Alla L. Zozulya

The role of regulatory T cells in multiple sclerosis

The dysregulation of inflammatory responses and of immune self-tolerance is considered to be a key element in the autoreactive immune response in multiple sclerosis (MS). Regulatory T (TREG) cells have emerged as crucial players in the pathogenetic scenario of CNS autoimmune inflammation. Targeted deletion of TREG cells causes spontaneous autoimmune disease in mice, whereas augmentation of TREG-cell function can prevent the development of or alleviate variants of experimental autoimmune encephalomyelitis, the animal model of MS. Recent findings indicate that MS itself is also accompanied by dysfunction or impaired maturation of TREG cells. The development and function of TREG cells is closely linked to dendritic cells (DCs), which have a central role in the activation and reactivation of encephalitogenic cells in the CNS. DCs and TREG cells have an intimate bidirectional relationship, and, in combination with other factors and cell types, certain types of DCs are capable of inducing TREG cells. Consequently, TREG cells and DCs have been recognized as potential therapeutic targets in MS. This Review compiles the current knowledge on the role and function of various subsets of TREG cells in MS and experimental autoimmune encephalomyelitis. We also highlight the role of tolerogenic DCs and their bidirectional interaction with TREG cells during CNS autoimmunity.

Dendritic Cell Transmigration through Brain Microvessel Endothelium Is Regulated by MIP-1α Chemokine and Matrix Metalloproteinases

Dendritic cells (DCs) accumulate in the CNS during inflammatory diseases, but the exact mechanism regulating their traffic into the CNS remains to be defined. We now report that MIP-1α increases the transmigration of bone marrow-derived, GFP-labeled DCs across brain microvessel endothelial cell monolayers. Furthermore, occludin, an important element of endothelial tight junctions, is reorganized when DCs migrate across brain capillary endothelial cell monolayers without causing significant changes in the barrier integrity as measured by transendothelial electrical resistance. We show that DCs produce matrix metalloproteinases (MMP) -2 and -9 and GM6001, an MMP inhibitor, decreases both baseline and MIP-1α-induced DC transmigration. These observations suggest that DC transmigration across brain endothelial cell monolayers is partly MMP dependent. The migrated DCs express higher levels of CD40, CD80, and CD86 costimulatory molecules and induce T cell proliferation, indicating that the transmigration of DCs across brain endothelial cell monolayers contributes to the maintenance of DC Ag-presenting function. The MMP dependence of DC migration across brain endothelial cell monolayers raises the possibility that MMP blockers may decrease the initiation of T cell recruitment and neuroinflammation in the CNS.

Pericyte–endothelial cell interaction increases MMP-9 secretion at the blood–brain barrier in vitro

The extracellular matrix (ECM) connecting brain capillary endothelial cells (BCEC) with the surrounding brain resident cells is an essential part of the blood-brain barrier (BBB). Represented by the basement membrane which joins BCEC with brain neuroglia (astrocytes and pericytes) it forms a neurovascular unit. Neuroglia-secreted matrix metalloproteinases (MMPs) control the ECM composition and are involved in the integrity and function of the BBB during cell diapedesis and BBB breakdown after ischemia and other CNS diseases. We examined the involvement of pericytes and astrocytes in endothelial MMP secretion and their effect on endothelial barrier properties in a primary cell culture model using porcine BCEC. We applied puromycin to eliminate pericyte growth and demonstrated a significant (to about 30%) reduction of endothelial MMP-9 production in pericyte-free cultures. In contrast, BCEC co-culture with pericytes resulted in an increased amount of endothelial MMP-9 and active MMPs measured by both zymography and fluorimetric assay. Astrocyte co-culture in a filter setup with BCEC allowing a cell-cell signaling via soluble factors revealed significantly reduced endothelial MMP activity. These data were directly correlated with improved BBB integrity under pericyte elimination and astrocyte co-culture conditions as indicated by transendothelial electrical resistance (TEER) values. Our data define pericyte interactions as a main inducer of endothelial MMP secretion and propose a new role for pericyte-endothelial cell crosstalk at the BBB in vitro.

A β-Lactam Antibiotic Dampens Excitotoxic Inflammatory CNS Damage in a Mouse Model of Multiple Sclerosis

In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), impairment of glial “Excitatory Amino Acid Transporters” (EAATs) together with an excess glutamate-release by invading immune cells causes excitotoxic damage of the central nervous system (CNS). In order to identify pathways to dampen excitotoxic inflammatory CNS damage, we assessed the effects of a β-lactam antibiotic, ceftriaxone, reported to enhance expression of glial EAAT2, in “Myelin Oligodendrocyte Glycoprotein” (MOG)-induced EAE. Ceftriaxone profoundly ameliorated the clinical course of murine MOG-induced EAE both under preventive and therapeutic regimens. However, ceftriaxone had impact neither on EAAT2 protein expression levels in several brain areas, nor on the radioactive glutamate uptake capacity in a mixed primary glial cell-culture and the glutamate-induced uptake currents in a mammalian cell line mediated by EAAT2. Moreover, the clinical effect of ceftriaxone was preserved in the presence of the EAAT2-specific transport inhibitor, dihydrokainate, while dihydrokainate alone caused an aggravated EAE course. This demonstrates the need for sufficient glial glutamate uptake upon an excitotoxic autoimmune inflammatory challenge of the CNS and a molecular target of ceftriaxone other than the glutamate transporter. Ceftriaxone treatment indirectly hampered T cell proliferation and proinflammatory INFγ and IL17 secretion through modulation of myelin-antigen presentation by antigen-presenting cells (APCs) e.g. dendritic cells (DCs) and reduced T cell migration into the CNS in vivo. Taken together, we demonstrate, that a β-lactam antibiotic attenuates disease course and severity in a model of autoimmune CNS inflammation. The mechanisms are reduction of T cell activation by modulation of cellular antigen-presentation and impairment of antigen-specific T cell migration into the CNS rather than or modulation of central glutamate homeostasis.

The role of dendritic cells in CNS autoimmunity

Multiple sclerosis (MS) is a chronic immune-mediated, central nervous system (CNS) demyelinating disease. Clinical and histopathological features suggest an inflammatory etiology involving resident CNS innate cells as well as invading adaptive immune cells. Encephalitogenic myelin-reactive T cells have been implicated in the initiation of an inflammatory cascade, eventually resulting in demyelination and axonal damage (the histological hallmarks of MS). Dendritic cells (DC) have recently emerged as key modulators of this immunopathological cascade, as supported by studies in humans and experimental disease models. In one such model, experimental autoimmune encephalomyelitis (EAE), CNS microvessel-associated DC have been shown to be essential for local antigen recognition by myelin-reactive T cells. Moreover, the functional state and compartmental distribution of DC derived from CNS and associated lymphatics seem to be limiting factors in both the induction and effector phases of EAE. Moreover, DC modulate and balance the recruitment of encephalitogenic and regulatory T cells into CNS tissue. This capacity is critically influenced by DC surface expression of co-stimulatory or co-inhibitory molecules. The fact that DC accumulate in the CNS before T cells and can direct T-cell responses suggests that they are key determinants of CNS autoimmune outcomes. Here we provide a comprehensive review of recent advances in our understanding of CNS-derived DC and their relevance to neuroinflammation.

Intracerebral dendritic cells critically modulate encephalitogenic versus regulatory immune responses in the CNS

Dendritic cells (DCs) appear in higher numbers within the CNS as a consequence of inflammation associated with autoimmune disorders, such as multiple sclerosis, but the contribution of these cells to the outcome of disease is not yet clear. Here, we show that stimulatory or tolerogenic functional states of intracerebral DCs regulate the systemic activation of neuroantigen-specific T cells, the recruitment of these cells into the CNS and the onset and progression of experimental autoimmune encephalomyelitis (EAE). Intracerebral microinjection of stimulatory DCs exacerbated the onset and clinical course of EAE, accompanied with an early T-cell infiltration and a decreased proportion of regulatory FoxP3-expressing cells in the brain. In contrast, the intracerebral microinjection of DCs modified by tumor necrosis factor α induced their tolerogenic functional state and delayed or prevented EAE onset. This triggered the generation of interleukin 10 (IL-10)-producing neuroantigen-specific lymphocytes in the periphery and restricted IL-17 production in the CNS. Our findings suggest that DCs are a rate-limiting factor for neuroinflammation.

Specific central nervous system recruitment of HLA-G+ regulatory T cells in multiple sclerosis†

We have recently described a novel population of natural regulatory T cells (Treg) that are characterized by the expression of HLA‐G and may be found at sites of tissue inflammation (HLA‐Gpos Treg). Here we studied the role of these cells in multiple sclerosis (MS), a prototypic autoimmune inflammatory disorder of the central nervous system (CNS).

An Imbalance of Two Functionally and Phenotypically Different Subsets of Plasmacytoid Dendritic Cells Characterizes the Dysfunctional Immune Regulation in Multiple Sclerosis

Plasmacytoid dendritic cells (pDCs) are instrumental in peripheral T cell tolerance and innate immunity. How pDCs control peripheral immunetolerance and local parenchymal immune response and contribute to the altered immunoregulation in autoimmune disorders in humans is poorly understood. Based on their surface markers, cytokine production, and ability to prime naive allogenic T cells, we found that purified BDCA-2+BDCA-4+ pDCs consist of at least two separate populations, which differed in their response to oligodeoxynucleotides and IFNs (IFN-β), and differently induced IL-17– or IL-10–producing T cells. To evaluate the potential immunoregulatory role of these two types of pDCs in multiple sclerosis (MS) and other human autoimmune disorders (myasthenia gravis), we studied the phenotype and regulatory function of pDCs isolated from clinically stable, untreated patients with MS (n = 16). Patients with MS showed a reversed ratio of pDC1/pDC2 in peripheral blood (4.4:1 in healthy controls, 0.69:1 in MS), a phenomenon not observed in the other autoimmune disorders. As a consequence, MS pDCs had an overall propensity to prime IL-17–secreting cells over IL-10–secreting CD4+ T cells. Immunomodulatory therapy with IFN-β induced an increase of the pDC1 population in vivo (n = 5). Our data offer a plausible explanation for the disturbed immune tolerance in MS patients and provide evidence that immunomodulatory therapy acts at the level of reconstituting homeostasis of pDC, thus reconstituting the disturbed balance.

T cell suppression by naturally occurring HLA-G-expressing regulatory CD4+ T cells is IL-10-dependent and reversible

CD4+ T cells constitutively expressing the immune‐tolerogenic HLA‐G have been described recently as a new type of nTreg (HLA‐Gpos Treg) in humans. HLA‐Gpos Treg accumulate at sites of inflammation and are potent suppressors of T cell proliferation in vitro, suggesting their role in immune regulation. We here characterize the mechanism of how CD4+ HLA‐Gpos Treg influence autologous HLA‐Gneg Tresp function. Using a suppression system free of APC, we demonstrate a T–T cell interaction, resulting in suppression of HLA‐Gneg Tresp, which is facilitated by TCR engagement on HLA‐Gpos Treg. Suppression is independent of cell–cell contact and is reversible, as the removal of HLA‐Gpos Treg from the established coculture restored the proliferative capability of responder cells. Further, HLA‐Gpos Treg‐mediated suppression critically depends on the secretion of IL‐10 but not TGF‐β.

B7-H1 restricts neuroantigen-specific T cell responses and confines inflammatory CNS damage : Implications for the lesion pathogenesis of multiple sclerosis

The co‐inhibitory B7‐homologue 1 (B7‐H1/PD‐L1) influences adaptive immune responses and has been proposed to contribute to the mechanisms maintaining peripheral tolerance and limiting inflammatory damage in parenchymal organs. To understand the B7‐H1/PD1 pathway in CNS inflammation, we analyzed adaptive immune responses in myelin oligodendrocyte glycoprotein (MOG)35–55‐induced EAE and assessed the expression of B7‐H1 in human CNS tissue. B7‐H1–/– mice exhibited an accelerated disease onset and significantly exacerbated EAE severity, although absence of B7‐H1 had no influence on MOG antibody production. Peripheral MOG‐specific IFN‐γ/IL‐17 T cell responses occurred earlier and enhanced in B7‐H1–/– mice, but ceased more rapidly. In the CNS, however, significantly higher numbers of activated neuroantigen‐specific T cells persisted during all stages of EAE. Experiments showing a direct inhibitory role of APC‐derived B7‐H1 on the activation of MOG‐specific effector cells support the assumption that parenchymal B7‐H1 is pivotal for delineating T cell fate in the target organ. Compatible with this concept, our data investigating human brain tissue specimens show a strong up‐regulation of B7‐H1 in lesions of multiple sclerosis. Our findings demonstrate the critical importance of B7‐H1 as an immune‐inhibitory molecule capable of down‐regulating T cell responses thus contributing to the confinement of immunopathological damage.