Wouter H. Gerritsen

Epstein Barr virus is not a characteristic feature in the central nervous system in established multiple sclerosis

Sir, Although recent studies have demonstrated a clear association of Epstein-Barr virus (EBV) infection with multiple sclerosis (Zaadstra et al. , 2008; Lunemann and Munz, 2009; Salvetti et al. , 2009), there has been much debate if and where the virus acts in the pathogenic cascade of multiple sclerosis and whether the virus needs to gain entry to the central nervous system (CNS). A recent study reported in Brain by Willis et al. (2009) showed that there is little evidence for the presence of EBV in the central nervous system of people with multiple sclerosis. These findings contrast greatly with the studies by Serafini et al. (2007) that have described abundant EBV positive cells in multiple sclerosis, and the presence of ectopic B cell follicles enriched with EBV infected cells in some patients. Willis and co-workers used a variety of validated methods to determine the presence of EBV in the CNS of patients with B cell infiltrates within the meninges and parenchyma. The paper described two crucial observations. Unlike Serafini et al. (2007), Willis et al. did not find the presence of EBV to be a characteristic feature of multiple sclerosis (aptly the title of the paper). Second, ectopic follicles, suggested by Serafini et al. to harbour EBV infected B cells, were not observed, despite scrutiny of meningeal tissues where these follicles should have been present. Clearly, the issue of whether EBV is indeed present in the CNS is crucial not only to determine the impact of the virus on the disease in the CNS, but also for diagnostic pathology in general. The studies reveal the vagaries of pathological detection methods for infectious agents such as EBV. Care in the practice and interpretation of such methodologies are of course key for correct diagnosis and proving pathogenic …

Clusters of activated microglia in normal-appearing white matter show signs of innate immune activation

BackgroundIn brain tissues from multiple sclerosis (MS) patients, clusters of activated HLA-DR-expressing microglia, also referred to as preactive lesions, are located throughout the normal-appearing white matter. The aim of this study was to gain more insight into the frequency, distribution and cellular architecture of preactive lesions using a large cohort of well-characterized MS brain samples.MethodsHere, we document the frequency of preactive lesions and their association with distinct white matter lesions in a cohort of 21 MS patients. Immunohistochemistry was used to gain further insight into the cellular and molecular composition of preactive lesions.ResultsPreactive lesions were observed in a majority of MS patients (67%) irrespective of disease duration, gender or subtype of disease. Microglial clusters were predominantly observed in the vicinity of active demyelinating lesions and are not associated with T cell infiltrates, axonal alterations, activated astrocytes or blood–brain barrier disruption. Microglia in preactive lesions consistently express interleukin-10 and TNF-α, but not interleukin-4, whereas matrix metalloproteases-2 and −9 are virtually absent in microglial nodules. Interestingly, key subunits of the free-radical-generating enzyme NADPH oxidase-2 were abundantly expressed in microglial clusters.ConclusionsThe high frequency of preactive lesions suggests that it is unlikely that most of them will progress into full-blown demyelinating lesions. Preactive lesions are not associated with blood–brain barrier disruption, suggesting that an intrinsic trigger of innate immune activation, rather than extrinsic factors crossing a damaged blood–brain barrier, induces the formation of clusters of activated microglia.

αB-Crystallin Is a Target for Adaptive Immune Responses and a Trigger of Innate Responses in Preactive Multiple Sclerosis Lesions

We present the first comparative analysis of serum immunoglobulinG reactivity profiles against the full spectrum of human myelin-associated proteins in multiple sclerosis patients and healthy control subjects. In both groups, serum antibodies display a consistent and prominent reaction to &agr;B-crystallin (CRYAB) versus other myelin proteins. As an apparently major target for the adaptive immune system in humans, CRYAB selectively accumulates in oligodendrocytes, but not in astrocytes, or axons in so-called preactive multiple sclerosis lesions. These are clusters of activated HLA-DR-expressing microglia in myelinated normal-appearing white matter with no obvious leukocyte infiltration. They are found in most multiple sclerosis patients at all stages of disease. In these lesion areas, CRYAB in oligodendrocytes may come directly in contact with activated HLA-DR+ microglia. We demonstrate that CRYAB activates innate responses bymicroglia by stimulating the secretion of leukocyte-recruiting factors, including tumor necrosis factor, interleukin 17, CCL5, and CCL1, and immune-regulatory cytokines such as interleukin 10, transforming growth factor-&bgr;, and interleukin 13. Together, these data suggest that CRYAB accumulation in preactive lesions may be part of a reversible reparative local response that involves both oligodendrocytes and microglia. At the same time, however, accumulated CRYAB may represent a major target for adaptive immune responses that could contribute to progression of preactive lesions to a stage of demyelination.

Critical appraisal of animal models of multiple sclerosis

Experimental autoimmune encephalomyelitis (EAE) is a spectrum of neurological disorders in laboratory animals that is used to model multiple sclerosis (MS). However, few agents have translated from efficacy in EAE to the treatment of human disease. Although this may reflect species differences in pathological disease mechanisms, importantly it may also relate to the practice of how drugs and models are currently used. This often bears very little resemblance to the clinical scenarios where treatments are investigated, such that lack of appreciation of the biology of disease may doom drugs to failure. The use of EAE is critically appraised with the aim of provoking thought, improving laboratory practise and aiding researchers and reviewers to address quality issues when undertaking, reporting and interpreting animal studies related to MS research. This is important as many researchers using EAE could and should do more to improve the quality of the studies.

Phagocytosis of neuronal debris by microglia is associated with neuronal damage in multiple sclerosis.

Neuroaxonal degeneration is a pathological hallmark of multiple sclerosis (MS) contributing to irreversible neurological disability. Pathological mechanisms leading to axonal damage include autoimmunity to neuronal antigens. In actively demyelinating lesions, myelin is phagocytosed by microglia and blood‐borne macrophages, whereas the fate of degenerating or damaged axons is unclear. Phagocytosis is essential for clearing neuronal debris to allow repair and regeneration. However, phagocytosis may lead to antigen presentation and autoimmunity, as has been described for neuroaxonal antigens. Despite this notion, it is unknown whether phagocytosis of neuronal antigens occurs in MS. Here, we show using novel, well‐characterized antibodies to axonal antigens, that axonal damage is associated with HLA‐DR expressing microglia/macrophages engulfing axonal bulbs, indicative of axonal damage. Neuronal proteins were frequently observed inside HLA‐DR+ cells in areas of axonal damage. In vitro, phagocytosis of neurofilament light (NF‐L), present in white and gray matter, was observed in human microglia. The number of NF‐L or myelin basic protein (MBP) positive cells was quantified using the mouse macrophage cell line J774.2. Intracellular colocalization of NF‐L with the lysosomal membrane protein LAMP1 was observed using confocal microscopy confirming that NF‐L is taken up and degraded by the cell. In vivo, NF‐L and MBP was observed in cerebrospinal fluid cells from patients with MS, suggesting neuronal debris is drained by this route after axonal damage. In summary, neuroaxonal debris is engulfed, phagocytosed, and degraded by HLA‐DR+ cells. Although uptake is essential for clearing neuronal debris, phagocytic cells could also play a role in augmenting autoimmunity to neuronal antigens.

Nrf2 and DJ1 are consistently upregulated in inflammatory multiple sclerosis lesions

Oxidative stress plays a major role in multiple sclerosis (MS), a chronic inflammatory central nervous system (CNS) disease. Invading leukocytes contribute to cell damage and demyelination by producing excessive amounts of cytotoxic mediators, including reactive oxygen species (ROS). To counteract the damaging effects of ROS the CNS is endowed with a repertoire of endogenous antioxidant enzymes, which are regulated by the transcription factor NF-E2-related factor 2 (Nrf2). Upon exposure to ROS, Nrf2 translocates to the nucleus allowing transcriptional activation of various antioxidant enzymes. DJ1 is a protein that is involved in the stabilization of Nrf2 and hence acts as a positive regulator of Nrf2-driven antioxidant protection. Here, we investigate the (sub)cellular localization of Nrf2 and DJ1 in various MS lesion stages and show that Nrf2 is strikingly upregulated in active MS lesions, in both the nucleus and the cytoplasm of infiltrating macrophages and to a lesser extent in reactive astrocytes. Simultaneously, DJ1 protein expression is predominantly increased in astrocytes in both active and chronic inactive MS lesions compared to control brain tissue and normal-appearing white matter. Together, our findings suggest that persistent Nrf2-mediated transcription occurs in active MS lesions, but that this endogenous response is insufficient to prevent ROS-induced cellular damage, which is abundant in inflammatory MS lesions.

Activation Status of Human Microglia Is Dependent on Lesion Formation Stage and Remyelination in Multiple Sclerosis

Abstract Similar to macrophages, microglia adopt diverse activation states and contribute to repair and tissue damage in multiple sclerosis. Using reverse transcription–quantitative polymerase chain reaction and immunohistochemistry, we show that in vitro M1-polarized (proinflammatory) human adult microglia express the distinctive markers CD74, CD40, CD86, and CCR7, whereas M2 (anti-inflammatory) microglia express mannose receptor and the anti-inflammatory cytokine CCL22. The expression of these markers was assessed in clusters of activated microglia in normal-appearing white matter (preactive lesions) and areas of remyelination, representing reparative multiple sclerosis lesions. We show that activated microglia in preactive and remyelinating lesions express CD74, CD40, CD86, and the M2 markers CCL22 and CD209, but not mannose receptor. To examine whether this intermediate microglia profile is static or dynamic and thus susceptible to changes in the microenvironment, we polarized microglia into M1 or M2 phenotype in vitro and then subsequently treated them with the opposing polarization regimen. These studies revealed that expression of CD40, CXCL10, and mannose receptor is dynamic and that microglia, like macrophages, can switch between M1 and M2 phenotypic profiles. Taken together, our data define the differential activation states of microglia during lesion development in multiple sclerosis–affected CNS tissues and underscore the plasticity of human adult microglia in vitro.

Reduced expression of PGC-1α partly underlies mitochondrial changes and correlates with neuronal loss in multiple sclerosis cortex.

There is growing evidence that mitochondrial dysfunction and associated reactive oxygen species (ROS) formation contribute to neurodegenerative processes in multiple sclerosis (MS). Here, we investigated whether alterations in transcriptional regulators of key mitochondrial proteins underlie mitochondrial dysfunction in MS cortex and contribute to neuronal loss. Hereto, we analyzed the expression of mitochondrial transcriptional (co-)factors and proteins involved in mitochondrial redox balance regulation in normal-appearing grey matter (NAGM) samples of cingulate gyrus and/or frontal cortex from 15 MS patients and nine controls matched for age, gender and post-mortem interval. PGC-1α, a transcriptional co-activator and master regulator of mitochondrial function, was consistently and significantly decreased in pyramidal neurons in the deeper layers of MS cortex. Reduced PGC-1α levels coincided with reduced expression of oxidative phosphorylation subunits and a decrease in gene and protein expression of various mitochondrial antioxidants and uncoupling proteins (UCPs) 4 and 5. Short-hairpin RNA-mediated silencing of PGC-1α in a neuronal cell line confirmed that reduced levels of PGC-1α resulted in a decrease in transcription of OxPhos subunits, mitochondrial antioxidants and UCPs. Moreover, PGC-1α silencing resulted in a decreased mitochondrial membrane potential, increased ROS formation and enhanced susceptibility to ROS-induced cell death. Importantly, we found extensive neuronal loss in NAGM from cingulate gyrus and frontal cortex of MS patients, which significantly correlated with the extent of PGC-1α decrease. Taken together, our data indicate that reduced neuronal PGC-1α expression in MS cortex partly underlies mitochondrial dysfunction in MS grey matter and thereby contributes to neurodegeneration in MS cortex.

The Microtubule Regulator Stathmin Is an Endogenous Protein Agonist for TLR3

TLR3 recognizes dsRNAs and is considered of key importance to antiviral host-defense responses. TLR3 also triggers neuroprotective responses in astrocytes and controls the growth of axons and neuronal progenitor cells, suggesting additional roles for TLR3-mediated signaling in the CNS. This prompted us to search for alternative, CNS-borne protein agonists for TLR3. A genome-scale functional screening of a transcript library from brain tumors revealed that the microtubule regulator stathmin is an activator of TLR3-dependent signaling in astrocytes, inducing the same set of neuroprotective factors as the known TLR3 agonist polyinosinic:polycytidylic acid. This activity of stathmin crucially depends on a long, negatively charged α helix in the protein. Colocalization of stathmin with TLR3 on astrocytes, microglia, and neurons in multiple sclerosis-affected human brain indicates that as an endogenous TLR3 agonist, stathmin may fulfill previously unsuspected regulatory roles during inflammation and repair in the adult CNS.

The link between small heat shock proteins and the immune system

There is now compelling evidence that members of the family of small heat shock proteins (HSP) can be secreted by a variety of different types of cells. Secretion of small HSP may at times represent altruistic delivery of supporting and stabilizing factors from one cell to another. A probably more general effect of extracellular small HSP, however, is exerted by their ability to activate macrophages and macrophage-like cells. When doing so, small HSP induce an immune-regulatory state of activation, stimulating macrophages to suppress inflammation. For this reason, small HSP deserve consideration as broadly applicable therapeutic agents for inflammatory disorders. In one particular case, however, adaptive immune responses to the small HSP itself may subvert the protective quality of the innate immune response it triggers. This situation only applies to alpha B-crystallin, and is unique for humans as well. In this special case, local concentrations of alpha B-crystallin determine the balance between protective innate responses and destructive adaptive responses, the latter of which are held responsible for the development of multiple sclerosis lesions. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.