Publications by authors named "Igal Ifergan"

32 Publications

Methodology for Assessment of Human T Cell Activation and Blockade.

Bio Protoc 2020 Jun 5;10(11):e3644. Epub 2020 Jun 5.

Dept. of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.

Methods to test both the functionality and mechanism of action for human recombinant proteins and antibodies have been limited by multiple factors. To test the functionality of a recombinant protein or antibody, the receptor, the receptor-associated ligand, or both must be expressed by the cells present within the culture. While the use of transfected cell lines can circumvent this gap, the use of transfected cell lines does not allow for studying the native signaling pathway(s) modulated by the specific recombinant protein or antibody in primary cells. The present protocol utilizes sort purified CD14 monocytes and T cells, both CD4 T cells and CD8 T cells, from healthy donors in a co-culture system. This methodology is particularly relevant for testing recombinant proteins or antibodies that are putative therapeutics for the treatment of autoimmune disease and cancer. While the current protocol focuses on co-cultures containing B7-H4 expressing monocytes plus either autologous CD4 T cells or CD8 T cells, the protocol can be modified for the user's specific needs.
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http://dx.doi.org/10.21769/BioProtoc.3644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842510PMC
June 2020

Potential for Targeting Myeloid Cells in Controlling CNS Inflammation.

Front Immunol 2020 6;11:571897. Epub 2020 Oct 6.

Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States.

Multiple Sclerosis (MS) is characterized by immune cell infiltration to the central nervous system (CNS) as well as loss of myelin. Characterization of the cells in lesions of MS patients revealed an important accumulation of myeloid cells such as macrophages and dendritic cells (DCs). Data from the experimental autoimmune encephalomyelitis (EAE) model of MS supports the importance of peripheral myeloid cells in the disease pathology. However, the majority of MS therapies focus on lymphocytes. As we will discuss in this review, multiple strategies are now in place to target myeloid cells in clinical trials. These strategies have emerged from data in both human and mouse studies. We discuss strategies targeting myeloid cell migration, growth factors and cytokines, biological functions (with a focus on miRNAs), and immunological activities (with a focus on nanoparticles).
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http://dx.doi.org/10.3389/fimmu.2020.571897DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573146PMC
October 2020

CRISPR screen in regulatory T cells reveals modulators of Foxp3.

Nature 2020 06 29;582(7812):416-420. Epub 2020 Apr 29.

Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

Regulatory T (T) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity. Conversely, T instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties, can promote autoimmunity and/or facilitate more effective tumour immunity. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective T therapies for autoimmune disease and cancer. The availability of new functional genetic tools has enabled the possibility of systematic dissection of the gene regulatory programs that modulate Foxp3 expression. Here we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse T cells and applied this technology to perform a targeted loss-of-function screen of around 500 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We identified several modulators of Foxp3 expression, including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin-modifying complex, was revealed to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested that Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. T-specific ablation of Usp22 in mice reduced Foxp3 protein levels and caused defects in their suppressive function that led to spontaneous autoimmunity but protected against tumour growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient T cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in T cells. These results reveal previously unknown modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for T immunotherapies for cancer and autoimmune disease.
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http://dx.doi.org/10.1038/s41586-020-2246-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305989PMC
June 2020

Herpesvirus Entry Mediator Binding Partners Mediate Immunopathogenesis of Ocular Herpes Simplex Virus 1 Infection.

mBio 2020 05 12;11(3). Epub 2020 May 12.

Department of Microbiology and Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA

Ocular herpes simplex virus 1 (HSV-1) infection leads to an immunopathogenic disease called herpes stromal keratitis (HSK), in which CD4 T cell-driven inflammation contributes to irreversible damage to the cornea. Herpesvirus entry mediator (HVEM) is an immune modulator that activates stimulatory and inhibitory cosignals by interacting with its binding partners, LIGHT (TNFSF14), BTLA (B and T lymphocyte attenuator), and CD160. We have previously shown that HVEM exacerbates HSK pathogenesis, but the involvement of its binding partners and its connection to the pathogenic T cell response have not been elucidated. In this study, we investigated the role of HVEM and its binding partners in mediating the T cell response using a murine model of ocular HSV-1 infection. By infecting mice lacking the binding partners, we demonstrated that multiple HVEM binding partners were required for HSK pathogenesis. Surprisingly, while LIGHT, BTLA, and CD160 mice did not show differences in disease compared to wild-type mice, BTLA LIGHT and CD160 LIGHT double knockout mice showed attenuated disease characterized by decreased clinical symptoms, increased retention of corneal sensitivity, and decreased infiltrating leukocytes in the cornea. We determined that the attenuation of disease in HVEM, BTLA LIGHT, and CD160 LIGHT mice correlated with a decrease in gamma interferon (IFN-γ)-producing CD4 T cells. Together, these results suggest that HVEM cosignaling through multiple binding partners induces a pathogenic Th1 response to promote HSK. This report provides new insight into the mechanism of HVEM in HSK pathogenesis and highlights the complexity of HVEM signaling in modulating the immune response following ocular HSV-1 infection. Herpes simplex virus 1 (HSV-1), a ubiquitous human pathogen, is capable of causing a progressive inflammatory ocular disease called herpes stromal keratitis (HSK). HSV-1 ocular infection leads to persistent inflammation in the cornea resulting in outcomes ranging from significant visual impairment to complete blindness. Our previous work showed that herpesvirus entry mediator (HVEM) promotes the symptoms of HSK independently of viral entry and that HVEM expression on CD45 cells correlates with increased infiltration of leukocytes into the cornea during the chronic inflammatory phase of the disease. Here, we elucidated the role of HVEM in the pathogenic Th1 response following ocular HSV-1 infection and the contribution of HVEM binding partners in HSK pathogenesis. Investigating the molecular mechanisms of HVEM in promoting corneal inflammation following HSV-1 infection improves our understanding of potential therapeutic targets for HSK.
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http://dx.doi.org/10.1128/mBio.00790-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218284PMC
May 2020

Intravenous Immunomodulatory Nanoparticle Treatment for Traumatic Brain Injury.

Ann Neurol 2020 03 22;87(3):442-455. Epub 2020 Jan 22.

Ken & Ruth Davee Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago.

Objective: There are currently no definitive disease-modifying therapies for traumatic brain injury (TBI). In this study, we present a strong therapeutic candidate for TBI, immunomodulatory nanoparticles (IMPs), which ablate a specific subset of hematogenous monocytes (hMos). We hypothesized that prevention of infiltration of these cells into brain acutely after TBI would attenuate secondary damage and preserve anatomic and neurologic function.

Methods: IMPs, composed of US Food and Drug Administration-approved 500nm carboxylated-poly(lactic-co-glycolic) acid, were infused intravenously into wild-type C57BL/6 mice following 2 different models of experimental TBI, controlled cortical impact (CCI), and closed head injury (CHI).

Results: IMP administration resulted in remarkable preservation of both tissue and neurological function in both CCI and CHI TBI models in mice. After acute treatment, there was a reduction in the number of immune cells infiltrating into the brain, mitigation of the inflammatory status of the infiltrating cells, improved electrophysiologic visual function, improved long-term motor behavior, reduced edema formation as assessed by magnetic resonance imaging, and reduced lesion volumes on anatomic examination.

Interpretation: Our findings suggest that IMPs are a clinically translatable acute intervention for TBI with a well-defined mechanism of action and beneficial anatomic and physiologic preservation and recovery. Ann Neurol 2020;87:442-455.
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http://dx.doi.org/10.1002/ana.25675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296512PMC
March 2020

Monocytes prime autoreactive T cells after myocardial infarction.

Am J Physiol Heart Circ Physiol 2020 01 6;318(1):H116-H123. Epub 2019 Dec 6.

Department of Pathology, Northwestern University, Chicago, Illinois.

In humans, loss of central tolerance for the cardiac self-antigen α-myosin heavy chain (α-MHC) leads to circulation of cardiac autoreactive T cells and renders the heart susceptible to autoimmune attack after acute myocardial infarction (MI). MI triggers profound tissue damage, releasing danger signals and self-antigen by necrotic cardiomyocytes, which lead to recruitment of inflammatory monocytes. We hypothesized that excessive inflammation by monocytes contributes to the initiation of adaptive immune responses to cardiac self-antigen. Using an experimental model of MI in α-MHC-mCherry reporter mice, which specifically express mCherry in cardiomyocytes, we detected α-MHC antigen in myeloid cells in the heart-draining mediastinal lymph node (MLN) 7 days after MI. To test whether monocytes were required for cardiac self-antigen trafficking to the MLN, we blocked monocyte recruitment with a C-C motif chemokine receptor type 2 (CCR2) antagonist or immune modifying nanoparticles (IMP). Blockade of monocyte recruitment reduced α-MHC antigen detection in the MLN after MI. Intramyocardial injection of the model antigen ovalbumin into OT-II transgenic mice demonstrated the requirement for monocytes in antigen trafficking and T-cell activation in the MLN. Finally, in nonobese diabetic mice, which are prone to postinfarction autoimmunity, blockade of monocyte recruitment reduced α-MHC-specific responses leading to improved tissue repair and ventricular function 28 days after MI. Taken together, these data support a role for monocytes in the onset of pathological cardiac autoimmunity following MI and suggest that therapeutic targeting of monocytes may mitigate postinfarction autoimmunity in humans. Our study newly identifies a role for inflammatory monocytes in priming an autoimmune T-cell response after myocardial infarction. Select inhibition of monocyte recruitment to the infarct prevents trafficking of cardiac self-antigen and activation of cardiac myosin reactive T cells in the heart-draining lymph node. Therapeutic targeting of inflammatory monocytes may limit autoimmune responses to improve cardiac remodeling and preserve left ventricular function after myocardial infarction.
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http://dx.doi.org/10.1152/ajpheart.00595.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6985803PMC
January 2020

B7-H4 Modulates Regulatory CD4 T Cell Induction and Function via Ligation of a Semaphorin 3a/Plexin A4/Neuropilin-1 Complex.

J Immunol 2018 08 13;201(3):897-907. Epub 2018 Jun 13.

Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611;

The potent immune regulatory function of an agonistic B7-H4-Ig fusion protein (B7-H4Ig) has been demonstrated in multiple experimental autoimmune models; however, the identity of a functional B7-H4 receptor remained unknown. The biological activity of B7-H4 is associated with decreased inflammatory CD4 T cell responses as supported by a correlation between B7-H4-expressing tumor-associated macrophages and Foxp3 T cells within the tumor microenvironment. Recent data indicate that members of the semaphorin (Sema)/plexin/neuropilin (Nrp) family of proteins both positively and negatively modulate immune cell function. In this study, we show that B7-H4 binds the soluble Sema family member Sema3a. Additionally, B7-H4Ig-induced inhibition of inflammatory CD4 T cell responses is lost in both Sema3a functional mutant mice and mice lacking Nrp-1 expression in Foxp3 T cells. These findings indicate that B7-H4Ig binds to Sema3a, which acts as a functional bridge to stimulate an Nrp-1/Plexin A4 heterodimer to form a functional immunoregulatory receptor complex resulting in increased levels of phosphorylated PTEN and enhanced regulatory CD4 T cell number and function.
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http://dx.doi.org/10.4049/jimmunol.1700811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894186PMC
August 2018

Peripherally derived T regulatory and γδ T cells have opposing roles in the pathogenesis of intractable pediatric epilepsy.

J Exp Med 2018 04 27;215(4):1169-1186. Epub 2018 Feb 27.

Department of Pediatrics, Feinberg School of Medicine, Northwestern University, Chicago, IL

The pathophysiology of drug-resistant pediatric epilepsy is unknown. Flow cytometric analysis of inflammatory leukocytes in resected brain tissues from 29 pediatric patients with genetic (focal cortical dysplasia) or acquired (encephalomalacia) epilepsy demonstrated significant brain infiltration of blood-borne inflammatory myeloid cells and memory CD4 and CD8 T cells. Significantly, proinflammatory (IL-17- and GM-CSF-producing) γδ T cells were concentrated in epileptogenic lesions, and their numbers positively correlated with disease severity. Conversely, numbers of regulatory T (T reg) cells inversely correlated with disease severity. Correspondingly, using the kainic acid model of status epilepticus, we show ameliorated seizure activity in both γδ T cell- and IL-17RA-deficient mice and in recipients of T reg cells, whereas T reg cell depletion heightened seizure severity. Moreover, both IL-17 and GM-CSF induced neuronal hyperexcitability in brain slice cultures. These studies support a major pathological role for peripherally derived innate and adaptive proinflammatory immune responses in the pathogenesis of intractable epilepsy and suggest testing of immunomodulatory therapies.
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http://dx.doi.org/10.1084/jem.20171285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881465PMC
April 2018

Pre-metastatic cancer exosomes induce immune surveillance by patrolling monocytes at the metastatic niche.

Nat Commun 2017 11 6;8(1):1319. Epub 2017 Nov 6.

Department of Cancer Biology, University of Texas MD Anderson Cancer Center, 1881 East Rd, Houston, TX, 77054, USA.

Metastatic cancers produce exosomes that condition pre-metastatic niches in remote microenvironments to favor metastasis. In contrast, here we show that exosomes from poorly metastatic melanoma cells can potently inhibit metastasis to the lung. These "non-metastatic" exosomes stimulate an innate immune response through the expansion of Ly6C patrolling monocytes (PMo) in the bone marrow, which then cause cancer cell clearance at the pre-metastatic niche, via the recruitment of NK cells and TRAIL-dependent killing of melanoma cells by macrophages. These events require the induction of the Nr4a1 transcription factor and are dependent on pigment epithelium-derived factor (PEDF) on the outer surface of exosomes. Importantly, exosomes isolated from patients with non-metastatic primary melanomas have a similar ability to suppress lung metastasis. This study thus demonstrates that pre-metastatic tumors produce exosomes, which elicit a broad range of PMo-reliant innate immune responses via trigger(s) of immune surveillance, causing cancer cell clearance at the pre-metastatic niche.
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http://dx.doi.org/10.1038/s41467-017-01433-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673063PMC
November 2017

Intravenous immune-modifying nanoparticles as a therapy for spinal cord injury in mice.

Neurobiol Dis 2017 Dec 18;108:73-82. Epub 2017 Aug 18.

Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. Electronic address:

Intravenously infused synthetic 500nm nanoparticles composed of poly(lactide-co-glycolide) are taken up by blood-borne inflammatory monocytes via a macrophage scavenger receptor (macrophage receptor with collagenous structure), and the monocytes no longer traffic to sites of inflammation. Intravenous administration of the nanoparticles after experimental spinal cord injury in mice safely and selectively limited infiltration of hematogenous monocytes into the injury site. The nanoparticles did not bind to resident microglia, and did not change the number of microglia in the injured spinal cord. Nanoparticle administration reduced M1 macrophage polarization and microglia activation, reduced levels of inflammatory cytokines, and markedly reduced fibrotic scar formation without altering glial scarring. These findings thus implicate early-infiltrating hematogenous monocytes as highly selective contributors to fibrosis that do not play an indispensable role in gliosis after SCI. Further, the nanoparticle treatment reduced accumulation of chondroitin sulfate proteoglycans, increased axon density inside and caudal to the lesion site, and significantly improved functional recovery after both moderate and severe injuries to the spinal cord. These data provide further evidence that hematogenous monocytes contribute to inflammatory damage and fibrotic scar formation after spinal cord injury in mice. Further, since the nanoparticles are simple to administer intravenously, immunologically inert, stable at room temperature, composed of an FDA-approved material, and have no known toxicity, these findings suggest that the nanoparticles potentially offer a practical treatment for human spinal cord injury.
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http://dx.doi.org/10.1016/j.nbd.2017.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675775PMC
December 2017

Targeting the GM-CSF receptor for the treatment of CNS autoimmunity.

J Autoimmun 2017 Nov 20;84:1-11. Epub 2017 Jun 20.

Departments of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, United States; Department of Interdepartmental Immunobiology Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, United States. Electronic address:

In multiple sclerosis (MS), there is a growing interest in inhibiting the pro-inflammatory effects of granulocyte-macrophage colony-stimulating factor (GM-CSF). We sought to evaluate the therapeutic potential and underlying mechanisms of GM-CSF receptor alpha (Rα) blockade in animal models of MS. We show that GM-CSF signaling inhibition at peak of chronic experimental autoimmune encephalomyelitis (EAE) results in amelioration of disease progression. Similarly, GM-CSF Rα blockade in relapsing-remitting (RR)-EAE model prevented disease relapses and inhibited T cell responses specific for both the inducing and spread myelin peptides, while reducing activation of mDCs and inflammatory monocytes. In situ immunostaining of lesions from human secondary progressive MS (SPMS), but not primary progressive MS patients shows extensive recruitment of GM-CSF Rα myeloid cells. Collectively, this study reveals a pivotal role of GM-CSF in disease relapses and the benefit of GM-CSF Rα blockade as a potential novel therapeutic approach for treatment of RRMS and SPMS.
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http://dx.doi.org/10.1016/j.jaut.2017.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5647260PMC
November 2017

Murine Corneal Inflammation and Nerve Damage After Infection With HSV-1 Are Promoted by HVEM and Ameliorated by Immune-Modifying Nanoparticle Therapy.

Invest Ophthalmol Vis Sci 2017 01;58(1):282-291

Department of Microbiology and Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States.

Purpose: To determine cellular and temporal expression patterns of herpes virus entry mediator (HVEM, Tnfrsf14) in the murine cornea during the course of herpes simplex virus 1 (HSV-1) infection, the impact of this expression on pathogenesis, and whether alterations in HVEM or downstream HVEM-mediated effects ameliorate corneal disease.

Methods: Corneal HVEM levels were assessed in C57BL/6 mice after infection with HSV-1(17). Leukocytic infiltrates and corneal sensitivity loss were measured in the presence, global absence (HVEM knockout [KO] mice; Tnfrsf14-/-), or partial absence of HVEM (HVEM conditional KO). Effects of immune-modifying nanoparticles (IMPs) on viral replication, corneal sensitivity, and corneal infiltrates were measured.

Results: Corneal HVEM+ populations, particularly monocytes/macrophages during acute infection (3 days post infection [dpi]) and polymorphonuclear neutrophils (PMN) during the chronic inflammatory phase (14 dpi), increased after HSV-1 infection. Herpes virus entry mediator increased leukocytes in the cornea and corneal sensitivity loss. Ablation of HVEM from CD45+ cells, or intravenous IMP therapy, reduced infiltrates in the chronic phase and maintained corneal sensitivity.

Conclusions: Herpes virus entry mediator was expressed on two key populations: corneal monocytes/macrophages and PMNs. Herpes virus entry mediator promoted the recruitment of myeloid cells to the cornea in the chronic phase. Herpes virus entry mediator-associated corneal sensitivity loss preceded leukocytic infiltration, suggesting it may play an active role in recruitment. We propose that HVEM on resident corneal macrophages increases nerve damage and immune cell invasion, and we showed that prevention of late-phase infiltration of PMN and CD4+ T cells by IMP therapy improved clinical symptoms and mortality and reduced corneal sensitivity loss caused by HSV-1.
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http://dx.doi.org/10.1167/iovs.16-20668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5256684PMC
January 2017

Biodegradable antigen-associated PLG nanoparticles tolerize Th2-mediated allergic airway inflammation pre- and postsensitization.

Proc Natl Acad Sci U S A 2016 May 18;113(18):5059-64. Epub 2016 Apr 18.

Department of Microbiology-Immunology and Interdepartmental Immunobiology Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611;

Specific immunotherapy (SIT) is the most widely used treatment for allergic diseases that directly targets the T helper 2 (Th2) bias underlying allergy. However, the most widespread clinical applications of SIT require a long period of dose escalation with soluble antigen (Ag) and carry a significant risk of adverse reactions, particularly in highly sensitized patients who stand to benefit most from a curative treatment. Thus, the development of safer, more efficient methods to induce Ag-specific immune tolerance is critical to advancing allergy treatment. We hypothesized that antigen-associated nanoparticles (Ag-NPs), which we have used to prevent and treat Th1/Th17-mediated autoimmune disease, would also be effective for the induction of tolerance in a murine model of Th2-mediated ovalbumin/alum-induced allergic airway inflammation. We demonstrate here that antigen-conjugated polystyrene (Ag-PS) NPs, although effective for the prophylactic induction of tolerance, induce anaphylaxis in presensitized mice. Antigen-conjugated NPs made of biodegradable poly(lactide-co-glycolide) (Ag-PLG) are similarly effective prophylactically, are well tolerated by sensitized animals, but only partially inhibit Th2 responses when administered therapeutically. PLG NPs containing encapsulated antigen [PLG(Ag)], however, were well tolerated and effectively inhibited Th2 responses and airway inflammation both prophylactically and therapeutically. Thus, we illustrate progression toward PLG(Ag) as a biodegradable Ag carrier platform for the safe and effective inhibition of allergic airway inflammation without the need for nonspecific immunosuppression in animals with established Th2 sensitization.
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http://dx.doi.org/10.1073/pnas.1505782113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4983813PMC
May 2016

Cutting Edge: MicroRNA-223 Regulates Myeloid Dendritic Cell-Driven Th17 Responses in Experimental Autoimmune Encephalomyelitis.

J Immunol 2016 Feb 18;196(4):1455-1459. Epub 2016 Jan 18.

Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611.

Myeloid cells play a crucial role in the induction and sustained inflammation in neuroinflammatory disorders, such as multiple sclerosis. miR-223, a myeloid cell-specific microRNA, is one of the most upregulated microRNAs in multiple sclerosis patients. We demonstrate that miR-223-knockout mice display significantly reduced active and adoptive-transfer experimental autoimmune encephalomyelitis that is characterized by reduced numbers of myeloid dendritic cells (mDCs) and Th17 cells in the CNS. Knockout mDCs have increased PD-L1 and decreased IL-1β, IL-6, and IL-23 expression, as well as a reduced capacity to drive Th17, but not Th1, cell differentiation. Thus, miR-223 controls mDC-induced activation of pathologic Th17 responses during autoimmune inflammation.
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http://dx.doi.org/10.4049/jimmunol.1501965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4744529PMC
February 2016

Experimental Autoimmune Encephalomyelitis in Mice.

Methods Mol Biol 2016 ;1304:145-60

Department of Microbiology-Immunology and Interdepartmental Immunobiology Center, Feinberg School of Medicine, Northwestern University, Chicago, IL, 60611, USA.

Experimental autoimmune encephalitis (EAE), the animal model of multiple sclerosis (MS), has provided significant insight into the mechanisms that initiate and drive autoimmunity. Several central nervous system proteins and peptides have been used to induce disease, in a number of different mouse strains, to model the diverse clinical presentations of MS. In this chapter, we detail the materials and methods used to induce active and adoptive EAE. We focus on disease induction in the SJL/J, C57BL/6, and BALB/c mouse strains, using peptides derived from proteolipid protein, myelin basic protein, and myelin oligodendrocyte glycoprotein. We also include a protocol for the isolation of leukocytes from the spinal cord and brain for flow cytometric analysis.
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http://dx.doi.org/10.1007/7651_2014_88DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4402278PMC
May 2016

Melanoma cell adhesion molecule identifies encephalitogenic T lymphocytes and promotes their recruitment to the central nervous system.

Brain 2012 Oct 13;135(Pt 10):2906-24. Epub 2012 Sep 13.

Neuroimmunology Research Laboratory, Centre of Excellence in Neuromics, Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM), Montréal, Québec, H2L 2W5, Canada.

In multiple sclerosis, encephalitogenic CD4(+) lymphocytes require adhesion molecules to accumulate into central nervous system inflammatory lesions. Using proteomic techniques, we identified expression of melanoma cell adhesion molecule (MCAM) on a subset of human effector memory CD4(+) lymphocytes and on human blood-brain barrier endothelium. Herein, we demonstrate that MCAM is a stable surface marker that refines the identification of interleukin 17(+), interleukin 22(+), RAR-related orphan receptor γ and interleukin 23 receptor(+) cells within the CD161(+)CCR6(+) subset of memory CD4(+) lymphocytes. We also show that MCAM(+) lymphocytes express significantly more granulocyte/macrophage colony stimulating factor and granzyme B than MCAM(-) lymphocytes. Furthermore, the proportion of MCAM(+) CD4(+) lymphocytes is significantly increased in the blood and in the central nervous system of patients with multiple sclerosis and experimental autoimmune encephalomyelitis animals compared with healthy controls or other neurological diseases, and MCAM expression is upregulated at the blood-brain barrier within inflammatory lesions. Moreover, blockade of MCAM or depletion of MCAM(+) CD4(+) T lymphocytes both restrict the migration of T(H)17 lymphocytes across blood-brain barrier endothelial cells and decrease the severity of experimental autoimmune encephalomyelitis. Our findings indicate that MCAM could serve as a potential biomarker for multiple sclerosis and represents a valuable target for the treatment of neuroinflammatory conditions.
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http://dx.doi.org/10.1093/brain/aws212DOI Listing
October 2012

Role of Ninjurin-1 in the migration of myeloid cells to central nervous system inflammatory lesions.

Ann Neurol 2011 Nov;70(5):751-63

Neuroimmunology Research Unit, Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Faculté de Médecine, Université de Montréal, Montreal, Quebec, Canada.

Objective: Blood-derived myeloid antigen-presenting cells (APCs) account for a significant proportion of the leukocytes found within lesions of multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). These APCs along with activated microglia are thought to be pivotal in the initiation of the central nervous system (CNS)-targeted immune response in MS and EAE. However, the exact molecules that direct the migration of myeloid cells from the periphery across the blood-brain barrier (BBB) remain largely unknown.

Methods: We identified Ninjurin-1 in a proteomic screen of human BBB endothelial cells (ECs). We assessed the expression of Ninjurin-1 by BBB-ECs and immune cells, and we determined the role of Ninjurin-1 in immune cell migration to the CNS in vivo in EAE mice.

Results: Ninjurin-1 was found to be weakly expressed in the healthy human and mouse CNS but upregulated on BBB-ECs and on infiltrating APCs during the course of EAE and in active MS lesions. In human peripheral blood, Ninjurin-1 was predominantly expressed by monocytes, whereas it was barely detectable on T and B lymphocytes. Moreover, Ninjurin-1 neutralization specifically abrogated the adhesion and migration of human monocytes across BBB-ECs, without affecting lymphocyte recruitment. Finally, Ninjurin-1 blockade reduced clinical disease activity and histopathological indices of EAE and decreased infiltration of macrophages, dendritic cells, and APCs into the CNS.

Interpretation: Our study uncovers an important cell-specific role for Ninjurin-1 in the transmigration of inflammatory APCs across the BBB and further emphasizes the importance of myeloid cell recruitment during the development of neuroinflammatory lesions.
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http://dx.doi.org/10.1002/ana.22519DOI Listing
November 2011

The Hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence.

Science 2011 Dec 1;334(6063):1727-31. Epub 2011 Dec 1.

Neuroimmunology Unit, Center of Excellence in Neuromics, Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Faculty of Medicine, Université de Montréal, Montréal, Quebec, Canada.

The blood-brain barrier (BBB) is composed of tightly bound endothelial cells (ECs) and perivascular astrocytes that regulate central nervous system (CNS) homeostasis. We showed that astrocytes secrete Sonic hedgehog and that BBB ECs express Hedgehog (Hh) receptors, which together promote BBB formation and integrity during embryonic development and adulthood. Using pharmacological inhibition and genetic inactivation of the Hh signaling pathway in ECs, we also demonstrated a critical role of the Hh pathway in promoting the immune quiescence of BBB ECs by decreasing the expression of proinflammatory mediators and the adhesion and migration of leukocytes, in vivo and in vitro. Overall, the Hh pathway provides a barrier-promoting effect and an endogenous anti-inflammatory balance to CNS-directed immune attacks, as occurs in multiple sclerosis.
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http://dx.doi.org/10.1126/science.1206936DOI Listing
December 2011

Central nervous system recruitment of effector memory CD8+ T lymphocytes during neuroinflammation is dependent on α4 integrin.

Brain 2011 Dec 4;134(Pt 12):3560-77. Epub 2011 Nov 4.

Neuroimmunology Research Unit, Centre for Excellence in Neuromics, CRCHUM-Notre-Dame Hospital, Université de Montréal, Montréal, QC, H2L 4M1, Canada.

Clonally expanded CD8(+) T lymphocytes are present in multiple sclerosis lesions, as well as in the cerebrospinal fluid of patients with multiple sclerosis. In experimental autoimmune encephalomyelitis, CD8(+) T lymphocytes are found in spinal cord and brainstem lesions. However, the exact phenotype of central nervous system-infiltrating CD8(+) T lymphocytes and the mechanism by which these cells cross the blood-brain barrier remain largely unknown. Using cerebrospinal fluid from patients with multiple sclerosis, spinal cord from experimental autoimmune encephalomyelitis and coronavirus-induced encephalitis, we demonstrate that central nervous system-infiltrating CD8(+) T lymphocytes are mostly of the effector memory phenotype (CD62L(-) CCR7(-) granzymeB(hi)). We further show that purified human effector memory CD8(+) T lymphocytes transmigrate more readily across blood-brain barrier-endothelial cells than non-effector memory CD8(+) T lymphocytes, and that blood-brain barrier endothelium promotes the selective recruitment of effector memory CD8(+) T lymphocytes. Furthermore, we provide evidence for the recruitment of interferon-γ- and interleukin-17-secreting CD8(+) T lymphocytes by human and mouse blood-brain barrier endothelium. Finally, we show that in vitro migration of CD8(+) T lymphocytes across blood-brain barrier-endothelial cells is dependent on α4 integrin, but independent of intercellular adhesion molecule-1/leucocyte function-associated antigen-1, activated leucocyte cell adhesion molecule/CD6 and the chemokine monocyte chemotactic protein-1/CCL2. We also demonstrate that in vivo neutralization of very late antigen-4 restricts central nervous system infiltration of CD8(+) T lymphocytes in active immunization and adoptive transfer experimental autoimmune encephalomyelitis, and in coronavirus-induced encephalitis. Our study thus demonstrates an active role of the blood-brain barrier in the recruitment of effector memory CD8(+) T lymphocytes to the CNS compartment and defines α4 integrin as a major contributor of CD8(+) T lymphocyte entry into the brain.
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http://dx.doi.org/10.1093/brain/awr268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7110084PMC
December 2011

B cell-derived IL-15 enhances CD8 T cell cytotoxicity and is increased in multiple sclerosis patients.

J Immunol 2011 Oct 12;187(8):4119-28. Epub 2011 Sep 12.

Département de Médecine, Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montréal, Québec H2L 4M1, Canada.

Multiple lines of evidence suggest that CD8 T cells contribute to the pathogenesis of multiple sclerosis (MS). However, the sources and involvement of cytokines such as IL-15 in activating these cells is still unresolved. To investigate the role of IL-15 in enhancing the activation of CD8 T cells in the context of MS, we determined cell types expressing the bioactive surface IL-15 in the peripheral blood of patients and evaluated the impact of this cytokine on CD8 T cell cytotoxicity and migration. Flow cytometric analysis showed a significantly greater proportion of B cells and monocytes from MS patients expressing IL-15 relative to controls. We established that CD40L activation of B cells from healthy donors increased their IL-15 levels, reaching those of MS patients. We also demonstrated an enhanced cytotoxic profile in CD8 T cells from MS patients upon stimulation with IL-15. Furthermore, we showed that IL-15 expressed by B cells and monocytes is sufficient and functional, enhancing granzyme B production by CD8 T cells upon coculture. Exposure of CD8 T cells to this cytokine enhanced their ability to kill glial cells as well as to migrate across an in vitro inflamed human blood-brain barrier. The elevated levels of IL-15 in patients relative to controls, the greater susceptibility of CD8 T cells from patients to IL-15, in addition to the enhanced cytotoxic responses by IL-15-exposed CD8 T cells, stresses the potential of therapeutic strategies to reduce peripheral sources of IL-15 in MS.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5052068PMC
http://dx.doi.org/10.4049/jimmunol.1100885DOI Listing
October 2011

Isolation of human brain endothelial cells and characterization of lipid raft-associated proteins by mass spectroscopy.

Methods Mol Biol 2011 ;686:275-95

Neuroimmunology Research Laboratory, CHUM-Hopital Notre-Dame Université de Montréal, Montréal, QC, Canada.

The blood-brain barrier (BBB) limits the movements of molecules, nutrients, and cells from the systemic blood circulation into the central nervous system (CNS), and vice versa, thus allowing an optimal microenvironment for CNS development and function. The brain endothelial cells (BECs) form the primary barrier between the blood and the CNS. In addition, pericytes, neurons, and astrocytes that make up the neurovascular unit support the BEC functions and are essential to maintain this restrictive permeability phenotype. To better understand the molecular mechanisms underlying BBB properties, we propose a method to study the proteome of detergent resistant microdomain, namely lipid rafts, from human primary cultures of BECs. This chapter describes a robust human BECs isolation protocol, standard tissue culture protocols, ECs purity assessment protocols, lipid raft microdomain isolation method, and a mass spectrometry analysis technique to characterize the protein content of membrane microdomains.
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http://dx.doi.org/10.1007/978-1-60761-938-3_13DOI Listing
June 2011

Preferential recruitment of interferon-gamma-expressing TH17 cells in multiple sclerosis.

Ann Neurol 2009 Sep;66(3):390-402

Neuroimmunology Research Unit, Center for Excellence in Neuromics, Montreal, Quebec, Canada.

Objective: There is substantial evidence supporting the role of interferon (IFN)-gamma-producing T helper (T(H)) 1 and interleukin (IL)-17-expressing T(H)17 lymphocytes in multiple sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (EAE). However, to date little is known about the potential cooperative interplay between these 2 cytokines. In the current study, we sought to evaluate the frequency of IFN-gamma-expressing T(H)17 lymphocytes in MS and EAE, and study their recruitment into the central nervous system (CNS).

Methods: Human T(H)17 lymphocytes were expanded in vitro from the blood of healthy controls and relapsing MS patients using IL-23. Immune cell migration to the CNS was assessed in vitro with primary cultures of human blood-brain barrier (BBB)-derived endothelial cells, and in vivo in EAE mice.

Results: We demonstrate that in response to IL-23, human memory lymphocytes expand into a T(H)17 phenotype, with a subpopulation of cells simultaneously expressing IFN-gamma and IL-17. We note that lymphocytes obtained from the blood of relapsing MS patients have an increased propensity to expand into IFN-gamma-producing T(H)17 cells and identify numerous T lymphocytes coexpressing IL-17 and IFN-gamma in brain tissue of MS patients. We also find lymphocytes expressing both the T(H)1- and the T(H)17-associated transcription factors ROR gamma t and T-bet, in situ and in vitro. We further provide in vitro and in vivo evidence that IFN-gamma(+) T(H)17 lymphocytes preferentially cross the human BBB and accumulate in the CNS of mice during the effector phase of EAE.

Interpretation: Our data underscore the involvement of IFN-gamma(+) T(H)17 lymphocytes in the pathology of MS and EAE and their preferential recruitment into the CNS during inflammatory events.
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http://dx.doi.org/10.1002/ana.21748DOI Listing
September 2009

Activation of kinin receptor B1 limits encephalitogenic T lymphocyte recruitment to the central nervous system.

Nat Med 2009 Jul 28;15(7):788-93. Epub 2009 Jun 28.

Cecilie Vogt Klinik, Charité-University Hospital Berlin, Max Delbrueck Center for Molecular Medicine and NeuroCure Research Center, Berlin, Germany.

Previous proteomic and transcriptional analyses of multiple sclerosis lesions revealed modulation of the renin-angiotensin and the opposing kallikrein-kinin pathways. Here we identify kinin receptor B1 (Bdkrb1) as a specific modulator of immune cell entry into the central nervous system (CNS). We demonstrate that the Bdkrb1 agonist R838 (Sar-[D-Phe]des-Arg(9)-bradykinin) markedly decreases the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in SJL mice, whereas the Bdkrb1 antagonist R715 (Ac-Lys-[D-betaNal(7), Ile(8)]des-Arg(9)-bradykinin) resulted in earlier onset and greater severity of the disease. Bdkrb1-deficient (Bdkrb1(-/-)) C57BL/6 mice immunized with a myelin oligodendrocyte glycoprotein fragment, MOG(35-55), showed more severe disease with enhanced CNS-immune cell infiltration. The same held true for mixed bone marrow-chimeric mice reconstituted with Bdkrb1(-/-) T lymphocytes, which showed enhanced T helper type 17 (T(H)17) cell invasion into the CNS. Pharmacological modulation of Bdkrb1 revealed that in vitro migration of human T(H)17 lymphocytes across blood-brain barrier endothelium is regulated by this receptor. Taken together, these results suggest that the kallikrein-kinin system is involved in the regulation of CNS inflammation, limiting encephalitogenic T lymphocyte infiltration into the CNS, and provide evidence that Bdkrb1 could be a new target for the treatment of chronic inflammatory diseases such as multiple sclerosis.
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http://dx.doi.org/10.1038/nm.1980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4903020PMC
July 2009

IFN-beta regulates CD73 and adenosine expression at the blood-brain barrier.

Eur J Immunol 2008 Oct;38(10):2718-26

Department of Medical Microbiology, MediCity Research Laboratory, University of Turku, Turku, Finland.

IFN-beta treatment reduces the relapse rate in MS but its mechanism of action remains incompletely understood. Our aim was to clarify the beneficial effect of IFN-beta in the treatment of MS. We assessed the influence of IFN-beta treatment on (i) CD73 expression on the surface of primary cultures of human blood-brain barrier endothelial cells (BBB-EC) and human astrocytes using immunofluorescence staining and flow cytometry, (ii) transmigration of CD4+ T lymphocytes using an in vitro model of BBB and (iii) CD73 enzyme activity, i.e. ecto-5'-nucleotidase activity in the serum of MS patients using a radiochemical assay. IFN-beta increases the expression of ecto-5'-nucleotidase both on BBB-EC and astrocytes. As a consequence, lymphocyte transmigration through BBB-EC is reduced. Importantly, this reduction can be reversed using alpha,beta-methyleneadenosine-5'-diphosphate, a specific inhibitor of ecto-5'-nucleotidase. CD73 is strongly expressed in microvasculature in samples of postmortem MS brain and, moreover, in the majority of MS patients there was a clear upregulation both in the soluble serum ecto-5'-nucleotidase activity and skin microvascular CD73 expression after IFN-beta treatment. Upregulation of ecto-5'-nucleotidase and a subsequent increase in adenosine production might contribute to the beneficial effects of IFN-beta on MS via enhancing the endothelial barrier function.
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http://dx.doi.org/10.1002/eji.200838437DOI Listing
October 2008

Activated leukocyte cell adhesion molecule promotes leukocyte trafficking into the central nervous system.

Nat Immunol 2008 Feb 23;9(2):137-45. Epub 2007 Dec 23.

Neuroimmunology Research Laboratory, Center for Excellence in Neuromics, Centre Hospitalier de l'Université de Montréal-Notre-Dame Hospital, Université de Montréal, H2L 4M1, Montréal, Québec, Canada.

Adhesion molecules of the immunoglobulin superfamily are crucial effectors of leukocyte trafficking into the central nervous system. Using a lipid raft-based proteomic approach, we identified ALCAM as an adhesion molecule involved in leukocyte migration across the blood-brain barrier (BBB). ALCAM expressed on BBB endothelium localized together with CD6 on leukocytes and with BBB endothelium transmigratory cups. ALCAM expression on BBB cells was upregulated in active multiple sclerosis and experimental autoimmune encephalomyelitis lesions. Moreover, ALCAM blockade restricted the transmigration of CD4+ lymphocytes and monocytes across BBB endothelium in vitro and in vivo and reduced the severity and delayed the time of onset of experimental autoimmune encephalomyelitis. Our findings indicate an important function for ALCAM in the recruitment of leukocytes into the brain and identify ALCAM as a potential target for the therapeutic dampening of neuroinflammation.
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http://dx.doi.org/10.1038/ni1551DOI Listing
February 2008

The blood-brain barrier induces differentiation of migrating monocytes into Th17-polarizing dendritic cells.

Brain 2008 Mar 20;131(Pt 3):785-99. Epub 2007 Dec 20.

Neuroimmunology Research Laboratory, Center for the Study of Brain Diseases, CHUM-Hôpital Notre-Dame, Université de Montréal, Montréal, Québec, Canada.

Trafficking of antigen-presenting cells into the CNS is essential for lymphocyte reactivation within the CNS compartment. Although perivascular dendritic cells found in inflammatory lesions are reported to polarize naive CD4+ T lymphocytes into interleukin-17-secreting-cells, the origin of those antigen-presenting cells remains controversial. We demonstrate that a subset of CD14+ monocytes migrate across the inflamed human blood-brain barrier (BBB) and differentiate into CD83+CD209+ dendritic cells under the influence of BBB-secreted transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor. We also demonstrate that these dendritic cells secrete interleukin-12p70, transforming growth factor-beta and interleukin-6 and promote the proliferation and expansion of distinct populations of interferon-gamma-secreting Th1 and interleukin-17-secreting Th17 CD4+ T lymphocytes. We further confirmed the abundance of such dendritic cells in situ, closely associated with microvascular BBB-endothelial cells within acute multiple sclerosis lesions, as well as a significant number of CD4+ interleukin-17+ T lymphocytes in the perivascular infiltrate. Our data support the notion that functional perivascular myeloid CNS dendritic cells arise as a consequence of migration of CD14+ monocytes across the human BBB, through the concerted actions of BBB-secreted transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor.
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http://dx.doi.org/10.1093/brain/awm295DOI Listing
March 2008

Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation.

Nat Med 2007 Oct 9;13(10):1173-5. Epub 2007 Sep 9.

Neuroimmunology Unit, Center for the Study of Brain Diseases, Centre Hospitalier de l'Université de Montréal-Notre-Dame Hospital, 1560 Sherbrooke Street East, Montreal, Quebec H2L 4M1, Canada.

T(H)17 lymphocytes appear to be essential in the pathogenesis of numerous inflammatory diseases. We demonstrate here the expression of IL-17 and IL-22 receptors on blood-brain barrier endothelial cells (BBB-ECs) in multiple sclerosis lesions, and show that IL-17 and IL-22 disrupt BBB tight junctions in vitro and in vivo. Furthermore, T(H)17 lymphocytes transmigrate efficiently across BBB-ECs, highly express granzyme B, kill human neurons and promote central nervous system inflammation through CD4+ lymphocyte recruitment.
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http://dx.doi.org/10.1038/nm1651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5114125PMC
October 2007

Statins reduce human blood-brain barrier permeability and restrict leukocyte migration: relevance to multiple sclerosis.

Ann Neurol 2006 Jul;60(1):45-55

Neuroimmunology Laboratory, Center for Research on Brain Diseases, Centre Hospitalier de l'Université de Montreal, CHUM Research Center, Quebec, Canada.

Objective: Dysregulation of the blood-brain barrier (BBB) and transendothelial migration of immune cells are among the earliest central nervous system changes partaking in lesion formation in both multiple sclerosis (MS) and its early clinical form, the clinically isolated syndrome. Evidence for the anti-inflammatory effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors within the central nervous system arose from studies demonstrating that statins improve clinical signs in the animal model of MS and reduce the number of gadolinium-enhancing lesions in MS.

Methods: We sought to describe the impact of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor treatment on the physiology and immunology of human BBB-derived endothelial cells (ECs).

Results: We demonstrate that lovastatin and simvastatin induce a 50 to 60% reduction in the diffusion rates of bovine serum albumin and [(14)C]-sucrose across human BBB-ECs in vitro through abrogation of isoprenylation processes, but independent of the expression of the tight junction molecules occludin, VE-cadherin, JAM-1, zonula occluden-1, and zonula occluden-2. Simvastatin and lovastatin were equipotent in reducing BBB permeability in vitro, with median effective concentration (EC(50)) of 9.5 x 10(-8) and 1.0 x 10(-7)M, respectively. We further demonstrate that lovastatin and simvastatin treatment of BBB-ECs significantly restricts the migration of clinically isolated syndrome-derived and MS-derived monocytes and lymphocytes across the human BBB in vitro, through a specific reduction in the secretion of the chemokines monocyte chemotactic protein-1/CCL2 and interferon-gamma-inducible protein-10/CXCL10 by BBB-ECs.

Interpretation: Our data parallel the previously reported magnetic resonance imaging-based radiological findings and suggest an effect of statins that could be beneficial in early MS, restricting the diffusion of molecular tracers and the migration of immune cells across the human BBB.
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http://dx.doi.org/10.1002/ana.20875DOI Listing
July 2006

Microglial expression of the B7 family member B7 homolog 1 confers strong immune inhibition: implications for immune responses and autoimmunity in the CNS.

J Neurosci 2005 Mar;25(10):2537-46

Department of Neurology, University of Homburg, D-66424 Homburg, Germany.

Inflammation of the CNS is usually locally limited to avoid devastating consequences. Critical players involved in this immune regulatory process are the resident immune cells of the brain, the microglia. Interactions between the growing family of B7 costimulatory ligands and their receptors are increasingly recognized as important pathways for costimulation and/or inhibition of immune responses. Human and mouse microglial cells constitutively express B7 homolog 1 (B7-H1) in vitro. However, under inflammatory conditions [presence of interferon-gamma (IFN-gamma) or T-helper 1 supernatants], a significant upregulation of B7-H1 was detectable. Expression levels of B7-H1 protein on microglial cells were substantially higher compared with astrocytes or splenocytes. Coculture experiments of major histocompatibility complex class II-positive antigen-presenting cells (APC) with syngeneic T cells in the presence of antigen demonstrated the functional consequences of B7-H1 expression on T-cell activation. In the presence of a neutralizing anti-B7-H1 antibody, both the production of inflammatory cytokines (IFN-gamma and interleukin-2) and the upregulation of activation markers (inducible costimulatory signal) by T cells were markedly enhanced. Interestingly, this effect was clearly more pronounced when microglial cells were used as APC, compared with astrocytes or splenocytes. Furthermore, B7-H1 was highly upregulated during the course of myelin oligodendrocyte glycoprotein-induced and proteolipid protein-induced experimental allergic encephalomyelitis in vivo. Expression was predominantly localized to areas of strongest inflammation and could be colocalized with microglial cells/macrophages as well as T cells. Together, our data propose microglial B7-H1 as an important immune inhibitory molecule capable of downregulating T-cell activation in the CNS and thus confining immunopathological damage.
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http://dx.doi.org/10.1523/JNEUROSCI.4794-04.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6725171PMC
March 2005

Type 2 monocyte and microglia differentiation mediated by glatiramer acetate therapy in patients with multiple sclerosis.

J Immunol 2004 Jun;172(11):7144-53

Neuroimmunology Unit, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.

Glatiramer acetate (GA) therapy of patients with multiple sclerosis (MS) represents a unique setting in which in vivo Th2 deviation of T cells is consistently observed and associated with clinical benefit in a human autoimmune disease. We postulated that APCs are important targets of GA therapy and demonstrate that treatment of MS patients with GA reciprocally regulates the IL-10/IL-12 cytokine network of monocytes in vivo. We further show that Th1- or Th2-polarized GA-reactive T cells isolated from untreated or treated MS patients mediate type 1 and 2 APC differentiation of human monocytes, based on their ability to efficiently induce subsequent Th1 and Th2 deviation of naive T cells, respectively. These observations are extended to human microglia, providing the first demonstration of type 2 differentiation of CNS-derived APCs. Finally, we confirm that the fundamental capacity of polarized T cells to reciprocally modulate APC function is not restricted to GA-reactive T cells, thereby defining a novel and dynamic positive feedback loop between human T cell and APC responses. In the context of MS, we propose that GA therapy results in the generation of type 2 APCs, contributing to Th2 deviation both in the periphery and in the CNS of MS patients. In addition to extending insights into the therapeutic mode of action of GA, our findings revisit the concept of bystander suppression and underscore the potential of APCs as attractive targets for therapeutic immune modulation.
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http://dx.doi.org/10.4049/jimmunol.172.11.7144DOI Listing
June 2004