Macrophage migration inhibitory factor is required for

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Macrophage migration inhibitory factor is required for NLRP3 inflammasome activation. Lang et. al. Supplementary Figures ...

Macrophage migration inhibitory factor is required for NLRP3 inflammasome activation Lang et. al.

Supplementary Figures





Compound

Binding Late (RU)

Stability Early (RU)

Stability Late (RU)

Binding to Reference Spot

COR123625

6.4

0.4

0.0

No

R50 Method

Steady State Method

R50 (µM)

KD (µM)

KD (µM)

ka (103M-1s-1)

kd (10-3 s-1)

Molecules bound/MIF timer

34 ± 9.9

10 ± 1.4

2.5 ± 1.2

0.6 ± 0.2

1.3 ± 0.3

1:1

Kinetics Method

Predicted Stoichiometry of Complex†

Assuming 3 molecules of MIF are required to form one binding site.

Supplementary Table 1. Binding and kinetics of COR123625 to immobilized MIF

A

ns

****

6000

B

**** ***

10000

***

ns

IL-6 (pg ml -1)

4000

2000

8000 6000 4000 2000 0

0 Control

LPS

control

LPS + nigericin

C

LPS

D

**** **** ****

**** ****

***

****

15000

IL-1β (pg ml-1)

400

****

10000

**

200

0

5000

+

LP S

+

AT P

ic in

LP S

ni ge r

ol on tr

+

AT P

ic in LP S

ni ge r

LP S LP S

+

C

on tr

ol

0

C

IL-1α (pg ml-1)

600

LPS + nigericin

LP S

TNF-α (pg ml-1)

WT MIF KO

Supplementary Figure 1. MIF is required for release of IL-1 family cytokines. (A and B) Primary murine WT and Mif-/- BMDM were left untreated (control), primed with LPS (10 ng ml-1) or treated with LPS followed by nigericin (5 µM) for 1 h. Levels of (A) TNF-a and (B) IL-6 in cell culture supernatants were assessed by ELISA. (C and D) Primary WT BMDC were left untreated, primed with LPS alone (10 ng ml-1), primed with LPS, then treated with COR123625 (50 µM) for 2 h before the addition of nigericin (5 µM) or ATP (10 mM) for 1 h. Levels of (C) IL-1α and (D) IL-1β in cell culture supernatants were assessed by ELISA. Data are expressed as means ± SEM of 3-4 mice. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 by one-way ANOVA of variance with a correction provided by the Tukey multiple comparisons test.

A

B Ile64 Ser63

Lys66

Pro1 Lys32

C

Thr30 Gly31

OD (475nm)

0.08

0.07

0.06 1.0

1.5

2.0

2.5

Ln Concentration COR [M]

D ***

*

**

E 5000

TNF-α (pg ml-1)

3000 2000 1000 0

****

4000 3000 2000 1000

µM 0

µM O R

10

50

µM O R

C +

LP S

LP S

+

C + LP S

C

O R

25

LP S

ol on tr

LP S n ig + e n LP + ric ig C er S in O ic + R in ni 2 ge 5 + µM C ric O in R 50 + C µM O R 10 0 µM ic in

LP S

LP S

+

ni ge r

LP S

+

C

on tr

ol

0

C

IL-1β (pg ml-1)

4000

**

Supplementary Figure 2. COR123625 impairs the biological actions of MIF. (A) Chemical structure of the MIF small molecule inhibitor COR123625. (B) The binding position of COR123625 within the tautomerase active site near Pro-1. (C) Concentrationdependent inhibition of MIF tautomerase activity by COR123625. (D) and (E) iBMDM were left untreated, primed with LPS alone (100 ng/ml) for 6 h, or treated with increasing doses of COR123625 (25-100 µM) for 1 h before the addition of LPS for 5 h, followed by nigricin treatment (10 µM) for 1 h. Levels of (D) IL-1β or (E) TNF-α in cell culture supernatants were assessed by ELISA. Data are expressed as means ± SEM of at least 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 by one-way ANOVA of variance with a correction provided by the Tukey multiple comparisons test.

A

B

****

6000

****

IL-6 (pg ml -1)

3000 2000 1000

4000

2000

O R C +

on tr

LP S LP S

LP S

+

C

O R

LP S

on tr ol C

ol

0

0

C

TNF-α (pg ml-1)

4000

Supplementary Figure 3. Inhibition of MIF does not affect the release of IL-6 or TNF-α. Primary murine WT BMDM were left untreated (control), primed with LPS (10 ng ml-1) for 6 h, or pre-treated COR123625 (50 µM) for 1 h followed by treatment with LPS for 5 h. Levels of (A) TNF-α or (B) IL-6 in cell culture supernatants were assessed by ELISA. Data are expressed as means ± SEM of 3-4 mice. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, by one-way ANOVA of variance with a correction provided by the Tukey multiple comparisons test.

100 50

100 50

10000 5000

LP S

i+

Im

Im

**** ****

60 40 20

ns ns

+ LP S

G

30000

IL-1β (pg ml-1)

50

20000

10000

saline

LPS

LP nig + n LPS S er ige + i ni cin rici n ge + ric IS in O1 + 4IP P

ol on tr

LP S

C

+ LP ni + n LP g S i S + eric ger ni i ge in + cin ric IS O in -1 + 4IP P

ol on tr C

****

80

LP S LP S

F IL-6 (pg ml -1)

5000

0

0

0

10000

THP-1 100

IL-1α (pg ml-1)

IL-1β (pg ml-1)

E

**** ****

15000

i+ Im i ni ni ge ge ric ric in in + C O R

ol on tr C

on tr ol P P 3C P 3C 4 + 4 + ni 3 C4 ni ge ge ric ric in in + C O R

C

****

20000

**** ****

0

0

THP-1

****

15000

150

0

D

***

on tr ol + L + LP PS ni n ge ig S e + r N ici rici ig n er + n C ic O in + R 4IP P

150

**

C

IL-1β (pg ml-1)

IL-1β (pg ml-1)

200

200

BMDM

C

LP S

**

250

B

*

IL-1β (pg ml-1)

A

****

WT MIF-/MIF-/- + COR

**

40

ns

30 20 10 0

saline

LPS

Supplementary Figure 4. Release of IL-1β is inhibited in response to different TLR ligands and different MIF inhibitors. Primary WT BMDM were left untreated, primed with LPS (10 ng ml-1), Imiquimod (Imi; 10 µg ml-1) or Pam3CSK4 (P3C4; 10 ng ml-1) overnight alone, or primed with TLR agonists followed by activation of the inflammasome with nigericin (5 µM) for 1 h, or primed with TLR agonists before treatment with MIF inhibitors COR123625 or 4-IPP (50 µM) for 2 h prior to inflammasome activation with nigericin. (A-C) Levels of ILβ in cell culture supernatants were assessed by ELISA. (D-E) THP-1 cells were left untreated, primed with LPS (10 ng/ml) overnight, primed with LPS followed by treatment with nigericin (5 µM) for 1 h, or primed with LPS then treated with MIF antagonists ISO-1 (50 µM) or 4IPP (50 µM) for 2 h prior to inflammasome activation with nigericin. (F) and (G) WT or MF/mice were injected intraperitoneally with vehicle control (saline), LPS alone (2 mg kg-1) or COR123625 (20 mg kg-1) in combination with LPS for 2 h. Serum levels of (F) IL-6 and (G) IL-1β were measured by ELISA. Data are expressed as mean ± SEM of at least 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by one-way ANOVA of variance with a correction provided by the Tukey multiple comparisons test.

B

100

ge ge lli lli n n + C O R

fla LP S

LP S

E

+

+ p( p( dA dA :d :d T) T) + C O R +

LP S

LP S

+

C

p( dA

on tr

ol LP S

:d T)

LP S

ol on tr C

D

LP S

0

0

0

500

fla

50

200

1000

+

100

300

ol

****

150

400

LP S

Mif-/-

on tr

200

***

1500

500

****

C

WT

IL-1α (pg ml-1)

250

C

*

IL-1α (pg ml-1)

IL-1α (pg ml-1)

A

WT Mif-/-

8000

IL-1β (pg ml-1)

2000

1000

0

****

4000 2000

LP S

+

si lic a

LP S

ol on tr

+

M

SU

LP S LP S

ol

0

on tr C

****

6000

C

IL-1β (pg ml-1)

***

****

3000

Supplementary Figure 5. NLRP3-dependent release of IL-1α and IL-1β. (A) Primary murine WT or Mif-/- BMDM were left untreated, primed with LPS alone (10 ng ml-1) or primed with LPS before transfection of poly (dA:dT) (p(dA:dT); 1 µg ml-1) for 5 h. IL-1α release was measured by ELISA. WT BMDM were left untreated, primed with LPS (10 ng ml-1) or primed with LPS before the addition of COR123625 (50 µM) for 2 h before transfection of (B) p(dA:dT) (1 µg ml-1) or (C) flagellin (250 ng/ml) for 5 h. IL-1α release was measured by ELISA. Primary murine WT and Mif-/- BMDM were left untreated (control), primed with LPS (10 ng ml-1) or primed with LPS followed by (D) MSU or (E) silica for 6 h. IL-1β release was measured by ELISA. Data are expressed as mean ± SEM of at least 3 mice. *P < 0.05, ***P < 0.001, ****P < 0.0001, by one-way ANOVA of variance with a correction provided by the Tukey multiple comparisons test.

Donor 2

100

THP-1

90 80 70

Donor 1 Donor 2

20

Donor 3

10

0

0 5 25 50

0 5 25 50

0 5 25 50

0 5 25 50

60

30

ISO-1 (µM)

0 5 25 50

Donor 1

0 5 25 50

110

% Plasmodium+ cells

B Trypan blue negative (%)

A

ISO-1 (µM)

Supplementary Figure 6. MIF inhibition does not induce cell death or prevent phagocytosis of RBC with Plasmodium falciparum. (A) Cell viability was assessed by PI staining and data presented as percentage of PI-negative cells. (B) Plasmodium falciparuminfected RBC were stained with DiffQuik and phagocytosis assessed by light microscopy. Data is presented as the percentage of Plasmodium infected cells. Data are expressed as means ± SEM from 3 separate donors.

A

B -/-

LPS

C

-

+

-

intracellular pro-IL-1β (relative to β-actin)

MIF

WT

+

39 28

pro-IL-1β

39

β-actin

1.5

WT MIF-/-

1.0

0.5

0.0

control

LPS

-

+

+

+

+

+

+

Nigericin

-

-

-

-

+

+

+

COR (before LPS)

-

-

+

-

+

-

-

COR (after LPS)

-

-

-

+

-

-

+

39

LPS

β-actin Lysates pro-IL-1β

28

28 19

pro-IL-1β Supernatants IL-1β p17

Supplementary Figure 7. Levels of intracellular pro-IL-1b are not affected by MIF. (A) Primary WT and MIF-/- BMDM were treated with LPS (10 ng ml-1) for 5 h. Western blot analysis of cellular lysates to assess levels of pro-IL-1β and β-actin was performed. (B) Densitometry was used to calculate expression of intracellular proteins shown in (A). Expression of pro-IL-1b was normalised to b-actin. n = 2 mice per group. (C) Primary murine BMDM were treated with LPS (10 ng ml-1) for 5 h before stimulation with nigericin (5 µM). Cells were treated with COR123625 (50 µM) either 1 h prior to LPS treatment or 1 h prior to nigericin treatment. Western blot analysis of cellular supernatants and lysates to assess levels of pro-IL-1β, mature IL-1β and β-actin was performed.

% cells with ASC specks

*** 40

*

- COR

****

****

+ COR 30

*

20

*

10 0

0

5

15

30

45

Time with nigericin (min)

B % cells with ASC specks

A

* 20

- COR

****

+ COR 15

***

10 5 0

0

1

2

4

Time with silica (h)

Supplementary Figure 8. Time course for NLRP3 inflammasome activation. ASCcerulean macrophages were primed with LPS (10 ng mL-1) overnight. Cells were then treated with (A) nigericin (10 µM) for 1 h or (B) silica (150 µg/ml) for 4 h in the presence or absence of COR123625 (50 µM). Data presented is the percentage of ASC-cerulean cells containing an ASC speck after treatment with nigericin. Data is mean ± SEM of three independent experiments.

A LPS

LPS + nigericin

**

**

50

20 µm

C

40 30 20 10

+ ni ni ge ge ric ric in in + W FA +

LP S

C

LP S

ol

0

on tr

% cells with ASC specks

B

LPS + nigericin + WFA

LP S

D

- COR **

2.15

+ COR

τ Av Int (ns)

** 2.10

2.05

2.00

0

15

30

60

Time with nigericin (min)

Supplementary Figure 9. MIF and Vimentin are required for NLRP3 activation. (A) ASC-cerulean macrophages were primed with LPS (10 ng/ml) overnight, primed with LPS followed by inflammasome activation with nigericin (5 µM) for 1 h, or primed with LPS followed by treatment with Withaferin A (5 µM) for 2 h prior to nigericin (5 µM) treatment for 1 h. Confocal images shown are representative of at least three independent experiments. (B) Data presented is the percentage of ASC-cerulean cells containing an ASC speck. Data shown is the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA of variance with a correction provided by the Tukey multiple comparisons test. (C) Stimulated emission depletion (STED) Super resolution imaging of vimentin (red) and NLRP3 (green) in WT BMDM treated with LPS and nigericin. (D) WT BMDM were primed with LPS (100 ng ml-1) for 5 h, +/- COR123625 (50 µM) for 2 h prior to nigericin (5 µM) treatment for 0-60 min h. Levels of interaction between NLRP3 and Vimentin were assessed by FLIM-FRET. Changes in the amplitude weighted average lifetime (t Av Amp) of the donor (A488) due to proximity to the acceptor (A568). n = 3 mice, **P < 0.01, one-way ANOVA of variance with a correction provided by the Tukey multiple comparisons test.

Supplementary Figure 10: Full length blots for Figure 4D NLRP3 - lysates

ASC - lysates 191

28

19

97

caspase-1 - lysates

IL-1β - lysates

39 39

28

14

β-actin - lysates

39 28

Note: some blots were cut to size to allow simultaneous probing with multiple antibodies.

Supplementary Figure 11: Full length blots for Figure 4D

caspase-1 - lysates

39 28

14

IL-1β - supernatants 39

28

14

Note: blots were cut above 45kDa

Supplementary Figure 12: Full length blots for Figure 5F

β-actin - lysates

caspase-1 - lysates

51 39 51 39

WT

Mif-/-

WT

caspase-1 - supernatants

Mif-/-

IL-1β - supernatants 28

14

51 39 28

WT

Mif-/-

14

WT

Mif-/-

Note: Samples were read on a luminescent image reader and blots were cut for specific sizes. Images on the left are direct photographs of the blot (ladder). Unrelated samples were removed from the blots above.

Supplementary Figure 13: Full length blots for Figure 6A

191 97

51

Note: blots were cut to size to allow simultaneous probing with multiple antibodies.

Supplementary Figure 14: Full length blots for Figure 6A

97

19 14

Supplementary Figure 15: Full length blots for Figure 8A

191 97

19 14

WB: MIF co-IP: MIF WB: NLRP3

WB: NLRP3

191 97

co-IP: NLRP3 WB: NLRP3

WB: NLRP3

14 14

co-IP: NLRP3 WB: MIF

WB: MIF

Supplementary Figure 16: Full length blots for Supplementary Figure 7A

IL-1β

β-actin

51 39 28

WT

Mif-/-

WT

Mif-/-

Supplementary Figure 17: Full length blots for Supplementary Figure 7C

51 39

β-actin

28 19

39

IL-1β (lysates)

28

28 19 14

IL-1β (supernatants)

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