Lipid autoreactivity in multiple sclerosis

Medical Hypotheses 74 (2010) 433–442

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Lipid autoreactivity in multiple sclerosis M.M. Blewett * Harvard College, 211 Currier Mail Center, 64 Linnaean Street, Cambridge, MA 02138-1502, United States

a r t i c l e

i n f o

Article history: Received 2 October 2009 Accepted 8 October 2009

s u m m a r y Lipids comprise over 70% of the myelin sheath but have been largely underinvestigated as autoantigens in multiple sclerosis (MS). This paper cites evidence for the involvement of lipid autoreactivity in MS and details how self lipid cross-reactivity may also contribute to the development of type 1 diabetes and autoimmune thyroid disorders (both of which have been associated with MS). A further analysis of myelin chemistry suggests several mechanisms by which infection may contribute to etiology and trigger lipid autoreactivity via molecular mimicry. This analysis may aid the development of new therapies for autoimmune diseases. Ó 2009 Elsevier Ltd. All rights reserved.

Introduction Multiple sclerosis (MS) is a demyelinating condition of the central nervous system (CNS) that affects roughly three million people worldwide. Symptoms include lack of coordination, numbness, slurred speech, and double vision [1]. MS is believed to be an autoimmune condition, perhaps triggered by an infection during adolescence. One major setback to developing new MS therapies is our limited understanding of the antigenic specificity of the autoimmune response. While protein autoantigens have been investigated for decades, the identity of the molecular target in myelin remains a topic of great debate. This paper presents an analysis of past autoantigen research and cites evidence for lipid autoreactivity in MS. Understanding the specificity of the humoral and cell-mediated responses in MS may, in turn, shed light on this disease’s etiology, a topic discussed in the latter section of this paper. The search for autoantigens The molecular target of the autoimmune attack in MS (the autoantigen) remains unknown. The majority of research to date has focused on protein autoantigens. Myelin basic protein (MBP) Arguably the most heavily investigated candidate autoantigen has been myelin basic protein (MBP) [2–4]. MBP is well recognized for its role in the rodent model of MS known as experimental allergic encephalomyelitis (EAE); administration of MBP-reactive T-cell clones leads to paralysis, as well as perivascular inflammation and * Tel.: +1 973 432 9821. E-mail address: [email protected]. 0306-9877/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2009.10.003

demyelination [5]. T-cell clones recognizing MBP are observed in both MS patients and healthy controls [3], though some authors speculate that these clones are activated (by infection, for example) only in MS patients [2,3]. Several additional factors complicate MBP’s role in the pathogenesis of MS. MBP is not located on the extracellular surface of the myelin sheath [6] and as a result is not readily accessible to antibodies in the brain or cerebrospinal fluid (CSF). Therefore an assault on MBP should come secondary to an initial attack on surface antigens; no demyelination is observed when anti-MBP antibodies are administered to aggregating brain cell cultures [7]. Anti-MBP antibodies isolated from MS patients are characterized by low-affinity interactions, and O’Connor et al. [8] have reported discrepancies between solid-phase and solution-phase detection methods. Proteolipid protein (PLP) Proteolipid protein (PLP) accounts for 30–50% of myelin protein, making it the most abundant protein in myelin [6] (Fig. 1). PLP is comprised of four membrane spanning domains and can induce EAE when emulsified in complete Freund’s adjuvant (CFA) [9– 11]. A number of groups have identified activated PLP-reactive Tcell lines in MS patients [12–17], though it is unclear at this point whether these T cells are pathogenic [18]; serum containing PLPreactive antibodies does not induce demyelination in vivo [19]. Myelin/oligodendrocyte glycoprotein (MOG) Myelin/oligodendrocyte glycoprotein (MOG) is exposed on the extracellular surface of the myelin sheath and, as the name suggests, this protein is also located on oligodendrocytes. MOG accounts for a small percentage (0.01–0.05%) of myelin protein [6]. Several reports describe elevated T- and B-cell responses against MOG in MS sera and/or CSF [20–25]. Again, though, the data are

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Fig. 1. Composition of the myelin sheath. Myelin lipids are listed first, followed by myelin proteins. Data are based on numbers from Basic Neurochemistry: Molecular, Cellular, and Medical Aspects (7th edition) by Siegel et al. [6]. Numbers were calculated according to an estimate of 70% for the lipid fraction of total human CNS myelin. Note that some plasmalogens are also included under the phosphatidylethanolamine subheading.

complicated by nonspecific reactivity [26,27]; Reindl et al. [28] found MOG-reactive antibodies in the CSF of 33% of MS patients studied and in 53% of individuals with other inflammatory neurological diseases. The authors point out that MS patients remain MOG seropositive indefinitely, whereas in control subjects, after nine months MOG-reactive antibodies are no longer detectable. In a 2004 editorial discussing the papers by Berger et al. [20] and Lampasona et al. [26], Reder and Oger [29] conclude, ‘‘The takehome message is that most of these findings represent nonsense antibodies that measure the antigenicity of the proteins involved more than a specific root cause of MS. The antibodies may reflect the increased immune response of MS patients more than the pathogenicity of the antibodies, yet still could have predictive value”. Lipids Often overlooked in the discussion of MS autoantigens are the lipids that comprise over 70% of the myelin sheath [6] (Fig. 2). Most of these are glycolipids or phospholipids whose hydrophilic head groups extend from the myelin sheath and are vulnerable to antibody recognition. The most abundant myelin glycolipid is galactocerebroside (GalC) [6], which at first seems to be a potential autoantigen [30]. However, because of the abundance of similar galactose-containing structures in the human body, negative selection during the self tolerance check most likely leads to the removal of GalC-specific cell lines [31]. Experimental data from MS patients The sulfated version of GalC, known as sulfatide, is more prone to immune attack than GalC and appears to be an immunodominant myelin lipid [32]; MS patients display significantly higher levels of intrathecal anti-sulfatide antibodies than do controls with other neurological diseases [33]. Though sensitive detection methods are required to measure serum antibody levels, anti-lipid antibodies from MS sera have been reported [34–36]. Relapsingremitting MS patients experiencing a first attack display increased serum levels of the ganglioside GM1 relative to healthy controls

[37]. Elevated CD1-restricted T-cell responses to sulfatide and gangliosides have also been described [38]. In 2006, Kanter et al. [39] reported increased lipid reactivity in the CSF of MS patients compared to controls with other neurological diseases. Specifically, the authors observed reactivity to sulfatide, asialo-GM1 (a ganglioside), oxidized cholesterol, oxidized phosphatidylcholine, and sphingomyelin. Lipid antigens such as these are recognized by CD1 molecules [40,41]. CD1 molecules are found on microglia and some B cells. These proteins recognize the hydrophobic alkyl chains of lipids via a deep binding cleft and then present the lipid antigens to T-cell receptors. Therefore, the relative abundance of CD1 molecules in active, but not silent, MS lesions [42] further implicates lipid reactivity in the pathogenesis of MS. Experimental data from animal models Jahng et al. [43] coadministered MOG and sulfatide in CFA and determined that 3–4% of the infiltrating T-cell population in mouse CNS comprised sulfatide-reactive T cells. CNS iNKT cell levels were nearly six times lower. Conversely, in both healthy and diseased peripheral organs, iNKT cells commonly outnumber sulfatide-reactive T cells in a ratio of 5:1 [32]. Repeated administration of iNKT cell agonist a-GalCer in fact leads to NKT cell depletion, presumably by inducing NKT cell anergy [44,45]. A similar mechanism involving self lipid NKT cell agonists may be at work in MS brain. Whether NKT cell deficiency precedes MS development or rather is a consequence of it is not clear. Lipid reactivity has been observed in SJL/J and C57BL/6 mice administered either PLP or MOG in CFA [39]; this finding suggests some involvement of the adjuvant mycobacterial component, as M. tuberculosis expresses phospholipids and sulfated glycolipids which are known to be recognized by CD1 [41]. Co-administration of sulfatide and PLP emulsified in CFA increases the severity of EAE [39]. Neurochemistry of lipids and the presence of lipids on oligodendrocytes Myelin lipids are viable autoantigens for a number of reasons. Sulfatide, the gangliosides, and cholesterol, for example, are

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(1)

(2) (7)

(3) (8)

(4) (9)

(5)

(10) (6)

Fig. 2. Representative lipids of the myelin sheath. The lipids are numbered according to their relative abundance, from 1 (least abundant) to 10 (most abundant). 1: asialoGM1, 2: Phosphatidylinositol, 3: Sulfatide, 4: Phosphatidylserine, 5: Sphingomyelin, 6: Phosphatidylcholine, 7: Plasmalogen, 8: Phosphatidylethanolamine, 9: Galactocerebroside, 10: Cholesterol. R and R’ are alkyl chains. The lengths of these hydrophobic alkyl chains can vary, as can their number of units of unsaturation. The head group of phosphatidylinositol as shown here is myo-inositol.

displayed on the extracellular surface of the myelin sheath [6]. Sulfatide is also found on oligodendrocytes [46], which means an autoimmune attack on sulfatide could both induce lesion formation and limit the body’s ability to remyelinate those lesions. Treatment of oligodendrocyte progenitor cultures with a monoclonal antibody that recognizes both galactocerebroside and sulfatide inhibits progenitor differentiation, rendering those oligodendrocytes nonfunctional until the antibody is removed [47]. Dyer and Benjamins [48] similarly found that anti-sulfatide antibodies can damage oligodendrocyte microtubules, though the adverse effects on the cytoskeleton are more pronounced for anti-galactocerebroside antibodies. Sulfatide and galactocerebroside are essential for the proper formation of CNS myelin, whereas PNS myelin appears normal in mice unable to synthesize these lipids [49]. Sulfatide also plays critical roles in oligodendroglial–axon interactions [50], in the distribution of Na+ and K+ channels [51], and in myelin maintenance [6,52]. Jeon et al. [53] reported that sulfatide can activate microglia and promote glial inflammatory responses. Summary Lipids comprise the majority of the dry weight of myelin, are often exposed on the extracellular surface of the myelin sheath, are recognized by CD1 molecules upregulated in active lesions, and have been shown to be immunogenic. Lipid autoreactivity may account for the paucity of remyelination in MS and for the apparent involvement of NKT cells in disease development and progression. Further research needs to be performed before the full scope of lipid autoreactivity in MS can be understood.

Associations between MS, type 1 diabetes, and autoimmune thyroid condition Lipid reactivity may be one of several common threads among MS and related autoimmune conditions including autoimmune thyroid condition and type 1 diabetes. Sulfatide is found in several areas of the body, including the myelin sheath [6], the thyroid [54], the pancreatic islets of Langerhans [55–57], the kidney [56], and the choroid layer of the eye [58]. Damage to the blood–brain barrier [59,60] could allow autoantibodies and self-reactive lymphocytes in the CNS to exit into the periphery and migrate through the body to other organs. Thus, sulfatide-reactive antibodies and T cells originating in MS brain could potentially cause damage in the thyroid and the pancreas. This reasoning may help explain the constellation of common factors among MS and neighbor autoimmune conditions. According to a study at the Children’s Hospital of Pittsburgh [61], MS prevalence may be as much as twenty times higher among women with type 1 diabetes than among healthy female adults. Nielsen et al. [62] made similar findings in a Danish population. Likewise, diabetes prevalence among a sample of Sardinian MS patients was approximately three times higher than diabetes prevalence among their healthy siblings and five times higher than among the general population [63]. On a macromolecular level, anti-sulfatide and anti-ganglioside antibodies have been reported in type 1 diabetes patients [56,64–66]. Several reports have also found autoimmune thyroid disorders to be more prevalent among MS patients [67]. Karni and Abramsky [68] found that thyroid disorders are three times more common

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among women with MS than among control women without inflammatory diseases. The immunosuppressants interferon beta [69,70] and Alemtuzumab [71,72] can induce autoimmune thyroid disorders in people suffering from MS. MS etiology These data raise an important question: what triggers autoreactivity against myelin lipids? Again there are many candidates. According to Sospedra and Martin [73], MS may develop by one (or a combination) of two general mechanisms: molecular mimicry or bystander activation. Molecular mimicry arises when an individual is exposed to a pathogen whose antigens share epitopes with self antigens. The immune response directed against that pathogen can evolve into an autoimmune attack against self antigens (in the myelin sheath, for example) [74–77]. Bystander activation involves a nonspecific inflammatory response to an infection. The high percentage of discordant monozygotic twins (one monozygotic twin has MS while the other does not) [78] implies that genetics alone does not determine whether an individual will develop the disease; an environmental agent likely plays a critical role. The identity of this agent has been a topic of great debate. One fundamental point to address is whether MS is caused by one agent or many. If a single agent is at the root of this disease, that pathogen must meet a number of criteria in order to produce the biochemical and symptomatic traits associated with MS. Another possibility is that a cocktail of agents is responsible for disease onset and progression. Furthermore, this set of causative pathogens may differ from patient to patient; the heterogeneity of MS progression suggests that our larger categorization of MS may house a number of conditions caused by distinct agents. MS may be an exception to Koch’s ‘‘one organism – one disease” paradigm [79]. Even so, other conditions like polio can be attributed to a single etiological agent yet produce highly varied disease progression. Clearly further research is required before we can understand the complex underpinnings of MS. If in fact molecular mimicry underlies the development of MS, the question remains as to how an investigator might go about identifying the causative agent(s). Searches for viral or bacterial DNA may be difficult, as a strong, and for many purposes dysregulated, immune response against that pathogen would most likely have eliminated it; for example, several notable reports have detected no Borrelia burgdorferi DNA in subgroups of Lyme arthritis patients [80,81] (B. burgdorferi is the causative pathogen of Lyme disease). Some autoimmune mechanism may account for the persistence of symptoms [82]. Therefore, the etiological agent’s only reliable footprint may be the specificity of the humoral and cellmediated immune responses. For this reason especially, a better understanding of target antigens in MS is critical to our understanding of this disease as a whole. Candidate etiological agents in MS Reviewed here are several candidate etiological agents of MS and a brief discussion of how each might contribute to the development and progression of MS, as well as how infection with some of these agents may give rise to lipid autoreactivity. The section begins with a discussion of candidate viral agents and ends with bacteria. Much of the etiological research in MS has involved viruses. Russell [83] gives the following data to justify this focus; (1) the latitude effect in MS epidemiology suggests involvement of an infectious agent (though admittedly this agent need not be a virus), (2) the measles virus has been associated with CNS infection and demyelination (subacute sclerosing panencephalitis, or SSPE), and (3) viruses have been shown to induce demyelination in animal

models. Other etiological agents not mentioned here have certainly been proposed; the hypotheses surrounding the pathogens described in this paper illustrate prior and new approaches to elucidating etiology. Epstein-Barr virus Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that can infect B cells. Unlike many other herpesviruses (one of which will be discussed later), EBV does not inhibit antigen expression when it infects cells in its lytic cycle; EBV-transformed cell lines display a number of EBV nuclear antigens known as EBNA’s. This virus can remain latent for the remainder of the host’s lifetime, though it is in some cases reactivated [84]. A number of data implicate EBV in MS. EBV-negative individuals have almost no risk of developing MS, and among EBV-positive individuals MS susceptibility is lower for those infected during infancy than for those infected during adolescence [85,86]. Acute EBV infection during adolescence usually manifests as infectious mononucleosis (IM). IM is more common among Caucasians than among blacks and Asians, and IM prevalence, like MS prevalence, increases with distance from the equator [86,87]. Arguably the most powerful pieces of data supporting a role for EBV in MS are the nearly 100% EBV seropositivity among adult MS patients [88] and the presence of EBV antibodies in patient serum five to twenty years before clinical onset of MS [85,89–92]. While EBV infection appears to predispose individuals to MS, the data suggest that EBV does not directly contribute to disease pathogenesis. Reports of increased CNS humoral response to EBV have been sidestepped by evidence of elevated responses to rubella, measles, Varicella zoster, and Chlamydia pneumoniae as well [85]. EBV-infected B cells and EBV-specific T cells do not appear to be pathogenic [79]. Pohl [85] concludes that intrathecal EBV antibodies may ‘‘merely be part of the MS-typical polyspecific CNS antibody production, directed against diverse common pathogens”. How EBV could initiate an autoimmune demyelinating condition is unclear. Homologies between EBV antigens and MBP have been identified, though T cells specific to these peptide sequences are observed in similar levels in MS patients and healthy controls [83]. Interestingly, some EBV-transformed B-cell lines from MS and other autoimmune disease patients produce anti-glycolipid antibodies. Sulfatide and GM1 are included among these target antigens [46,93]. One explanation for the role of EBV is that IM disrupts IL-15 signaling (which is critical for NK and NKT cell development and function) [84,94], therefore limiting the immune system’s ability to respond to the true etiological agent(s) of MS. Incomplete removal of a pathogen by NKT cells may open the door for increased antibody production against that pathogen; Discussed later in this paper are cases in which elevated IgG against foreign agents may lead to autoreactivity. However, the effects of IM on IL-15 signaling are currently controversial [95]. NKT cell populations appear to be depleted and/or defective in MS patients and in EAE [96–98]. Chung et al. [99] reported that EBV infection can downregulate surface expression of CD1d; CD1d (one of four principal members of the CD1 family) is required for removal of those pathogens whose antigens are NKT cell agonists. Vaccinia virus and lymphocytic choriomeningitis virus, both mouse viruses, are known to downregulate CD1d expression. Reducing CD1d numbers may allow these viruses to evade NKT cell detection [100]. EBV may therefore contribute to MS etiology through several possible mechanisms. Human herpesvirus 6 Human herpesvirus 6 (HHV-6) is another ubiquitous virus that has been linked to MS. It belongs to the beta subfamily of herpesvi-

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ruses which stop antigen expression once they have infected cells. The HHV-6 etiological hypothesis arguably made its debut in 1995 when Challoner et al. [101] observed HHV-6 DNA in 78% of MS and 74% of control brains. While this difference is not significant, the authors also noted that viral protein expression is elevated in the MS group compared to controls. From this, they and others [102] speculated that HHV-6 infection is active in MS patients and latent in the control group. Some groups have reported increased seropositivity [103] and intrathecal antibody production against HHV-6 in MS patients relative to controls [103,104], though other groups have found only statistically insignificant differences between MS and control groups [105,106]. As with EBV, how HHV-6 could trigger an autoimmune demyelinating condition is not known at this point. Homologies have been identified between HHV-6 proteins and MBP. However, by one report, T cells specific to these peptide sequences were found in 36% of MS patients and 26% of controls. Stimulation with MBP led to cross-reactive T-cell lines more readily in MS patients than in controls, though the opposite was true for stimulation with viral extract [102,107]. Nevertheless, another finding by Challoner et al. [101] has gone relatively uncontested: HHV-6 DNA is present exclusively in oligodendrocytes from MS patients. HHV-6 viruses may infect glial precursor cells and prevent their proper differentiation into mature myelinating oligodendrocytes [102]. Therefore, oligodendroglial HHV-6 infection may partly explain the paucity of remyelination in MS. Even so, great caution must be exercised when addressing the role of ubiquitous viruses in MS so as not to confuse causation with nonspecific consequences of a heightened immune response [101,102]. B. burgdorferi Background B. burgdorferi, the causative agent of Lyme disease, can be cleared from the body with timely administration of antibiotics. In other cases, B. burgdorferi can remain latent for several years and infect the CNS. Neurological Lyme disease, known as neuroborreliosis, affects roughly 10–15% of Lyme disease patients [108], and can be difficult to distinguish from MS. Both diseases can result in regions of MRI hyperintensity [109–111]; in an MRI study involving Lyme disease patients, Kruger et al. [112] found ‘‘multiple sclerosis-like lesions” that remained two to four years after antibiotic treatment. MS and neuroborreliosis are related by a number of factors. Both conditions have been associated with facial palsy [113,114], myalgia, and vision loss [108,115,116]. MS and neuroborreliosis can lead to CSF oligoclonal banding in a large percentage of those affected (observed in up to 95% of MS patients [117] and 80–90% of neuroborreliosis patients [115]). Neuroborreliosis, like MS, can follow either a remitting or progressive disease course [108]. According to a 2006 study by Alonso et al. [118], individuals who had taken penicillin within three years of the study date had a statistically reduced risk of developing MS; penicillin is an effective treatment for early B. burgdorferi infection. Several investigations have found comparable B. burgdorferi antibody levels in sera from MS and control groups. Coyle [119] found serum B. burgdorferi antibodies in one out of 89 MS patients and in two out of 11 control patients. Schmutzhard et al. [120] made a similar finding; 14.2% of MS patients and 20.1% of controls displayed serum B. burgdorferi antibodies. The difference in IgG levels between the two study groups was not statistically significant. It should be noted that in a study of five neuroborreliosis patients, none displayed serum B. burgdorferi IgG [121]. Instead, antibodies against B. burgdorferi were identified in the CSF as might be expected for a CNS condition. Out of 75 neuroborreliosis patients,

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Stiernstedt et al. [116] noted that 18 (24%) displayed positive CSF serology but negative serum serology, while another nine (12%) were negative in both CSF and serum serology. Intrathecal antibody synthesis can usually be confirmed only when the CSF antibody titres per unit immunoglobulin are at least twice those in the serum [122], and Kaiser [115] has specified that only a possible, rather than definite, diagnosis of neuroborreliosis can be made if CSF analysis is not performed. Even so, a spinal tap to obtain CSF is unappealing for a number of reasons, and with the advent of MRI, CSF analysis has been on the decline. These data highlight the complexity of chronic B. burgdorferi infection and its potentially autoimmune nature. B. burgdorferi surface chemistry Careful analysis of B. burgdorferi surface lipids reveals a possible mechanism by which this bacterium may evade immune detection and eventually induce an autoimmune attack against the myelin sheath. Unlike most Gram-negative bacteria, B. burgdorferi lacks lipopolysaccharide (LPS) in its outer membrane [123]. Without this highly immunogenic molecule, the job of recognizing and removing B. burgdorferi (or other LPS-lacking Gram-negative bacteria) is left largely to NKT cells [124–126]; again, this population is known to be depleted in MS patients, though whether these reduced NKT cell numbers are causal or somehow an artifact of a dysregulated immune response is not clear. Many of the LPS substitutes in B. burgdorferi are lipids that bear strong structural similarities to abundant lipids in the myelin sheath (Table 1). BbGL-1 contains a cholesterol moiety; Kanter et al. [39] observed reactivity to oxidized cholesterol derivatives in MS CSF. The B. burgdorferi glycolipid BbGL-2 contains a galactose head group and a diacylglycerol moiety [127]. The galactose head group is found in sulfatide and GM1, both lipids recognized by antibodies in MS CSF. BbGL-1 and BbGL-2 both stimulated antibody production in mice and are surface exposed [127]. B. burgdorferi also display phosphatidylcholine [128,129]; Kanter et al. [39] observed reactivity to oxidized phosphatidylcholine derivatives and to sphingomyelin (a structural relative of phosphatidylcholine) in MS CSF (Table 1). Meanwhile Garcia Monco et al. [130] reported reactivity against gangliosides (such as GM1) in neuroborreliosis patients. B. burgdorferi and NKT cells The B. burgdorferi antigen BbGL-2 is a known agonist of NKT cells [125], a finding which may shed light on the larger purpose of the NKT cell population. The lower NKT cell levels observed in MS patients theoretically make this cohort more prone to B. burgdorferi infection, again assuming that NKT cell levels are reduced before onset of disease. Because deficient or defective NKT cells have also been linked to type 1 diabetes and other autoimmune conditions [131–133], one function of these cells may be to prevent self reactivity by recognizing glycolipids that share epitopes with self antigens. IM during adolescence might deplete an individual’s NKT cell and/or CD1d populations and predispose that individual to B. burgdorferi infection. Of course, much research is required before these hypotheses can be proven. Diagnostic difficulties It should be noted that our understanding of MS fundamentally relies on the diagnostic criteria used to make an MS diagnosis. Poser et al. [1] have specified that a diagnosis of clinically definite MS can be made only when an individual displays two separate lesions (via CT or MRI, for example) and has experienced two attacks separated by at least one month. The authors also instruct doctors to properly rule out other diseases which could produce CNS lesions and which may appear similar to MS symptomatically. However, Lyme disease has been labeled the ‘‘great imitator” [134,135] for

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Table 1 Comparison of B. burgdorferi and myelin lipid antigens. In the left column is: BbGL-1 (1a), BbGL-2 (2a), and phosphatidylcholine (3a). Kanter et al. [39] observed reactivity to structurally similar myelin lipids: oxidized cholesterol (1b), asialo-GM1 (2b), sulfatide (2c), oxidized phosphatidylcholine (3b), and sphingomyelin (3c). In the oxidized phosphatidylcholines, one of the alkyl chains terminates in an aldehyde or carboxylic acid rather than in a methyl group as in phosphatidylcholine.

its ability to mimic other conditions, and eliminating this disease from consideration is often not straightforward. K.D. Reed [136] explains, ‘‘In contrast to kits used for human immunodeficiency virus testing, there is little standardization among the numerous commercial kits marketed for LD [Lyme disease] diagnosis in the United States and Europe. When results from different laboratories for well-characterized proficiency samples are compared, significant differences in sensitivities and specificities of ELISA [enzyme-linked immunosorbent assay] and IFA [indirect fluorescent antibody] have been observed.” Given the variability of serum B. burgdorferi assays currently used in clinical practice [82,137–140], it is plausible that a certain number of neuroborreliosis patients have received an MS diagnosis. In addition, only 40–60% of Lyme disease patients display erythema chronicum migrans (ECM), the Bull’s eye rash that has come to be characterized with Lyme disease [115]. Case studies by Schmutzhard et al. [141] and Karussis et al. [142] highlight the difficulties doctors can face in distinguishing these two conditions. In the case reported by Karussis et al. [142], a 46-year-old male presented with paresthesias in his hands, legs, and face. Other symptoms included blurred vision and difficulty speaking and walking. MRI showed multiple hyperintense lesions. A serum ELISA for B. burgdorferi antibodies was negative and the patient met Poser’s [1] criteria for definite MS. After several years of increasing disability, the patient was again tested for Lyme disease. B. burgdorferi antigen was detected in the CSF and the patient was subsequently given a diagnosis of neuroborreliois and administered ceftriaxone for three weeks. Unfortu-

nately the patient’s condition continued to deteriorate even after taking antibiotics, though curiously no B. burgdorferi antigens could later be detected in his brain specimen. The doctors who treated this patient are highly trained physicians and researchers with many years of experience. The challenges they faced identifying the underlying cause of disease reveal the nontrivial risk of misdiagnosis. An evolving autoimmune response Neuroborreliosis sufferers may account for at least a small percentage of MS patients. However, in a sample of 38 probable or definite MS patients, Schmutzhard [135,141] identified elevated B. burgdorferi antibody titers in the CSF of only two. This low frequency of detectable intrathecal antibody production in MS patients suggests either that more sensitive detection methods are required (the study was performed over 20 years ago) or that the majority of MS patients may be infected with some other etiologic agent. Interestingly, Oschmann et al. [143] noted that the specificity of the immunologic response in neuroborreliosis patients changes over time. In Stage II patients (with an intermediate disease course), the response is directed primarily against the p41 antigen. Stage III patients with a more advanced disease state (disease duration over six months) display a higher number of oligoclonal bands (more than four for every patient). Reactivity to a separate set of proteins, as well as to a 5 kDa glycolipid, was observed. Among Stage III patients, the authors also observed IgG against other antigens besides the 12 B. burgdorferi proteins/glycolipids

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used in the study. The 5 kDa glycolipid may correspond to a bacterial antigen that bears structural similarities to myelin antigens. This study suggests that the CNS immune response to B. burgdorferi evolves over time and is characterized by a changing antigenic specificity. For this reason and others, studies reporting seronegativity to B. burgdorferi must be interpreted with caution. Again, one must be very careful about assessing the roles of certain etiologic agents based on empirical findings. If the bacteria itself has been eliminated and yet an autoimmune response persists (as has been proposed for neuroborreliosis), in the long-term one would likely not see elevated B- or T-cell levels against bacterial antigens that do not bear homologies to self antigens. Rather, as the study by Oschmann et al. [143] suggests, the late stage immune response is likely directed against those self antigens that can be recognized by cross-reactive B cells and T cells. A very interesting study would involve observing the evolution of the antigenic specificity in MS patients over time; early stage MS patients might display elevated antibody levels against a foreign pathogen, while late stage MS patients might display higher affinity B- and T-cell responses against structurally similar myelin antigens. Summary Lipid reactivity can be difficult to study experimentally, which may be one reason why this area has been largely underinvestigated. Therefore, in the future investigators might consider researching whether oligoclonal banding in MS includes antibodies against myelin antigens that have common epitopes with bacterial lipids. It is unclear whether we will ever be able to definitively link B. burgdorferi to the majority of MS cases. Even so, neuroborreliosis remains worthy of further study due to its many commonalities with MS. The model of lipid autoreactivity proposed here for neuroborreliosis may contribute to our understanding of this debilitating infectious condition. Such lipid autoreactivity triggered by B. burgdorferi infection might be directly responsible for disease progression in a subset of patients diagnosed with MS; autoreactivity in the remainder of people affected by MS could develop by a similar mechanism. Other bacterial candidates Induction of autoimmunity via bacterial molecular mimicry has been demonstrated previously. Perhaps the most notable example is the involvement of Campylobacter jejuni in Guillain–Barré syndrome (GBS) [144–146]. C. jejuni, a Gram-negative bacterium, displays lipopolysaccharides that are similar to gangliosides in the peripheral nervous system. This can lead to the development of GBS or the related disease known as Fisher’s syndrome. The peripheral demyelination observed in GBS can cause muscle weakness, numbness, and lack of coordination. Most of the candidate etiologic agents considered in MS have been viruses, with the exception of Chlamydia pneumoniae. Bacteria should be further investigated for a number of reasons. Recent evidence has shown that the demyelination in MS is mediated in part by a subset of T helper cells called TH-17 [147]. TH-17 cells respond primarily to extracellular bacteria [148], including B. burgdorferi [149]. The known agonists of NKT cells are also lipids belonging to bacteria such as B. burgdorferi, Sphingomonas spp., and Mycobacteria spp. [100]. Penicillin use reduces the risk of developing MS [118], and, as just mentioned, Gram-negative bacteria are known to trigger autoimmune conditions via molecular mimicry in diseases such as GBS. Bacteria often display lipid antigens [150], presenting opportunities for dangerous cross-reactivity with the largely lipid-comprised myelin sheath.

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Final etiologic considerations Given these data, we can expand on the etiology discussion given at the beginning of this section. In order for a pathogen to cause a condition similar to MS via molecular mimicry, it must not only display antigens that are structurally similar to the myelin sheath but it must also be able to penetrate the blood–brain barrier and enter the CNS. A number of bacteria display glycolipid and phospholipid antigens that likely share epitopes with myelin antigens; however, not all of these bacteria are pathogenic. Despite the heterogeneity of MS progression, there are a number of parameters that can be associated with the large majority of cases. For example, MS is a CNS condition mediated primarily by T cells resulting in demyelination, disproportionately affecting women, and whose prevalence increases with distance from the equator. Any student of MS could quickly add more items to this list. The true etiological agent must account for these facts. This line of reasoning can eliminate a number of possible agents from consideration. Given the complexity of biological systems, unraveling the roles of the remaining candidate etiological agents will require careful experimentation; again, investigation into the antigenic specificity of the humoral and cell-mediated immune responses may provide the data required to finally identify the causal pathogens. Conclusion Decades of research have shown MS to be a complex and multifaceted disease, and a number of factors likely contribute to its etiology. Our only reliable indication of prior infection may be the antigenic specificity of the autoimmune response; even then, the autoimmune response is likely directed against only a subset of the foreign agent’s antigens, specifically those that share epitopes with self lipids. Despite many investigative efforts, no myelin protein has stood the test of time (and the scientific community) to be labeled a definite autoantigen. Attention has recently turned to the lipids that make up over 70% of the myelin sheath. Kanter et al. [39] found CSF reactivity to lipids which bear strong similarity to B. burgdorferi lipids, including sulfatide, asialo-GM1, oxidized cholesterol, oxidized phosphatidylcholine, and sphingomyelin. The lipid autoreactivity model outlined here may explain the biochemical basis of chronic neuroborreliosis and the limited efficacy of antibiotics for these patients. The same or similar lipid autoreactivity may underlie type 1 diabetes, autoimmune thyroid condition, and MS. If further research can solidify the role of lipid autoreactivity in MS, muchneeded breakthroughs in autoimmune therapies can follow. Conflicts of interest statement The author has no financial or personal relationship (e.g., employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, grants or other funding) with any people or organizations that could inappropriately influence (bias) the author’s work. Acknowledgement I would like to thank H. Yang for careful proofreading of this manuscript. References [1] Poser CM, Paty DW, Scheinberg L, McDonald WI, Davis FA, Ebers GC, et al. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann Neurol 1983;13:227–31.

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