**2. Blood–brain barrier and inflammation**

**1. Introduction**

32 Trending Topics in Multiple Sclerosis

symptoms among patients.

gateway, of immune cells to the CNS is desired.

Multiple sclerosis (MS) is a common neurological disease that is estimated to inflict more than 2.5 million patients worldwide [1–4]. MS is associated with chronic inflammation of the central nervous system (CNS) with myelin antigens as the immune target during the inflammation processes. The clinical manifestations of MS are variable and often include both symptoms of upper motor neurons, such as hyperreflexia, ataxia, spasticity, and visual defects, and lower motor neuron symptoms, such as sensory neuron defects and paralysis [5]. Given the uniform presence of myelin antigens in the CNS, however, variable local, rather than systemic clinical symptoms raise the possibility that there exists specific sites in the CNS that are vulnerable to immune cells thus triggering auto‐reactivity. As discussed below, we observed using a murine model of MS, experimental autoimmune encephalomyelitis (EAE), that regional neural stimulations permit entry of immune cells into the CNS, which may explain the variable MS

Genetic susceptibility of MS is well studied, and major histocompatibility complex (MHC) genes and genes associated with CD4+ helper T‐cell activation and homeostasis are identified as susceptible genes. The strongest genetic linkage was found at certain alleles of MHC class II, which suggests a relationship between autoreactive CD4+ T cells and MS development in humans [6]. Consistently, autoreactive CD4+ T cells have important roles in the development and relapse of animal models of MS [4, 7–13]. In addition to MHC genes, several genome‐wide association studies (GWAS) of MS patients have identified addition‐ al genetic loci including interleukin (IL)‐17, IL‐2 receptor (CD25), and IL‐7 receptor (CD127), which are important for CD4+ T‐cell effector function, activation, and survival [14–16]. These lines of genetic evidence suggest that blocking CD4+ T‐cell entry into the CNS, thereby blocking subsequent inflammation in the CNS, would be an effective way for the treat‐ ment of MS. Indeed, drugs that target T‐cell migration such as fingolimod (FTY720) and natalizumab (anti‐VLA4 antibody) have shown clinical success in the treatment of MS. Fingolimod is an orally available sphingosine‐1‐phosphate receptor modulator, which reduces CD4+ T‐cell invasion to the CNS due to the inhibition of lymphocyte egress from lymph nodes [17]. Natalizumab, on the other hand, targets alpha4 integrin (a subunit of VLA4), which is required for the migration of CD4+ T cells to inflamed CNS [4, 18]. The prominent clinical effects of these drugs provide a proof of concept for therapeutic strat‐ egies to suppress the invasion of autoreactive CD4+ T cells into the CNS. Because side effects such as progressive multifocal leukoencephalopathy warrant caution for the use of fingoli‐ mod and natalizumab [19, 20], a novel strategy including a blockade of the entry site, or

It is widely known that the CNS is an immune‐privileged site, protected by the blood– brain barrier (BBB), which restricts the exchange of substances and cell migration into and out of the organ. However, CNS invasion by immune cells occurs in not only neuropatho‐ logic conditions including MS, but also normal healthy conditions. We have been studying where and how immune cells can enter the CNS using EAE models. In this chapter, our The BBB is a specialized blood vessel formed by several cell types including vascular endo‐ thelial cells, pericytes, and astrocyte endfeet. In addition, tight junctions are critical for barrier development and are established through interactions of tight junction molecules, such as ZO‐ 1, claudins, and occludins, between the vascular endothelial cells to cause a size exclusion of about 500 daltons [21, 22]. Despite this rigid barrier system, autoreactive T cells can enter to the CNS and cause autoimmune diseases such as MS. The breakdown of the BBB is also observed in neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease [23]. Because neuroinflammation is associated with almost all diseases in the CNS [24], the relationship between a dysfunctional BBB and inflammatory cytokines has been well studied. For example, TNFαincreases BBB permeability during sepsis, and IL‐1β regulates the BBB via reactivation of the HIF‐VEGF axis in MS [25, 26]. IFNγ and TNFα decrease the expression of TWIK‐related potassium channel‐1 in human brain microvascular endothelial cells, which leads to increase the transmigration of various immune cells across the endothelial cells in vitro [27]. IL‐17A, which is a signature cytokine of Th17 cells, also contributes to BBB leakage in vitro and in vivo. EAE was significantly suppressed in IL‐17A‐deficient mice [28], and IL‐17A‐ deficient CD4+ T cells hardly invade the CNS through the fifth lumbar (L5) spinal cord unlike normal ones [12]. IL‐17A‐mediated BBB dysfunction involves the formation of reactive oxygen species by cellular oxidases, which down‐regulate tight junction molecules and the activation of the endothelial contractile machinery in vitro [29, 30]. Seeing that GWAS and animal model data have revealed that autoreactive CD4+ T cells producing many cytokines play a central role in MS, understanding the in vivo behavior of disease‐causing CD4+ T cells offers crucial insights about the pathophysiology of MS. Intravital two‐photon imaging using a rat transfer EAE model showed that there are at least three stages for CNS invasion by autoreactive CD4+ T cells: intraluminal scanning, perivascular scanning, and CNS‐surface scanning [31]. This study also suggested that the reactivation of CNS‐invading CD4+ T cells by perivascular/ meningeal antigen‐presenting cells is followed by parenchymal infiltration of pathogenic CD4+ T cells [31]. Chemokine guidance and cellular adhesion events of pathogenic CD4+ T cells are also critical for the CNS invasion [12, 31]. Myelin autoantigen candidates associated with MS including Myelin Basic Protein (MBP), myelin‐oligodendrocyte protein (MOG) and proteolipid proteins (PLP) [32–35] are present throughout CNS white matter, while MS lesions are often observed by MRI as focal plaques [36]. These facts suggest that there exists an additional signal that directs pathogenic CD4+ T cells to initiate inflammatory reactions at particular region(s) of the CNS. We recently demonstrated that this signal is provided by several types of neural activations. We also defined a molecular mechanism of the resulting neuro‐immune interactions for the development and relapse of murine models of MS, as discussed in the following sections.

### **3. Gravity‐mediated gateway reflex**

We first considered the initial site where the autoreactive CD4+ T cells invade the CNS. In EAE models, mice or rats are generally immunized with various CNS antigens such as mye‐ lin‐oligodendrocyte protein (MOG) emulsified with complete Freund's adjuvant (CFA) in the presence of pertussis toxin administration, which are critical for the generation of autor‐ eactive CD4+ T cells and the induction of MS‐like symptoms, including a progressive para‐ lysis that usually begins at the tip of the tail. EAE can also be induced in wild‐type animals by the transfer of autoreactive CD4+ T cells isolated from other EAE mice. This transfer EAE model is induced without antigens, CFA, or pertussis toxin treatment, making this model suitable for investigating the behavior of the disease‐causing CD4+ T cells in detail. Whole‐ mount sagittal sections of adult mice at a preclinical stage of the transfer EAE model (day 4 or 5 after MOG‐reactive CD4+ T‐cell transfer) revealed that MOG‐reactive pathogenic CD4+ T cells preferentially accumulated at the L5 spinal cord, but not in the brain at this early stage. This observation suitably explains the first clinical symptom of typical EAE, a weak‐ ness of the tail tip. A closer analysis of the L5 spinal cord revealed that the accumulation of MOG‐reactive CD4+ T cells occurred around the dorsal vessels. Various chemokines includ‐ ing Th17‐attracting CCL20 were up‐regulated at the L5 dorsal vessels during EAE compared with dorsal vessels from other spinal cords. Indeed, anti‐CCL20 antibody treatment or trans‐ fer of MOG‐reactive CD4+ T cells lacking its receptor, CCR6, compromised the L5 accumula‐ tion of these CD4+ T cells and subsequent development of EAE, which is consistent with a published study [37]. Interestingly, the selective up‐regulation of chemokines at the L5 dor‐ sal vessels was observed even at steady state, albeit to a lesser degree than in the EAE condi‐ tion. These results suggested that the L5 dorsal vessels are the first gateway for pathogenic CD4+ T cells in this transfer EAE system (**Figure 1**), and some physiological effects under steady state that modulate chemokine levels at the L5 dorsal vessels are enhanced during EAE.

Further investigation revealed that the selective up‐regulation of chemokines at the L5 dorsal vessels at steady state were the result of neuro‐immune interactions. It is known that neurons in the dorsal root ganglion (DRG) beside the L5 spinal cord are connected to the soleus muscles, the main anti‐gravity muscles [38]. Consistent with a constant stimulation of the soleus muscles by Earth's gravity, the L5 DRG is the largest among all DRGs in mice and humans [39]. We hypothesized that constant stimulation by gravity might mediate the uniqueness of the L5 spinal cord in establishing the gateway of immune cells including autoreactive CD4+ T cells. Consistent with this hypothesis, we found two peaks of CCL20 expression at the dorsal vessels of the cervical and lumbar cords, although CCL20 expression was higher at the latter. To examine the effect of gravity, mice were suspended by their tails so that their hind legs were released from gravity. This treatment resulted in a reduced accumulation of MOG‐specific CD4+ T cells at the L5 cord. Instead, these CD4+ T cells invaded the cervical cords as if a new gateway was created by the additional gravitational burden on the forefeet muscles due to the tail suspension [12]. Consistently, the tail suspension downregulated CCL20 levels in the L5 dorsal blood vessels, but up‐regulated CCL20 in the cervical cords. Moreover, electric stimu‐ lations to the soleus muscles in tail‐suspended mice restored CCL20 levels and CD4+ T‐cell accumulation at the L5 spinal cord [12]. These results suggest that a certain neural activation triggered by gravity contributes to the gateway at the L5 dorsal blood vessels for autoreactive CD4+ T cells to infiltrate the CNS (**Figure 1**).

**3. Gravity‐mediated gateway reflex**

34 Trending Topics in Multiple Sclerosis

EAE.

We first considered the initial site where the autoreactive CD4+ T cells invade the CNS. In EAE models, mice or rats are generally immunized with various CNS antigens such as mye‐ lin‐oligodendrocyte protein (MOG) emulsified with complete Freund's adjuvant (CFA) in the presence of pertussis toxin administration, which are critical for the generation of autor‐ eactive CD4+ T cells and the induction of MS‐like symptoms, including a progressive para‐ lysis that usually begins at the tip of the tail. EAE can also be induced in wild‐type animals by the transfer of autoreactive CD4+ T cells isolated from other EAE mice. This transfer EAE model is induced without antigens, CFA, or pertussis toxin treatment, making this model suitable for investigating the behavior of the disease‐causing CD4+ T cells in detail. Whole‐ mount sagittal sections of adult mice at a preclinical stage of the transfer EAE model (day 4 or 5 after MOG‐reactive CD4+ T‐cell transfer) revealed that MOG‐reactive pathogenic CD4+ T cells preferentially accumulated at the L5 spinal cord, but not in the brain at this early stage. This observation suitably explains the first clinical symptom of typical EAE, a weak‐ ness of the tail tip. A closer analysis of the L5 spinal cord revealed that the accumulation of MOG‐reactive CD4+ T cells occurred around the dorsal vessels. Various chemokines includ‐ ing Th17‐attracting CCL20 were up‐regulated at the L5 dorsal vessels during EAE compared with dorsal vessels from other spinal cords. Indeed, anti‐CCL20 antibody treatment or trans‐ fer of MOG‐reactive CD4+ T cells lacking its receptor, CCR6, compromised the L5 accumula‐ tion of these CD4+ T cells and subsequent development of EAE, which is consistent with a published study [37]. Interestingly, the selective up‐regulation of chemokines at the L5 dor‐ sal vessels was observed even at steady state, albeit to a lesser degree than in the EAE condi‐ tion. These results suggested that the L5 dorsal vessels are the first gateway for pathogenic CD4+ T cells in this transfer EAE system (**Figure 1**), and some physiological effects under steady state that modulate chemokine levels at the L5 dorsal vessels are enhanced during

Further investigation revealed that the selective up‐regulation of chemokines at the L5 dorsal vessels at steady state were the result of neuro‐immune interactions. It is known that neurons in the dorsal root ganglion (DRG) beside the L5 spinal cord are connected to the soleus muscles, the main anti‐gravity muscles [38]. Consistent with a constant stimulation of the soleus muscles by Earth's gravity, the L5 DRG is the largest among all DRGs in mice and humans [39]. We hypothesized that constant stimulation by gravity might mediate the uniqueness of the L5 spinal cord in establishing the gateway of immune cells including autoreactive CD4+ T cells. Consistent with this hypothesis, we found two peaks of CCL20 expression at the dorsal vessels of the cervical and lumbar cords, although CCL20 expression was higher at the latter. To examine the effect of gravity, mice were suspended by their tails so that their hind legs were released from gravity. This treatment resulted in a reduced accumulation of MOG‐specific CD4+ T cells at the L5 cord. Instead, these CD4+ T cells invaded the cervical cords as if a new gateway was created by the additional gravitational burden on the forefeet muscles due to the tail suspension [12]. Consistently, the tail suspension downregulated CCL20 levels in the L5 dorsal blood vessels, but up‐regulated CCL20 in the cervical cords. Moreover, electric stimu‐ lations to the soleus muscles in tail‐suspended mice restored CCL20 levels and CD4+ T‐cell

**Figure 1.** Gravity‐mediated gateway reflex. Constant gravity‐mediated stimulation of the soleus muscles induces the activation of sensory nerves, whose cell bodies are located at the dorsal root ganglion (DRG) of the fifth lumbar (L5) spinal cord. Signals via L5 DRG neurons travel to sympathetic neurons located nearby, resulting in norepinephrine (NE) secretion at the dorsal vessels there. NE enhances the inflammation amplifier (Amp) in the L5 dorsal vessel endo‐ thelium, causing an up‐regulation of CCL20 and subsequent accumulation of pathogenic CD4+ T cells.

Sympathetic nerve activation was involved in the gravity‐mediated chemokine expressions at the L5 dorsal vessels. c‐Fos expression, which is a marker of neural activation, was higher in L5 sympathetic ganglions than in L1 sympathetic ganglions. Blood flow speeds at the L5 dorsal vessels, but not at other blood vessels including L1 dorsal vessels, femoral artery, and portal vein, became slower when mice were tail suspended, while electronic stimulation of the soleus muscles increased the flow speed, suggesting an involvement of autonomic nerves including sympathetic ones in this response [12]. Furthermore, pharmacological blockade of adrenergic receptors significantly inhibited CCL20 levels, NF‐κB activation and MOG‐reactive pathogenic CD4+ T‐cell accumulation at the L5 dorsal vessels, and also suppressed clinical signs of EAE [12]. Thus, sensory nerve activation due to anti‐gravity responses leads to a sympathetic nerve stimulation that creates a gateway for immune cells to pass through the CNS via the L5 dorsal vessels [12]. This neuro‐immune response, which leads to change in the status of the vascular endothelium, is named the "gateway reflex" [40–42]. Other examples of the gateway reflex are described below.

#### **4. Electric stimulation‐mediated gateway reflex**

Neural activations can be artificially induced by various methods including electric stimula‐ tions and treatment with reagents. We wondered if electric stimulation of different muscles could create gateways in blood vessels at distinct positions via regional neural activations. As discussed earlier, weak electric stimulation to the soleus muscles restored chemokine expres‐ sion at the L5 dorsal vessels of tail‐suspended mice, because the cell body of sensory neurons in the soleus muscles is mainly located in the L5 DRG. We applied this methodology to other muscles. As expected, electric stimulations to thigh muscles including the quadriceps, which are known to be regulated by L3 DRG neurons, led to an increased expression of chemokines in L3 dorsal vessels in mice, while electric stimulations of upper regions, such as forefoot muscles including the epitrochlearis and triceps brachii, resulted in an up‐regulation of chemokines in the fifth cervical to fifth thoracic cord dorsal vessels, which is where the DRG

**Figure 2.** Electric stimulation‐mediated gateway reflex. Artificially induced neural activation by weak electric stimula‐ tion followed by sensory‐sympathetic cross talk can trigger the gateway reflex. Electric pulses to the triceps induce chemokine up‐regulation at the dorsal vessels of the fifth cervical (C5) to fifth thoracic (T5) spinal cord through activa‐ tion of the inflammation amplifier (Amp). Similarly, stimulation of the quadriceps induces a gateway at the dorsal ves‐ sels of the third lumbar (L3) cord, whereas the gateway dorsal vessels of the L5 cord are created by electric pulses to the soleus muscles.

neurons of the C5‐T5 spinal cords project to/from these muscles. These results established the electric stimulation‐mediated gateway as another example of the gateway reflex (**Figure 2**). In addition, they offer an important implication that the gateway reflex can be controlled by artificially stimulating specific neurons, providing a promising opportunity for a novel therapeutic strategy against inflammatory diseases in the CNS.
