Endothelial TWIK-related potassium channel-1 is a critical regulator of immune cell trafficking into the CNS

Transcription

1 Endothelial TWIK-related potassium channel- is a critical regulator of immune cell trafficking into the C Stefan Bittner, Toias Ruck, Michael K Schuhmann, Alexander M Herrmann, Hamid Moha ou Maati, Nicole Boak, Kerstin Göel, Friederike Langhauser, David Stegner, Petra Ehling, Marc Borsotto, Hans-Christian Pape, Bernhard Nieswandt, Christoph Kleinschnitz, Catherine Heurteaux, Hans-Joachim Galla, Thomas Budde, Heinz Wiendl & Sven G. Meuth Supplementary Information Nature Medicine: doi:./nm.

2 K + lockers Current density (pa pf ) Current density (pa/pf) c Current density (pa pf ) Current density (pa/pf) mv AA K + lockers K + lockers + AA K+ lockers (n = ) K+ lockers + AA (n = ) K + lockers K + lockers + ALA K+ lockers (n = ) K+ lockers + Ala (n = ) na ms Memrane potential (mv) Memrane potential (mv) Memrane potential (mv) Memrane potential (mv) mv K K+ + lockers AA AA na ms K + K+ lockers Ala ALA na ms mv mv d Current density (pa/pf) e Current density (pa pf ) Current density (pa pf ) Current density (pa/pf) K + lockers K+ lockers (n = ) K+ lockers + Riluzole (n = ) K + lockers + K+ lockers (n = ) K+ lockers + AA (n = ) K+ lockers + AA + Spadin (n = ) K + lockers K + lockers + AA K + lockers + AA + spadin Memrane potential (mv) Memrane potential (mv) Memrane potential (mv) Memrane potential (mv) AA K + lockers K+ lockers Riluzole na ms na ms AA + Spadin K+ lockers K + lockers AA AA + spadin mv mv f Current increase (%) Trek / - - AA µm ALA µm µm Supplementary Figure Nature Medicine: doi:./nm.

3 Supplementary Figure Electrophysiological characterization of human TREK/HEK (htrek- /HEK) cells and mouse rain microvascular endothelial cells (MBMEC). (a) Pharmacological activation of htrek currents with µm arachidonic acid (AA) in whole-cell patch-clamp configuration on a htrek/hek cell line. Currents were recorded in the presence of a cocktail of potassium channel inhiitors (K + lockers). Representative current traces are shown. () Currentvoltage relationship curves otained efore (open circles) and after (closed circles) current activation y AA µm (n = ). Lower panel: Typical ramp traces otained in control conditions and in the presence of µm AA. (c) Current-voltage relationships otained efore (open circles) and after (closed circles) current activation y alpha-linolenic acid (ALA) µm (n = ). Lower panel: Typical ramp traces otained in control conditions and in the presence of µm ALA. (d) Current-voltage relationships otained efore (open circles) and after (closed circles) current activation y µm (n = ). Lower panel: Typical ramp traces otained in control conditions and in the presence of µm. (e) After the pre-activation of the current y µm AA, the inhiition of the current y µm of spadin was measured. Current-voltage relationships otained in the presence of K + lockers (closed circles), K + lockers + AA (open circles) and K + lockers + AA + spadin µm (closed triangles). Lower panel: Typical ramp traces otained in the presence of K + lockers (lack line), K + lockers + AA (lue line) and K + lockers + AA + spadin µm (red line). (f) Application of AA, ALA and on MBMEC from wild type () and Trek / mice (n =, respectively). Relative current change is shown. p <.. Nature Medicine: doi:./nm.

4 e Cells per image Trek / EAE score 7 D D/Trek / Time after adoptive transfer c d IFN-γ production (ng ml ) Cell / ead ratio ATP release (cpm x ) Trek / f,,,..... IL-7 production (ng ml ) Trek / Trek / Trek / Trek / g Counts CDc IFN-γ production (pg ml ) T cells DCs CD CD CD Trek / Trek / Trek / Trek / Trek / CD MHCII. CD + CD + Fluorescence intensity Nature Medicine: doi:./nm. Supplementary Figure

5 Supplementary Figure MBEMC, lymphocytes and dendritic cells from Trek / and mice exhiit a comparale immunological profile. (a) MBMEC cell layers prepared from or Trek / mice displayed similar densities. Representative images of MBMEC layers are shown with cell densities (numer of cells per image) expressed in the ar chart. (,c) and Trek / splenocytes showed comparale levels of IFN- and IL-7 production () and similar proliferation rates (c) after stimulation with CD/CD eads. (d) Migratory capacity of and Trek / CD + and CD + T cells across transwell filters devoid of ECs (n = ). (e) Adoptive transfer EAE scores in recipients of in vitro stimulated D or D/Trek -/- cells (n = vs 7). (f) Co-culture experiment of dendritic cells (DCs) and T cells from immunized (n = ) and Trek / mice (n = ) isolated at disease maximum and vice versa. Co-cultures were assessed for IFN production after MOG stimulation. (g) Characterization of CD + CDc + antigen presenting cells in and Trek / mice y flow cytometry for the surface markers MHCII, CD, CD and CD. No significant differences were oserved (n = ). ns = not significant. Nature Medicine: doi:./nm.

6 Fluorescence intensity ICAM.... c Occludin Occludin, DAPI Trek / Fluorescence intensity..... Fluorescence intensity VCAM.... Trek / d ZO- ZO-, DAPI Trek / Fluorescence intensity..... Trek / Trek / Trek / Supplementary Figure An upregulation of ICAM and VCAM ut no changes for Occludin and ZO- can e oserved in EAE lesions from Trek / mice compared to mice. (a,) Fluorescence intensity of (a) ICAM and () VCAM expression in EAE lesions from and Trek / mice (n =, p <.). Representative results are also displayed in Fig. c,d. (c,d) Representative results and evaluation for (c) Occludin and (d) ZO- expression in EAE lesions from and Trek / mice (n = ). ns = not significant. Nature Medicine: doi:./nm. Supplementary Figure

7 EAE score 7 Isotype control + anti-icam + anti-vcam Time after immunization EAE score 7 Trek / Isotype control + anti-icam + anti-vcam Time after immunization c IFN-γ production (ng ml ) Trek / Isotype control + anti-vcam + anti-icam Supplementary Figure EAE course and evaluation of anti-icam and anti-vcam treated and Trek / mice. (a,) EAE course in (a) and () Trek / mice receiving locking antiodies against VCAM or ICAM or isotype controls on day and day 9 (n = per group). (c) ELISAs for IFN-γ in isotype- or antiody-treated Trek / splenocytes at disease maximum. Nature Medicine: doi:./nm. Supplementary Figure

8 .. Relative fluorescence intensity..... Relative fluorescence intensity..... Trek / Trek / Supplementary Figure Trek deletion has no impact on the permeaility of MBMEC for molecular tracers in vitro. (a,) No significant difference was found for permeaility for (a) Dextran kda-fluorescein and () Dextran 7 kda-tr over minutes (n =, respectively). Later time points up to hours also revealed no significant differences (data not shown). Nature Medicine: doi:./nm. Supplementary Figure

9 Trek / DAPI, DHE relative intensity Trek / µm c Infllamed / nonininflamed endothelial cells.... Trek / µm Nitrotyrosine-pos. cells per slice Trek / Supplementary Figure Reactive oxygen species/reactive nitrogen species (ROS/R) production is unchanged upon Trek deletion in endothelial cells. (a,) EAE lesions in and Trek / were characterized y H&E and LFB staining (not shown) and further characterized y staining for DHE (a) and nitrotyrosine (). Representative pictures are displayed on the left side and evaluation on the right side. Scale ar = µm. (c) In vitro assessment of ROS production in MBMEC revealed no differences etween and Trek / mice (n=). DAPI = ',-diamidino-- phenylindole; ns = not significant. Nature Medicine: doi:./nm. Supplementary Figure

10 GFAP DAPI GFAP GS DAPI GFAP DAPI GFAP GS DAPI numer of terminal ranches.9. Trek / length of processes (µm) Trek / GFAP EAAT DAPI GFAP EAAT DAPI c TNF-α (pg ml ) IL (pg ml ) Trek / Trek / Trek / d GFAP, TREK GFAP, TREK e GFAP-positive cells in inflammatory lesions (per mm ) Trek / Supplementary Figure 7 Trek deletion has no impact on astrocyte morphology and function. (a) Histologic staining of cultured astrocytes for glial firillary acidic protein (GFAP), glutamine synthetase (GS), and excitatory amino acid transporter (EAAT) revealed comparale results. () Morphologic analysis of astrocytes showed no differences in the numer of terminal ranches and the lengths of cell processes. (c) Stimulation of cultured astrocytes with IL-β resulted in a comparale production of TNF-α and IL-. (d) Representative GFAP staining of TREK-expressing astrocytes in EAE lesions from immunized mice. (e) Quantification of astrocyte numers in EAE lesions of and Trek / mice showed a similar numer of GFAP-positive cells. ns = not significant. Nature Medicine: doi:./nm. Supplementary Figure 7

11 c d e EAE score EAE score Relative expression Thymidine uptake (cpm) Relative expression Cell / ead ratio 7 7,,,,... Control (prophylactic) Trek / Time after immunization Naïve Naïve all cells (therapeutic) Control ICAM VCAM EAE EAE CD + rluzole EAE Naïve CD + T cells Occludin T cells IFN-γ production (ng ml ) CD +.. Control Naïve Naïve EAE T cells ZO-. EAE IL-7 production (ng ml ) EAE T cells Control. Naïve Naïve EAE EAE PECAM T cells Claudin- T cells Supplementary Figure Nature Medicine: doi:./nm.

12 Supplementary Figure TREK activation reduces immune cell trafficking in vivo and ameliorates the disease course of EAE. (a) The TREK activator ( mg/kg, twice daily) significantly ameliorated the course of EAE when administered prophylactically (starting from the day of immunization) or therapeutically (starting after the first signs of EAE) compared with untreated mice (single horizontal line: control versus (prophylactic); doule horizontal line: control versus (therapeutic); n = mice per group). () Riluzole administration in Trek / mice resulted in a significantly delayed disease onset, while it had no influence on the EAE course eyond day (n = mice per group). (c) Proliferation rate and cytokine production of splenocytes isolated from -treated or untreated mice at maximum extent of EAE showed no significant (ns) differences. (d) MBMEC from naïve mice were compared with MBMEC isolated at the maximum extent of EAE from immunized untreated mice and -treated mice for the expression of EC markers. T cells served as negative controls. (e) Flow cytometry assessment of C-invading cells (left panel; one representative example out of three) and representative histologic staining of inflammatory foci in the spinal cord (H&E stain, lack arrows). P <.; Scale ar represents µm. ICAM = intercellular adhesion molecule ; IFN-γ = interferon-γ; IL-7 = interleukin-7; PECAM = platelet endothelial cell adhesion molecule ; VECAM = vascular cell adhesion molecule. p <.. Nature Medicine: doi:./nm.

13 Daily food intake per g mouse per day (g g ).... P L ATP release (cpm x ) P L IFN-γ production (ng ml ) IL-7 production (ng ml ) P L P L Supplementary Figure 9 Evaluation of dietary enrichment with linseed oil or palmseed oil for treated EAE animals with regard to food intake and T cell effector function. (a) Daily food intake per mouse per day was unchanged in the palm seed oil (P) and linseed oil (L) group. (). Splenocytes isolated at disease maximum showed a comparale proliferation rate (left panel) and IFN-γ- and IL- 7-production (middle and right panels, respectively) upon MOG restimulation. ns = not significant. Nature Medicine: doi:./nm. Supplementary Figure 9

14 Ct value Trek ND ND ND ND ND ND Trek Traak Task Task Task Tresk d Migrating cells (all cells = %) Monocytes Lymphocytes NK CD + CD + B cells c Relative expression Relative expression... Unstim LPS IFN-γ TNF-α IFN-γ +TNFα... RNA level Protein level Unstim LPS IFN-γ TNF-α IFN-γ +TNF-α e Cell / ead ratio (inflamed cells =.) AA µm + ALA µm Supplementary Figure Characterization of HBMEC in vitro. (a) Gene expression of different K P channels in HBMEC (n = ). () Trek gene expression in HBMEC under different inflammatory stimuli, normalized to unstimulated (Unstim) cells (n = per group). (c) TREK protein expression in HBMEC under different inflammatory stimuli. The densitometric quantification is shown (n = per group). (d) Relative migration of monocytes and lymphocytes under non-inflamed and inflamed conditions. Lymphocytes are further divided into sutypes. (e) The TREK activators arachidonic acid (AA) and alpha-linolenic acid (ALA) cause a comparale reduction of immune cell migration in vitro. LPS = lipopolysaccharide; nd = not detectale; NK = natural killer. p <.. Nature Medicine: doi:./nm. Supplementary Figure

15 Unstim Counts Inflam MFI FL-H Phalloidin-TRITC Supplementary Figure Reduction of actin polymerization under inflammatory conditions is restored y TREK activation. The measurement of F-actin content of HBMEC stained with phalloidin-tritc using flow cytometry is shown in the histogram and quantified in the ar chart. Unstim = unstimulated; Inflam = inflamed; ALA = linolenic acid; AA = arachidonic acid. P <.. Nature Medicine: doi:./nm. Supplementary Figure

16 Cytokine production (ng ml ) MCP- IL- c IFN-γ production (ng ml ) Cytokine production (ng ml ) IP- ND ND ND... RANTES ND ND ND IL- production (ng ml ) Relative expression ICAM ND ND ND Relative expression... PECAM Relative expression Claudin- Relative expression..... ZO- Relative expression.... Occludin Supplementary Figure Nature Medicine: doi:./nm.

17 Supplementary Figure TREK modulation has no influence on the cytokine profile of HBMEC and PBMC or on EC markers. (a) TREK modulation did not affect the secretion of MCP-, IL-, IP- and RANTES y HBMEC. () Expression of ICAM, PECAM, claudin-, ZO- and occludin was not altered y TREK modulation. (c) The cytokine profile of T cells was not changed y TREK modulation. AA = arachidonic acid, ALA = alpha-linolenic acid. n = ; ns = not significant. Nature Medicine: doi:./nm.

18 Fluorescence intensity..... TREK HD NAWM MS lesion DAPI ICAM TREK MS lesion DAPI VCAM TREK MS lesion c Fluorescence intensity ICAM d Fluorescence intensity 7 VCAM HD NAWM MS lesion HD NAWM MS lesion Supplementary Figure TREK downregulation is associated with ICAM and VCAM upregulation in human MS lesions. (a) Fluorescence intensity of TREK expression in C tissue of healthy control sujects (HD) and normal appearing white matter (NAWM) and lesions of MS sujects (MS lesion; n =, respectively). Representative pictures are depicted in Fig. g. () Immunohistochemical costaining for ICAM, VCAM and TREK in MS lesions (representative results). (c,d) Fluorescence intensity of (c) ICAM and (d) VCAM expression (n = ). Nature Medicine: doi:./nm. Supplementary Figure

19 splenos Endos undiff END. DAPI CD vwf 7 ß actin CD ß actin Supplementary Figure Purity controls after MBMEC preparation. (a) Western lot analysis from freshly isolated splenocytes, freshly isolated MBMEC (Endos undiff) and from the endothelial cell line END. for the rain endothelial cell marker von Willerand factor (vwf) and the immune cell marker CD. Purity controls were performed on a regular asis and one representative example is shown. () Immunocytochemical staining of freshly isolated MBMEC from immunized mice at disease maximum for CD (left: DAPI; right: CD. Inset shows a staining of a splenocyte for visual comparison in the same scale). Scale ar represents µm. Nature Medicine: doi:./nm. Supplementary Figure

20 thymidine uptake (cpm),,,,, Trek / unstim MOG eads Supplementary Figure Comparison of proliferation assays. Thymidine-uptake assays and ATP release assays showed comparale results as depicted in Figure f. Nature Medicine: doi:./nm. Supplementary Figure