Multiple sclerosis (MS), the most common neurological disease affecting the young to middle-aged population, is a chronic inflammatory disease characterized by demyelination and degeneration of axons in the central nervous system (CNS) (ffrench-Constant, 1994; Lassmann, 1998). While the fundamental trigger has yet to be identified, MS is characterized pathologically by multiple inflammatory lesions of white matter that are separated both temporally and spatially. Alterations in vascular properties play a critical role in the initiation and maintenance of disease pathogenesis by permitting inflammatory leukocytes to infiltrate the CNS (Gay and Esiri, 1991; Kirk et al., 2003; Roscoe et al., 2009; Seabrook et al., 2010). Blood vessels in the CNS have the unique properties of high electrical resistance and low permeability. This blood-brain barrier (BBB) serves to protect neural cells from both the intrusion of circulating peripheral immune cells and potentially harmful blood components (Ballabh et al., 2004; Brett et al., 1995; Huber et al., 2001; Pardridge, 2003; Wolburg and Lippoldt, 2002).

While the molecular basis of the BBB depends largely on the establishment of tight junction protein complexes which form between neighboring endothelial cells and the influence of pericytes and astrocyte end-feet (Daneman et al., 2010; del Zoppo and Milner, 2006; Osada et al., 2011; Wolburg and Lippoldt, 2002), endothelial adhesion to the extracellular matrix (ECM) proteins of the vascular basement membrane (BM) also plays an important instructive role. ECM proteins regulate vascular function, both during development and in the adult (Davis and Camarillo, 1995; Grant and Kleinman, 1997; Grant et al., 1989; Ingber and Folkman, 1989; Smyth et al., 1999). In the CNS, the vascular BM contains high levels of the ECM proteins laminin and collagen IV and of all structures within the CNS, blood vessels express by far, the highest levels of all integrins examined, including those of the β1 class and β4 (del Zoppo and Milner, 2006; Milner and Campbell, 2002; Welser-Alves et al., 2013), supporting the idea that they play important roles in regulating vascular function. Integrins are expressed at the cell surface as αβ heterodimers (Hemler, 1999). We have focused our attention on the β4 integrin for several reasons. First, α6β4 integrin's ligand, laminin is a major component of the vascular BM and transgenic mice deficient in astrocyte or pericyte laminin show defective BBB integrity (Chen et al., 2013; Menezes et al., 2014). Second, by contrast to the laminin receptors α6β1 integrin and dystroglycan, which are widely expressed on all types of blood vessel, α6β4 integrin expression is restricted to arterioles under normal conditions (Welser-Alves et al., 2013) though interestingly, under neuroinflammatory conditions, α6β4 integrin is more widely expressed on a greater number of blood vessels including capillaries, both in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE) (Welser et al., 2017) and in transgenic mice overexpressing interleukin (IL)-6 in astrocytes (GFAP-IL6 transgenic mice) (Milner and Campbell, 2006). Fourth, perhaps hinting at its function in endothelial cells, in the epidermal layer of the skin, α6β4 integrin plays a critical role in promoting mechanical stability and barrier integrity (Dowling et al., 1996; Nakano et al., 2001; van der Neut et al., 1996).

Based on these findings we postulated that endothelial upregulation of α6β4 integrin under inflammatory conditions may be an inducible protective mechanism that stabilizes the BBB under conditions of stress. As a first step in testing this hypothesis we evaluated the progression of EAE in transgenic mice lacking β4 integrin in endothelial cells (β4-EC-KO). This revealed that β4-EC-KO mice show worse clinical disease and leukocyte infiltration, supporting a protective function for β4 integrin in enhancing BBB integrity and suppressing inflammation (Welser et al., 2017). Having found that loss of endothelial β4 integrin results in worse EAE outcome, the goal of the current study was to test the opposite, namely: determine if constitutive overexpression of endothelial β4 integrin would enhance BBB integrity and thereby protect against the development and progression of EAE. To achieve this, we used the Cre-Lox system to generate a novel transgenic knock-in mouse (β4-EC-KI) in which all endothelial cells constitutively express high levels of β4 integrin. We then evaluated how this intervention influences (i) CNS vascular structure and activation, both in the brain and spinal cord, and (ii) the clinical and histological progression of EAE, with a specific focus on BBB integrity, vascular inflammation, leukocyte infiltration, and microglial and astrocyte activation.