Immune modulation in MS models by peptidoglycan, CD97 and CD44

Multiple sclerosis (MS) is a chronic in.ammatory disease of the central nervous system (CNS). We are interested in the mechanisms involved in the immunopathogenesis of MS, especially in microbial signals able to break T cell tolerance, and in the function of molecules on the activated cells in costimulation, migration, adhesion and apoptosis. Peptidoglycan (PG) is a major cell wall component of Gram-positive bacteria, that activates the innate immune system by binding to TLR2/6, Nod1 (Card4) and Nod2 (Card 15) receptors. The enzymes lysozyme and NAMLAA are produced by phagocytic cells and can degrade PG. However, after bacterial phagocytosis, PG can persist intracellularly when the digestion is incomplete. During chronic in.ammation, PG-containing antigen presenting cells (APC) accumulate at in.ammatory sites, as previously demonstrated in patients with colitis, arthritis and MS. At these in.ammatory sites or in peripheral lymphoid tissues PG may serve as a costimulatory factor in autoimmune disease by overriding tolerance against self-antigens. To obtain more insight into the functional relevance of PG in MS, we compared the presence of PG and its degrading enzymes in the CNS of two distinct non­human primate EAE models (chapter 2). As in brain tissue of MS patients we here demonstrate that EAE-affected brain tissue from marmoset and rhesus monkeys contained elevated numbers of cells with PG, compared to control immunized animals. Interestingly, chronic EAE in marmoset monkeys was accompanied by a modest number of PG-containing cells in the brain, whereas brain tissue from rhesus monkeys that had developed acute EAE contained abundant numbers of PG-containing cells. Lysozyme was only sporadically expressed in EAE-affected brain tissue from both monkey species. In contrast, NAMLAA was expressed on many perivascular cells in EAE-affected brain tissue from rhesus monkeys, but only by few perivascular cells in marmoset monkeys. Double-labeling revealed that NAMLAA was mostly expressed by neutrophils. Thus, in EAE-affected brain tissue, PG was present within signi.cantly higher numbers of APC compared to controls. Conversely, lysozyme was mainly absent in normal and in.amed brain tissue, and NAMLAA was only abundantly expressed during acute CNS in.ammation in rhesus monkeys. These data suggest that PG persists inside APC in the CNS and may contribute to the in.ammation by stimulating TLR/Nod receptors. To further elucidate the functional contribution of PG to autoimmune disease we investigated the hypothesis that PG acts as a costimulatory factor for disease development in MS, by using mouse EAE (chapter 3). EAE is normally induced by s.c. immunization of autoantigens admixed in a strong adjuvant with attenuated Mycobacterium tuberculosis bacteria. In.ammatory Staphylococcus aureus PG could replace whole M. tuberculosis in EAE induction. We demonstrate that S. aureus PG was transported to the spleen and draining lymph nodes after immunization with S. aureus PG-containing adjuvant. In the draining lymph nodes S. aureus PG was localized within dendritic cell (DC) clusters. Clusters of PG-containing cells were also present in the spleen of rhesus monkeys that developed EAE (chapter 2). Compared to control mice that were immunized with autoantigen in incomplete Freund’s adjuvant (IFA), PG increased autoantigen­speci.c T cell proliferation and Th1 cell development, when the autoantigen-IFA mix was supplemented with PG. By using bone marrow-derived DC, S. aureus PG was shown to stimulate antigen uptake by DC, DC maturation, and subsequent differentiation and proliferation of Th1 cells. Taken together, proin.ammatory PG may stimulate autoimmune-mediated processes in MS, either in the CNS or in the periphery, by accelerating (auto)antigen uptake, initiating DC maturation, and polarizing and expanding (auto)antigen-speci.c T cells. CD97 and CD44 are membrane-expressed molecules required for cell-cell and cell-matrix interactions. These molecules contribute to disease development in experimental models for other chronic in.ammatory diseases, e.g. arthritis and colitis and may determine interactions between leukocytes, endothelium and the extracellular matrix (ECM) of the CNS during MS and EAE. Here we assessed whether CD97 and CD44v isoforms are involved in the immunopathogenesis of MS and EAE. CD97 is a member of the 7-span transmembrane receptor family that is expressed on leukocytes early after activation. CD97 is involved in migration and adhesion, by binding to CD55 (decay accelerating factor), a5/ß1 and avß3 integrins and ECM components. The CD97-ligand CD55 is expressed on several cell types, protecting them from complement-mediated damage. Here we examined the expression of CD97 and CD55 by immunohistochemistry in MS brain tissue. Chapter 4 shows that cells in normal white matter did not express CD97, whereas many MF or microglial cells and T cells in MS brain lesions expressed CD97. Endothelial cells in normal white matter only modestly expressed CD55. In contrast, in MS brain lesions, endothelial cells expressed high levels of CD55 and different leukocyte subsets also expressed CD55. In active lesions, expression of CD55 was predominantly detected on MF or microglial cells. In these lesions, a substantial proportion of cells also expressed CD97. The soluble form of CD97 was found in serum, but not in CSF of a signi.cant number of MS patients. These .ndings suggest that local CD97-CD55 interactions may contribute to the immunopathogenesis of MS. CD55 expressed on brain endothelial cells may serve to protect the vessels from complement-mediated damage. Additionally, CD97­expressing cells may utilize CD55 on activated brain endothelial cells to facilitate adhesion and transmigration into the CNS. To determine whether CD97-CD55 interactions are important in disease development, we blocked the interaction between CD97 and CD55 by CD97 Mab treatment in the priming phase of mouse EAE. This treatment regimen did not affect the onset and severity of disease. However, these data do not exclude the possibility that CD97-CD55 interactions are involved in EAE development, since the treatment duration might have been suboptimal. Alternatively, CD97 may participate in MS and EAE by binding to integrins or ECM components. By using mice with a genetic deletion for CD97, we can elucidate whether CD97 functionally contributes to EAE development. CD44 and its variant isoforms (CD44v1-v10) are required for many different biological processes such as lymphocyte activation, costimulation, adhesion/ extravasation into in.ammatory sites and apoptosis. The CD44 molecule also serves as a docking site for mediators of in.ammation by binding cytokines, growth factors and chemoattractants. One gene encodes different CD44v isoforms, which emerge as a result of complex RNA splicing. We determined whether CD44v isoforms are involved in the immunopathogenesis of MS and EAE (chapter 5). It appeared that only CD44v10 was expressed by normal white matter endothelium, whereas both CD44v3 and v10 were expressed by cells in perivascular in.ltrates and by microglia or MF in active lesions. Due to technical dif.culties, we could not assess expression of CD44v7. All other isoforms were not expressed in MS and control brain. Also in mouse EAE, brain-in.ltrating cells expressed CD44v10 and were already detected before clinical signs. The number of leukocytes that expressed CD44v10 paralleled the extent of clinical EAE symptoms. CD44v10-expressing CD4+ T cells persisted in EAE brain until late time points after immunization, whereas these cells were absent in control-immunized mice. Single deletion for either CD44v7 or CD44v10 resulted in reduced EAE, which was accompanied by reduced in.ltration of the spinal cord. Adoptive transfer experiments demonstrated that CD44v7 exerted its effect on the donor lymphocyte compartment as well as on the recipients APC. No clear differences were detected in autoantigen-speci.c T cell proliferation and cytokine production around the day of onset. In the remission phase of EAE, CD44v7-deleted lymph node cells showed a reduced proliferation compared to wild type cells. At present, it is unclear by which mechanism CD44v7 and CD44v10 reduced EAE. Currently we are exploring the possibility that de.ciency of CD44v7 or v10 promote clearance of in.ammation by the induction of apoptosis. Additionally, CD44v10 may also affect cell migration, as indicated in animal models for Th1-mediated DTH responses. It remains to be determined whether CD44v3 can functionally contribute to EAE. Taken together these data show that a single bacterial component, PG, can create an in.ammatory environment. During an autoimmune attack, PG-containing cells most likely enter the in.ammatory site and contribute to the pathological process. Intracellular PG may activate cells by engaging TLR or Nod receptors. PG promotes antigen uptake, DC maturation and proin.ammatory cytokine production. In the presence of autoantigens, PG induces autoantigen-speci.c Th1 cell priming and expansion. This is relevant in MS, since elevated numbers of PG-containing APC are found in the CNS, where autoantigens are released during in.ammatory attacks. Moreover, autoantigens and PG are present within APC of peripheral lymphoid organs, where similar events may take place. CD97 and CD44v isoforms may facilitate cellular activation, costimulation, adhesion, extravasation and survival. By their interactions with other cellular ligands and different ligands in ECM these molecules likely contribute to tissue damage and chronic in.ammation in MS. We here show that CD97 and CD44v molecules are expressed in MS brain lesions. EAE models demonstrated that by targeting CD44v7 or CD44v10 the clinical symptoms of the disease can be reduced. Total prevention of CD97 expression, by genetic deletion or long-lasting antibody treatment against ligand-binding CD97EGF domains may give similar effects. Eventually, altering the immune response by the modulation of PG, CD97 and CD44v isoforms in EAE will give more insights in the possibilities for therapeutic interventions in MS.