Leah Alabanza

Neuropilin-1 attenuates autoreactivity in experimental autoimmune encephalomyelitis

Neuropilin-1 (Nrp1) is a cell surface molecule originally identified for its role in neuronal development. Recently, Nrp1 has been implicated in several aspects of immune function including maintenance of the immune synapse and development of regulatory T (Treg) cells. In this study, we provide evidence for a central role of Nrp1 in the regulation of CD4 T-cell immune responses in experimental autoimmune encephalitis (EAE). EAE serves as an animal model for the central nervous system (CNS) inflammatory disorder multiple sclerosis (MS). EAE is mediated primarily by CD4+ T cells that migrate to the CNS and mount an inflammatory attack against myelin components, resulting in CNS pathology. Using a tissue-specific deletion system, we observed that the lack of Nrp1 on CD4+ T cells results in increased EAE severity. These conditional knockout mice exhibit preferential TH-17 lineage commitment and decreased Treg-cell functionality. Conversely, CD4+ T cells expressing Nrp1 suppress effector T-cell proliferation and cytokine production both in vivo and in vitro independent of Treg cells. Nrp1-mediated suppression can be inhibited by TGF-β blockade but not by IL-10 blockade. These results suggest that Nrp1 is essential for proper maintenance of peripheral tolerance and its absence can result in unchecked autoreactive responses, leading to diseases like EAE and potentially MS.

Extracellular adenosine signaling induces CX3CL1 expression in the brain to promote experimental autoimmune encephalomyelitis

BackgroundMultiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE) are debilitating neuroinflammatory diseases mediated by lymphocyte entry into the central nervous system (CNS). While it is not known what triggers lymphocyte entry into the CNS during neuroinflammation, blockade of lymphocyte migration has been shown to be effective in controlling neuroinflammatory diseases. Since we have previously shown that extracellular adenosine is a key mediator of lymphocyte migration into the CNS during EAE progression, we wanted to determine which factors are regulated by adenosine to modulate EAE development.MethodsWe performed a genetic analysis of wild type and CD73−/− (that are unable to produce extracellular adenosine and are protected from EAE development) to identify factors that are both important for EAE development and controlled by extracellular adenosine signaling.ResultsWe show that extracellular adenosine triggered lymphocyte migration into the CNS by inducing the expression of the specialized chemokine/adhesion molecule CX3CL1 at the choroid plexus. In wild type mice, CX3CL1 is upregulated in the brain on Day 10 post EAE induction, which corresponds with initial CNS lymphocyte infiltration and the acute stage of EAE. Conversely, mice that cannot synthesize extracellular adenosine (CD73−/− mice) do not upregulate CX3CL1 in the brain following EAE induction and are protected from EAE development and its associated lymphocyte infiltration. Additionally, blockade of the A2A adenosine receptor following EAE induction prevents disease development and the induction of brain CX3CL1 expression. The CX3CL1 induced during EAE is found on the choroid plexus, which is the barrier between the blood and cerebral spinal fluid in the brain and is a prime entry point into the CNS for immune cells. Furthermore, CX3CL1 expression can be induced in the brains of mice and in choroid plexus cell line following A2A adenosine receptor agonist administration. Most importantly, we show that CX3CL1 blockade protects against EAE development and inhibits lymphocyte entry into the CNS.ConclusionsWe conclude that extracellular adenosine is an endogenous modulator of neuroinflammation during EAE that induces CX3CL1 at the choroid plexus to trigger lymphocyte entry into the brain.

Human brain endothelial cells are responsive to adenosine receptor activation

The blood–brain barrier (BBB) of the central nervous system (CNS) consists of a unique subset of endothelial cells that possess tight junctions which form a relatively impervious physical barrier to a large variety of blood components. Until recently, there have been no good in vitro models for studying the human BBB without the co-culture of feeder cells. The hCMEC/D3 cell line is the first stable, well-differentiated human brain endothelial cell line that grows independently in culture with characteristics that closely resemble those of resident human brain endothelial cells. As our previously published findings demonstrated the importance of adenosine receptor (AR) signaling for lymphocyte entry into the CNS, we wanted to determine if human brain endothelial cells possess the capacity to generate and respond to extracellular adenosine. Utilizing the hCMEC/D3 cell line, we determined that these cells express CD73, the cell surface enzyme that converts extracellular AMP to adenosine. When grown under normal conditions, these cells also express the A1, A2A, and A2B AR subtypes. Additionally, hCMEC/D3 cells are responsive to extracellular AR signaling, as cAMP levels increase following the addition of the broad spectrum AR agonist 5′-N-ethylcarboxamidoadenosine (NECA). Overall, these results indicate that human brain endothelial cells, and most likely the human BBB, have the capacity to synthesize and respond to extracellular adenosine.

Thrombin induces an inflammatory phenotype in a human brain endothelial cell line.

In this study, we utilized the human brain endothelial cell line, hCMEC/D3, to determine the effects of the coagulation factor, thrombin, on the human blood-brain barrier (BBB). We show that thrombin increased the mRNA and cell surface levels of ICAM-1 and VCAM-1 in hCMEC/D3 cells. Thrombin similarly upregulated several chemokines implicated in human neurological conditions. Additionally, the paracellular permeability of the human BBB in vitro was also increased following thrombin treatment. Overall, this study demonstrates that thrombin can effectively induce an inflamed phenotype in an in vitro human BBB.

Inhibition of Endogenous Activated Protein C Attenuates Experimental Autoimmune Encephalomyelitis by Inducing Myeloid-Derived Suppressor Cells

Activated protein C (PC) is an anticoagulant involved in the interactions between the coagulation and immune systems. Activated PC has broad anti-inflammatory effects that are mediated through its ability to modulate leukocyte function and confer vascular barrier protection. We investigated the influence of activated PC on the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. We modulated activated PC levels in the circulation during EAE induction through systemic administration of a mAb against PC/activated PC (anti-PC). We initially hypothesized that inhibition of activated PC may result in a heightened inflammatory environment, leading to increased EAE pathogenesis. Contrary to this hypothesis, mice treated with anti-PC Ab (anti-PC mice) exhibited attenuated EAE. Interestingly, despite reduced disease severity and minimal pathogenic conditions in the CNS, anti-PC mice exhibited considerable leukocyte infiltration in the brain, comparable to control mice with severe EAE. Furthermore, CD4+ T cells were diminished in the periphery of anti-PC mice, whereas various CD11b+ populations were increased, notably the myeloid-derived suppressor cells (MDSCs), a CD11b+ subset characterized as potent T cell suppressors. MDSCs from anti-PC mice exhibited increased expression of T cell suppressive factors and effectively inhibited T cell proliferation. Overall, our findings show that activated PC inhibition affected EAE pathogenesis at multiple fronts, specifically increasing vascular barrier permeability, as evidenced by considerable leukocyte infiltration in the brain. Additionally, inhibition of activated PC modulated the functional responses of CD11b+ cells, leading to the expansion and increased activation of MDSCs, which are suppressive to the CD4+ T cells required for EAE progression, thereby resulting in attenuated EAE.