New developments in understanding and treating neuroinflammation
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- Infante-Duarte, C., Waiczies, S., Wuerfel, J. et al. J Mol Med (2008) 86: 975. doi:10.1007/s00109-007-0292-0
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We are currently witnesses to and authors of a paradigm shift in neuropathology. While classical acute and chronic neuroinflammatory diseases such as meningitis or multiple sclerosis (MS) present aspects of neurodegeneration, the disease course of progressive degenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), or stroke-mediated neuronal deficit are demonstrably affected by inflammation. These insights have immediate consequences both for research methods and for the development of novel, more efficient therapies for these diseases. In this review, we analyze the inflammatory and degenerative pathological mechanisms in the brain with particular emphasis on the classical chronic inflammatory disease MS. We demonstrate that the latest pathological considerations not only require the application of advanced research technologies to investigate new pathomechanistic pathways, but also affect the investigation, development, and monitoring of novel potential therapeutic tools.
KeywordsMultiple sclerosisNeuroinflammationNeurodegenerationT cell
Inflammation vs degeneration
Traditionally, disorders of the central nervous system (CNS) have been categorized as inflammatory and noninflammatory. While multiple sclerosis (MS) represented the classical example of a chronic inflammatory disease, other CNS disorders such as Parkinson’s disease (PD) and Alzheimer’s disease (AD) were conventionally considered noninflammatory neurodegenerative processes. However, this classical paradigm has been reexamined , partly due to new findings, partly due to a reinterpretation of century-old data in light of new methodologies, and also due to a revision of the notion of neuroinflammation itself. For a very long time, neuroinflammation was considered to be a process accompanied by a huge invasion of the CNS by blood leukocytes . According to this interpretation, CNS inflammation occurred exclusively in infectious processes of the CNS or under pathological conditions that promote blood–brain barrier (BBB) alterations and massive leukocyte infiltration, such as in MS. By contrast, the recent neuropathological concept has rediscovered long-standing investigations and redefines the presence of reactive microglia as a primary form of localized inflammation, which is a pathological hallmark of, among others, AD [3, 4] and PD [5–7]. Also, supporting the inflammatory aspect of both disorders, it has been suggested that certain nonsteroidal antiinflammatory drugs (NAID) reduce disease risk [8–10]. However, recent studies show either no general effects of NAID in PD or AD [11, 12] or a sex-related beneficial effect only in PD . Nevertheless, it is becoming widely acknowledged that brain inflammation (and thus glia activation) is involved in the pathology of neurodegenerative disorders and should be therapeutically targeted . Stroke is another example in which inflammation is now thought to exacerbate the tissue injury caused by the infarction. The interaction between the immune and nervous systems is, in the case of stroke, far from clear. It can involve many aspects, including protection, damage, or even neuroregeneration that make the development of therapeutic strategies particularly complex .
Similarly, the neurodegenerative aspects of acute and chronic inflammatory CNS disorders such as meningitis and MS were in part disregarded for a long time. The impact and mechanisms of neuronal damage in meningitis have been extensively investigated in recent years [16, 17]. By contrast, in MS, a disease that currently represents our primary research focus, evidence for neuronal damage was already provided more than a century ago . The redefinition of MS pathology has immediate implications: it has now become evident that reliable monitoring of the disease course and accurate treatment strategies can only be guaranteed if both inflammatory and neurodegenerative aspects are considered and targeted .
The immune system and the CNS
In the last few decades, it has become evident that the immune and nervous systems cannot be considered as two compartments, separated by a barrier, and not communicating. The interaction of immune cells and nervous cells is part of the general host defense and is essential in protecting the CNS from chronic infection and pathogen-induced damage. The normal CNS is continuously patrolled by activated, and possibly also inactivated, lymphocytes that, under “healthy” conditions, do not lead to inflammation or alter BBB integrity [20, 21]. However, when those lymphocytes, in the context of a local infection or autoimmune process , reencounter their specific antigens in the CNS, which are probably presented by perivascular antigen-presenting cells [22, 23], they may initiate a classical (auto)inflammatory response that promotes BBB disruption and the invasion of high numbers of activated leukocytes into CNS parenchyma —both key manifestations of the earlier disease stage in MS and its animal model, experimental autoimmune encephalomyelitis (EAE) . In this context, recent findings by our group and others prove T cells in the CNS to have highly dynamic properties [25, 26].
MS has so far been considered as a demyelinating disorder of the CNS that principally affects young adults and is twice or in some countries more than three times more common in females than in males . However, the etiology of MS is still unclear. Epidemiological data suggest that while genetic factors appear to determine disease susceptibility , environmental factors, such as exposure to sunlight or to certain infectious agents in early life, determine disease development [29, 30]. MS is thought to be a T cell-mediated autoimmune disease . The role of inflammatory autoreactive CD4-positive T helper (Th) cells, producing cytokines such as IFN-gamma and IL-17, has been extensively proven in models of EAE [32–35]. In patients, evidence for the involvement of T cells and proinflammatory cytokines in disease pathogenesis has come mostly from studies of lesions [36–39]. Furthermore, the “accidental” T cell response and consequent disease exacerbation observed in patients treated with an altered peptide ligand confirmed the active participation of T cells in human disease development . According to the autoimmune theory of MS, it is believed that myelin-specific T cells are responsible for the initiation, coordination, and perhaps perpetuation of the autoimmune reaction, leading to oligodendrocyte damage and demyelination . Although the concept of autoimmunity as a primary trigger in lesion formation is still a matter of discussion, it is now widely accepted that immune-mediated inflammation contributes to MS pathogenesis. This fact has recently been corroborated by the promising therapeutic effect in MS of two novel drugs: firstly, the alpha4 integrin antagonist natalizumab, which inhibits the migration of activated lymphocytes and monocytes into the CNS parenchyma [41–43]; and secondly, the sphingosine 1-phosphate (S1P) receptor modulator fingolimod, which captures lymphocytes within the lymph nodes and inhibits recirculation .
So far, it has been reported that axonal loss might be triggered by deprivation of oligodendrocyte-derived trophic factors , by nitric oxide (NO)-mediated disruption of axonal conduction , or by deficient mitochondria that are unable to deliver the increased amount of energy demanded in chronically demyelinating axons . This brings us to the question of whether axonal pathology is responsible for the loss of neurons observed in MS. It has been shown that damage of axons might promote neuronal death (retrograde degeneration). However, the fact that gray matter pathology was observed early in the disease, in the absence of white matter involvement, points to the occurrence of primary neuronal loss independently of demyelination or axonal damage as well  (Fig. 1). One of the main goals of our ongoing research is to elucidate the mechanisms involved in neuronal apoptosis. Using animal models, we recently demonstrated that CD4-positive T helper cells are able to cover long distances in the brain to interact with neurons and to induce their death . Because neurons do not express MHC class II molecules and neuronal damage has been reported to be antigen-independent [25, 64], we believe that CD4-positive T cells might damage neurons in a bystander fashion [1, 18]. In addition, our current data show that non-CNS specific CD4-positive T cells have the capacity not only to enter the CNS but also to promote alterations in the permeability of cerebral blood vessels without promoting any neuronal pathology . Based on these data, we speculate that in a compromised brain, such as in MS , perturbations of the BBB caused by nonspecific effector T cells may have further pathological consequences. Currently, we are investigating the role of those unspecific trigger mechanisms, which might support damage in the inflamed brain in vivo. Furthermore, we demonstrated that, in mouse, the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a death ligand secreted by or expressed on T cells, is a specific death-inducing effector molecule in the brain, which triggers bystander apoptosis of neurons in the inflamed CNS in vivo and promotes EAE . Apart from CD4-positive T cells, CD8-positive cytotoxic T cells potentially cause neuronal death in the CNS. In contrast to MHC class II, the expression of MHC class I seems to be upregulated on neurons under inflammatory conditions. Therefore, an antigen-dependent fatal interaction of CD8-positive cells and the target neurons is conceivable .
Independently of the killing activity of T lymphocytes, it has been suggested that activated microglia detected close to neurons may be involved in neuronal death . In this context, we have recently defined a possible effector molecule, 7-ketocholesterol (7-KC), which might provide a link between demyelination, activation of microglia, and subsequent neuronal loss. 7-KC is a breakdown product of the oxidation of myelin lipids. On the one hand, we showed that 7-KC is present in the CSF and CNS of MS patients and correlates with disability in EAE, and on the other hand, we demonstrated that 7-KC is able to activate microglia and to indirectly promote microglia-mediated neuronal death .
Future studies will elucidate which processes are fundamental to the CNS, and thus shared by the MS pathology and other diseases such as stroke and classical neurodegeneration . There is already evidence that the death ligand TRAIL , inflammatory cytokines such as interleukin-1 (IL-1) , and free radicals such as superoxide and NO  play a central role in different CNS pathologies. NO is produced in increased amounts in damaged mitochondria and, at the same time, impairs mitochondrial function . NO, detected in brains of patients with neurodegeneration and MS, has been suggested to contribute to ongoing neuronal and oligodendroglial death, as it inhibits mitochondrial respiration, probably leading to intracellular accumulation of Ca2+ .
Newly approved and experimental therapies in MS
We, like many other research groups, are focusing our efforts on establishing the optimal strategy for treating MS patients. Our current understanding of the pathogenesis outlined above in which neuroinflammation and neurodegeneration may occur in parallel and in series indicate that therapies should ideally target both processes simultaneously (Fig. 1).
Drugs currently available for the treatment of MS reduce the risk of relapse, but only slightly prolong the progression of disability. The main treatment strategies tackled so far primarily involve measures to prevent damage from occurring by local inflammation. Clinical approaches initially involved the application of drugs, which unspecifically suppress the immune system (immunosuppressants) such as the glucocorticoids and the antineoplastic agent mitoxanthrone, both of which reduce the progression of disease but are associated with harmful side effects. Eventually, drugs that modulate the immune system (immunomodulators) in a pleiotropic manner, including the interferon beta (IFN-beta) group and glatiramer acetate (GA, a short synthetic peptide mix mimicking MBP), were also shown to subdue local inflammation and reduce the relapse rate in MS. These disease-modifying agents (DMA) have been approved during the past 10 years for the treatment of MS . Other promising drugs acting similarly and, possibly, more efficiently (such as statins or flavanoids, which will be discussed further on) are still awaiting clinical trials for efficacy and safety testing. To enhance the efficacy of currently approved DMA (that have undergone thorough postmarketing surveillance), it may also be beneficial to apply add-on treatments to base DMA.
Newly approved and forthcoming therapies
Neuroinflammation in MS is dominated by the infiltration of activated autoreactive T cells into the CNS. Indeed, most of the new pharmacological agents currently being introduced as therapy options for MS, such as natalizumab and fingolimod (FTY720), specifically target T cell migration.
Fingolimod is a derivative of myriocin (a constituent of the fungus Isaria sinclairii) and mimics the activity of the phospholipid S1P . The beneficial effect of the oral administration of fingolimod to MS patients was recently shown in a phase II clinical trial . The salutary mechanisms seem to be the sequestration of newly generated autoreactive T cells in the medullary sinuses of lymph nodes  and the ligation to S1P1 on endothelial cells, which results in Rac-dependent tightening of endothelial-cell junctions . In addition, fingolimod binds with highest affinity to S1P5 receptors , which, in the CNS, are exclusively expressed on oligodendrocytes. The S1P5 receptor is involved in oligodendrocyte survival  and in the inhibition of the motility of oligodendrocyte precursors, creating a possible setback in the remyelination process .
S1P is not only involved in lymphocyte trafficking and oligodendrocyte mobilization but also in other physiological events, such as vascular tone, heart rate regulation, and contraction of airway smooth muscle cells . Thus, apart from the observed lymphopenia in patients on fingolimod , a higher incidence of adverse events in patients receiving the agonist is to be expected . Notwithstanding these uncertainties, the results of the preliminary study by Kappos et al. provide us with a strong incentive for long-term follow-up trials on a large scale. Indeed, this treatment strategy is currently being checked in a multicentered placebo-controlled phase III clinical trial in relapsing–remitting MS.
Natalizumab, a humanized recombinant monoclonal antibody (mAb) that binds to the alpha4 chain of the alpha4beta1(VLA-4) and alpha4beta7 integrins, is a selective adhesion molecule inhibitor that has already shown impressive results in RRMS patients by dramatically reducing infiltration lymphocytes in the CSF [41, 43, 81]. Recently, natalizumab has also been shown to robustly prevent new lesion formation as shown by MRI analysis . Apart from natalizumab, various new mAb therapies have been developed to deplete harmful immune cell populations in the periphery. These therapies include alemtuzumab, targeting CD52, which is found on mononuclear cells, and rituximab, targeting CD20 and thereby specifically depleting premature and mature B cells. Alemtuzumab (Campath-1H) was originally tested in SPMS in a phase I clinical trial, and although results were initially encouraging [83, 84], in the long-term, only a minority of patients benefited. Alemtuzumab did, however, markedly reduce the relapse rate and EDSS score over a period of 2 years in RRMS patients  and a phase II study in RRMS showed dose-dependent reduction in the risk for relapse and for progression of disability (reduction of 87% and 66%, respectively, at the highest dose) compared with the control group treated with Rebif (data presented by Coles et al. at the 59th Annual Meeting of the American Academy of Neurology, 2007, Abstract S12.004). Similarly, preliminary results from an ongoing phase II/III clinical trial in PPMS with rituximab only revealed a temporary suppression of B cell activation . Rituximab was, however, shown to be beneficial in neuromyelitis optica , which in view of recent data on the occurrence of aquaporin-4 antibodies, can be considered a distinct disease entity . As yet unpublished results from a phase II trial with RRMS patients indicate that rituximab reduces MRI lesion load by 90% and relapse rate by 58% (data presented by Hauser et al. at the 59th Annual Meeting of the American Academy of Neurology, 2007, Abstract S12.003).
Overall, selective therapies including the mAb therapies represent a new stage in MS therapy. However, even these promising therapies may have their setbacks. For instance, the rare appearance of progressive multifocal leukoencephalopathy (PML) after natalizumab  and Graves’ disease or idiopathic thrombocytopenic purpura after alemtuzumab  cautions us to apply each therapeutic agent, especially such selective agents with extreme care and consideration to each individual patient . One further difficulty with drugs that selectively target one molecule is a situation of redundancy where the organism will find a means of compensating for deleted targeted protein or cell. This might be one reason why the CCR1 blockade did not show any significant beneficial effect in a phase II clinical trial in patients with RRMS , although on the basis of data obtained in animal models [93–95], this therapy had been considered very promising.
Therefore, there is an emerging school of thought which argues that complex and heterogeneous diseases such as MS would better targeted with “dirty” promiscuous drugs rather than selective therapies .This leaves us with the still open question as to whether we should further characterize the partially effective pleiotropic drugs, identifying their “pitfalls” in the process, or whether we should concentrate on identifying the many targets significant in the pathology of complex diseases such as MS to design new “multiple ligands.”
Apart from the development of selective therapies that target one or more molecules, another therapeutic goal in patient health care is to identify therapeutic agents that may be administered via a noninvasive, i.e., oral, route to enhance patient compliance. Examples range from pilot trials to phase III studies.
Flavonoids: antiinflammatory and neuroprotective
Flavonoids, including EGCG (epigallocatechin-3-gallate), are polyphenolic plant metabolites that have been shown to have versatile biological activities, including antimicrobial  and antitumor properties [98–100]. While it is clear that they promote apoptosis and cell cycle arrest in transformed cells [101–104], polyphenols such as EGCG have also been shown to protect nontransformed cells from apoptosis [105, 106]. Indeed, this substance, which exhibits several mechanisms of action, has been shown to have protective effects in autoimmune animal models. It reduced disease severity in collagen-induced arthritis by significantly lowering the levels of inflammatory cytokines and mediators . We reported that EGCG is capable of targeting both inflammation and direct neuronal injury in EAE: the antioxidative properties of EGCG directly protected living brain tissue against neuronal injury by N-methyl-d-aspartate (NMDA) or the death ligand TRAIL, and blocked the formation of neurotoxic reactive oxygen species in neurons . Neuroprotective effects by polyphenols have also been reported in neurodegenerative diseases such as PD [109, 110] and AD  or transient global ischemia . Our recent findings , and work by Hendriks et al. , suggest that flavonoids may be therapeutic candidates for the treatment of MS due to their versatility in combining antiinflammatory and neuroprotective properties. Phase II trials, including one conducted by us, are scheduled to begin shortly.
HMG CoA reductase inhibitors: beyond lipid lowering
The β-3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors (HMGCRI), known as statins, have been used for several years as the gold standard for lipid-lowering therapy. These drugs were initially designed to lower cholesterol levels by specifically inhibiting the catalytic conversion of HMG-CoA to l-mevalonate, which is a precursor to cholesterol and also to intermediary lipid donors such as farnesyl pyrophosphate (Fpp) or geranylgeranyl pyrophosphate (GGpp). These isoprenoids are important for the posttranslational modification or isoprenylation of GTP-binding proteins of the Ras superfamily . These include members such as the prototype Ras and Rap and also the Ras homologue (Rho) family members, including Rho, Rac, and Cdc42, all of which are important for signaling in inflammatory processes. Indeed, the outcome of manipulating cholesterol and isoprenoid synthesis with HMGCRI, and thereby intracellular signaling, has been studied in brain pathology, including neuroinflammation . Originally, various groups, including ours, showed that the HMGCRI atorvastatin is therapeutically effective in EAE [116, 117], the salutary effects probably being due either to regulatory mechanisms preventing the expansion of harmful autoreactive cells already outside the CNS compartment  or to the blocking of leukocytes from leaving the BBB vasculature into the CNS [116, 119]. Evidence to support the latter scenario is that HMGCRI reduce levels of chemokine receptors, T cell adhesion molecules, and matrix metalloproteinase (MMP)-9 by peripheral immune cells derived from MS patients  and inhibit the migration of these cells through BBB-derived endothelial cells . Furthermore, for statins, which are bioavailable in the brain, the antioxidative properties of these drugs  might hold the key to preventing cholesterol oxidation and formation of harmful oxysterol that induces microglia-mediated neuronal damage , thus providing further rationale for the attenuating neuroinflammatory processes in the EAE. The beneficial effect of HMGCRI in the EAE, which readily diffuse into the CNS compartment, has also been linked to the enhanced survival of oligodendrocyte progenitor cells and their differentiation to remyelinating oligodendrocytes in vivo . Thus, HMGCRI represent another versatile tool, which might target both neuroinflammation and direct neuronal damage simultaneously. This has already led to positive outcomes in MS treatment in two small open-label trials, which were not free of criticism due to their size and design [124, 125]. These promising data are currently undergoing evaluation in larger-scale controlled clinical trials, including our own.
New techniques to monitor neuroinflammation
Our understanding of the interactions between immune cells and cells of the CNS has been considerably improved in the last few years. However, the data obtained so far have resulted from in vitro models or from the ex vivo evaluation of CNS tissue, which can lead to more conflict and controversy than clarity regarding the in vivo situation. Nevertheless, the new era of methodological approaches to molecular imaging has the potential to elucidate the dynamics of the pathological processes involved. Molecular imaging is a noninvasive methodology that combines molecular and cell biology tools for the in vivo imaging of biological processes such as location and degree of gene and protein expression, signal transduction pathways, cell migration patterns, and cellular interactions at the site of pathology in the CNS. Molecular imaging technologies, such as MRI, positron emission tomography (PET), multiphoton microscopy (MPM), bioluminescence, or fluorescence, are applied broadly across the life sciences, from stem cell transplantation research, through cancer investigation, to the treatment of CNS diseases. As far as we are concerned, the necessity to develop these techniques is of utmost importance because the brain is not as accessible as other living organs.
Multiphoton microscopy is a relatively novel imaging technique. It is based on the probability that two or more low-energy photons are absorbed quasisimultaneously by a fluorophore, exciting it to the same level as one high energy photon. Multiphoton excitation was first theoretically predicted by the German physicist Maria Göppert-Mayer more than 75 years ago , but its application in microscopy in the biological sciences would not come until much later, and was first described by Denk et al. in 1990 . The principal advantages of using two or more photons vs one are, on the one hand, the reduced phototoxicity caused in the specimen by low-energy wavelengths (especially important in living tissue) and, on the other hand, the fact that low energy penetrates deeper into the tissue. In addition, multiphoton excitation occurs only at the focal plane and does not affect the surrounding tissue, instead limiting photobleaching and damage to the imaged area . As mentioned above, we have used MPM to investigate the activity of T cells in living brain tissue  and to determine the behavior of nonneural specific T cells within the CNS in vivo . Instances of the use of live imaging in neurology so far include the investigation of neuronal activity , even in freely moving animals , and of microglia function  and the monitoring of T cell behaviors in neuroinflammation . Intravital MPM can be expected to provide an answer to our many questions regarding the role of different T cell types during CNS inflammation, the mechanisms driving T cell trafficking within the CNS, and the ways in which T cells interact with CNS cells.
Another approach, which may be transferred to the clinic more rapidly, is MR cellular and molecular imaging. MRI is a technique providing high spatial, temporal, and spectral information on anatomy, metabolism, and tissue function in vivo. Due to its noninvasive nature and the fact that it is virtually free of side effects, MRI has already become one of the most important and most frequently used imaging tools in medicine. Today, high magnetic field strengths and dedicated rodent scanners allow for sufficient spatial resolution in vivo, even in murine animal models, such as EAE mice. Novel MRI diffusion imaging techniques aim at differentiating between axonal damage and neurodegeneration . Recent advances in MR sequences have been applied to enhance the image resolution or signal to noise ratio . Using new contrast agents, we recently showed very early BBB breakdown preceding the clinical onset of disease in EAE, thereby mimicking multiple sclerosis . One of these agents, Gadofluorine M, was also successfully used to detect peripheral nerve degeneration . Another class of contrast agents, superparamagnetic iron oxide particles, is now widely employed in labeling phagocytic cells in MRI . A novel very small iron oxide particle (VSOP) currently under our investigation is being used to label various cell types, such as macrophages or T cells ex vivo, which are subsequently transferred to an animal and can be traced longitudinally in vivo. It is interesting to note that VSOP also binds specifically to proteoglycane structures, which appear to be extensively deposited in active inflammatory lesions, providing a unique tool for the molecular targeting of extracellular matrix alterations and basal membrane damage. By applying MR spectroscopy, magnetization transfer imaging or diffusion tensor techniques, even occult tissue damage and reduced nerve fiber integrity can be quantified in vivo [133, 137]. In the future, novel MR contrast techniques applying hyperpolarized nuclei such as C-13 will be able to provide metabolic information regarding, e.g., glykogen or pyrovate metabolism . The latest approach to developing multimodal imaging devices, such as integrated MRI–PET scanners or combined immunofluorescence and MR contrast markers, will help us to bridge the gap between the macroscopic, microscopic, and molecular levels, thus completing our pathophysiological understanding by supplying essential information, which has so far eluded us.
The Cecilie Vogt Clinic is a center for translational neurology. One of its focuses is the investigation of inflammatory disorders of the brain such as MS. This disease is a major cause of severe disability in young adults in the Western world, and consequently has a serious socioeconomic impact. Our clinic aims not only to clarify disease pathophysiology using state-of-the-art technology, but also to apply this knowledge to the development of effective, safer, more efficient therapies, and reliable markers for diagnosis, prognosis, and therapy response. We take basic findings from the bench and apply them directly to the patient’s care. This allows for an optimal coordination of theoretical and clinical knowledge, leading to new concepts in neurological treatment and providing the opportunity for patients to take part in clinical studies with novel therapeutic tactics.
We gratefully acknowledge the funding from the Institut für MS-Forschung (IFMS) Göttingen/Gemeinnützige Hertie-Stiftung (GHA), the Deutsche Forschungsgemeinschaft (DFG), the Bundesministerium für Bildung und Forschung (BMBF), and the Charité–Universitätsmedizin Berlin. We thank Andrew Mason for reading the manuscript as a native English-speaker, and Volker Siffrin and Ulf Schulze-Topphoff for the helpful discussions.