Brain barriers control immune cell trafficking to the CNS. Britta Engelhardt Theodor Kocher Institute University of Bern - Switzerland

Transcription

1 Brain barriers control immune cell trafficking to the CNS Britta Engelhardt Theodor Kocher Institute University of Bern - Switzerland

2 The brain barriers 2

3 The BBB establishes a physical and metabolic barrier brain blood tight junction

4 The blood-brain barrier at the level of capillaires and post-capillary venules Engelhardt and Sorokin, 2009 Engelhardt and Ransohoff, Trends Immunol,

5 Encephalitogenic T cells cross the blood-brain barrier and cause experimental autoimmune encephalomyelitis Meyermann, Wekerle 1986

6 How do encephalitogenic T cells cross the BBB during initiation of EAE? I. Rolling II. Activation III. Adhesion IV. Diapedesis

7 Intravital microscopy: Visualization of the interaction of circulating T cells with the spinal cord microvasculature in vivo Image processor Video timer SIT camera VCR Mercury Lamp Monitor Animal preparation microscope OFF LINE ANALYSIS Vajkoczy et al. 2001, J Clin Invest. 108:

8 Th1 cell interaction with the healthy BBB is unique: lack of rolling - predominant involvement of α4-integrins - extended duration of diapedesis I. III. IV. II. Capture Activation Adhesion Diapedesis > 4 hours GPCR How about Th17 cells?

9 Migration of autoaggressive CCR6 + T H17 T cells across the BCSFB initiates EAE Reboldi et al. Nature Immunol, 2009 and Engelhardt, Clinic Exp Neuroimmunol, 2010

10 T cell recruitment across the inflamed BBB during EAE I. II. III. IV. Tethering Rolling/Capture? A c t i v a t i o n A d h e s i o n T r a n s m i g r a t i o n

11 Anti-α4-integrin therapy of relapsing-remitting multiple sclerosis targets T cell trafficking across the BBB Natalizumab α α4 4 β1/β7

12 Intravital microscopy: Visualization of the interaction of human T cells with the inflamed spinal cord microvasculature in vivo Image processor Video timer SIT camera VCR Mercury Lamp Monitor Animal preparation microscope OFF LINE ANALYSIS Coisne et al., 2009 JI Cutting Edge

13 Observation of the interaction of human T lymphocytes with the inflamed BBB during EAE - the animal model for multiple sclerosis IgG4 natalizumab Coisne et al., 2009 JI Cutting Edge 13

14 Natalizumab blocks the adhesion but not the initial contact of T cells to the inflamed BBB in vivo I. II. III. IV. Rolling/Capture Activation Adhesion Diapedesis PSGL-1 P-selectin GPCR Coisne et al., Cutting Edge J Immunol, 2009

15 T cell interaction with the BBB under physiological shear in vitro Arrest/Adhesion - Crawling - Diapedesis 15

16 Multi-step recruitment of T cells across the inflamed BBB I. II. III. IV. V. Capture/Rolling Activation Arrest/Adhesion Crawling Diapedesis PSGL-1 P-selectin GPCR

17 ICAM-1 and ICAM-2 are required for T cell polarization on the BBB Steiner et al., J, 2010

18 ICAM-1 and ICAM-2 are required for T cell polarization and directed T cell crawling on the BBB in vitro wild-type endothelium ICAM-1+2 -/- endothelium Steiner et al., J Immunol, 2010

19 ICAM-1 and ICAM-2 are essential for post-arrest T cell polarization and crawling against the flow I. II. III. IV. Capture/Rolling Activation Adhesion Crawling?? Arrest V. Diapedesis? GPCR PSGL-1 P-selectin ICAM-1 ICAM-2 LFA-1 ICAM-1/-2

20 Where do T cells crawl to? I. II. III. IV. Capture/Rolling Activation Adhesion Crawling?? Arrest V. Diapedesis? GPCR PSGL-1 P-selectin ICAM-1 ICAM-2 LFA-1 ICAM-1/-2

21 Hypothesis: The BBB directs T cells to rare transcellular sites of diapedesis Transcellular? Paracellular?

22 VE-cadGFP-C57BL/6 knock-in mice: Live cell imaging of endothelial junctions 22

23 T cells cross the BBB via the transcellular and paracellular pathway in vitro Transcellular Paracellular 23

24 Which cell decides on the T cell diapedesis pathway? I. II. III. IV. V. Capture/Rolling Activation Adhesion Crawling Diapedesis? Arrest paracellular transcellular PSGL-1 GPCR P-selectin ICAM-1 ICAM-2

25 The BBB determines the cellular pathway of T cell diapedesis TNF-α IL-1β Abadier et al, EJI,

26 Hypothesis: IL-1β induces BBB leakiness thus promoting rapid paracellular T cell diapedesis Transcellular Paracellular intact BBB leaky BBB

27 IL-1β but not TNF-α impairs barrier characteristics of brain endothelial cells in vitro TEER (TNF-α 1.0) electrode TEER TNF-α * IL-1β perm. (3kDa Dextran) (TNF-α 1.0) Permeability 2.0 *** TNF-α IL-1β ~ 40 min electrode 2 Abadier et al, EJI, 2015

28 Hypothesis: IL-1β induces BBB leakiness thus promoting rapid paracellular T cell diapedesis Transcellular Paracellular intact BBB leaky BBB

29 The cellular pathway of T cell diapedesis across the BBB is regulated independently of barrier integrity Paracellular diapedesis Transcellular diapedesis TNF-α IL-1β high Abadier et al, EJI, 2015

30 Remember: ICAM-1 and ICAM-2 are essential for post-arrest T cell polarization and crawling against the flow I. II. III. IV. Capture/Rolling Activation Adhesion Crawling?? Arrest V. Diapedesis? GPCR PSGL-1 P-selectin ICAM-1 ICAM-2 LFA-1 ICAM-1/-2

31 Inflammatory conditions inducing different cell surface levels of endothelial ICAM ** * ** ns 31 ICAM-1 cell surface Relative values (unstimulated =1.0) ng/ml: *** VCAM-1 cell surface Relative values (unstimulated =1.0) ng/ml: TNF-α IL-1β TNF-α IL-1β Abadier et al, EJI, 2015

32 Cell surface levels of endothelial ICAM-1 influence T cell crawling on and diapedesis across the BBB T cell arrest Post arrest T cell behaviour Arrested T cells per FOV 120 ns ns ns ng/ml: TNF-α IL-1β % of arrested T cells 100 ** ** * 80 ** ng/ml: TNF-α IL-1β Detach Crawling Stationary Diapedesis Abadier et al, EJI,

33 Cell surface levels of endothelial ICAM-1 - rather than BBB integrity or inflammatory stimulus - direct the cellular pathway of T cell diapedesis across the BBB I. II. III. IV. Capture/Rolling Activation Adhesion Crawling? Arrest ICAM-1 lo paracellular V. Diapedesis ICAM-1 high transcellular PSGL-1 GPCR P-selectin ICAM-1 ICAM-2

34 What happens in the absence of ICAM-1 and ICAM-2? I. II. III. IV. Capture/Rolling Activation Adhesion? Arrest paracellular V. Diapedesis transcellular? PSGL-1 GPCR P-selectin

35 ICAM-1/ICAM-2 independent T cell diapedesis across the IL-1β-stimulated BBB Arrested T Cells per FOV Cytokine: ** TNFα IL1β pmbmecs: I1 null /I2 -/- I1 null /I2 -/- 80 ** 60 ** Cytokine: TNFα IL1β pmbmecs: I1 null /I2 -/- I1 null /I2 -/- % of arrested T cells Detach Recurrent arrest/stationary Diapedesis Abadier et al, EJI,

36 Cellular pathway of T cell diapedesis across the BBB in the absence of endothelial ICAM-1 and ICAM-2 I1 null /I2 -/- //VE-Cadherin-GFP BBB Abadier et al., unpublished 36

37 ICAM-1 null /ICAM-2 -/- C57BL/6 mice develop ameliorated EAE Abadier et al, EJI, 2015

38 The brain barriers control immune cell trafficking to the CNS I. II. III. IV. Capture/Rolling Activation Adhesion Crawling? Arrest ICAM-1 lo paracellular V. Diapedesis ICAM-1 high transcellular PSGL-1 GPCR P-selectin ICAM-1 ICAM-2

39 The B Lab

40 Acknowledgements... ICAM-in vivo studies Scott Barnum University of Brimingham, Alabama VE-cadherin-GFP studies Dietmar Vestweber Münster, Germany In vitro flow studies Michael Abadier and Ruth Lyck Transgenic mice Urban Deutsch Intravital microscopy Jens Stein