-deficient mice develop a more severe form of clinical disease, increased perivascular inflammation and demyelination, and increased mRNA expression of proinflammatory cytokines following EAE induction [55]. In the present study, EAE induction did not increase IL-4 expression in striatum or motor cortex of Nox2 +/+ mice.
However, Nox2 deletion significantly increased IL-4 expression in both brain structures after Both IL-4 and IL-10 are likely to be involved on the protective mechanism observed following Nox2 deletion.

Ethical approval
All procedures performed in studies involving animals were in accordance with the ethical standards of the University of Sao Paulo.