Constitutive Interferon Maintains GBP Expression Required for

Cell Reports, Volume 24

Supplemental Information

Constitutive Interferon Maintains GBP Expression Required for Release of Bacterial Components Upstream of Pyroptosis and Anti-DNA Responses Beiyun C. Liu, Joseph Sarhan, Alexander Panda, Hayley I. Muendlein, Vladimir Ilyukha, Jörn Coers, Masahiro Yamamoto, Ralph R. Isberg, and Alexander Poltorak

Gated on GFP+ cells Uninf WT

sdhA

40

30

30

20

20

10

10

40 30 20 10

Ifnb induction 4hr

15000

2000

10000

1000

5000

40

20

E

tim LP S W sd T hA

ns

4hr

GBP2 p43 Prop38 Casp11

GAPDH

U

2hr

W do T tA sd hA

W do T tA sd hA

0

W do T tA sd hA

0

nf

B6 Casp11-/-

W T sd hA sdh sd + A hA N e + c1 zV A D

Ifnb Transcriptional Induction kinetics

ni

**** *** ns

0

3000

U

60

Uninf WT dotAsdhA LPS cGAMP

Ifnb/Gapdh

sdhA

Casp11-/-

sdhA

D

WT

W T hA s d h sd + A hA Ne + c1 zV A D

B6

0

****

Propidium Iodide (6 hours p.i.) ***

sd

WT (B6)

sdhA

10hr post infection

% Propidium Iodide (+)

Casp11-/ - sdhA B6 sdhA B6 WT Uninf

50

0

WT

C

Gated on GFP+ cells

GFP+ (Infected) ** **

60

50

*

40

0

B

GFP+ (Infected)

GFP- (Bystander) 50

% Cytotoxicity

Gated on live cells

% Propidium Iodide (+)

A

8hr

Figure S1. (Related to Figure 1) Maximum cell death (A, B) Macrophages were inoculated with L. WT induced death pneumophila GFP+ and at 6 hpi (A) or 8 hpi (B), (WT infection) 80 -20hr 80 cellular GFP-associated signal and propidium -4hr iodide (PI) incorporation were measured by flow 60 -1hr 60 cytometry. Histograms show one representative ** 0hr experiment. Bar graphs show compiled data from ** 40 No IFN 40 n>3 experiments of PI incorporation in the indicated gates after challenge of either B6 or 20 20 Casp11-/- macrophages. (C) B6 and Casp11-/- macrophages were 0 0 challenged with the L. pneumophila WT and Δ sdhA strains in the presence of Nec1 (RIPK1/3 IFN priming time Hours post infection inhibitor) or zVAD (pan-Caspase inhibitor). PI incorporation at 6 hpi is show. (D) B6 macrophages were challenged with WT, dotA- mutant, or ΔsdhA mutants for the indicated times and Ifnb mRNA was measured by qRT-PCR. (E) B6 macrophages were challenged with WT, dotA-, or ΔsdhA strains for 10 hours. Whole cell lysates were probed by Western blot for Gbp2, pro-Casp11 and GAPDH. 10 hour LPS incubation and cGAMP transfection are displayed. (F) B6 macrophages were pre-treated with 100U/ml of IFNβ at the indicated times points prior to, or concurrent with L. pneumophila WT challenge (see timeline in Fig 1G). Displayed is PI incorporation as a function of time (left), and maximum cell death measured at 6 hpi (right). 0h r -1 hr -4 hr -2 0h r

6

5

4

3

2

1

0

% Cytotoxicity

% Cytotoxicity

F

B

Isg15/Gapdh

1

1

1

0.100

0.100 0.10

0.010

1

2

3

4

5

1

1

0.1000

0.1000

0.0100

0.0100

G Ig

--

1

1

0.1000

0.1000

0.0100

0.0100

0.0010

0.0010

0.0001

0.0001

1 0.10000

Gbp7/Gapdh

0.01000

hr

/-

hr

3-

B Ifn 6 ar If /G nb b p /c

3-


/-

hr 3-


hr

3-

hr

0.00001 /-

0.0001 3-

0.0001

0.00010


0.0010

/-

0.0010

/-

0.00100


mRNA transcripts of chromosome-5 encoded GBPs

0.0100

0.0100

0.0100

0.0010

0.0010

0.0010

0.0010

0.0001

0.0001

0.0001

0.0001 hr

3-

hr

B

3-

hr


0.0100

/-

0.1000

Ifn 6 ar Ifn /G bb p /c

0.1000

/-

0.1000


0.1000

/-

1


1

/-

Gbp9/Gapdh

1

3-

Gbp8/Gapdh

Gbp6/Gapdh

1

3-

Relative to GAPDH

B6

Gbp5/Gapdh

Gbp3/Gapdh

Gbp2/Gapdh

Gbp4/Gapdh

Relative to Gapdh

B6

mRNA transcripts of chromosome-3 encoded GBPs Gbp1/Gapdh

D

IF

B6

hr

C

IF

N

6

Hours post infection

A R Ifn ar -/-

0

A R Ifn ar -/ -

A R Ifn ar -/-

0.001

IF

Ig

--

G

0.01

N

0.010 0.001

0

10

G

20

Mx1/Gapdh

10

--

Relative to B6

% Cytotoxicity

Casp11-/- IgG Casp11-/- IFNAR B6 IgG B6 IFNAR

40

Irf7/Gapdh 10

N

sdhA induced death 60

Ig

A

Figure S2. (Related to Figure 2) (A) B6 and Casp11-/- Macrophages inoculated with L. pneumophila ΔsdhA after 20 hr treatment with noted antibodies. PI incorporation used to measure cell death. (B) qRT-PCR of Irf7, Isg15, Mx1 expression in the presence or absence of constitutive IFN signaling. (C, D) qRT-PCR showing the baseline expression of noted genes in macrophages from various mouse strains. The CT values of genes of interest are normalized to sample-intrinsic Gapdh CT values. Dotted red line indicates background amplification from knock-out macrophages.

IFN rescue of baseline ISG expression 1000

100

100

10

10

1 0.100

10 1

1

0.10

0.010

Ifnb-/- + IFN (IU/ml)

10 0

4. 0

2. 0

1. 0

0. 5

0. 0

10 0

4. 0

2. 0

1. 0

0. 5

0. 0

10 0

4. 0

2. 0

1. 0

0. 5

0. 0

B 6 Ifn ar -/ -


B 6 Ifn ar -/-

0.01

0.1

0.001

B 6 Ifn ar -/ -

Relative to B6

Mx1/Gapdh

Isg15/Gapdh

Irf7/Gapdh 100


Figure S3. (Related to Figure 3) Ifnb-/- macrophages were treated for 20 hours with various doses of recombinant IFNβ as in Fig 3B, C, D. Transcript levels were measured by qRT-PCR. Dotted black line indicates steady-state expression of genes of interest in B6 macrophages.

Infect with WT L. pneumophila

100U/ ml IFN

removed IFN 20hr

Cell death induced by SdhA-sufficient L. pneumophila +IFN No IFN

20 0

20 10

1.5hr 2.5hr 3.5hr

3.5hpi, no IFN

IFN + WT L. pn.

E 100

% Aberrant morphology

Gbp chr3-/Casp11-/-

Within cytosolic population + IFN , 3hpi *** *** 100

80 60 40 20 0

B

6 c

G

bp

3 hr

80 60 40 20 0

-/-

B

6

ch

G

bp

r3

-/-

-/ -

inset

p1 1

merge

as

anti-L.p.

sdhA WT

C

GFP-L.p.

**

30

B6

nucleus

**

4

3

2

1

0


D

40

0

-20

B6 Gbpchr3-/Casp11-/-

**

% rod shaped

% Cytotoxicity

40

Cytosolic permeable bacteria 50

% Cytosolic Permeable

60

C

-/ -

B

Measure cell death

0hr

p1 1

-20hr

as

Plate BMDMs

C

A

Figure S4. (Related to Figure 4) (A) Macrophages were pre-treated for 20 hrs with 100U/ml of IFN prior to inoculation with WT L. pneumophila. (B) Kinetics of WT L. pneumophila-induced cell death in the absence and presence of IFN pre-activation. (C) Percentage of cytosolic-accessible L. pneumophila based on antibody staining without detergent permeabilization. (D) Representative images of cytosolic WT bacteria in noted macrophages at 3 hpi. Images were taken using 63x lens, scale bar = 5m. (E) Quantification of cytosolic accessible WT bacteria scored for aberrant (left) or rod-shaped (right) morphology at 3 hpi.

60

60

0

5

4

3

2

1

0

5

4

0

3

0

2

0

1

0

0

0

5

20

4

20

3

20

2

20

1

20

0

40

5

40

4

40

3

40

2

40

1

sdhA WT

5

60

80

sdhA WT

4

60

80

sdhA WT

BAL 5

3

80

sdhA WT

BAL 4

2

80

sdhA WT

60

0

% Cytotoxicity

80

BAL 3

1

BAL 2

BAL 1

Figure S5 (Related to Figure 5) Individual kinetics of ex vivo human bronchoalveolar lavage (BAL) cells infected with L. pneumophila WT or ΔsdhA, from 5 independent donors.

% ASC speck within ROI

**

10

WT

*

8

ΔsdhA **

WT + zVAD *

6

**

4 2 0

2hr

4hr

6hr


8hr

ΔsdhA + zVAD

Figure S6. (Related to Figure 6) B6 macrophages were challenged with the indicated strains of L. pneumophila in the presence of zVAD as indicated. ASC signal positivity is quantified within areas positive for anti-Legionella antibody (ROI).

Lung IL-1β 5hr 15

*

10

10

ns

ng/ml

ng/ml

ns

5

5

T

T

6

6

B

B

6

Lung CXCL1 5hr

Lung CXCL1 5hr p=0.025

50

30

30

100 10-1 10-2

Gbp7 Gbp3 Gbp8

Gbp2 Gbp9 Gbp4 Gbp5

0.1 10

*

Gbp6 Gbp10

1

Gbp1 Gbp11

10-3 10-310-210-1100 101 102 103 104 105

0.1

10

* 1

B6

Ifnar-/-

Ifnb-/-

Δ G sd B G P chr h A B 3Pc /hr W 3T /Δ sd hA

0.1

W 6 B

sd 6

W 6

B

B

101

T

0

Ifn hA Ifn ar-/ ar -/- WT Δ sd hA

0

T

10

1

102

B6 FPKM

20

10

103

6

20

104

Mx1/Hprt

40

ng/ml

40

* **

B

50

ng/ml

Δ sd

W

Δ sd G B hA Pc hr G 3B /Pc W hr T 3/Δ sd hA

W 6 B

B

D

G B hA Pc hr G 3B /Pc W hr T 3/Δ sd hA

0

0

Lung ISG expression 10

105

*

15

C Irf7/Hprt

20

Lung

B

Isg15/Hprt

Lung IL-1α 5hr

Ifnar-/- FPKM

A

Supporting Figure 7 (Related to Figure 7) (A) Lung IL-1α and IL-1β were measured by ELISA. Each dot represents an animal, results are pooled from 2 experiments in which tissues were harvested between 12 and 18hrs post infection. (B) Whole genome RNA-sequencing was performed on the indicated tissue and cellular populations. Fragments per kilobase mapped (FPKM) is plotted. Each dot is an individual gene. Gray dots represent genes where the expression difference between B6 and Ifnar-/- tissue is less than 2 fold. Yellow dots represent genes in which the expression difference between B6 and Ifnar-/- tissue is more than 2 fold. Blue dots represent the 11 Gbp genes encoded on the mouse genome, dots labeled. Magenta represent classical ISGs Stat1, Irf7, Isg15, Isg20, Mx1, Mx2. (C) Various ISGs expression levels measured by qPCR from uninfected lungs from B6, Ifnar-/- and Ifnb-/- animals. Each dot represents an animal. (D) Lung CXCL1 was measured by ELISA. Each dot represents an animal, results are pooled from 2 experiments in which tissues were harvested within a 4-6 hour window post infection.