Leukocyte infiltration into spinal cord of EAE mice is attenuated by removal of endothelial leptin signaling
Introduction
Leptin, a 16 kD soluble protein produced mainly by adipocytes, can reach the CNS by specific transport systems at the blood–brain barrier (BBB) and blood–spinal cord barrier (BCSB) (Banks et al., 1996, Pan and Kastin, 2001, Tu et al., 2008). Leptin is proinflammatory and facilitates autoimmunity. Studies involving leptin treatment, antagonism, and knockout mice have collectively shown that leptin worsens experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS) (Matarese et al., 2001a, Matarese et al., 2001c, Sanna et al., 2003). In particular, a serum leptin surge precedes the onset of EAE, and leptin-deficient (ob/ob) mice and leptin receptor ObRb-deficient (db/db) mice are resistant to the induction and progression of EAE. Leptin treatment by peripheral delivery increases susceptibility of mice to EAE. In active EAE brain lesions, leptin is also produced by pathogenic T helper (Th1) cells and macrophages (Sanna et al., 2003). Studies from ob/ob and db/db mice that have defective T cell responses show that leptin stimulates Th1 response but inhibits Th2 response (Lord et al., 1998). However, astrocyte-specific leptin receptor knockout (ALKO) mice show worsened EAE and greater leukocyte infiltration in spinal cord and brain (Mishra et al., 2013), indicating a cell-specific leptin action in the CNS.
The BBB and BSCB are three-dimensional, multicellular neurovascular interfaces. The comprising microvascular endothelial cells are joined by tight junction (TJ) complexes. The TJ complex consists of three major classes of transmembrane proteins: occludins, claudins, and junctional adhesion molecules, and it is reinforced by TJ-associated adaptor proteins, such as zonula occludin (ZO)-1, ZO-2, and ZO-3 that serve as scaffolding proteins to secure transmembrane TJ protein localization on the membrane (Liebner et al., 2000b, Wolburg and Lippoldt, 2002, Abbott et al., 2010). Altered expression of occludin, junctional adhesion molecules, and ZO-1 has been documented in acute and progressive forms of MS, together with changes in the vascular basement membrane and pericytes (Kirk et al., 2003, Alvarez et al., 2011). Inflammation and BSCB breakdown are also major features in different EAE models (Juhler et al., 1984, Pan et al., 1996). In EAE, the reduction of occludin, claudin-1, claudin-3, claudin-5, and ZO-1 coincides with the onset of inflammation and slightly precedes signs of disease and increased permeability of the BSCB (Wolburg et al., 2003, Bennett et al., 2010).
The BBB endothelia express leptin receptors (Hileman et al., 2002, Pan et al., 2008b). Leptin transport involves its specific receptors at the BBB and BCSB (Banks et al., 1996, Pan and Kastin, 2001, Tu et al., 2008), and megalin at the blood-cerebrospinal fluid barrier (Kurrimbux et al., 2004, Dietrich et al., 2008). In mice with EAE, the transport of leptin does not diminish, but rather shows a selective upregulation (Hsuchou et al., 2013b). This is similar to the change of transport of tumor necrosis factor α (TNF) (Pan et al., 1996) but opposite from that of interleukin (IL)-15 (Hsuchou et al., 2009b). Regulated transport may underlie differential effects of leptin in autoimmunity in the periphery and CNS (Mishra et al., 2013), a phenomenon seen for many peptides (Pan and Kastin, 2009). During the course of transcytosis, leptin also modifies cellular signaling with generation of secondary mediators by the BBB (Pan et al., 2011). However, the role of endothelial leptin signaling in the incidence and progression of EAE is not clear. Here, we used endothelial specific leptin receptor mutant (ELKO) mice to determine whether endothelial leptin signaling promotes EAE.
Section snippets
EAE induction
All experimental procedures followed a protocol approved by the Institutional Animal Care and Use Committee. The generation and characterization of the ELKO mice are described in detail elsewhere (Hsuchou et al., 2011, Pan et al., 2012). The ELKO mice were studied along with their littermate controls on a C57BL/6 strain background (wildtype, WT). All mice were group-housed and fed ad lib in a specific pathogen-free animal facility. Female ELKO and WT mice (8–10 week old) were used to induce EAE
Dynamic changes of ObR mRNA expression in enriched brain and spinal cord microvessels during the course of EAE
The effect of EAE evolution on mRNA expression of ObR isoforms at the BBB and BSCB was estimated by randomized block analysis, similar to repeated measures ANOVA (n = 3/time point). Although direct comparison among ObR isoforms suffered from differences of amplification efficiency of these splicing variants, a significant effect of time after EAE induction was evident. In enriched cerebral microvessels, there was an initial trend of increase of ObRb, ObRc, and ObRe on day 6 and a later decrease
Discussion
Leptin receptors in the endothelial cells of the BBB and BSCB are known to mediate leptin transport from blood to the CNS. However, the roles of endothelial leptin signaling in EAE progression have not been addressed. Results from this study show that leptin receptors at the BSCB show dynamic changes with an initial elevation preceding EAE onset and subsequent attenuation. In ELKO mice lacking endothelial leptin signaling, there was less disease burden, impairment of BSCB function, leukocyte
Conflict of Interest
There is no conflict of interest.
Acknowledgment
Grant support was provided by NIH (DK54880 and DK62249 to AJK, and NS62291 to WP). The ObR-floxed mice used for endothelial excision of exon 17 originated from Dr. Streamson Chua Jr. (Albert Einstein Medical College) and was cross-bred to pure C57 strain background by Dr. Silvana Obici’s lab (University of Vermont). Cryosectioning and flow cytometry analysis were performed in the Cell Biology and Bioimaging core facility with a service charge.
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