Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation

Abstract

T(H)17 lymphocytes appear to be essential in the pathogenesis of numerous inflammatory diseases. We demonstrate here the expression of IL-17 and IL-22 receptors on blood-brain barrier endothelial cells (BBB-ECs) in multiple sclerosis lesions, and show that IL-17 and IL-22 disrupt BBB tight junctions in vitro and in vivo. Furthermore, T(H)17 lymphocytes transmigrate efficiently across BBB-ECs, highly express granzyme B, kill human neurons and promote central nervous system inflammation through CD4+ lymphocyte recruitment.

Figure 1

T_H17 lymphocytes migrate efficiently across the BBB in vitro and in vivo and kill human neurons. (a) Human CD4⁺CD45RO⁺ T_H17, CD4⁺ T_H1 (both generated in vitro, see Supplementary Methods) and ex vivo CD4⁺ lymphocytes were allowed to migrate across human BBB-ECs in a modified Boyden chamber assay (ref. and Supplementary Methods) for 18 h. Significantly more T_H17 lymphocytes migrated than either T_H1 or ex vivo CD4⁺ lymphocytes. (b) CD4⁺CD45RO⁺ T_H17 lymphocytes were allowed to migrate across human BBB-ECs for 18 h. Cells were stained for IL-17 and IL-22 before and after migration. The cytokine profile revealed the preferential migration of IL-17⁺ and IL-22⁺ lymphocytes. (c) Immune cells from lymph nodes and spleen of MOG_35–55-immunized 2D2 mice were polarized toward T_H1 or T_H17 and transferred to Rag1^−/− mice, and CD45^hiCD4⁺ lymphocytes were isolated from the CNS 7 d after transfer. Shown is a representative flow cytometry dot plot of CNS cell content from Rag1^−/− mice injected with either T_H1- (left) or T_H17-polarized (right) 2D2 lymphocytes (n = 4 mice per group). (d) Human CNS postmortem material from unaffected individuals (control, non-inflamed; above) and heavily infiltrated CNS material from individuals with multiple sclerosis (below) were immunostained for CD45RO (red), IL-17 (green) and nuclear stain TO-PRO3 (blue). Confocal microscopy imaging confirmed the presence of IL-17⁺CD45RO⁺ cells (yellow) in infiltrated multiple sclerosis lesions but not in control CNS. Bar, 75 μm. (e) Similarly, IL-22⁺CD45RO⁺ staining was observed in multiple sclerosis lesions, but not in control CNS material. (f) Human CD4⁺CD45RO⁺ T_H17–polarized lymphocytes were stained for CD45RO, IL-17, IL-22 and granzyme B. Both IL-17– and IL-22–producing lymphocytes expressed granzyme B (22.5% and 17.3%, respectively). (g) More than 60% of IL-17⁺IL-22⁺ lymphocytes highly expressed granzyme B. Granzyme A and perforin were not detected in T_H17 cells, whether or not these cells produced IL-22 (data not shown). (h) The cytotoxic activity of T_H17 lymphocytes was assessed using neuron-enriched cultures obtained from human fetal CNS material and compared to that of unactivated T lymphocytes. All data shown are representative of the mean ± s.e.m. of three independent experiments.

Figure 2

IL-17 and IL-22 receptors are expressed on human brain endothelium, and their activation permeabilizes the BBB. (a) Unactivated human BBB-ECs grown in primary culture were stained for IL-17R and IL-22R, revealing their expression on the surface of 23.2% and 16.6% of BBB-ECs, respectively. (b) Human CNS postmortem material from unaffected individuals (control, non-inflamed; above) and heavily infiltrated CNS material from individuals with multiple sclerosis (below) were immunostained for IL-17R (green), caveolin-1 (red) and nuclear stain TO-PRO3 (blue). Confocal microscopy imaging confirmed the expression of IL-17R on caveolin-1⁺ endothelium in inflamed CNS material. IL-17R expression was undetectable in control CNS material. Bar, 75 μm. (c) Similarly, IL-22R staining was observed on endothelial cells in multiple sclerosis lesions, but not in controls. (d) Human BBB-ECs were grown in Boyden chambers and treated with IL-17 (top) or IL-22 (bottom). Permeability of the monolayers was monitored with fluorescent BSA, showing that BBB-EC monolayer permeability increased after treatment with either IL-17 or IL-22. (e) Western blot for the tight-junction proteins occludin, ZO-1 and junction adhesion molecule (JAM)-1 from human BBB-ECs revealed disruption of occludin and ZO-1 by IL-17 (100 ng/ml, 18 h). (f) Western blot for tight-junction proteins in spinal cord homogenates of MOG_35–55-immunized EAE mice revealed a similar reduction in occludin and ZO-1. In situ immunostaining for ZO-1 (red) and nuclear stain TO-PRO3 (blue) in normal-appearing cerebellar white matter (control) and in infiltrated and demyelinated cerebellar lesions from C57BL/6 mice immunized with MOG_35–55 (EAE, grade 4). Confocal microscopy imaging confirmed disruption of ZO-1 around infiltrated vessels. Bar, 75 μm. (g) Freshly isolated peripheral blood human CD4⁺ lymphocytes were allowed to migrate for 18 h across IL-17– (100 ng/ml) or IL-22–treated (100 ng/ml) human BBB-ECs. Both cytokines promoted migration of human ex vivo CD4⁺ lymphocytes across human BBB-ECs, as compared to control. (h) CCL2 (or MCP-1) secretion by human BBB-ECs was assessed by ELISA in untreated and IL-17– or IL-22–treated cultures (100 ng/ml, 18 h). Both IL-17 and IL-22 upregulate CCL2 secretion by human BBB-ECs. All data shown represent the mean ± s.e.m. from three independent experiments performed in triplicate.