Clinical implications of leukocyte infiltration at the choroid plexus in (neuro)inflammatory disorders

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Clinical implications of leukocyte infiltration at the choroid plexus in (neuro)inflammatory disorders Delphine Demeestere1,2, Claude Libert1,2,3 and Roosmarijn E. Vandenbroucke1,2,3 1 2

Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium

The choroid plexus (CP) is a highly vascularized organ located in the brain ventricles and contains a single epithelial cell layer forming the blood– cerebrospinal fluid barrier (BCSFB). This barrier is crucial for immune surveillance in health and is an underestimated gate for entry of immune cells during numerous inflammatory disorders. Several of these disorders are accompanied by disturbance of the BCSFB and increased leukocyte infiltration, which affects neuroinflammation. Understanding the mechanism of immune cell entry at the CP might lead to identification of new therapeutic targets. Here, we focus on current knowledge of leukocyte infiltration at the CP in inflammatory conditions and its therapeutic implications. Introduction The central nervous system (CNS) is an immunologically privileged site that is maintained by three different brain barriers: the blood–brain barrier (BBB), the BCSFB (see Glossary), and the arachnoid barrier [1]. Barriers comprising fenestrated endothelium and tightly regulated epithelium, such as the BCSFB, are believed to serve as active and selective immune-skewing gates in the steady state, whereas endothelial barriers, such as the BBB, are considered as absolute immunological barriers that block leukocyte entry into the parenchyma [2]. Indeed, immune surveillance is assumed to occur primarily through the BCSFB [2,3]. In neuroinflammatory conditions, such as traumatic brain injury (TBI) and multiple sclerosis (MS), disturbance of these barriers exacerbates CNS inflammation, which is thought to damage the brain parenchyma [2,4–6], and entry of circulating immune cells into the CNS is believed to worsen the damage [2,5,7–12]. Conversely, in some cases, infiltrating leukocytes can be beneficial by supporting CNS repair, for example in spinal cord injury (SCI) [13,14]. Hence, there is a thin line between a beneficial inflammatory response that is necessary for CNS repair and a pathological neuroinflammatory state [4]. Functional characterization of adhesion molecules, chemokines, and chemokine receptors that control leukocyte trafficking might indicate their potential usefulness as therapeutic targets for inflammatory diseases of the CNS [15]. In addition, therapeutic strategies should specifically

Delphine Demeestere is a PhD student at the Flemish Institute for Biotechnology (VIB) in Ghent, Belgium. She studied Biomedical Sciences at the Faculty of Medicine and Health Sciences of Ghent University (Belgium) and graduated in 2011. Her Master thesis was on epilepsy and her current research interests include the choroid plexus in inflammatory disorders. Claude Libert is a group leader at the Flemish Institute for Biotechnology (VIB) in Ghent. He is a Master in Sciences and obtained his PhD at the University of Ghent (UGhent) under the guidance of Walter Fiers. After a post-doc in Italy, he became a group leader at VIB in 1997 and a professor at the University of Ghent in 2003. His major research interest is acute inflammation and the cross-talk between several important players in inflammation including TNF, IFN, matrix metalloproteinases and glucocorticoids. Roosmarijn Vandenbroucke is postdoctoral scientists in Prof. Dr. Libert’s group at the Inflammation Research Center (IRC) in Ghent, Belgium. She graduated as Master in Biotechnology at Ghent University and obtained her PhD in Pharmaceutical Sciences in 2008 on non-viral nucleic acid delivery systems. Her current research focusses on the role of the choroid plexus in inflammatory disorders, including sepsis, aging and neurodegenerative diseases.

Corresponding author: Vandenbroucke, R.E. ([email protected]) 3

These authors contributed equally to this article.

1359-6446/ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drudis.2015.05.003

www.drugdiscoverytoday.com 1 Please cite this article in press as: Demeestere, D. et al. Clinical implications of leukocyte infiltration at the choroid plexus in (neuro)inflammatory disorders, Drug Discov Today (2015), http://dx.doi.org/10.1016/j.drudis.2015.05.003

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Future research should focus more on the underestimated role of the choroid plexus as an active immune-skewing gate in (neuro)inflammatory diseases because better understanding of this process will enable the discovery of new drug targets and therapeutic approaches.

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GLOSSARY

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Blood–cerebrospinal fluid barrier (BCSFB) the CP contains an epithelial monolayer, the choroid plexus epithelium (CPE), that forms the BCSFB. The BCSFB is a physical barrier between the blood and the CSF resulting from the presence of tight junctions (TJs) between adjacent cells, which prevent paracellular transport. Strictly speaking, this term also covers the arachnoidea, one of the brain meninges that similarly forms a brain barrier. Choroid plexus (CP) located in the ventricles of the brain and produces CSF. It comprises a layer of cuboidal CPE cells that form the BCSFB surrounding a core of fenestrated capillaries and loose connective tissue. The CP also acts as a filter, removing (toxic) metabolic waste, foreign substances, and excess neurotransmitters (and their metabolites) from the CSF, thereby helping to maintain the extracellular environment, which is required for optimal brain function. Central memory T cells (TCM cells) antigen-experienced CD4+ and CD8+ T cells that lack immediate effector functions but can mediate rapid recall responses. These cells rapidly develop the phenotype and function of effector memory T cells (TEM cells) after restimulation with an antigen and can circulate through tissues and secondary lymphoid organs. Chemokines cytokines secreted by different cell types, such as immune cells and epithelial cells, that are essential in immune cell influx via the process of chemotaxis. Effector memory T cells (TEM cells) terminally differentiated T cells that lack lymph node-homing receptors but express receptors that enable homing to inflamed tissues. TEM cells can exert immediate effector functions without the need for further differentiation. Epiplexus cell (Kolmer cell) macrophages located at the apical side of the CPE facing the CSF; they are assumed to perform an important immune surveillance function as local APCs [15]. Experimental autoimmune encephalomyelitis (EAE) an experimental model of the human autoimmune disease, MS. In this model, animals immunized with myelin or peptides derived from myelin develop a paralytic disease with inflammation and demyelination in the brain and/or SC (CNS). Helper T cells (Th) mature Th cells express CD4 and have a role in adaptive immunity by secreting cytokines. Proliferating helper T cells can differentiate into two major subtypes: Th1 cells, which are triggered by IL12 and IL2 and produce IFN-g as effector cytokines, and Th2 cells, which are activated by IL4 and release IL4, IL5, IL9, IL10, and IL13 as effector cytokines. M1 and M2 macrophages terms used to describe a continuum of macrophage activity in which classically activated M1 macrophages are pro-inflammatory and alternatively activated M2 macrophages are antiinflammatory. M1 activity is associated with a Th1 response and tissue damage, whereas M2 promotes a Th2 response and tissue repair. Multiple sclerosis (MS) a neurological disease characterized by focal demyelination in the CNS and by leukocyte infiltration. Polymorphonuclear cells (PMNs) leukocytes with a specifically shaped multi-lobed nucleus, also called granulocytes, and categorized as neutrophils, basophils, and eosinophils. Subarachnoid space (SAS) the anatomic space between the pia mater and arachnoid membrane (two brain meninges). It

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contains spongy tissue comprising arachnoid trabeculae and intercommunicating channels filled with CSF. Arachnoid trabeculae comprise delicate connective tissue filaments that extend from the arachnoid mater and blend into the pia mater. Spinal cord injury (SCI) severe traumatic injury to the SC that usually results in sensation disability and paralysis. Traumatic brain injury (TBI) the most common type of TBI is contusion caused by a sudden acceleration or deceleration of the head and characterized by a brief disturbance of neural function.

target leukocyte infiltration that is detrimental, while leaving beneficial immunosurveillance untouched [16]. Furthermore, a better understanding of the role of immune cells in different pathologies could be used to optimize the delivery of therapeutic agents to the site of interest. For example, monocytes and/or macrophages are being engineered for the delivery of antiretroviral drugs and other agents to sites within the CNS [17]. Natural delivery of these cells to damaged regions, guided by secreted chemokines is expected to deliver therapeutic agents selectively and efficiently [17,18]. In this way, a more targeted therapy is possible by bypassing the BCSFB in a ‘natural’ way [18]. Here, we discuss current knowledge on leukocyte infiltration at the CP in inflammatory conditions and its therapeutic implications.

Leukocyte infiltration at the choroid plexus The CP in immunosurveillance The brain has three important brain barriers: (i) the BBB; (ii) the BCSFB; and (iii) the arachnoidea (Fig. 1). The tight junctions (TJs) of the CP epithelium (CPE) form the BCSFB by limiting paracellular transport of molecules and immune cells [2,15,19,20]. Besides this physical barrier, the CP also forms an enzymatic barrier (e.g., drug conjugation) with an important transport function (e.g., efflux transporters) that is important in detoxification [21– 24]. The CP, a highly vascularized organ located in the brain ventricles, produces cerebrospinal fluid (CSF) [15,20]. CPE cells are polarized with microvilli at the apical side and a basolateral labyrinth, both increasing the cell surface area. In contrast to the BBB, formed by the nonfenestrated endothelial cells that contain TJs, the choroidal endothelium is fenestrated and lacks TJs, allowing free communication of the CP stroma with the peripheral blood [15,20]. Recently, the transcriptomes of healthy human and mouse CPE were analyzed [25]. Despite the few differences with respect to transport and metabolic functions, the transcriptomes of human and mouse CPE are similar and display common functionalities, proving the relevance of mouse models to the study of the CP in human neuroinflammatory diseases [25]. The BCSFB is believed to be a ‘permissive’ epithelial gate that enables selective immune cell trafficking and provides active immune-regulating mechanisms to skew immune cells toward specific effector responses (Fig. 2) [2]. These effector responses are characterized by regulatory T cells (Treg), T helper 2 cells (Th2), and alternatively activated macrophages (M2) [2]. In humans, indirect evidence for the entry of T cells into the CNS through the BCSFB came from the observation that T cells in healthy individuals in the ventricular and lumbar CSF are mainly central memory T (TCM) cells, which are cluster of differentiation 4

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(c)

Pia mater

Third ventricle

Neuron

Astrocyte Blood

Capillary endothelial cells

Brain tissue

(b)

Lateral ventricle

Blood–brain barrier (BBB)

(a)

Pericyte Microglia

Fourth ventricle Ventricle

Ependymal cells CSF Epiplexus cell

Basal lamina Blood Fenestrated capillary endothelial cells

Choroid plexus

Blood–CSF barrier (BCSFB)

CPE cell

TJs

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FIGURE 1

Three main brain barriers limit the free movement of cells and molecules between the brain and the periphery. The blood–brain barrier (BBB) (a) is formed by the brain capillaries that comprise endothelial cells containing tight junctions (TJs). The blood–cerebrospinal fluid barrier (BCSFB) (b) comprises the choroid plexus epithelium (CPE), which contains fenestrated endothelium. The CPE is polarized, in that the apical microvilli are in close contact with the cerebrospinal fluid (CSF), whereas the basolateral labyrinth can communicate with the CP stroma. The arachnoid barrier (c) comprises TJ-containing epithelial cells of the arachnoidea, which is one of the brain meninges. Abbreviation: SAS, subarachnoid space.

(CD4+) [20,26,27]. These TCM cells were distinct from the T cell populations in the circulation and in the inflamed brain parenchyma in experimental autoimmune encephalomyelitis (EAE) and MS [20,26,27]. This indicates that the entry of TCM cells into the CSF via the healthy CP is regulated [20]. Immune cells first migrate from the blood across the fenestrated endothelium into the CP stroma to the basolateral side of the CPE cells [15]. Then, after traveling through the stroma, they cross the CPE and enter the CSF at the apical side [20,28]. The healthy CSF contains, besides these CD4+ T cells, very few natural killer (NK) cells and B cells [15]. Furthermore, it has almost no neutrophils but contains small subsets of monocytes [15,18]. Macrophages at the apical side of the CPE, called epiplexus or Kolmer cells, are thought to contribute to the immune component of the BCSFB [29]. Moreover, the presence of dendritic cells (DCs) and CD3+ T cells has been

documented in normal, healthy stroma of the CP [18,30,31]. Also, the CP is rich in anti-inflammatory CX3CR1hi Ly6Clow (M2) monocytes and constitutively expresses the CX3C chemokine receptor (CX3CR) 1 ligand CX3CL1, which is crucial for recruitment and survival of these monocytes [2]. Although leukocytes enter the CSF, under normal conditions they do not invade the parenchyma [2]. Three cell types within the CP have a prominent role in controlling immune cell transmigration to the CSF: the epithelial cells that form the actual barrier, the resident immune cells in the stromal matrix, including macrophages and dendritic cells, and the endothelial cells of the fenestrated capillaries. They can all express adhesion-related molecules, such as integrins and selectins, and contribute to the secretion of chemokines [18]. However, adhesion molecules are barely detected at the CP endothelium,

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Arachnoid trabeculae

SAS

Arachnoid barrier

Dura mater

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Brain tissue

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Macrophage

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T cell (e) Epiplexus cell

Ependymal cell CSF

CPE cell with TJs

Ventricle

(d)

(c)

(b) (a)

Choroid plexus

Stroma

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FIGURE 2

Immunosurveillance at the choroid plexus (CP) and leukocyte transmigration during inflammation. Immune cells from the peripheral blood can freely enter the CP stroma via the fenestrated endothelium (a). Once they reach the basolateral side of the choroid plexus epithelium (CPE), they can migrate to the apical side and into the cerebrospinal fluid (CSF). Depending on the type of immune cell and on host cell factors, they migrate through the tight junctions (TJs) in-between the CPE cells via the paracellular route (b) or through the CPE via the transcellular route (c). When leukocytes enter the CSF during inflammatory conditions (d), they can migrate further into the brain parenchyma by crossing the ependymal cell layer (e) and/or they can migrate to the arachnoidea via the CSF flow. Under normal conditions, immune cells do not infiltrate the brain parenchyma, whereas the CP contains epiplexus cells at the apical side and resident macrophages, dendritic cells (DCs), and T cells in the stroma for immunosurveillance.

suggesting that, in comparison to their role at the BBB, they have a minor role in leukocyte transmigration at the CP. The expression of adhesion molecules and chemokines by the CPE allows substantial control of leukocytes that transmigrate, so targeting the CPE specifically might be the best strategy to modulate leukocyte infiltration during pathological conditions. Finally, resident stromal macrophages and DCs, which function in antigen presentation at the CP, could transmigrate into the cerebral ventricles or migrate back into the peripheral bloodstream after immune activation to serve a variety of immune functions [18].

Key molecules in leukocyte trafficking at the CP Many adhesion molecules, chemokines, and chemokines receptors have been implicated in leukocyte trafficking across the BCSFB [15]. Despite the absence of adhesion molecules at the choroid 4

capillaries during inflammation, leukocytes can freely enter the CP stroma through the fenestrated endothelium [32]. By contrast, during inflammation, the CPE expresses the adhesion molecules intracellular adhesion molecule 1 (ICAM-1), vascular cellular adhesion molecule 1 (VCAM-1), and mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) on its apical surface [4,32]. This localization implies that they are not available for basolateralto-apical migration of immune cells (blood-to-CSF direction), which is contradictory because large numbers of leukocytes are found in the CSF during neuroinflammation [32,33]. However, neuroinflammation can cause the loss of TJ functionality, possibly leading to the loss of CPE polarization and rearrangement of adhesion molecules [34,35]. In this way, immune cells could infiltrate from the basolateral to the apical surface via these adhesion molecules. By contrast, adhesion molecules at the apical

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side of the CP might allow T cells to crawl along the apical surface of the CPE, enabling them to perform immunosurveillance by scanning the apical CPE surface and CSF for potential threats [20]. Additionally, these adhesion molecules might mediate the adhesion of the epiplexus cells that present CNS antigens to T cells during immunosurveillance, ensuring their strategic localization behind the BCSFB [20]. Another adhesion molecule, epithelial V-like antigen (EVA), an immunoglobulin-like adhesion molecule, was identified as a regulator of BCSFB integrity [28]. EVA is expressed at high levels on human CPE cells and, as shown by a monoclonal blocking antibody, it seems to have an important role in CD4+ T lymphocyte adhesion in vitro [28]. Moreover, lymphocyte-deficient mice have reduced lymphocyte immune surveillance associated with decreased expression of EVA at the CP and increased BCSFB permeability [28]. This suggests that lymphocyte immunosurveillance is involved in maintaining the BCSFB. Besides expressing adhesion molecules, the CP synthesizes and releases cytokines [e.g., interleukin 1b (IL1b) and tumor necrosis factor a (TNFa)] and chemokines [e.g., C-X-C motif chemokine ligand (CXCL10) and monocyte chemoattractant protein 1 (MCP1)], which are crucial in the recruitment of immune cells during systemic inflammation in response to bacterial and viral triggers, as well as in the response to injuries and ischemia [36–41]. Therefore, targeting these chemokines would interfere with leukocyte infiltration at the CP and might be useful for the treatment of various ischemic, traumatic, neurodegenerative, and infectious brain disorders [15,16,36]. Macrophages and/or/DCs, CPE cells, and parenchymal cells might all contribute to the secretion of these chemokines [18]. For example, the chemokine CCL20, a ligand for CCR6, is expressed by the CP and has been suggested to mediate the initial step of Th17 cell invasion across the BCSFB into the CNS during EAE [42]. Chemokines are crucial for integrindependent adhesion and transmigration of naive and memory lymphocytes, but the role of extracellular chemokines in the transmigration of lymphocytes is under discussion [43,44]. Research indicates that Th1 and type 1 cytotoxic T cells express integrins that bypass chemokine signals [43]. Furthermore, those cells established a stable arrest on inflamed endothelial barriers, such as the BBB, and the transendothelial migration of these lymphocytes was promoted by multiple endothelium-derived inflammatory chemokines, which were stored in vesicles docked on actin fibers beneath the plasma membranes and locally released within tight lymphocyte-endothelium synapses [43]. Thus, effector T lymphocytes can cross inflamed barriers through contactguided consumption of intraendothelial chemokines without surface-deposited chemokines or extraendothelial chemokine gradients [43]. Hence, this alternative route of leukocyte infiltration at the BBB should also be explored at the BCSFB.

Dual role of leukocyte infiltration Immune cells have a dual role in different pathological conditions; they can worsen neuroinflammation or support recovery. For example, the effects of CCR6 deficiency on the occurrence of neuroinflammatory disease are divergent [15]. The chemokine receptor CCR6 is used by both pathogenic Th17 cells and protective Treg cells [15]. In one study, CCR6-deficient Th17 cells accumulated in the CP and impaired disease initiation, showing the

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beneficial effect of inhibiting the infiltration of immune cells via CCR6 [42]. However, in another study, entry of CCR6-deficient Treg cells into the CNS also decreased, but disease was worsened, indicating that blocking CCR6 can also have adverse effects [14,45]. Another example is seen in interferon g receptor-deficient (IFN-gR / ) mice, in which trafficking of both CD4+ T cells and monocytes to the CSF was impaired during SCI [46]. These mice had a worse recovery following injury, indicating that leukocyte trafficking of certain immune cells is beneficial in SCI [46]. In general, monocytes and macrophages are thought to have an essential role in restoring the functional integrity of the BCSFB by phagocytosing dismantled structural elements and extracellular debris in the injured CP, again implying that infiltrating leukocytes can have a beneficial function [47–49]. By contrast, invasion of inflammatory cells after ischemic injury or TBI can be detrimental to neuronal survival and functional recovery after injury, demonstrating the complexity of immune cell trafficking across the CP during various inflammatory conditions and the need for a specific therapeutic approach [8,10–12].

Leukocyte infiltration at the CP in inflammatory diseases Immune cell trafficking into the CSF varies greatly in response to disease and trauma [18]. However, the cells that traffic into the CSF do not reflect the composition of the immune cells in the blood, again highlighting the importance of the CP as an active immuneskewing gate [18]. Additionally, the profile of immune cells in the CSF can vary during the disease course, pointing to the diagnostic potential of examining the cellular composition of CSF as well as the therapeutic potential of intervening in leukocyte trafficking in a specific and directed way [18]. When therapeutically targeting leukocyte trafficking to the brain, it is important to define whether the event of interest is migration across the BBB to the parenchyma (via the CP to the CSF) or through the arachnoidea to the subarachnoidal space (SAS), because this will affect both the efficacy and safety of the intervention [20]. The relative contribution of these various sources is poorly understood, indicating the importance of studying the different trafficking sites in various inflammatory diseases. Here, we discuss the current knowledge on the involvement of leukocyte migration at the CP during peripheral inflammation, neuroinflammatory diseases, and acute CNS trauma, and the potential drug targets, which are summarized in Table 1 and Fig. 3.

Peripheral inflammation The CP links peripheral inflammation to neuroinflammation by responding to cytokines generated systemically [36,38,40,41]. Systemic inflammatory response syndrome (SIRS) is an exaggerated or unbalanced peripheral immune response leading to organ dysfunction and possibly death. SIRS includes sepsis, which is a well-known peripheral inflammatory disease. However, peripheral immune activation also occurs in autoimmune diseases, such as joint inflammation in rheumatoid arthritis, as well as in metabolic disorders, such as diabetes and obesity [36,50]. In addition, systemic inflammation is starting to be recognized as a prominent feature of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases [51]. Clearly, peripheral inflammation is a broad concept because of its involvement in numerous diseases. A

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TABLE 1

Overview of important observations at the CP regarding leukocyte infiltration during (neuro)inflammatory diseases, which could lead to the identification of possible therapeutic targets

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Pathology

Model

Species

Target molecule

Observation

Potential therapeutic strategy

Refs.

Peripheral inflammation

Hind paw inflammation Systemic LPS (i.p.)

Rat

MCP1

Block MCP1

[55]

Mouse; rat Mouse

MCP1 Leptin

High basal expression, elevated at CP during peripheral inflammation Elevated expression at CP Reduction of leptin correlated with decreased ICAM-1 expression and reduced neutrophil recruitment to brain

Block MCP1 Reduce leptin; block ICAM-1; reduce neutrophil influx

[54] [56]

Bacterial meningitis

In vitro CPE S. suis

Porcine

CD11b/CD18 (Mac1)

Inhibition of transmigration of neutrophils with integrin-specific antibodies

Inhibit neutrophil influx; integrin-blocking Abs; block CD11b/CD18

[59]

TBI

Controlled cortical impact model

Rat

JNK

Rat

ICAM-1

Block JNK; inhibit neutrophil chemoattractants; reduce neutrophil influx Inhibition of ICAM-1 (Ab);block neutrophil recruitment

[101]

Feeney’s weightdrop model

Selective JNK inhibitor (i.p.) decreases posttraumatic production of neutrophil chemoattractants by CP and reduces influx of neutrophils ICAM-1-inhibiting antibody blocked significant portion of neutrophil recruitment 24-h post trauma

Moderate contusive centralized injury

Mouse

IFN-gR; ICAM-1; CXCL9; CXCL10

IFN-gR-deficient mice fail to elevate expression of ICAM-1, CXCL9 and 10 at CP, resulting in reduced T cell and monocyte entry, and have impaired recovery

[46]

Moderate contusive centralized injury

Mouse

VCAM-1; VLA4; CD73

Blocking VCAM-1/VLA4 interaction or interrupting CD73 activity with blocking antibody or chemical compound inhibits M2 macrophage recruitment, leading to impaired recovery of motor function

Stimulate IFN-gR pathway; induce ICAM1; stimulate production of CXCL9 and 10; stimulate T cell and monocyte entry (only first phase?) Block VCAM-1/VLA4; block CD73 activity; stimulate M2 macrophages

Passive EAE

Mouse

CCL20/CCR6 interaction

CCR6-deficient mice protected against EAE by inhibition of migration of Th17 cells across BCSFB CCR6-deficient mice develop more severe chronic EAE Increased expression at CP

Block CCR6; inhibit Th17 influx

[75]

Stimulate CCR6

[14,45]

Block adhesion molecules Mimic Sdc-1 function

[33]

SCI

MS

Active EAE (MOG) Active EAE

Mouse

Active and passive EAE

Mouse

ICAM-1; VCAM-1; MadCAM1 Sdc-1

Sdc-1 KO mice have enhanced disease severity and impaired recovery

[104]

[13]

[76]

Abbreviations: KO, knockout; VLA, very late antigen.

variety of experimental models, particularly of fever, hypothalamus–pituitary–adrenal gland (HPA) axis activation and sickness behavior, have been used to investigate the communication between the peripheral immune system and the brain [52]. Nonetheless, the response to bacterial lipopolysaccharides (LPS) is the most widely used model for mimicking peripheral inflammation because it produces a robust response [52]. It has been shown that systemic inflammation caused by intraperitoneal (i.p.) injection of LPS induced BCSFB leakage that was dependent on matrix metalloproteinase (MMP) activity [38]. Moreover, systemic immune activation by i.p. injection of Staphylococcus aureus enterotoxin B (SEB) was shown to increase T cell numbers at the CP [53]. It is known that both MCP1 and leptin, a hormone that has a role in satiety, are involved in leukocyte recruitment at the CP, but the link between them remains unclear [36]. Systemic injection of LPS and inflammation of the hind paw induced by bilateral injection of carrageenan Lambda type IV into the plantar surface both led to elevated expression of the 6

chemokine MCP1 at the CP [54,55]. Furthermore, systemic or central (intracerebroventricular; i.c.v.) administration of leptin upregulated ICAM-1 expression on the meninges and CP [56]. ICAM-1 is involved in neutrophil infiltration into the brain [57]. Following i.p. injection of a septic LPS dose, leptin was also directly or indirectly involved in neutrophil infiltration by acting on the leptin receptors on the CP and leptomeninges [56]. Reduction of leptin expression by food deprivation or antibody neutralization, as well as in leptin-deficient mice, reduced LPS-induced leukocyte recruitment to the brain [56]. Clearly, both leptin and MCP1 could mediate some deleterious effects of systemic inflammation on the brain by controlling leukocyte infiltration, making them possible targets in the development of therapeutic strategies (Fig. 3). Not only acute inflammatory stimuli, but also chronic peripheral inflammation can affect the CP [39,41]. Gene expression profiling of the mouse CP after a single systemic LPS injection or after multiple injections showed upregulation of adhesion molecules [ICAM-1, MAdCAM-1, and glycosylation-dependent cell

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CCL20

Neutrophil

Leptin receptor

Neutrophil (CD11/CD18)

MadCAM-1

Neutrophil (Mac-1)

ICAM-1

T cell

VCAM-1

Th17 cell (CCR6)

CD73

T cell (α4 integrin)

Bacteria

Treg cell

Virus

Th1 cell (VLA4)

Choroid plexus epithelial cell with TJs

M2 macrophage

Ependymal cell

Chemokines CXCL10 CXCL11 Leptin

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MCP1 IFNγ

(f) (e) (a) Choroid plexus (d) (b)

(c)

Ventricle

Brain tissue Drug Discovery Today

FIGURE 3

Overview of possible therapeutic targets regarding leukocyte infiltration across the blood–cerebrospinal fluid barrier (BCSFB) in different (neuro)inflammatory conditions. Systemic inflammation (a) is accompanied by neutrophil influx induced by the release of the chemokine monocyte chemoattractant protein 1 (MCP1). Leptin can induce intercellular adhesion molecule (ICAM)-1 expression by binding the leptin receptors at the choroid plexus (CP). In experimental autoimmune encephalitis (EAE) (b), it is suggested that the initial step is characterized by T helper (Th)-17 cell invasion, which depends on the interaction between chemokine (C-C motif) ligand 20 (CCL20) and C-C chemokine receptor type 6 (CCR6). The subsequent CCR6-independent parenchymal invasion of T cells occurs at the blood– brain barrier (BBB) and is mediated by a4-integrin. Syndecan-1 (Sdc-1) has a role in the interaction of ICAM-1 with the leukocyte integrin CD11/CD18. Bacterial meningitis (c) is dominated by neutrophils that can migrate depending on the surface integrin receptor Mac-1. The bacteria causing meningitis can enter the central nervous system (CNS) from the bloodstream via the BCSFB and/or BBB. Viral meningitis (d) is associated with T cell migration at the BBB and BCSFB. The chemokines CXCL10 and CXCL11 are upregulated in the CSF of children with viral meningitis. Interferon (IFN)-g has been implicated in spinal cord injury (SCI) (e) by (in)directly affecting ICAM-1 expression, which results in T cell and macrophage entry into the CSF. Th1 cells are believed to activate the recruitment of M2 macrophages, and this depends on the interaction between vascular cell adhesion protein (VCAM)-1 and very late antigen-4 (VLA4) and on CD73 activity. These macrophages regulate the recruitment of regulatory T (Treg) cells and are essential for recovery. In traumatic brain injury (TBI) (f), neutrophils infiltrate the CP, BBB, and the brain parenchyma via ICAM-1 and by chemokine production. Abbreviations: PMN, polymorphonuclear cells; TJ, tight junction.

adhesion molecule I (GlyCAM-1)] and chemokines [CC chemokine ligand 5 (CCL5), CCL2, CCL7, and CXCL1], indicating a role for leukocyte trafficking in both acute and chronic inflammation [39,41]. However, the number of cells in the CSF did not increase

after chronic peripheral inflammation, implying that, during repeated exposure to low-grade peripheral inflammation, entry of immune cells does not occur or is limited [39]. Whether leukocyte influx into the CSF occurs during acute peripheral inflammation

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was not investigated in this study. It should be noted that the gene expression analysis was performed on the complete CP and, consequently, the results apply to the CPE cells as well as to other cells, such as endothelial cells and macrophages.

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Neuroinflammation CNS inflammation has numerous causes, including bacterial infections and MS [50]. Neuroinflammation is accompanied by BBB disturbances, allowing the influx of inflammatory cells and tissuedestructive products [50]. Moreover, inflammatory cytokines are elevated in both serum and CNS [50]. Given that the role of the BCSFB in infiltration of leukocytes during neuroinflammation is understudied and might be underestimated, we summarize the current knowledge on this topic with a focus on meningitis and MS, to highlight possible shortcomings.

Meningitis Bacterial meningitis, also called septic meningitis, is a life-threatening disease in which bacteria cross either the endothelium of the BBB or the epithelium of the BCSFB [58]. For example, Neisseria meningitidis is a Gram-negative, invasive pathogen that frequently colonizes the nasopharynx [58]. However, in a small percentage of patients, it enters the bloodstream and penetrates the CNS via the BCSFB and BBB to cause meningitis [58]. The presence of N. meningitidis in vessels close to the CP suggests that bacteria reach the CSF through the CPE, but there is still no direct evidence for this [58]. Additionally, it has been suggested that Streptococcus suis, which also causes a disease resembling bacterial meningitis, enters the brain via the BCSFB [59]. In vitro studies using integrin-specific antibodies that inhibit transmigration of neutrophils across CPE cells after S. suis infection showed that its entry is dependent on CD11b/CD18 (Mac-1) [59]. This suggests that Mac-1, a surface integrin receptor, is a potential drug target in septic meningitis (Fig. 3). PMNs (e.g., neutrophils) and monocytes dominate bacterial meningitis, whereas lymphocytes (especially T lymphocytes) are increased in viral CNS infections [18,60–64]. Enterovirus (EV) is the most common pathogen causing viral meningitis, which is less fatal than septic meningitis and is associated with T cell migration at the BBB and BCSFB [44,65]. In the CSF of children with aseptic EV meningitis, concentrations of the chemokines CXCL10 and CXCL11 are increased, implying leukocyte infiltration [44]. However, EV30 infection of an in vitro CPE culture led only to a minor enhancement of T lymphocyte transmigration [44]. From a therapeutic point of view, an interesting study was done in a cytokine-induced meningitis model, in which mice were systemically injected with monoclonal antibody BV11 to block the junctional adhesion molecule (JAM) [66]. Antibody-treated mice displayed attenuated disease, by inhibition of leukocyte accumulation in the CSF and brain parenchyma infiltration [66]. Given that JAMs are selectively concentrated at the TJs of both endothelial and epithelial cells, the individual roles of the BBB and BCSFB are not clear. Moreover, the overall individual contributions of the two barriers to the development of viral and bacterial meningitis are unknown. Furthermore, most studies investigating meningitis use in vitro models, so there is a lack of in vivo evidence for immune cell trafficking at the brain barriers, in particular the BCSFB, during this disease. 8

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MS EAE is a T cell-mediated model for MS that is induced either by immunization with myelin antigens (active EAE), such as myelin oligodendrocyte glycoprotein (MOG), or by transfer of myelin antigen-specific CD4+ Th cells to susceptible animals (passive EAE), including Lewis rats and certain strains of mice [16]. These autoaggressive CD4+ T cells are activated outside the CNS, then migrate into the CNS, where they initiate the molecular and cellular events leading to edema, inflammation, and demyelination in the CNS white matter [33]. In EAE, two T cell entry events are involved: (i) T cells cross the CP to reach the meninges, where they are restimulated by local myeloid antigen-presenting cells (APCs); and (2) meningeal infiltration is amplified and the parenchyma is invaded from the meninges, followed by activation of the BBB [15]. This indicates that the BCSFB should be targeted to prevent the initiation of MS, whereas its therapeutic treatment should focus on the BBB. For example, inhibition of EAE development was achieved by blocking a4-integrin-mediated T cell migration across the BBB [16,67]. Although this treatment was effective in the clinic for relapsing-remitting MS, it was associated with a risk of developing progressive multifocal leukoencephalopathy (PML), which is a fatal disease of the CNS caused by viral infection of the oligodendrocytes [68–71]. This risk increased with the duration of the therapy, which means that therapeutic targeting of a4-integrins may ultimately impair the beneficial immunosurveillance of the CNS [16]. It is unknown whether similar pathogenic processes occur in EAE animal models and patients with MS, but it is well documented that immune cells, such as T cells (including various Th subsets), B cells, and macrophages, are present in the CSF of patients with MS [35,72]. Alterations at the level of the BCSFB leading to an inflammatory environment in the CSF may initiate disease and precede relapses [35]. Nonetheless, the specific role of the CPE in the development of clinical symptoms and immune cell trafficking in MS remains unknown [35]. The CP expresses CCL20, a CCR6 ligand that is believed to mediate the early step of CNS invasion by pathogenic Th17 cells across the BCSFB in EAE and is thought to trigger the subsequent CCR6-independent parenchymal invasion, which is associated with disease onset and peak severity [2,42,73,74]. However, the role of this initial Th17 cell invasion via the CP to the brain remains controversial. No IL17-producing CD4+ T cells were found at the CP and the blood under naı¨ve conditions and IL17 could not induce trafficking molecules, except for CCL20 [46]. This indicates that the initial entry of Th17 cells to the brain does not automatically involve the CP. Furthermore, CCR6-deficient mice were protected against EAE because CCR6/CCL20-mediated migration of Th17 cells into the inflammatory tissue and across the BCSFB is reduced [42,75]. Thus, it has been hypothesized that CCL20/CCR6 binding is only relevant for EAE induction by the first wave of pathogenic Th cells through the CP, suggesting that targeting CCR6 or CCL20 would only be useful if activated T cells cross the BCSFB during MS progression [16,42]. Conversely, another study reported a worse outcome of EAE in CCR6 / mice [14,45]. Moreover, CCR6 is expressed on other cell types, thus, blocking CCR6 or its ligand is not necessarily protective in EAE and might also impair migration of protective immune cells into the CNS, leading to adverse effects [14,16,45].

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During EAE, the adhesion molecules ICAM-1 and VCAM-1 are increased, whereas the expression of MAdCAM-1, which is absent under basal conditions, is initiated at the apical side of the CPE [33]. This polarized localization of these adhesion molecules at the CPE indicates its importance in immunosurveillance [20,33]. Syndecan-1 (CD138) is a cell-surface heparin sulfate proteoglycan expressed by epithelial cells, such as the CPE [76]. It modulates the activity of chemokines, cytokines, integrins, selectins, and other adhesion molecules, and is assumed to be a negative regulator of various inflammatory processes [76,77]. Syndecan-1 deficient (Sdc-1 / ) mice show exacerbated disease symptoms in several inflammatory models, such as hypersensitivity and dextran sulfate sodium (DSS)-induced colitis, in which enhanced immune cell infiltration was reported [77,78]. In Sdc-1 / mice, EAE is more severe and recovery is impaired [76]. An in vitro adhesion assay showed that the increased adhesion of Sdc1 / leukocytes to ICAM-1 was inhibited by a CD18-blocking antibody, which interferes with the binding of ICAM-1 to its leukocyte integrins (CD11/ CD18) [77]. Therefore, Sdc-1 and CD11/CD18 have been proposed as novel MS targets in anti-inflammatory therapy [77]. The ultimate goal in the battle against MS is to prevent disease onset, for example by interfering with the CCR6/CCL20 interaction. One technique that might identify patients at risk of developing MS is an MRI-based method in which very small superparamagnetic iron oxide particles (VSOPS) are used to track phagocytic cells, namely macrophages [79]. These VSOPS can discriminate between inflammatory events occurring in EAE and, more importantly, they can detect CNS alterations that precede immune cell infiltration and clinical onset [79]. However, more research is needed to clarify the sensitivity and specificity of these VSOPS in predicting the onset of MS. Nevertheless, using this technique to identify patients at risk of developing MS could give us a window of opportunity to prevent the disease.

Acute CNS trauma Acute CNS injuries, including TBI and SCI, have a similar pathophysiology and are common causes of human disabilities and deaths [80–84]. The pathophysiological events are still not fully elucidated and effective pharmacotherapies are not available [85– 87]. Acute CNS trauma causes neuropathology, including infiltration of immune cells, neuronal and glial cell death, mitochondrial dysfunction, and inhibition of axon regeneration [88]. The result is a tissue environment that favors cell death and inhibits repair [89–91]. Innate immune cells, such as phagocytic macrophages, might have an important role in repair by removing debris. Therefore, activating repair mechanisms by targeting the infiltration of macrophages could be of therapeutic importance. By contrast, invasion of other inflammatory cells after the insult can have a detrimental effect on the functional recovery after injury, indicating that blocking the infiltration of nonphagocytes (e.g., neutrophils, monocytes, T cells, etc.) can be beneficial [7,8,92]. Nonetheless, it remains difficult to categorize a certain immune cell as beneficial or detrimental in a certain disease state because they can be both, depending on localization and/or timing.

SCI SCI is severe traumatic injury to the spinal cord (SC) that usually results in sensation disability and paralysis [87,93]. There is

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currently no effective therapy [87]. Pathologically, SCI can be divided into primary injury, in the form of tissue damage and bone fractures directly caused by intense forces, and secondary injury phases, which includes the inflammatory lesion resulting from the primary injury [94–96]. Given that the primary injury is always unexpected, the SC can be protected or rescued only during the secondary injury phase [95]. However, the mechanisms underlying secondary injury are not fully defined [93–96]. Recovery following SCI is impaired in IFN-gR / mice, and this impairment is linked to reduced expression of the trafficking molecule ICAM-1 and the chemokines CXCL9 and -10 by the CP, which results in reduced T cell and monocyte entry into the CSF [46]. There was no systemic reduction in immune cell numbers, indicating that it is leukocyte trafficking at the CP that is beneficial during SCI [46]. In such cases, stimulation of leukocyte migration at the CP might be beneficial. Outside the CNS, the phenotype of macrophages derived from circulating monocytes homing to injured tissues reflects two phases [13]. The initial phase is characterized by CX3CR1loLy6chi macrophages corresponding to the ‘classically activated’ (M1) cells, which have been shown to have pro-inflammatory, phagocytic, and proteolytic functions essential for the digestion of damaged tissue and removal of debris [13]. The second phase is associated with CX3CR1hiLy6clo macrophages, ‘alternatively activated’ (M2) macrophages, which are anti-inflammatory and participate in tissue regeneration, growth, angiogenesis, and matrix deposition, thereby supporting tissue remodeling [97,98]. It is still unclear whether these distinct macrophage populations are generated by monocyte recruitment in two waves or result from in situ phenotypic conversion of the already recruited cells [97,98]. In the traumatized SC, two similar monocyte populations corresponding to M1 and M2 macrophages have been found [13]. M1 macrophages were found to be derived from macrophages that entered the traumatized SC via the CCL2 chemokine, through the adjacent leptomeninges, whereas M2 macrophages came from monocytes that trafficked through the CP [13]. The study further demonstrated that the CP entry route is an M2-supporting milieu [13]. After SCI, the M2 macrophages, which are essential for recovery, were recruited via the CP to the site of injury without breakdown of the BBB [13]. Blocking the VCAM-1–VLA4 interaction with a blocking antibody or interrupting CD73 activity by a chemical compound inhibited the recruitment of M2 macrophages and impaired the recovery of motor function [13]. By contrast, stimulation of M2 macrophage recruitment via the CP might have therapeutic effects. In a recent study, mice deficient in IFN-g-producing T cells failed to activate the CP and their ability to recruit inflammation-resolving M2 monocytes after SCI was limited [99]. These monocytes regulate the recruitment of Treg cells, which are necessary for tissue remodeling, to the SC parenchyma [99]. In conclusion, that study demonstrated that both Th1 effector cells and Tregs are needed at discrete locations and with distinct kinetics for recovery following SCI [99]. This again highlights the importance of developing a temporally and spatially controlled therapeutic intervention. TBI The most common type of TBI is contusion, which is characterized by a brief disturbance of neural function provoked by a sudden acceleration or deceleration of the head and is

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associated with a brief loss of consciousness and amnesia [100]. TBI frequently results in neuroinflammation, which includes neutrophil invasion [7,8]. After TBI, neutrophils infiltrate the CP and accumulate in the CSF space near the injury, from where they can migrate into the brain parenchyma [7]. An association has been demonstrated between the cortical production of neutrophil and monocyte chemoattractants, the magnitude of influx of inflammatory cells, and the extent of neural tissue loss after TBI [8]. Given that post-traumatic neuroinflammation progresses slowly, there is a window for anti-inflammatory intervention in TBI [101]. Different treatments to counteract neutrophil influx into the injured brain exhibited several beneficial effects [101–103]. For example, in a stroke model, application of an antibody against the cytokineinduced neutrophil chemoattractant (CINC) reduced the infarction size 7 days after cerebral ischemia-reperfusion (I/R) [103]. In rabbits subjected to cerebral I/R, using an antibody to neutralize IL8, a potent neutrophil chemotactic cytokine, significantly reduced infarct size compared with a control antibody [102]. In both latter studies, it is unclear whether neutrophils invaded predominantly at the BCSFB or at the BBB. However, one study showed that intraperitoneal administration of a selective c-Jun N-terminal kinase (JNK) inhibitor in vivo decreased post-traumatic production of neutrophil chemoattractants at the CP and reduced the influx of neutrophils across the BCSFB after TBI [101]. During the acute inflammatory reaction in TBI, the adhesion molecule ICAM-1 is upregulated at the BBB and its upregulation persists for a long time [104]. A murine monoclonal antibody that inhibits the adhesive function of ICAM-1 blocked a substantial portion of neutrophil recruitment 24 h after trauma (peak infiltration at 48 h) [104]. Given that neutrophils were shown to emigrate predominantly from vessels within the leptomeninges and CP, neutrophil infiltration into the brain seems to occur at the arachnoidea and the BCSFB, respectively [104]. Targeting these adhesive interactions between neutrophils and ICAM-1 could be used therapeutically to prevent the acute inflammatory response in TBI [104]. Although indirect evidence indicates that the CP is one of the entry gates for neutrophils during TBI, it is unclear which brain barrier primarily mediates the inhibition of neutrophil infiltration when administering this ICAM-1-blocking antibody. Given that the magnitude of influx of inflammatory cells is associated with the extent of neural tissue loss after TBI, inhibition of neutrophil infiltration by this antibody should be protective [8].

Therapeutic approaches and caveats It is increasingly believed that the CP has a leading role as an immune-skewing gate in immune cell entry into the brain. Efforts to identify new therapeutic targets should include greater focus on the role of the CP. However, special care should be taken when targeting inflammatory cells at the brain barriers. For example, blocking a4-integrin-mediated T cell migration across the BBB was protective and, therefore, was translated to the clinic for treatment of relapsing-remitting MS [16]. However, serious adverse events that were probably related to impairment of the beneficial immunosurveillance were observed [16]. Consequently, the goal is to identify the detrimental and beneficial roles of the different immune cell subsets and their mechanisms of action in each neuroinflammatory disease separately, at the time point at which therapeutic intervention is possible. In this way, not only new 10

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therapeutic targets can be proposed, but the occurrence of certain adverse effects could also be predicted. Therefore, therapy targeting immune cells should be specific, local, and well timed. In most neuroinflammatory conditions, for example after TBI, chemokine expression at the CP increases gradually [7,105,106]. Moreover, an increase in chemokine expression was found after TBI in both the ipsilateral CP and the ipsilateral cerebral cortex that peaked at the same time after injury [7]. Nevertheless, the time course of neutrophil influx into these brain regions differed, which suggests that factors other than chemokine synthesis, such as different time courses for induction and expression of cell adhesion molecules on the CPE and brain capillaries, account for different kinetics of neutrophil migration to the CP and traumatized brain parenchyma [7]. Furthermore, it was shown in CP explants that labeled macrophages and/or DCs rapidly expanded and migrated to the apical surface of the epithelium, and that this migration was dependent on the presence of epithelial cells but not on the experimental chemokine gradient [18]. Both observations indicate the importance of an orchestrated interplay between chemokines, adhesion molecules, and other factors produced by the inflammatory, epithelial, and endothelial cells of the CP. A better understanding of these interactions will give more insight into how leukocyte infiltration could be modulated for therapeutic purposes. Although integrins, selectins, and chemokines are the most frequently targeted molecules to interfere with leukocyte infiltration, other targets should be considered. Adhesion molecules expressed by the CPE enable attachment and subsequent transmigration of inflammatory cells. Moreover, MMPs can degrade components of the extracellular matrix (ECM) (increasing the accessibility of the CPE), TJs (allowing paracellular migration), and/or chemokines (leading to their activation or deactivation), and thereby mediate both transcellular and paracellular diapedesis. For example, it was shown in an in vitro model of the BCSFB that TNFa induces the expression of adhesion molecules (ICAM-1 and VCAM-1) and MMPs, accompanied by barrier disruption[107]. This effect was attenuated by administration of a broad-spectrum MMP inhibitor [107]. Furthermore, it was reported that systemic inflammation induced MMP-dependent BCSFB leakage as a result of cleavage of collagen type I, a component of the ECM [38]. MMPs also cleave a broad range of other substrates, including TJs and chemokines, so the physiological relevance of MMP inhibition to leukocyte transmigration remains speculative [108]. Given that MMPs are responsible for both in vitro and in vivo BCSFB disruption, one can expect an increase in paracellular transmigration of leukocytes at the CP in cases of increased MMP activity. Additionally, MMPs also indirectly facilitate higher influx of leukocytes into the CP stroma by modulating the ECM, thereby making the CPE more accessible. For example, a recent study in parasitic meningitis supports a role for an increased MMP9 activity in fibronectin proteolysis, leading to BCSFB permeability and an augmented eosinophilic cell count in the CSF [109]. Given that TJs are possible substrates for MMPs, and considering the link between TJ disturbance and BCSFB impairment, the role of TJs in leukocyte transmigration should be explored further. An in vivo study showed that mice deficient in the TJ molecule claudin 3 (CLDN3) have a weakened barrier and experience a more rapid onset of exacerbated clinical signs upon the induction of EAE [35].

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Aggravation of the course of the disease coincided with enhanced levels of leukocyte infiltration in the CSF of these mice [35]. For those reasons, MMPs, ECM molecules, and TJs should be considered as possible (in)direct therapeutic targets in leukocyte migration. Improving the stability of ECM and/or TJ components, such as by preventing its breakdown, could diminish BCSFB disruption, hereby decreasing detrimental leukocyte infiltration and improving clinical outcome. Both the ECM and TJs have gained more interest as therapeutic targets in cancer and MS [110–114]. Targeting of the cell–ECM interface was successfully done in cancer because abnormal expression of integrins and their ECM ligands promotes cell proliferation, migration, and differentiation [113]. Blocking peptides and antibodies against integrins, which are transmembrane receptors for ECM proteins, are used in clinical trials for prostate cancer [113]. Additionally, tumor growth was suppressed in mice by targeting monoclonal cytotoxic antibodies to cells that specifically express CLDN3 and -4, because these TJ components are increased in tumors [115–117]. In EAE, therapeutic stabilization of the BBB by a synthetic small molecule inhibitor for protein kinase Cb, which is currently under investigation in clinical trials for cancer treatment, was shown to suppress transmigration of activated T cells across the BBB and was accompanied by the induction of TJ molecules (ZO1, CLDN3, and -5) [111]. Considering the possibility of targeting TJs or ECM molecules in cancer therapy and EAE, the lack of knowledge on targeting them specifically at the CP in view of leukocyte transmigration requires attention. Furthermore, even though this is beyond the scope of this review, we want to highlight briefly the importance of the BCSFB in viral entry. First, paracellular viral entry occurs via passive diffusion and/or via infected-leukocytic trafficking (‘Trojan Horse’) with or without brain barrier breakdown [118–123]. This indicates that leukocyte influx at the brain barriers, including the BCSFB, could be therapeutically targeted in viral CNS infection to prevent the influx of virus particles or virus-infected leukocytes. Second, TJ components can have a direct role in viral entrance. For example, CLDN1 is used as a co-receptor during viral infection to mediate entry in the liver [124–126]. During rotavirus infection, JAM-A was shown to function as a co-receptor for virus entrance in epithelial kidney cells [126]. This points toward a possible, yet unstudied, therapeutic approach in which TJ proteins could be targeted to prevent viral entry at the BCSFB during viral infection in the brain (e.g., viral meningitis). As a result of an increase in the identification of new immune cell markers and the ‘discovery’ of new leukocyte subtypes, the complexity of immune cell classification is increasing and impeding the discovery of new therapeutic targets in leukocyte infiltration [127]. Some markers only reflect the activation state of a certain kind of immune cell, making it more difficult to categorize a certain subtype as beneficial or detrimental [88,97]. For example, the characterization of M1 macrophages as ‘bad’ and M2 as ‘good’ is too simplistic because both types have important functions [88]. However, this nomenclature is widely used to describe macrophage activation states and is an imperfect classification that reflects a continuum of macrophage activation states [88,128]. Most likely, prolonged imbalance in their ratios causes pathology [88]. Thus, therapeutic targeting should focus on the ‘rebalancing’ of these two phenotypes in the injured CNS [88].

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To target the CP therapeutically, attempts are being made to identify peptides that specifically target the CPE [129,130]. This could be useful for the delivery of compounds that reduce brain inflammation and leukocyte entry into the brain [130]. Direct therapeutic targeting of the CPE might be achieved by conjugating such peptides with the ligand of interest. Moreover, the CP produces CSF, thereby offering an opportunity to manipulate CP cells to deliver compounds to the brain through the CSF [36]. By targeting the CP, the molecules released might have a more-limited brain distribution, which restricts possible adverse effects. In another CPbased therapeutic approach, stem cells were used to generate CPE cells that can integrate into the host CP after intracerebroventricular injection [131]. Generating and engineering CPE cells for the production of high levels of therapeutic proteins or peptides for a natural, safe, and long-term delivery into the CNS could be an attractive possibility for treating a range of (chronic) CNS diseases [131,132]. Besides CPE cells, monocytes and macrophages could also be useful for drug delivery because they are being engineered for the delivery of antiretroviral drugs and other agents to sites within the CNS [17]. This natural targeting strategy enables a selective and effective delivery of therapeutic agents, guided by secreted chemokines to damaged regions [17,18]. Finally, when developing new therapeutic strategies that target CNS leukocyte infiltration in neuroinflammatory diseases for older people, one should consider the process of aging, which is defined as a slow deterioration of homeostatic functions throughout the lifespan of an organism [133]. In the adult brain, there is a balance between pro- and anti-inflammatory cytokines, but with increased age this balance is shifted toward a Th2-like pro-inflammatory state [133,134]. This chronic state of low-grade neuroinflammation in the aged brain makes it more vulnerable to the disruptive effects of both intrinsic and extrinsic factors, such as disease, infection, and stress, and requires special attention when developing new therapeutics for the aged population [133]. Recently, a type I IFN (IFN-I) signature, induced by brain-derived signals present in the CSF, was documented in the aged CP [135]. Given that IFN-I (negatively) affects type II IFN (IFN-g)-dependent genes, this leads to a reduced expression of homing and trafficking molecules that are required for leukocyte entry at the CP into the CSF under physiological conditions during aging [46,135]. This reduction in CP activity was also present in IFN-gR / mice [46,135]. Also, blocking IFN-I with neutralizing antibodies restored IFN-g-dependent CP activity and, consequently, immune surveillance [46,135]. In agreement with this, the absence of IFN-g is reported to be detrimental in EAE and SCI by inhibiting leukocyte infiltration in the CNS [46,136,137]. Furthermore, IFN-g is needed to suppress infiltration of Th17 and is believed to decrease susceptibility in EAE development [138]. By contrast, IFN-b treatment improves EAE by impeding leukocyte influx [139]. For these reasons, identification of particular IFN effector genes responsible for the beneficial therapeutic properties is necessary. Given that these IFN effector genes might differ between various cell types and considering the lack of information concerning the role of IFNs at the CP in the context of leukocyte infiltration in neuroinflammatory diseases, this poses another interesting field of investigation that could point toward new therapeutic targets. However, care should be taken when targeting the IFN pathway, because the IFN-related reduction in immune surveillance at the

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CP during aging is accompanied by brain functional deterioration, which could be (partially) restored by neutralizing IFN-I signaling [135]. In addition, transgenic mice deficient in IFN-II signaling have impaired physiological leukocyte trafficking across the CP associated with cognitive decline and restricted hippocampal neurogenesis [135]. Therefore, one should keep in mind that chronic administration of drugs targeting the IFN pathway could influence the physiological immune surveillance at the CP and/or cause adverse effects, such as cognitive deterioration.

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Here, we discussed different aspects that should be considered when developing strategies modulating leukocyte migration, such as the dual role of leukocytes. Given that the CPE is in close contact with the CP stroma on one side and the CSF on the other side, communication across these interfaces should receive more attention. Further characterization of immune cell influx mechanisms and functions of leukocyte subtypes and their adhesion molecules, chemokines, chemokine receptors, and other leukocyte migration-related molecules, such as MMPs and TJs, will lead to improved therapeutic strategies in (neuro)inflammatory diseases.

Concluding remarks and future perspectives A large body of evidence indicates that targeting leukocyte infiltration is a promising strategy in the treatment of a range of (neuro)inflammatory diseases. Immune cells transmigrate into the brain via different routes (BBB, arachnoidea, and CP), but the role of the CP is heavily understudied. Most research has been done on modulating immune cell trafficking across the endothelial BBB in view of therapy, so information on the BCSFB is limited [16,66,102,103].

Acknowledgements D. D., C. L., and R. E. V. are supported by the Agency for Innovation by Science and Technology (IWT), Research Foundation – Flanders (FWO), the Concerted Research Actions (GOA) of Ghent University, and the Belgian Science Policy (Interuniversity Attraction Pools – IAP7/07). We thank Amin Bredan for careful editing of the manuscript.

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120 Verma, S. et al. (2010) Reversal of West Nile virus-induced blood–brain barrier disruption and tight junction proteins degradation by matrix metalloproteinases inhibitor. Virology 397, 130–138 121 Garcia-Tapia, D. et al. (2006) Replication of West Nile virus in equine peripheral blood mononuclear cells. Vet. Immunol. Immunopathol. 110, 229–244 122 Fletcher, N.F. et al. (2011) The neuropathogenesis of feline immunodeficiency virus infection: barriers to overcome. Vet. J. 188, 260–269 123 Bragg, D.C. et al. (2002) Infection of the choroid plexus by feline immunodeficiency virus. J. Neurovirol. 8, 211–224 124 Evans, M.J. et al. (2007) Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446, 801–805 125 Farquhar, M.J. et al. (2012) Hepatitis C virus induces CD81 and claudin-1 endocytosis. J. Virol. 86, 4305–4316 126 Torres-Flores, J.M. et al. (2015) The tight junction protein JAM-A functions as coreceptor for rotavirus entry into MA104 cells. Virology 475, 172–178 127 Ruck, T. et al. (2013) CD4+ NKG2D+ T cells exhibit enhanced migratory and encephalitogenic properties in neuroinflammation. PLOS ONE 8, e81455 128 Mosser, D.M. and Edwards, J.P. (2008) Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8, 958–969 129 Baird, A. et al. (2011) Targeting the choroid plexus–CSF–brain nexus using peptides identified by phage display. Methods Mol. Biol. 686, 483–498 130 Gonzalez, A.M. et al. (2011) Targeting choroid plexus epithelia and ventricular ependyma for drug delivery to the central nervous system. BMC Neurosci. 12, 4 131 Watanabe, M. et al. (2012) BMP4 sufficiency to induce choroid plexus epithelial fate from embryonic stem cell-derived neuroepithelial progenitors. J. Neurosci. 32, 15934–15945 132 Lehtinen, M.K. et al. (2013) The choroid plexus and cerebrospinal fluid: emerging roles in development, disease, and therapy. J. Neurosci. 33, 17553–17559 133 Sparkman, N.L. and Johnson, R.W. (2008) Neuroinflammation associated with aging sensitizes the brain to the effects of infection or stress. Neuroimmunomodulation 15, 323–330 134 Baruch, K. et al. (2012) CNS-specific immunity at the choroid plexus shifts toward destructive Th2 inflammation in brain aging. Proc. Natl. Acad. Sci. U. S. A. 110, 2264–2269 135 Baruch, K. et al. (2014) Aging-induced type I interferon response at the choroid plexus negatively affects brain function. Science 346, 89–93 136 Mangalam, A.K. et al. (2015) Absence of IFN-gamma increases brain pathology in experimental autoimmune encephalomyelitis-susceptible DRB1*0301. DQ8 HLA transgenic mice through secretion of proinflammatory cytokine IL-17 and induction of pathogenic monocytes/microglia into the central nervous system. J. Immunol. 193, 4859–4870 137 Zhang, L. et al. (2011) Type I IFN promotes IL-10 production from T cells to suppress Th17 cells and Th17-associated autoimmune inflammation. PLoS ONE 6, e28432 138 Berghmans, N. et al. (2011) Interferon-gamma orchestrates the number and function of Th17 cells in experimental autoimmune encephalomyelitis. J. Interferon Cytokine Res. 31, 575–587 139 Cheng, W. et al. (2014) IFN-beta inhibits T cells accumulation in the central nervous system by reducing the expression and activity of chemokines in experimental autoimmune encephalomyelitis. Mol. Immunol. 64, 152–162

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