Mechanistic insights into corticosteroids in multiple sclerosis: War horse or chameleon?☆

Clinical Neurology and Neurosurgery 119 (2014) 6–16

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Mechanistic insights into corticosteroids in multiple sclerosis: War horse or chameleon?夽 Stephen Krieger a,1 , Shawn F. Sorrells b,2 , Molly Nickerson c,3 , Thaddeus W.W. Pace d,∗ a

Corinne Goldsmith Dickinson Center for MS, Icahn School of Medicine at Mount Sinai, New York, New York, USA Department of Neurosurgery, The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, California, USA c Questcor Pharmaceuticals, Hayward, California, USA d College of Nursing and College of Medicine (Department of Psychiatry), University of Arizona, Tucson, Arizona, USA b

a r t i c l e

i n f o

Article history: Received 17 June 2013 Received in revised form 19 November 2013 Accepted 27 December 2013 Available online 10 January 2014 Keywords: Corticosteroids Glucocorticoids Multiple sclerosis Relapses Exacerbations

a b s t r a c t Objectives: Relapse management is a crucial component of multiple sclerosis (MS) care. High-dose corticosteroids (CSs) are used to dampen inflammation, which is thought to hasten the recovery of MS relapse. A diversity of mechanisms drive the heterogeneous clinical response to exogenous CSs in patients with MS. Preclinical research is beginning to provide important insights into how CSs work, both in terms of intended and unintended effects. In this article we discuss cellular, systemic, and clinical characteristics that might contribute to intended and unintended CS effects when utilizing supraphysiological doses in clinical practice. The goal of this article is to consider recent insights about CS mechanisms of action in the context of MS. Methods: We reviewed relevant preclinical and clinical studies on the desirable and undesirable effects of high-dose corticosteroids used in MS care. Results: Preclinical studies reviewed suggest that corticosteroids may act in unpredictable ways in the context of autoimmune conditions. The precise timing, dosage, duration, cellular exposure, and background CS milieu likely contribute to their clinical heterogeneity. Conclusion: It is difficult to predict when patients will respond favorably to CSs, both in terms of therapeutic response and tolerability profile. There are specific cellular, systemic, and clinical characteristics that might merit further consideration when utilizing CSs in clinical practice, and these should be explored in a translational setting. © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

1. Introduction High-dose steroids have been the “war horse” therapy in the treatment of autoimmune diseases and the standard of care for decades [1]. Indeed, much benefit has been realized for patient care since the introduction of steroids into clinical practice in the 1940s [2]. However, preclinical findings over the last several years

夽 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ∗ Corresponding author at: College of Nursing and College of Medicine (Department of Psychiatry), University of Arizona, 1305 N Martin Avenue, Tucson, AZ 85721, USA. Tel.: +1 520 626 3520; fax: +1 520 626 6424. E-mail address: [email protected] (T.W.W. Pace). 1 Icahn School of Medicine at Mount Sinai, 5 East 98th Street, Box 1138, New York, NY 10128, USA. 2 Department of Neurosurgery, University of California, San Francisco, 35 Medical Center Way RMB 933A, Box 0525, San Francisco, CA 94143, USA. 3 Questcor Pharmaceuticals, Inc., 26118 Research Road, Hayward, CA 94545, USA.

suggest a re-evaluation of the therapeutic benefits and adverse effects of steroid therapy, in particular the use of high-dose steroids for the treatment of relapses in multiple sclerosis (MS), may be warranted. In some cases standard practice of care persists, despite advances in preclinical research that might suggest alternative approaches to optimal patient care. The relationship between MS and various comorbid conditions, including psychiatric illnesses with likely endocrine-immune pathophysiological underpinnings, further encourage a re-examination of the use of high-dose steroids for MS. Such a critical look at steroids may not only be beneficial for understanding when steroids may be most effective for MS and other autoimmune disorders, but also when other therapies should be considered. The goal of the current article is to offer a “fresh” perspective for clinical neurologists and clinician scientists on the convergence of recent preclinical science and mainstream medicine related to the use of corticosteroids (CSs) in MS care. Our hope is that this opinion piece will foster further preclinical and clinical research on the use of high-dose steroids for treatment of MS. Synthetic CS analogs have been developed to harness the anti-inflammatory properties of endogenous cortisol, and we first

0303-8467/$ – see front matter © 2014 The Authors. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.clineuro.2013.12.021

S. Krieger et al. / Clinical Neurology and Neurosurgery 119 (2014) 6–16

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Fig. 1. Circulating corticosteroid (CS) concentrations over the normal diurnal cycle, in response to acute psychological stress, and after a single 1 g intravenous methylprednisolone (IVMP) treatment (depicted over a 48-h period). Measurements from original sources converted to nmol/L for comparison. The panel on the right is a magnification of the lower section of the graph on the left. Diurnal variations of CS concentrations were assessed in healthy volunteers by immunoassay of serum cortisol levels at various timepoints to create a daily physiological profile of cortisol (purple) [17]. A maximal CS response to stress was measured in serum samples taken from individuals during acute military exercises and measured by radioimmunoassay (green) [18]. Stress-induced circulating CS concentrations do not always reach this level, and can be substantially lower than the values represented here depending on the stressor. More moderate stressors such as a laboratory psychosocial stress challenge elicit an acute stress response of approximately 350 nmol/L [19]. Supraphysiological concentrations of CSs are seen following treatment with a single dose of 1 g IVMP. Plasma MP was measured by reverse phase high-performance liquid chromatography (yellow) [20]. Of note, others have found even higher circulating concentrations of MP [21], and MP is known to exert greater bioactivity compared to endogenous CSs [22]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

consider how they are believed to work in MS. We then discuss relevant preclinical and clinical reports that reveal important information regarding CS treatment in MS, including potential limitations of treating patients with CSs. Finally, possible lessons from the overlap of MS and major depression will be explored. 2. Relapse treatment Acute relapses in MS are defined by newly-emerging neurologic deficits that last for longer than 24 h [3]. Characteristics of these relapses (also referred to as “attacks” or exacerbations) include optic neuritis, limb weakness, numbness, or brainstem episodes, including imbalance, vertigo, diplopia, and loss of facial strength or sensation. Relapses typically develop over a day or days, persist at their symptomatic peak for days to weeks, and self-resolve over a period of weeks to months. Approximately 50% of relapses leave behind residual loss of neurologic function [4], contributing to the step-wise accrual of disability in MS. Various chronic symptoms, including fatigue, depression, and cognitive difficulties, are common in MS, particularly in the progressive stage of the illness. These symptoms are less common in the context of acute relapses, but greater awareness has led to their recognition as acute manifestations of MS in some instances. On a pathophysiological level, acute relapses are considered to be a clinical manifestation of new inflammatory activity in the central nervous system (CNS). Multiple sclerosis lesions generally occur around small venous vessels, and are thought to be driven by autoreactive T-cells that cross the blood brain barrier (BBB) and orchestrate an attack on myelin [5]. In many patients, the disease progresses to a neurodegenerative phase and the immune response to the acute injury progresses to a more chronic inflammatory state that includes activated immune responses from local immune cells, T-cells, B-cells, macrophages, and microglia [5–8]. As our understanding of the specific immunopathology that contributes to MS lesions has expanded, so has our appreciation for active inflammatory lesions being associated with axonal transection and loss [9]. Incomplete recovery from early relapses is common [10] and has been associated with a worse prognosis [11]. This suggests

that neuronal vulnerability after a relapse has important clinical implications. The clinical management of MS includes interventions that are intended to: (1) prevent relapses and the accumulation of disability; (2) hasten resolution of acute relapses (i.e., “abortive” therapy); and (3) manage MS-related symptoms. Preclinical and clinical research of MS therapies has focused primarily on the first of these through the development of disease-modifying therapies (DMTs). While DMTs are the standard of care in MS for chronic treatment to prevent relapses, none do so completely [12], making the prompt recognition and treatment of relapse a primary challenge in MS therapy. Abortive treatment of acute relapses is used to bring about a faster resolution of relapse symptoms than would be expected to occur naturally. Corticosteroids, which include the stress hormone cortisol, have robust anti-inflammatory properties and have been a mainstay of treatment in MS ever since the discovery in the 1940s that their therapeutic effect in the context of autoimmune conditions was beneficial. High-dose CSs are currently the mainstay treatment for acute MS relapses [13–15]. The current standard treatment for acute relapses in MS is a supraphysiological dose of intravenous (IV) methylprednisolone (MP) 1 g/day for 3–5 days, which was derived from the Optic Neuritis Treatment Trial [16]. This dose of MP is much greater than the amount of endogenous CS produced during normal circadian cycles or in response to stress (Fig. 1) [17–22]. A few key studies have examined the tolerability and efficacy of high-dose steroids versus placebo in MS relapse (Table 1; Durelli [23], Milligan [24], Beck [16], Barnes [25], Sellebjerg [26]; for more detailed reviews of relapse treatments and high-dose steroids, see Berkovich 2012 [27], Filipini [28], Miller [29], Burton [30]). Less widely employed treatments for MS relapses include plasmapheresis, IVIg, and Acthar Gel. These treatments are well beyond the scope of the current discussion, and are reviewed elsewhere [12,31]. It is widely known that CS treatments can lead to both desirable and undesirable effects depending on the type of steroid and the situation in which it is used. Although CSs are frequently effective for shortening relapses in MS, they also often exhibit

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Table 1 Key studies of high-dose corticosteroids in the treatment of multiple sclerosis exacerbation. Reference

Patient allocation (n)

Efficacy

Side effects (n)

Durelli et al. (1986) [23]

IVMP high dose (13 + 10 cross-over from placebo)

At day 1–3, 13 reported subjective improvement At day 3–6, 12 reported clinical improvement + additional improvement in 9 crossed from placebo group At day 4–8, 6 reported subjective improvement By day 11, clinical improvement was reported in 4 At week 4, 10 had decrease in disability score

Unusual taste in mouth after infusion (5) Elevated mood and insomnia (12) Slight weight gain and edema (10)

Placebo (10)

Milligan et al. (1987) [24]

IVMP high dose (13)

Placebo (9) a

Beck et al. (1992). [16]

IVMP high dose (144)

IVMP high dose (38) Oral MP low dose (42) Oral MP high dose (26)

At day 30, 123 did not have normal visual acuity At 6–24 months, 35 were diagnosed with MS At day 30, 125 did not have normal visual acuity At 6–24 months, 28 were diagnosed with MS At week 4, median change of 0.5 on EDSS At week 4, median change of 0.5 on EDSS At week 8, 17 defined as responders

Placebo (25)

At week 8, 8 defined as responders

Oral prednisone low dose (151)

Placebo (143)

Barnes et al. (1997) [25] Sellebjerg et al. (1998) [26]

At week 4, 2 had decrease in disability score At day 30, 112 did not have normal visual acuity At 6–24 months, 20 were diagnosed with MS

None reported

No serious adverse events; reports of flushing, transient ankle swelling and metallic taste in the mouth during infusion None reported Serious adverse events: acute transient depression requiring medication (1); acute pancreatitis (1) Minor side effects (not quantified): sleep disturbances, mild mood change, stomach upset, facial flushing, weight gain Minor side effects (not quantified): sleep disturbances, mild mood change, stomach upset, facial flushing, weight gain None reported

No serious side effects; adverse events not reported No serious side effects; adverse events not reported Insomnia (17), gastrointestinal symptoms (10), hot flashes (7), palpitations (7), dysphoria (6), edema (5), musculoskeletal pain (4), acne (3), weight gain (3), headache (2), metallic taste (2) Insomnia (2), gastrointestinal symptoms (2), palpitations (1), dysphoria (1), edema (2), musculoskeletal pain (1), headache (1), metallic taste (1)

Expanded Disability Status Scale (EDSS), IV, intravenous; MP, methylprednisolone. a The Optic Neuritis Treatment Trial (ONTT) (Beck et al.) is included for comparison because it had a significant impact on current standard of care regarding the use of high-dose steroids, although the patients in this study had solely optic neuritis and not other MS relapses.

side effects that require active management [32,33]. Gastrointestinal side effects include abdominal discomfort, gastritis and peptic ulcer, and unpleasant metallic taste. Changes in mood and energy levels range from mild insomnia to euphoria to frank psychosis. Sodium and water retention leading to weight gain and peripheral edema are common transient side effects, as is concomitant potassium wasting and hypokalemia. Other important but transient metabolic effects include elevated blood glucose levels, and systemic side effects such as elevated blood pressure and acne. Cardiac arrhythmias and conduction disturbances have been reported in MS patients treated with high-dose IVMP [34]. In patients with underlying medical comorbidities that may amplify the adverse event profile, high-dose steroids must be used with caution; these comorbidities include diabetes, mood disorders such as major depression, a history of psychosis, and cardiac arrhythmias or bradycardia [15]. In such cases, in-patient monitoring during steroid dosing may be warranted. Steroids should be used carefully in patients with a history of glaucoma or osteoporosis, or in a setting of recent or recurrent infection. In order to understand these undesirable outcomes during CS therapy, it is important to consider that synthetic CSs may exert their effects via mechanisms different from those involved with endogenous cortisol. Although there is a general consensus that CSs are effective in the context of MS relapses, there is a relative absence of information about whether or not CS treatment brings about a more complete resolution of symptoms than would ultimately occur without treatment. Furthermore, our recent review of the literature did not uncover evidence indicating that not treating a relapse would lead

to more frequent or severe subsequent relapses. Given this uncertainty, and the side effect profile and potential toxicities of CSs, it is not standard practice to treat all relapses. However, relapses that are disabling or cause functional impairment are generally treated [12]. Mild relapses, such as an area of tingling or paresthesias in the absence of significant sensory loss, may be simply allowed to recover without CS treatment. Another application of CSs that is particularly relevant to this discussion is the use of monthly high-dose steroids for 1–3 days. This is sometimes referred to as “pulse dosing” and is utilized in response to uncontrolled disease that may not qualify for the clinical definition of relapse. One study that examined the effects of repeated high-dose IVMP over a 5-year period showed that the treatment resulted in less brain atrophy than placebo [35]. A subsequent study, however, found that IVMP resulted in a decrease in brain volume over the subsequent 8 weeks after steroid administration [36]. Patients with secondary progressive MS showed a greater decline in brain volume following IVMP than relapsing-remitting MS (RRMS) patients, suggesting pathological heterogeneity in the development of steroid-related outcomes as the disease course progresses. Clinical outcomes for MS treated with repeated pulse dosing of IVMP have similarly been variable. A small cross-over study in RRMS showed that the addition of a single monthly dose of 500 mg IVMP followed by a 3-day oral taper reduced the number of gadolinium-enhancing lesions on monthly MRI scans for 12 months [37]. The larger NORMIMS study evaluated monthly oral dosing of 200 mg MP daily for 5 days versus placebo as an add-on therapy to

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interferon beta-1a in RRMS, with yearly relapse rate as the primary outcome [38]. The study showed that this pulse-dosing strategy yielded a significant reduction in relapse rate. It bears mention that sleep disturbances and other neuro-psychiatric symptoms were the most frequent adverse events in the methylprednisolone group. In contrast, the MECOMBIN study also studied the addition of pulsedosed MP (500 mg orally per day for 3 days monthly) or placebo to interferon beta-1a, and found that the addition of monthly pulses of MP did not prevent or delay disability progression more than interferon beta-1a treatment alone [39]. These results are thus inconclusive and longer-term outcomes are unavailable at this time. In keeping with the heterogeneous nature of MS, on an individual basis there is great variability in the severity of relapses, their extent and timing of recovery, and the degree to which relapse recovery is responsive to CS therapy. Because steroid treatment is often not initiated as soon as relapse symptoms begin, and studies have utilized a wide range of relapse durations and timing of clinical endpoints, it is difficult to know precisely how fast, or how completely steroids attenuate the clinical symptoms of MS relapse. Corticosteroid treatment is often employed repeatedly in a single patient over the course of their disease. The unpredictability of response exists both within and between patients, as the clinical response to CS treatment for one relapse does not necessarily predict response for the same patient in subsequent relapses. Although a history of failing to respond to steroids cannot rule out their use to treat a subsequent relapse, failing to respond can be taken in to account when considering the benefit/side effect risk calculation. This is because the clinical response to steroid treatment often decreases over time [40]. The mechanism of this therapeutic decrement is not clear. One possible reason for decreased effectiveness of CSs is “steroid resistance.” However, steroid resistance in MS has not been thoroughly studied, and some patients may be inherently less responsive to CSs. Alternatively, CSs may become less effective as MS becomes less of an inflammatory-mediated disease with de novo inflammatory attacks, and more a disease of neurodegeneration and failure of compensatory strategies. A clearer picture of why CSs lose efficacy during MS would thus improve not only our understanding of the efficacy of steroid treatments, but also our understanding of the various ways in which this disease may progress. It would be helpful to better understand the extent to which CSs provide clinical benefit in different phases of MS. It would also be worthwhile to know whether or not the suppression of inflammation is universally effective, or if it occurs at the cost of collateral CS-mediated damage in the CNS. The remainder of the article will briefly review the mechanisms likely involved with the clinical “unknowns” involving CSs in MS, including individual variability in responsiveness, therapeutic decrement, steroid resistance, on-going neurodegeneration, and the role of comorbid conditions such as major depression in patients with MS who are non-responsive to CS. 3. The anti-inflammatory mechanisms of CSs The best understood mechanisms of CS function are mediated via the genomic actions of intracellular glucocorticoid receptors (GRs). Like other nuclear hormone receptors, these receptors remain inactive in the cytosol until they bind ligand, either the endogenous CS cortisol or a synthetic CS. Activated receptors then translocate to the nucleus where they act as transcription factors by binding to discrete regulatory regions of DNA called the glucocorticoid response element (GRE) to modulate the expression of genes that affect activities ranging from immune function to metabolism [41,42]. There are two CS-responsive intracellular receptors: the GR and the mineralocorticoid receptor (MR). These receptors differ both in their affinity for different CSs as well as their central and peripheral distributions. Glucocorticoid receptor

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is found in almost all areas of the brain and body, while MR is confined to the hippocampal formation and lateral septum in the brain and the kidney [42,43]. Furthermore, the MR has a high binding affinity for the endogenous CS, cortisol (approximately Kd = 0.5–1.0 nM), resulting in the vast majority of MR being tonically bound by cortisol, even at basal physiological concentrations. In contrast, GR exhibits a much lower affinity for cortisol (approximately Kd = 2.5–5.0 nM) and is only activated by cortisol during periods of moderate to severe stress or at the circadian cycle peak [42]. Because most synthetic CSs preferentially bind the GR when used at therapeutic doses, the majority of studies on autoimmune disorders and intracellular receptors for CSs have focused on this particular receptor. However, it is important to note that several synthetic CSs do exert an effect at the MR [44]. Genomic actions mediated via GR signaling are widely believed to be responsible for the anti-inflammatory actions of CSs (Fig. 2). For example, expression of genes coding for the anti-inflammatory cytokine interleukin (IL)-10 and the protein inhibitor ␬-B (I␬B)␣ increases when GR binds to a GRE upstream of either [45]. The genomic effects of CSs are made less predictable by the fact that GR is subject to alternative splicing, leading to receptor variants with different functionality [46]. Homodimers of GR␣ are the “classic” receptor isoform; however, low-level expression of an alternative splice form GR␤ [47] that is also able to dimerize with GR␣ [48] results in GR␣/␤ heterodimers that are less active [49]. The balance of GR isoforms has been shown to be important for illnesses with pathophysiological processes known to involve CSs. For example, CS resistance in leukemia has been linked to high expression levels of GR␤ relative to GR␣ in cultured patient lymphocytes [50]. Additionally, increased expression of GR␤ has been identified in rheumatoid arthritis, a disorder that critically involves inflammatory excess [51]. Finally, medically healthy patients with major depression have been reported to have a lower GR␣ to GR␤ ratio [52]. These data suggest that the balance of GR␣ and GR␤ may be important for understanding disease states involving inflammatory immune alterations. Indeed, one explanation for the decrement in clinical response to CSs in MS, particularly as the disease progresses [40], may be that resistance to CSs results from a shift in the relative balance of GR␣ and GR␤. Although preliminary studies have not supported this hypothesis [53], more work in this area is needed. Activated GRs have also been shown to influence cell function in ways not involving direct DNA binding, but instead via extensive protein–protein interactions within many other molecular signaling pathways [41] (Fig. 2). In contrast to the direct genomic effects of CSs, these nongenomic effects of CSs, also mediated by the GR, have been far less studied. However, it is likely that a significant portion of CS effects in health and disease are mediated by nongenomic effects involving protein–protein interactions between the GR and other signaling pathways, including effects of CSs on immune function [41]. For example, activated GRs have been shown to bind to the transcription factor nuclear factor-␬B (NF-␬B) in the nucleus, which in turn blocks NF-␬B from binding to DNA and impairs enhancement or inhibition of genes that are normally regulated by NF-␬B to increase inflammation [54]. This nongenomic effect of GR on the NF-␬B pathway may be one of the primary mechanisms by which CSs exert anti-inflammatory effects. Conversely, increased activity of a number of signaling pathways involved with the inflammatory system have been shown to impair GR function, including p38 mitogen-activated protein kinase (MAPK) and Janus kinase-signal transducers and activators of transcription (JAK-STAT) [55,56]. This reveals the possibility that excessive activation of these signaling pathways may lead to glucocorticoid resistance [57]. Corticosteroids, particularly at very high doses, may also exert nongenomic effects on immune function that do not involve intracellular receptors such as GR (Fig. 2). Instead, preclinical studies

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Fig. 2. Corticosteroids (CSs) act on multiple intracellular signaling pathways to modify inflammatory gene transcription. Although they canonically work by binding to the cytosolic glucocorticoid nuclear hormone receptor (GR), CSs also have rapid effects through poorly understood membrane-bound receptors, and many of their downstream effects cross-talk with other aspects of the inflammatory signaling cascade. Corticosteroids bind to GR, resulting in dissociation of heat shock protein (HSP, here HSP 90) complexes and subsequent receptor phosphorylation. Glucocorticoid receptor then translocates to the nucleus where it dimerizes and either interacts with other transcription factors or binds to glucocorticoid response elements (GREs) upstream of GR-regulated genes including those that code for proteins critical to inflammatory pathway function (e.g., inhibitor ␬-B [I␬B]). The proinflammatory transcription factor nuclear factor-kappa B (NF-␬B; shown here as the P65 and P50 Rel subunits) is normally activated by extracellular inflammatory signals such as cytokines (via their receptors, here the receptors for interleukin [IL]-1 or interferon [IFN]-␣) or direct pathogenic cues (e.g., lipopolysaccharide) acting through toll-like receptors (TLRs; here TLR4). Activation of cytokine receptors and TLRs also results in the activation of other inflammatory signaling pathways, including the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathways, the activator-protein-1 (AP-1) pathway, and the p38 mitogen-activated protein kinase (MAPK) pathway. Activity of inflammatory signaling pathways is modified by CSs at several levels. For example, GR can bind directly to NF-␬B to block activation of genes coding for proinflammatory cytokines with response elements (REs) for NF-␬B. Glucocorticoid receptor can also impact NF-kB pathway activity by increasing transcription of I␬B through binding to the GRE upstream of the gene coding for I␬B. Also shown here is GR-mediated expression of MAPK phosphatase 1 (MKP1), which negatively regulates MAPK pathways including ERK. Glucocorticoid receptor also interacts with the AP-1 (comprised of Fos and Jun subunits) through inhibitory protein–protein interactions, which prevents binding of AP-1 to its RE.

suggest that the rapid therapeutic effects of CSs, including pulse therapies (e.g., 1 g IVMP for 1–3 days administered once a month as preventative therapy) for MS, may act on cells by changing physiochemical properties of their membranes [41]. For example, cortisol has been shown to increase membrane fluidity [58]. Immune cell apoptosis induced by high-dose glucocorticoids may involve effects of CSs on mitochondrial membrane permeability [59]. In rat thymocytes, several synthetic CSs commonly used in MS relapse treatment have been shown to lead to differential changes in intracellular calcium concentrations, despite having similar genomic receptor affinities [60]. Corticosteroids may also exert their effects through membrane-bound CS receptors (Fig. 2), including a variant of GR that resides within the cell membrane. The membrane GR has been associated with caveloae contained in the cell membrane, which also are associated with G-protein coupled receptor signaling [58]. The specific concentration of CSs that a particular cell receives is also critical in determining CS actions. Unfortunately, several factors make it difficult to determine the actual dose of CSs that is “felt” at a cellular level from a given delivery regimen. First, CSs are subject to multiple levels of regulation (e.g., differential binding

to carrier proteins in the blood and local inactivation by degrading enzymes) from the time they are released into the blood until when they enter target cells, so concentrations of CSs in blood do not necessarily predict GR signaling within a single cell of a complex tissue [61]. A better understanding of the local tissue concentrations of CSs resulting from different administration regimens is therefore required. Second, GR signaling is likely to carry different consequences depending on the cell type in which it occurs, based on the widespread GR expression throughout many CNS cell types, and variability in GR isoforms. Third, CS concentrations are sensitive to multiple factors, including the timing and duration of CS exposure relative to circadian peaks and troughs in endogenous CS production, whether the CSs are synthetic and substrates for multi-drug resistant transporters or endogenously released from the adrenal glands post-injury, and the specific tissue in which CSs are acting. Finally, CSs affect not only the genes that they directly activate, but also may have concentration-dependent effects on other transcription factors and the genes they regulate (i.e., nongenomic effects). When using synthetic CSs in therapeutic settings, supraphysiological dosing strategies are typically employed, which may eliminate concerns about differential dosing through a ceiling effect, but

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differences in the timing of their administration may still be critical. Both genomic and nongenomic CS effects are likely relevant in the context of MS, as the full constellation of CS actions are likely to determine the clinical response. Given our currently limited understanding of how intracellular GRs bind to proteins associated with other signaling pathways, as well as the possible effects of CSs mediated by membrane-bound receptors, the role of nongenomic effects of CSs are much harder to elucidate in the context of MS pathophysiology. However, these nongenomic effects and the actions of CSs directly on cell membranes may explain some of the unexpected outcomes seen with CS therapy. It is quite possible that the effects of high-dose glucocorticoids are mediated through nongenomic mechanisms that do not involve GR [60]. More research is required to understand the relative importance of these different mechanisms of CSs in MS, where CS treatment is a mainstay therapy.

4. Revising our understanding of CSs The more than half-century old view that CSs universally suppress immune responses has been updated in the past decade, beginning in part with studies on various aspects of the biological response to stress. Work from several laboratories strongly suggests that at the beginning of the stress–response, CSs have permissive, immunostimulatory effects [62]. The early immunostimulatory effect of stress is partly due to the short-lived activation of the sympathetic nervous system [63]. However, acute exposure to CSs can also increase the early activation of the immune response to injury [64]. In the later phases of the injury, increasing concentrations of CS secreted in response to stress constrain and facilitate recovery of the immune response exerting the classically described immunosuppressive effect. These findings have important implications for how we understand and use CSs in the context of MS. Importantly, this updated view that includes CS-activated immune responses does not challenge the traditional view that stress levels of endogenous CSs and pharmacological levels of synthetic CSs are immunosuppressive. However, contemporary investigations have identified situations where even high-dose, chronic CS treatments failed to suppress, and in some cases increased, immune responses to CNS inflammation [65]. The common finding from these studies was that even sustained occupancy of GR can have proinflammatory effects on CNS inflammation, predominantly when CS exposure occurs prior to injury [66–72]. These studies indicate that for the MS patient, factors that may be important during disease treatment are: stressors, mood disorders, and genetic variability. These three factors may all converge to create an endogenously complex CS exposure pattern that is likely to impact the individual response to therapeutic CSs. Recent studies suggest that the most important factors determining whether CSs will decrease inflammation, increase inflammation, or fail to have an effect all together include: (1) the type of CNS injury; (2) the timing and duration of CS exposure; and (3) the amount of CS delivered. Different types of inflammatory injury are likely to have different pathophysiological processes, with differential steroid sensitivity (even relapse-to-relapse). Currently available rodent models in which CSs have been tested that might serve as a model of some aspects of the acute inflammatory injury that occurs during an MS relapse are lipopolysaccharide (LPS)-induced inflammation, focal excitotoxic injury, and experimental autoimmune encephalomyelitis (EAE). More appropriate rodent models like EAE are being studied to generate a better understanding of how CSs affect processes that are more specific to MS [73,74].

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The timing of CS exposure is one of the most important parameters that can lead to an unexpected inflammatory response to injury. Exposure to CSs prior to injury is particularly likely to lead to future exacerbated inflammatory responses. In animal models, a single exposure to stress-level CSs has been reported to stimulate both CNS and peripheral immune responses [74–76], provided that it occurs prior to or at the same time as the inflammatory challenge. In rats, CS exposure stimulated cytokine release if it occurred 2 h ahead of peripheral LPS challenge, but was suppressive if CSs were given 1 h after the challenge [75,76]. The enhanced inflammatory response in the brain is relatively long-lived, being observable for at least 4 days following the acute stressor [77]. In an EAE model, CSs given at the time of immunization worsened disease symptoms and was associated with increased infiltration of leukocytes into the CNS [74]. Stress levels of CSs beginning 12 h prior to immune stimulation via LPS treatment in healthy human adults potentiate plasma IL-6 and tumor necrosis factor (TNF)-␣ [78–80]. These findings indicate that prior stress can have stimulatory effects on subsequent immune responses, an effect that has been described as CSs priming the inflammatory response to injury [81]. Several studies have found that CSs given at the same time as or following an inflammatory challenge are more likely to have classic anti-inflammatory effects [82,83]. In the context of MS, treatment with CSs does not often precede acute inflammatory injury. However, treatment of relapses with CSs may precede (and could therefore possibly potentiate) certain aspects of the chronic inflammatory state that develops with recurring injury. This hypothesis remains untested in existing rodent models. Given the sensitivity of the relationship between CSs and the immune response, it is possible that chronic inflammation, significant life stressors, and acute injury may have unexpected responses to CSs, particularly when they are given at supraphysiological doses having both genomic and nongenomic effects. Another scenario where this may be of importance is when a patient fails one round of CS therapy and is retreated with CSs. Although these theories require more research, we suggest that patients with certain characteristics, such as CS resistance or CS system response to frequent exposure to stressors, may warrant some additional consideration before being retreated with CSs. Because of their clinical relevance, the properties of synthetic CSs are pervasively explored in the literature, albeit with limitations regarding supraphysiological dosing strategies. Care must be taken when considering the actions of these compounds, as they are frequently quite different than the effects of endogenous CSs. Methylprednisolone, for example, is 5 times more potent than cortisol at the GR [22]. A concerted effort is needed to understand the differences between how endogenous and synthetic CSs affect inflammation in the CNS. Much work remains to be done to fully understand what factors dictate whether CSs exert proor anti-inflammatory effects in the CNS. For now, we can define several trends among the diverse CS effects on inflammation: low-to-moderate doses, acute exposure, and CS presence prior to inflammation all tend to augment the inflammation, whereas high doses, chronic exposure, and CS presence after inflammation tend to suppress it [64,65,84–86]. However, the rules about how these parameters interact are still undetermined and there are virtually no data available describing the CNS effects of supraphysiological doses (e.g., 1 g IVMP). The evidence discussed thus far suggests that in the context of stress and immune challenge, CSs play a complex role in orchestrating an optimal and timely response that maintains control over the potentially damaging effects of an excessive immune response, such as that found in patients with MS. More generally, these studies indicate that clinical assumptions about the universal immunosuppressive effects of CSs should be reexamined in the most objective ways possible.

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5. Side-effects associated with compromised remyelination and neuron death Although CSs are the standard therapy used to treat MS exacerbations, several lines of evidence suggest that treatment with CSs may not always produce the expected clinical outcomes. Several studies have reported mixed results with respect to the extent and timing of MS relapse improvement [10,87,88]. Although more recent insights into the effects of CSs on inflammation suggest that CS therapies may sometimes activate or fail to inhibit the immunopathophysiological underpinnings of MS in certain situations, other unexpected effects of CSs may also involve noninflammatory endpoints. Indeed, CSs have been shown to have potent direct effects on neuronal survival and remyelination, irrespective of their effects on immune cells. As discussed, CSs have also been found to lead to the expression of psychiatric abnormalities, including depression, mania, and psychosis [89], suggesting that CSs have potent and rapid effects on neurotransmitter systems that critically regulate mood and behavior. Taken together, these studies suggest that CSs could exert powerful and unexpected effects in the CNS besides those related to macrophages, microglia, and autoreactive T-cells. Corticosteroids may also have potent effects on spinogenesis, or the formation of dendritic spines on neurons. Both stress-induced cortisol and exogenous corticosterone treatment resulted in decreased spinogenesis in the basolateral amygdala [90], while others found similar results of decreased spinogenesis and dendritic arborization in the hippocampus and frontal cortex, but not in the amygdala [91,92]. Corticosteroids are well known to be important for learning processes at lower physiological concentrations, and have been shown to inhibit learning at higher concentrations [93,94]. Corticosteroid treatments have been known for decades to exacerbate neuron death during a wide variety of necrotic neurologic injuries, including hypoxia–ischemia [95] and excitotoxicity [96]. Furthermore, CS effects on immune cells may actually contribute to this worsened neuron death [97]. One important preclinical study suggests that CSs may also increase neuron death in MS [98]. This study investigated the effects of short-course, high-dose MP treatment on retinal ganglion cells in the rat model of EAE. While imperfect, the generation of myelin-reactive T-cells in EAE is nonetheless an instructive animal model of MS, and the short, but high-dose paradigm used in that study is analogous to current clinical practice. This study quantified optic neuritis by recording visual evoked potentials, or the change in electrical activity within retinal ganglion cells, from before to immediately after presentation of a light stimulus. The high-dose MP treatment did not improve visual function in EAE animals as measured by evoked potentials, and instead promoted increased apoptosis of retinal ganglion cells, the neurons that form axons of the optic nerve. This deleterious effect of MP on cell survival was found to be nongenomic, not dependent on the GR, and dependent on MAPK pathways. This work suggests that for some neurons, or in certain brain regions, high-dose CSs can be neurotoxic in the context of MS disease activity independent of GR-mediated effects on inflammatory activity. In addition to effects on neurons, CSs also impact oligodendrocytes and interfere with remyelination and repair processes after damage by autoreactive T-cells. Recovery after MS exacerbations requires remyelination, as oligodendrocytes attempt to “replace” myelin destroyed by autoreactive T-cells. This type of formation has been described as shadow plaques [5]. Recently, Clarner and colleagues used the non-immune cuprizone model of MS to explore the impact of 9 or 21 days of high-dose MP on various markers of myelination in the corpus callosum [99]. Although previous studies have explored the effects of CSs on remyelination in the spinal cord with conflicting results [100–102], Clarner and colleagues are the

first to do so systematically in the brain. As expected, cuprizone treatment resulted in decreased expression of the oligodendrocyte differentiation marker, proteolipid protein (PLP), in the corpus callosum. Methylprednisolone treatment for 9 days after cuprizone was found to further reduce the expression of PLP compared to cuprizone alone. This effect remained when animals were treated with MP for 21 days instead of just 9 days and suggests that CS treatments, especially at high doses such as those given to MS patients, can potentially impede remyelination. Given the ethical constraints of conducting equivalent studies in humans, and despite requiring careful interpretation with the proper caveats, these preclinical models are of considerable importance for understanding how CSs may act in MS patients. 6. Autoimmunity, major depression, and CSs Multiple sclerosis and major depression are highly comorbid conditions, suggesting a possible link between the two that may critically involve resistance to CSs [103]. Depression is one of the many clinical aspects of MS that might warrant special attention; it is an example of how preclinical data can provide insight into the interplay between mood, immunology, and the CNS that is a critical part of the clinical puzzle. Often unrecognized and untreated, major depression represents a significant threat to long-term quality of life for patients with MS. Untreated depression may also threaten compliance, with adverse effects on overall treatment outcome. Patients with MS have been found to have a lifetime risk of depression of 25–50% [104,105], and a recent large community-based study found that 40% of patients with MS have clinical depression symptoms [106]. Although it was originally thought that DMTs such as interferon (IFN)-1b could be depressogenic [107], a longitudinal study suggests this may not be the case [108]. Instead, prior depression may be most predictive of the development of major depression after starting DMTs [109,110]. Aspects of the disease that may contribute to this higher rate of major depression among MS patients include three primary factors: (1) direct effects of damage from the disease; (2) the psychological effects of the unpredictable nature of the disease; and (3) the chronic inflammation associated with MS. Multiple sclerosis lesions often occur in brain regions that are essential for healthy mood regulation and are altered in major depression, including the prefrontal cortex, the hippocampus, and the amygdala. Indeed, brain morphometric or functional changes in MS patients that are associated with major depression have been assessed using diffusion tensor imaging, quantitative magnetic resonance imaging (MRI), and atrophy measured by MRI [104,111,112]. Secondly, the chronic psychological stress aspect of MS may also play an important role in the high rate of depression in these patients. Patients with MS are often confronted with the unpredictable nature of the illness, which may encourage feelings of loss of control, or a “learned helplessness” state [113]. Thirdly, research into the relationship between major depression and the immune system suggests that imbalances in immune function in MS may contribute to the development of major depression in patients. Although major depression is widely believed to involve imbalances in brain neurotransmitter systems [114], as well as over- or under-activation of discrete brain areas that are responsive to traditional antidepressant treatments [115], ongoing work on the pathophysiology of depression suggests that a significant portion of depression cases also involve alterations in immune function [116,117]. This opens up the possibility that for some MS patients also suffering from depression, a somewhat common underlying pathophysiological mechanism of immune dysregulation may exist for both conditions. Major depression has also been associated with CS resistance in medically healthy patients, as evidenced by abnormal

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Fig. 3. The pathophysiology of multiple sclerosis (MS) involves many inter-related and co-existing stages, depicted in a simplified step-wise manner in the top panel. Acute relapses are a manifestation of new inflammatory activity in the CNS, driven by autoreactive T-cells that orchestrate an attack on myelin-producing oligodendrocytes (top left panel). Both the loss of oligodendrocytes and impaired remyelination (top middle panel) may lead to the accumulation of relapse-related disability. Inflammatory lesions are also associated with axonal transection and loss, which may occur early in the disease course and worsen over time (top right panel). Incomplete relapse recovery suggests that neuronal vulnerability after a relapse has important clinical implications. High-dose corticosteroids (CSs) are frequently employed to reduce acute CNS inflammation (bottom left panel). This short-term benefit may however come at the expense of optimal remyelination, as animal models have demonstrated CSs may potentiate oligodendrocyte loss in certain brain areas (bottom middle panel) [99]. Further animal model data suggest CSs limit the development of neurons [90], and may promote increased apoptosis of the neurons that form axons of the optic nerve (bottom right panel) [98]. This deleterious effect of CSs on neuron survival suggests that for some neurons, or in certain brain regions, high-dose CSs can be neurotoxic. We hypothesize that there are important clinically-relevant implications associated with the use of high-dose CSs. As the MS disease process evolves, the unexpected effects of high-dose CSs on oligodendrocytes and neurons may potentiate disability, depression, and steroid resistance.

responses to the dexamethasone (DEX) suppression test and the DEX- adrenocorticotropic hormone (ACTH) releasing test [118]. Medically healthy depressed patients have also been found to have reduced sensitivity to cutaneous CSs [119]. In MS patients with depression, impaired CS sensitivity has been identified using the DEX suppression test [120]. Depressed MS patients have been found to exhibit increased evening circulating concentrations of cortisol, which also suggests reduced CS sensitivity [121]. Depressed patients with MS have been reported to have elevated cortisol concentrations that are associated with reduced hippocampal volume [107]. The hippocampus is essential for normal regulation of hypothalamic-pituitary adrenal (HPA) axis function [42], and the loss of hippocampal control over the hypothalamus could result in increased output of cortisol from the HPA axis. Finally, patients with depression have been shown to have reduced GR␣ in circulating lymphocytes, which could contribute to CS resistance in this subgroup of immune cells [52]. Observations of increased or inappropriate inflammatory activity in depressed patients combined with decreased CS sensitivity in patients with both major depression and MS may give unique insight into the overlapping pathophysiologies of the disorders. It has been proposed that altered immune function in major depression, including increased inflammation, may take place subsequent to CS resistance [57]. High-dose CS treatment, such as the IVMP therapy used for MS exacerbations, may encourage a reduced

sensitivity to CSs because of GR autoregulation. This, in turn, could promote less GR-mediated inhibition of inflammatory signaling pathways and an overall inflammatory excess, contributing to the severity of major depression in these patients. The MS patient with depression is thus one example of a clinical phenotype where the application of high-dose steroids should be done thoughtfully. 7. Conclusion: implications for clinical practice and future research In current clinical practice, it may be difficult to predict which MS patients will respond favorably to CSs and those who might respond sub-optimally, both in terms of therapeutic response and tolerability profile. In this opinion piece we have covered some of the diverse mechanisms that underscore this heterogeneous clinical response to exogenous CSs in patients with MS. We propose that there are specific cellular, systemic, and clinical characteristics that might merit consideration when utilizing supraphysiological doses of CSs in clinical practice. These characteristics must next be tested in a translational setting to determine how they might apply to patients. We also hope to encourage the measurement of long-term physiologic nuances in clinical studies of high-dose CSs, rather than just short-term measures of inflammation. The variable impact of CSs at a cellular level, mediated by genomic effects and mutable expression of different GR isoforms,

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could be examined as a potential contributor to CS responsiveness and heterogeneity of response in relapsing MS patients. Although some of the genomic and nongeomic effects of CSs might be desirable, many of the effects of supraphysiological doses of CSs (e.g., 1 g of IVMP as used to treat MS relapses) are poorly understood and these treatments often present with serious side effects. Aside from the well-described side effects of metabolic syndrome, bone loss, HPA axis dysregulation, and extreme mood shifts [89], high-dose CSs may have additional effects that are important in MS, such as the nongenomic interference with neurorepair processes [98,99]. Unfortunately, the majority of research on genomic and nongenomic effects of CSs to date involves cell culture systems that may poorly model a process that could vary from tissue to tissue and almost certainly is affected by cells acting in vitro vs. in vivo. Exploring these cellular contributions to CS sensitivity across different cell types and organ systems may yield ideas for side effect prediction and mediation. Biomarker assays for CS sensitivity, such as heat shock protein (HSP) 90 levels or GR␤ expression, may provide insight into individual CS responsiveness and allow for more rational patient selection for CS treatment and re-treatment over the course of a patient’s MS. It is clear that the clinical impact of exogenous high-dose CSs is more complex than a simple dose-response relationship. The precise timing, dosage, duration of therapy, cellular exposure, and background CS basal milieu all contribute to the pro- or anti-inflammatory properties of CSs (Fig. 3) [90,98,99]. Although activation state of immune cells is likely to be central to understanding how immune cells function in the context of MS and how CSs may act in MS patients, a strong need exists to expand our understanding of how CSs may either protect or harm neurons, or encourage or inhibit remyelination, especially in the setting of an MS exacerbation. The notion that exogenous CSs can exert a proinflammatory effect where anti-inflammatory effects are expected, and could be potentially neurotoxic where neuroprotection is the goal, runs contrary to the accepted dogma guiding the therapeutic use of CSs in MS relapse. Given the high rates of comorbid depression in MS and the complex interplay between the disease state, mood regulation, chronic inflammation and CS resistance, the use of exogenous CSs in MS patients with depression merits special consideration. It is perhaps in this population that the chameleon-like qualities of CSs are most evident. Corticosteroid resistance in depressed patients is well established, and may manifest as increased inflammatory activity. In this setting, supraphysiological CS dosing may in turn both worsen MS and undercut normal CNS function required for mood regulation, potentiating depression. Future investigations should work to determine if MS combined with major depression is associated with resistance to therapy with CSs and may reflect a clinical phenotype to specifically consider when treating MS relapses. Conflict of interest Dr. Krieger discloses: Consulting/advisory board work: Acorda Therapeutics, Bayer HealthCare Pharmaceuticals, Genzyme, Questcor Pharmaceuticals, Inc., Sanofi, Teva Neuroscience. Support for research: Bayer HealthCare Pharmaceuticals. Dr. Sorrells has nothing to disclose. Dr. Nickerson is an employee of Questcor Pharmaceuticals, Inc. Dr. Pace discloses: Advisory board work: Questcor Pharmaceuticals, Inc. Acknowledgments The authors wish to thank Courtney Breuel for editorial assistance and George Baquero for creation of the illustrations, both

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