VIEWING INTO THE CNS INFLAMMATION

Abstract


Experimental Autoimmune Encephalomyelitis (EAE) has been used for human autoimmune disease, Multiple Sclerosis (MS), which is characterized by inflammation in the Central Nerve System (CNS). In this review, we focus one of EAE variants, transfer EAE induced by adoptive transfer of encephalitogenic autoantigen specific T-cell clone. We describe about the model and introduce the application of this model for experimental research and therapeutic development. In addition, we introduce two-photon intravital imaging, which enable to observe and track fluorescently labeled encephalitogenic T-cells in living animal in real time. Further, by using activation sensors, which detect cellular activation during intravital imaging, we show migrating T-cells become activate upon contacting with local antigen presenting cells. The obtained results reveal that the transfer EAE model is useful to understand the mechanisms of initiating CNS inflammation.

Full Text

Introduction. Almost 150 years have passed since Charcot described the pathogenic lesions of MS as cir-cumscript plaques distributed through the brain and spinal cord white matter, a picture which led him to name the disease scléroseen plaques. Subsequent microscopic studies established the key changes dominating the pathological plaque tissue. These included degeneration of myelin and axons, as noted by Charcot himself, and inflammatory round cell infiltrations often around small blood vessels described about the same time by Rindfleisch (quoted from [1]). It took until recently to gain insight into the connection between neurodegeneration and inflammation. We now understand that most of the inflammatory cells are CD8+ T-cells along with CD4+ T-cells, macrophages and some sprinkling of B-cells. Importantly, analyses of CD8+ T-cell receptors hint to active proliferative response driven by some stimulus. Current hypothesis: T-cells attack brain autoantigens, kindle a cascade of events that ultimately culminates in the pathognomonic myelin and axon destruction. While this pathogenesis appears conclusive, it violates one basic tenet of brain physiology, namely the immune privileged status of the CNS tissues. It is undisputed that the CNS parenchyma is secluded from the peripheral blood circulation by a tight endothelial 50 МЕДИЦИНСКИЙ АКАДЕМИЧЕСКИЙ ЖУРНАЛ, 2014 г., ТОМ 14, № 4 Blood-Brain Barrier (BBB). How could the inflammatory cells be able to cross this forbidding barricade? Studies of suitable animal models have revealed the complex mechanisms involved in the passage of inflammatory cell into the CNS. In the following we shall display our current understanding of these events and describe the technologies that have been used in our experimentation. T-cell transferred Experimental Autoimmune Encephalomyelitis and some applications. The term of Experimental Autoimmune Encephalomyelitis (EAE) denominates a set of animal models featuring brain autoimmune inflammation mediated by activated autoreactive T-lymphocytes. The variety of EAE models is determined by different way of induction in different animals [2]. Although none of EAE model perfectly matches MS pathology, at least, each model recapitulates particular aspects of human disease. Therefore, depending on the purpose, the best suited model version must be selected. In our studies, we used mainly transfer EAE in the Lewis rats to study the function of autoreactive T-cells in early stages of CNS inflammation. This model was developed by Ben-Nun, Wekerle and Cohen who described isolation encephalitogenic T-cells from healthy, antigen primed animals, expansion of these cells in vitro as pure cell lines, and, revealing the encephalitogenicity of cells after adoptive transfer of cells [3]. Transfer-EAE was decisively refined by Flügel et al. by labeling encephalito-genic T-cells with the genetic marker Green Fluorescent Protein (GFP) [4]. Indeed, previously encephalitogenic T-cells were labeled with small molecular dyes. However, since encephalitogenic T-cells were vividly dividing, it was not possible to achieve stable labeling for more than some days. In rat transfer EAE system, T-cells need 3-4 days until they penetrate into the CNS and induce inflammation, the T-cells cannot be analyzed by ex vivo study. Flügel et al. overcame this by introducing GFP by retroviral gene transfer. Such engineered T-cells express GFP stably and can be analyzed at single cell level for their activation status and expression of cell surface molecules [5]. The model showed excellent reproducibility on the T cell infiltration kinetic into the CNS and following time kinetic of disease course. Therefore, this is the optimal model to study T cell infiltration. The transfer EAE models were used to evaluate the pathogenic potential of human autoantibodies. For example, Mathey et al. evaluated the effect of autoantibody which mimics autoantibody in the patients of MS [6]. They transferred anti-Myelin Oligodendrocyte Glycoprotein (MOG) specific T-cells to open the Blood-Brain Barrier as prerequisite of CNS inflammation and EAE induction. When animals showed the first sign of clinical, autoantibody was injected, resulting exacerbated clinical severity. A similar system was used by Bradl et al to evaluate of anti-aquaporin-4 antibodies purified from patients of neuromyelitis optica [7]. The injected antibodies exacerbated clinical symptoms. These results proved the usefulnessof the EAE model to evaluate the effect of pathogenic autoantibodies. The model was further used to validate the therapeutic effect of candidate drugs.The most spectacular success of transfer-EAE based studies was the discovery of Natalizumab, an anti-integrin α4 blocking antibody, which was humanized and approved for MS therapy. Administration of anti-integrin α4 antibody suppressed the development of clinical EAE effectively. As mentioned later, intravital imaging showed that this is due to inhibition of autoreactive T-cell infiltration into the CNS [8]. Another case is immunospecific treatment via injection of soluble autoantigens. At the different time point after adoptive transfer of GFP labeled Myelin Basic Protein (MBP) specific T-cells (TmbP-GFP cells), soluble MBP was infused intravenously. When MBP was given before onset of EAE, clinical symptoms were almost completely diminished [9], whereas the treatment after onset exacerbated the clinical severity [10]. Analyses of ex vivo isolated TMBP-GFP cells showed that soluble antigen treatment before onset trapped T-cells in spleen and prevent their penetration into the CNS, resulting prevention of EAE. On the other hand, treatment after onset of EAE also stopped and activated T-cells, this time not in the spleen but in the CNS. As result of activation, TmbP-GFP cells produced massive amount of inflammatory cytokines, which exacerbated the clinical severity. This emphasizes the importance of the timing for soluble antigen treatment and the of T-cell activation in the CNS. The encephalitogenicity of autoantigen specific T-cells critically depends on their antigen specificity and their genetic background. For example, MBP specific T-cells in Lewis rat as well as MOG specific T-cells in DA rat are highly encephalitogenic. However, MOG specific T-cells in Lewis rats are weakly encephalitoge-nic. By comparing these cell lines by ex vivo study, we found that the T-cell activation in the CNS is crucial to induce inflammation [11]. Indeed, when calcium signaling in the T-cells, which is important intracellular signaling for T-cell activation, were blocked pharmacologically, EAE clinical severity was ameliorated [12]. Finally transfer EAE allowed important insights into the clinical changes surrounding CNS inflammation. The adoptive transfer of TMBP-GFP cells induces massive acute inflammation in the CNS, but different brain regions are affected with different intensity. By using this system, Perekrest et al. examined the function of orexin- МЕДИЦИНСКИЙ АКАДЕМИЧЕСКИЙ ЖУРНАЛ, 2014 г., ТОМ 14, № 4 51 producing neurons (Perekrest et al., in press). We found that during the acute phase of EAE the number of orexin producing neuron decreased, whereas the orexin mRNA increased, indicating that CNS inflammation definitely influence the orexin production and usage. Since orexin is known to influence sleeping disorder and fatigue, this may explain some of clinical problems observed in MS patients. Intravital imaging of T cell infiltration into the CNS. In transfer EAE model, disease is induced exclusively by encephalitogenic T-cells. This raises the question of how these autoreactive T-cells infiltrate the CNS and induce inflammation. To this end, we performed intravital imaging of T-cells in the living animal at the single cell level. Genetically labelled TmbP-GFP cells were imaged by two-photon microscopy within the living animals during pre-clinical and acute phase of EAE [8] (Figure). penetrate deeper and induce less phototoxicity. In our experimental set-up, the penetration depth reaches 200 μm from the surface, which covers leptomeninges and upper part of white matter parenchyma of rat spinal cord. The penetration depth depends on intensity of labeling, giving laser power, and scattering of tissue. In the best case, penetration depth can reach more than 1 mm. These futures makes two-photon microscope as optimal technique for intravital imaging. In our intravital imaging studies, we observed TmBP-GFP cells appearing in spinal cord leptomeninge-al blood vessels as early as day 1 after transfer, which is far before onset of clinical signs. The number of TmbP-GFP cells increased within next 24 hours, but the vast majority of T MBP-GFP cells remained within the vessels. Three-dimensional reconstitution revealed that those cells adhered and migrated on the intraluminal surface of leptomeningeal vessels with an average speed of Figure. Panoramic picture of spinal cord leptomeninges after adoptive transfer of TMBP-GFP cells at the preclinical phase. Figure legend: majority of TMBP-GFP cells (green) locate within the blood vessels (red). Collagen fibers (blue). To perform stable and long-term imaging, the animal were intubated and anesthetized by isoflurane [13]. O2, CO2 and isoflurane concentration in the inspiratory and expiratory gas as well as airway pressure were monitored and recorded during entire imaging session. In addition, body temperature was kept between 35-37 C degrees by using heating blanket combined with monitoring system. Furthermore, electrocardiography was recorded and tail vein was catheterized for injection. For the T cell imaging at the spinal cord, laminectomy was performed at the upper part of lumber spinal cord after fixed animal in the custom made stage. This set-up allows stable imaging for many hours. For acquiring the image, we made use of two-photon microscope, which has been reviewеd in detail previously [14, 15]. Briefly, two-photon microscopy uses two-fold longer wavelength to excite fluorochrome than conventional microscopy. The longer waved excitation light can 13 μm/min.Interestingly, the TmbP-GFP cells preferentially migrated againstthe blood stream, a behavior that is still without explanation. We examined the molecular mechanisms behind the intraluminal crawling by infusion of blocking antibodies during intravital imaging. This approach allowed comparing T cell motility before and after treatment in same condition. Among a panel of antibodies tested, an anti-integrin a4 antibody showed the most remarkable effect. Within a few minutes after infusion, the antibody diminished intraluminal crawling completely. The TMBP-GFP cells could not adhere on the intraluminal surface of endothelial cells and washed out by blood stream. In contrast of intraluminal cells, extravasated TMBP-GFP cells were not affected. A similar antibody, Natalizumab, had been humanized and approved for MS treatments asTysabriR [16]. Importantly Natalizumab showed beneficial effects on MS patients, presumably, by bloc- 52 МЕДИЦИНСКИЙ АКАДЕМИЧЕСКИЙ ЖУРНАЛ, 2014 г., ТОМ 14, № 4 king intraluminal crawling and following extravasation as shown in animal model. Of note, an anti-LFA-1 antibody (integrin aL) which alone did not showed strong effect, enhances the effect of anti-integrin a4. As a next step, crawling TmBP-GFP cells cross the BBB. The BBB is composed of three main layers, a tight endothelial tube sealed by tight junctions, an endothelial basement and a parenchymal membrane. This barrier safeguards the special properties of the CNS tissues as an immune privileged microenvironment. According to intravital imaging date, TMBP-GFP cells pass through BBB within 10-20 minutes. TmbP-GFP cells adhere at one place, changing their shape, and extravasated. At the same time, there is frequently leakage of blood plasma, as indicated by the leakage of fluorescent dextran, a marker for visualization of blood vessels. Today it is still an open question, whether T-cells extravasate from specific points or the molecular mechanism for extravasation. Once extravasated, the T-cells continue crawling randomly on outer surface of the blood vessels, until they interact with local antigen presenting phagocytes, APCs. In our experimental set-up, APCs were labeled with phagocytosis of fluorescent dextran conjugates. Ex-vivo analyses showed that they express MHC class II as a prerequisite to present antigen to CD4 T-cells. Additionally, APC expressed macrophage-like cell surface marker, which is different from a previous study which showed dendritic markers [17]. The interactions between infiltrated TmbP-GFP cells and APC were dynamic and duration can reach more than 60 minutes. Since intravital imaging is limited to a maximal depth of 200 μm, imaging of the deeper spinal cord parenchyma was performed using acute slice explants [18].Two-photon imaging detected two different T-cellular motility modes in the CNS parenchyma. We distinguished on the one hand highly motile cells, which moved spontaneously through the tissue, and on the other hand there were stationary cells which were fixed at a particular point swaying theirbodies. We concluded that the motile T-cells are looking for their specific antigens, whereas the stationary cells are committed in antigens recognition. The reasons were as follows: 1) TowGFP cells, which do not find antigen in the native CNS showed extensively the motile mode. 2) Blocking of MHC class II shifted the proportion of stationary cells towards the motile cells. 3) In contrast, addition of specific antigen increased stationary cells and decreased motile cells. We detected T-cell arrest both in the spinal cord parenchyma [18] as well as in the leptomeninges [8], which presumably represent presentation/recognition of endogenous antigen by APCs in both compartments. As described above, we also found that the autoimmune T-cells were re-activated in the CNS, which is an essential prerequisite for CNS inflammation [11]. However, for technical limitations, it was not possible to directly connect between T-cell arrest/interaction with APC and their activation. Therefore, we proceed to establish intravital imaging of T-cell activation. Visualizing T cell activation in vivo. To detect T-cell activation at real-time, we used two distinct intracellular signaling events within T-cells. One is the elevation of intracellular calcium concentration which is contributed firstly by release of calcium stored in endoplasmic reticulum and secondly by the influx of extracellular calcium after opening calcium channel on cell surface [19]. The second event used is the translocation of Nuclear Factor of Activated T-cells (NFAT). The elevated intracellular calcium activates calcineurin pathway and dephosphorylated NFAT, which induce quick relocation of NFAT from cytosol to nucleus. Conventional studies used small synthetic calcium sensing molecules, such as Indo and Fura to monitor intracellular calcium levels. These were of little use here, as T-cells actively pump out these dyes. In addition, T-cells, especially the freshly activated effector T-cells used for transfer-EAE, proliferate vividly. As a result, calcium sensing dyes becomes invisible within a few hours. Therefore, to detect the change of intracellular Ca2+, we used a Fluorescent Resonance Energy Transfer (FRET)-based calcium sensing protein. The protein consists of a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) connected by the calcium sensing domain derived from troponin C (20). However, although the initial indicator protein readily detected changes of intracellular Ca in neuronal cells, it was not expressed in T-cells neither in transgenic mice, nor after retroviral transduction [21]. After a systematic search of this failure, we found that high homology between CFP and YFP sequence as well as tandem repeat sequence in calcium sensing domain cause homologous recombination during retrovirus production. Therefore, to reduce the homology, we performed codon diversification, by changing nucleotides, but keeping amino acids sequence unchanged. The codon diversified protein, named Twitch1, was successfully expressed in mouse T-cells by retroviral gene transfer and T-cells were stably express Twitch1 for longer time. Twitch 1 expressing MOG specific T-cells (TMOG-Twitch1 cells) or OVA specific T-cells (TOVA-Twitch1 cells) were adoptively transferred in recipient animals. At first, we performed intravital imaging within the peripheral lymph node to test the functionality of these T-cells [21]. Both TMOG-Twitch1 cells and TOVA-Twitch1 cells moved within the popliteal lymph node and occasionally showed short-lasting calcium spikes, which were caused by antigen independent stimula- МЕДИЦИНСКИЙ АКАДЕМИЧЕСКИЙ ЖУРНАЛ, 2014 г., ТОМ 14, № 4 53 tion. Intravenous application of soluble antigen, which provides massive antigen dependent stimulation, changed the situation dramatically. Now the T-cells were arrested and showed saturated high intercellular calcium levels. These results indicated that the Twitch sensor properly detected T-cell activation in vivo. As a next step, we followed T-cell activation following more physiological stimulation. Therefore, after adoptive transfer of TMOG-Twitch1 cells or TOVA-Twitch1 cells together with EAE induction by active immunization of MOG emulsified in CFA was imaged. Intravital imaging at the spinal cord leptomeninges was performed at the peak of disease when T-cells infiltrated into the CNS. Tow Twitch1 cells in the CNS showed occasionally short-lasting calcium spikes which were also observed in peripheral lymph nodes, and presumably were antigen independent. In contrast, TMOG-Twitch1 cells showed more frequent and longer-lasting calcium spikes as well as reduced T-cell motility. Also such spikes were often observed when T-cells interacted with local APCs. Similar as observed in peripheral lymph node, the application of soluble antigen further enhanced T-cell arrest and saturated calcium signaling in T-cells, indicating that local APC are not saturated by endogenous antigen in line with our previous observation [8]. As another, complementary sensor, we chose NFAT. This transcription factor was truncated to minimize artefacts potentially caused by overexpression of exogenous protein and fused with GFP for visualization (NFAT-GFP) [22]. Retroviral transduction of this gene construct results in successfully expression of NFAT-GFP in the T-cells without interfering with the T-cells’ function and encephalitogenic potential. We compared NFAT expressing highly encephalitogenic MBP specific T-cells (TmBP-NFAT-GFP cells) and weakly encephalitogenic MOG specific T-cells (TmoG-NFAT-GFP cells) in the Lewis rats. Intravital imaging showed that, regardless of T-cell antigen specificity, T-cells during intraluminal crawling as well as rolling maintained NFAT in their cytosol, indicating that these T-cells were not activated within the vessels. In contrast to intraluminal T-cells, in a substantial part of extravasated TmbP-NFAT-GFP cells NFAT was translocated to the nucleus, indicating an activated status. More directly, intravital imaging at the spinal cord leptomeninges showed NFAT translocation from cytosol to nucleus upon interaction of the T-cells with local APC. The freshly activated T-cells often continue its interaction for a while before they dissociate from the APC to continue migration. We commonly observed that certain APCs activated more than one T-cell either simultaneously or sequentially, while other APCs fail to activate T-cells. These results confirm that some special APC present endogenous antigen to infiltrated autoreactive T-cells. NFAT translocation was related to the pathogenic activity of autoimmune T-cells. Weakly encephalitogenic TmOG-NFAT-GFP cells rarely showed nuclear localized NFAT-GFP after extravasation. This is in line with our previous observation that MOG specific T-cells were not activated within the CNS [11]. As result of failed activation, the MOG specific T-cells do not induce severe clinical EAE. Similar application of NFAT as activation sensors were performed by Lodygin et al. which also detect activation of highly encephalitogenic T-cells within the spinal cord leptomeninges by using intravital imaging [23]. By using two distinct activation sensors, we directly identified T-cell activation in the spinal cord as critical event for triggering CNS inflammation. The two sensors used were not redundant but rather were complementary indicators. Calcium elevation describesvery early events after TCR mediated stimulation and very sensitive to detect weak and short lasting stimulation, whereas NFAT translocation integrates these early signals that ultimately lead to full cell activation. The combination of two sensors deepens the understanding of activation. For example, it will be useful to study whether T-cell can accumulate weak stimulation to get final activation. Importantly, both sensors can be applied other types of cells, which will certainly help to study and visualize entire immune cell networks. Conclusion. Brain autoimmunity starts out in the peripheral immune system, where self-reactive T-cells, which are regular components present in the healthy immune repertoire, are activated to expose their autoimmune potential. Migrating through peripheral immune compartments, these activated T-cells undergo changes [5] that render them competent for entry into the CNS tissues shielded by a complex microvascular blood-brain barrier. This passage is a complicated step-by step process [13]. Circulating autoimmune T-cells first attach to the endothelium of CNS blood vessels and roll along this surface driven by blood stream. Then, with their attachment becoming firmer, the T-cells start to crawl, now often against the blood stream, until they find or create a gap through the endothelial sheath. Beyond the vessel, the T-cells establish contacts with local antigen presenting phagocytes, which guide them towards the CNS parenchyma and instruct them to invade the tissue. T-cell interactions with antigen presenting phagocytes cause sustained calcium signals resulting in full activation detected by translocation of NFAT. These observations were made with a combination of advanced imaging technologies in living animals and the use of newly developed functional genetic fluorochrome makers allowing the staging at real-time of cell activation processes. Funding. This project was supported by DFG research grant (NK) and DFG Heisenberg fellowship 54 МЕДИЦИНСКИЙ АКАДЕМИЧЕСКИЙ ЖУРНАЛ, 2014 г., ТОМ 14, № 4 (NK), DFG Transregio CRC/TR128 (HW), DFG collaborative research center CRC571 (project C6 for NK, B6 for HW), SyNergyprogram (HW), KKNMS (NK), Novartis Foundation for Therapeutic Research (NK), Hertie Foundation (HW), Else Kröner-Fresenius Foundation (NK), LudwigMaximilian University Munich and Max-Planck Society.

About the authors

Naoto Kawakami

Ludwig-Maximilians University; Max-Planck Institute of Neurobiology

Email: Naoto.kawakami@med.uni-muenchen.de
Institute of Clinical Neuroimmunology

Hartmut Wekerle

Max-Planck Institute of Neurobiology

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