Elsevier

Brain, Behavior, and Immunity

Volume 40, August 2014, Pages 61-73
Brain, Behavior, and Immunity

Leukocyte infiltration into spinal cord of EAE mice is attenuated by removal of endothelial leptin signaling

https://doi.org/10.1016/j.bbi.2014.02.003Get rights and content

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  • EAE induced dynamic changes of leptin receptor mRNA expression at the BBB and BSCB.

  • Without endothelial leptin signaling, the ELKO mice had lower EAE disease burden and better preserved BSCB functions.

Abstract

Leptin, a pleiotropic adipokine, crosses the blood-brain barrier (BBB) and blood–spinal cord barrier (BSCB) from the periphery and facilitates experimental autoimmune encephalomyelitis (EAE). EAE induces dynamic changes of leptin receptors in enriched brain and spinal cord microvessels, leading to further questions about the potential roles of endothelial leptin signaling in EAE progression. In endothelial leptin receptor specific knockout (ELKO) mice, there were lower EAE behavioral scores in the early phase of the disorder, better preserved BSCB function shown by reduced uptake of sodium fluorescein and leukocyte infiltration into the spinal cord. Flow cytometry showed that the ELKO mutation decreased the number of CD3 and CD45 cells in the spinal cord, although immune cell profiles in peripheral organs were unchanged. Not only were CD4+ and CD8+ T lymphocytes reduced, there were also lower numbers of CD11b+Gr1+ granulocytes in the spinal cord of ELKO mice. In enriched microvessels from the spinal cord of the ELKO mice, the decreased expression of mRNAs for a few tight junction proteins was less pronounced in ELKO than WT mice, as was the elevation of mRNA for CCL5, CXCL9, IFN-γ, and TNF-α. Altogether, ELKO mice show reduced inflammation at the level of the BSCB, less leukocyte infiltration, and better preserved tight junction protein expression and BBB function than WT mice after EAE. Although leptin concentrations were high in ELKO mice and microvascular leptin receptors show an initial elevation before inhibition during the course of EAE, removal of leptin signaling helped to reduce disease burden. We conclude that endothelial leptin signaling exacerbates BBB dysfunction to worsen EAE.

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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