Claudin-1 induced sealing of blood-brain barrier tight junctions ameliorates chronic experimental autoimmune encephalomyelitis

Abstract

In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), loss of the blood-brain barrier (BBB) tight junction (TJ) protein claudin-3 correlates with immune cell infiltration into the CNS and BBB leakiness. Here we show that sealing BBB TJs by ectopic tetracycline-regulated expression of the TJ protein claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and furthermore did not influence the onset and severity of the first clinical disease episode. However, expression of claudin-1 did significantly reduce BBB leakiness for both blood borne tracers and endogenous plasma proteins specifically around vessels expressing claudin-1. In addition, mice expressing claudin-1 exhibited a reduced disease burden during the chronic phase of EAE as compared to control littermates. Our study identifies BBB TJs as the critical structure regulating BBB permeability but not immune cell trafficking into CNS during EAE, and indicates BBB dysfunction is a potential key event contributing to disease burden in the chronic phase of EAE. Our observations suggest that stabilizing BBB barrier function by therapeutic targeting of TJs may be beneficial in treating MS, especially when anti-inflammatory treatments have failed.

Fig. 1

Claudin-1 is not expressed in parenchymal CNS microvessels. mRNA expression level of Claudin-1 and Claudin-5 in parenchymal CNS microvascular endothelial cells was tested by quantitative RT-PCR with the s16 ribosomal protein (Rps16) set to 1.0. As a source for RNA mouse brain microvessels were used immediately after their isolation. Data express mean values of 5 qPCRs carried out in triplicates ± standard error. b.d. below limit of detection

Fig. 2

Expression of claudin-1 in the brains of Tie-2 tTA//TRE-claudin-1 double and single transgenic mice. Immunofluorescence staining for claudin-1⁺ (red) and PECAM-1⁺ (green) in frozen brain sections of double transgenic Tie-2 tTA//TRE–claudin-1 mice and single transgenic littermates from line 23949 is shown. Expression of claudin-1 was detected in PECAM-1 + parenchymal blood vessels in the brain of Tie-2 tTA//TRE-claudin-1 double transgenic mice but not in single transgenic littermates. In addition, diffuse claudin-1 immunostaining was detected in PECAM-1⁺ vascular endothelial cells within the leptomeninges and in cell-junctions of choroid plexus epithelial cells in Tie-2 tTA//TRE-claudin-1 double transgenic and single transgenic mice. Scale bar 20 μm

Fig. 3

TET-regulated claudin-1 ameliorates chronic EAE Active EAE was induced in Tie2-tTA//TRE-claudin-1 double transgenic mice and single transgenic littermates from lines 23949 and 23974, respectively, by subcutaneous immunization with MOG_aa35–55 in CFA. a Data show mean disease scores of 4 animals per group ± SEM, evaluated daily following induction of EAE. Black line with open squares indicate single transgenic mice line 23974; dark line with filled squares indicate double transgenic mice line 23974; gray line with open circles indicate single transgenic mice line 23949; light gray line with filled circles indicate double transgenic mice line 23949. One representative experiment out of six comparing a total of 20 Tie2-tTA//TRE-claudin-1 double transgenic mice with 19 single transgenic mice from line 23974 and 31 double transgenic mice with 33 single transgenic mice from line 23949 is shown. b The area under the curve (AUC) was calculated from the EAE clinical course of each mouse between day 30 and day 50 post-immunization. Shown are the mean AUC values for each group ± SEM. Statistical analysis of AUC values with the Mann–Whitney test revealed significantly reduced EAE severity in double transgenic (filled bars) compared to single transgenic (open bars) mice in both TRE-claudin-1 lines. *P < 0.05; **P < 0.005

Fig. 4

TET-regulated claudin-1 does not influence the number of inflammatory cuffs in the brain. Inflammatory cuffs were counted in a total of 10 double transgenic (dtg) and 15 single transgenic (stg) Tie2-tTA//TRE-claudin-1 mice of lines 23974 and 23949 in 168 versus 147 brain sections, respectively. A total of 1020 versus 1338 parenchymal cuffs (P), 474 versus 510 leptomeningeal cuffs (M) and 197 versus 170 periventricular cuffs (V) in double transgenic versus single transgenic mice, respectively, are included into the analysis. Each dot represents the mean number of inflammatory cuffs/per brain section of one mouse. Horizontal lines depict median for each group. Mann–Whitney U analysis revealed no significant differences comparing the number of cuffs in stg versus dtg mice

Fig. 5

TET-regulated claudin-1 induces BBB tightening for Hoechst dye in chronic EAE. At the end of an EAE experiment (day 50 p.i.), Hoechst dye was injected intravenously into 6 single and 4 double transgenic Tie2-tTA//TRE-claudin-1 mice. Brain cryosections of these mice were stained for Claudin-1 (red) and CD45 (green). Leakage of the nuclear dye Hoechst can be observed in blue. a The mean percentage (±SD) of inflammatory cuffs per brain localized around Hoechst-impermeable BBB vessels is significantly increased in double transgenic compared to single transgenic mice. b The following parenchymal cuffs are shown: inflammatory cuffs in single and double transgenic mice that localized around BBB vessels that do not stain positive for TET-regulated laudin-1 and are either permeable for Hoechst dye or not; and inflammatory cuffs in double transgenic mice localized around claudin-1-expressing BBB vessels which are either permeable for Hoechst dye or not. Scale bar 20 μm. c The mean ratio (±SD) of Hoechst-impermeable to Hoechst-permeable inflammatory cuffs localized around claudin-1 negative vessels (in single and double transgenic mice) or claudin-1 positive vessels (in double transgenic mice) demonstrates highly significant tightening of the BBB for Hoechst dye in the presence of TET-regulated claudin-1. *P < 0.05; ***P < 0.001

Fig. 6

TET-regulated claudin-1 induces BBB tightening for endogenous fibronectin in EAE. Brains of 9 single and 6 double transgenic TET-claudin-1 mice at day 50 post induction of aEAE were prepared, cryo-sectioned and stained for CD45 (green) and claudin-1 (red) as well as for CD45 (green) and fibronectin (red) on consecutive sections. a The mean percentage (±SD) of inflammatory cuffs per brain that have formed around fibronectin-impermeable BBB vessels is significantly increased in double transgenic compared to single transgenic mice. b Shown are representative parenchymal cuffs in a double transgenic mouse. One has formed around a claudin-1^neg BBB vessel and is leaky for plasma fibronectin (upper row), one stains positive for claudin-1 and is impermeable for plasma fibronectin (lower row). Note that fibronectin-labeling also stains the basement membranes of BBB vessels. Scale bar 50 μm. c The mean ratio (±SD) of fibronectin-impermeable to fibronectin-permeable inflammatory cuffs that formed around claudin-1^neg vessels (in single and double transgenic mice) or claudin-1^pos vessels (in double transgenic mice) demonstrates highly significant tightening of the BBB for plasma fibronectin in the presence of transgenic claudin-1. *P < 0.05; **P < 0.005; ***P < 0.001