Supplemental Information The Protein ATG16L1 Suppresses ...

Immunity Volume 39

Supplemental Information

The Protein ATG16L1 Suppresses Inflammatory

Cytokines Induced by the Intracellular Sensors

Nod1 and Nod2 in an Autophagy-Independent Manner

Matthew T. Sorbara, Lisa K. Ellison, Mahendrasingh Ramjeet, Leonardo H. Travassos, Nicola L. Jones,

Stephen E. Girardin, and Dana J. Philpott

Inventory of Supplemental Information 4 Supplemental Figures:

Figure S1: Knockdown of ATG5, ATG9a, and ATG16L1 in ModeK Cells, Related to Figure 2. Figure S2: The increased cytokine response in ATG16L1-‐deficient cells in response to cyto-‐invasion is not dependent on increased bacterial load, related to Figure 3. Figure S3: Knockdown of ATG16L1 but not ATG5 or ATG9a triggers increased Nod responses in ModeK cells, related to Figure 4. Figure S4: A truncated ATG16L1 lacking the N-‐terminal binds Nod1 and Nod2 but not ATG5, related to Figure 6.

Supplemental Experimental Procedures References for Supplemental Experimental Procedures 3 Supplemental Tables, related to Supplemental Methods:

Table S1: shRNA sequences used for ATG5, ATG9a and ATG16L1 knockdown Table S2: Primers used to generate pLJM1-‐ATG16L1 constructs. Table S3: Primer sequences for qPCR

Supplemental Experimental Procedures

shRNAs, expression constructs, and Lentivirus Production: HEK293T cells were

seeded at a density of 3 x 106 per 10 cm plate. The following day, cells were

transfected with 2.25 μg of psPax2, 0.75 μg pMDG, and 3 μg of either pLKO-‐shRNA

plasmid or pLJM1 constructs (See below) using Fugene HD (Roche) according to the

manufacturer’s protocols. shRNA sequences are listed in Table S1. 48 hours after

transfection, the supernatant of the cultures was collected, filtered to remove cell

debris, and buffered with 100 mM HEPES.

Knockdown procedure: ModeK, MEF, or MDAMC cells were seeded in 6 well

plates at densities of 4 x 105, 2.5 x 105, or 2 x 105 cells/well, respectively. 1 day later,

1 mL of lentivirus preparation was added to the cells in the presence of 8 μg/mL

polybrene, and the cells were centrifuged at 3500 rpm for 90 minutes. 24 hours

post-‐transduction, cells were split into 10 cm plates in media containing 8 μg/mL,

3µg/mL, or 2 μg/mL puromycin for ModeK, MEF and MDAMC cells, respectively.

Cells were used at day 5 post-‐transduction. Knockdown cells were prepared

independently for each infection and stimulation experiment to avoid serially

passaging cells deficient in autophagy.

Rescue Plasmid construction: Flag-‐tagged ATG16L1 and mCherry constructs

were amplified using the primers listed in Table S2. Amplified inserts were cloned

into the AgeI and EcoRI sites of pLJM1. The 300A and 300T variants of ATG16L1

were subcloned from previously described plasmids (Raju et al., 2012). Positive

clones were verified by sequencing.

Cell Culture and Bacteria Strains: S. flexneri M90T (Shigella), S. flexneri M90T-‐RFP,

S. flexneri BS176 (non-‐invasive) were grown in TSB broth. For M90T-‐RFP TSB broth

was supplemented with 100μg/mL ampicillin. L. monocytogenes 10403S (Listeria)

and the isogenic Δhly,plcAB (DP-‐2319 – endosome-‐confined strain) were grown in

BHI broth. Salmonella enterica serovar Typhimurium strain (Salmonella) SL1344

and the isogenic ΔphoP, and ΔSPI-‐1/pInv strains were grown in LB broth.

Protein Blots: Cells were lysed in RIPA buffer and separated by SDS-‐PAGE and

transferred to PVDF membranes. Membranes were blocked with 5% milk in TBS-‐T,

and incubated overnight with the following primary antibodies: 1:1000 rabbit anti-‐

ATG16L1 for human ATG16L1(Novus Biologicals), 1:2500 rabbit anti-‐ATG16L1 for

mouse ATG16L1 (Cosmo Bio Co), 1:1000 rabbit anti-‐ATG5 (Novus Biologicals),

1:1000 rabbit anti-‐ATG9a (Novus Biologicals) 1:10000 mouse anti-‐α Tubulin

(Sigma), 1:1500 rabbit anti-‐LC3 (Cell Signaling), 1:3000 mouse anti-‐Flag (Sigma),

1:10000 rabbit anti-‐Flag (Sigma), 1:2500 mouse anti-‐HA (Abcam), 1:1000 rabbit

anti-‐Rip2 (Santa Cruz Biotechnology), 1:1500 rabbit anti-‐p62 (Cell Signaling)

1:1000 rabbit anti-‐K63 Ubiquitin (Millipore). Following washing and incubation

with appropriate HRP-‐conjugated secondary antibodies, blots were developed with

ECL-‐Prime (GE Healthcare).

Immunofluorescence and Quantifications: For immunofluorescence, cells were

seeded in 24 well plates on glass coverslips. Cells were washed 2x with PBS, and

fixed for 15 minutes with 4% PFA in PBS. Fixed cells were mounted with Prolong

Gold Antifade reagent with DAPI (Invitrogen), and imaged and analyzed using a

Carlo Zeiss Axiovert 200 microscope with a 63X oil immersion objective and

Volocity software (Quorum Technologies). For quantifications, at least 25 cells were

counted from each of three independent experiments per condition.

Luciferase Assays: ModeK cells were transfected with 75 ng of NF-‐κB driven

luciferase (Igκ-‐luc, Invitrogen), 25 ng of constitutive β-‐gal, and 200 ng of empty

pcDNA3. 24 hours post-‐transfection, cells were stimulated (as described in

Experimental Procedures), and 4.5 hours post-‐stimulation luciferase activity was

measured.

qRT-‐PCR and ATG16L1 Rescue Analysis: RNA was isolated from infected cells

with the RNeasy Kit (Qiagen) and treated with the TURBO DNA-‐free kit (Ambion).

Reverse transcription was performed using the SuperScript III First-‐Strand

Synthesis Kit (Invitrogen), and cDNA was treated with RNASE H (New England

Biolabs). All procedures were performed according to the manufacturers protocols.

qPCR was performed with Power SYBR Green (Applied Biosystems), using primers

listed in Table S3. Values presented are normalized to Rpl19 for murine samples

and β-‐actin for human samples and each condition’s unstimulated control.

Rescue Analysis: to calculate rescue percentages the following formula was used:

rescue%=(3’UTR response – rescue response)/(3’UTR response – control response).

References Raju, D., Hussey, S., Ang, M., Terebiznik, M.R., Sibony, M., Galindo-‐Mata, E., Gupta, V., Blanke, S.R., Delgado, A., Romero-‐Gallo, J., et al. (2012). Vacuolating Cytotoxin and Variants in Atg16L1 That Disrupt Autophagy Promote Helicobacter pylori Infection in Humans. Gastroenterology 142, 1160-‐1171.

Supplementary Figures

Figure S1: Knockdown of ATG5, ATG9a, and ATG16L1 in ModeK Cells, related to

Figure 2. (A) Left panel: Lysates of cells transduced with the indicated shRNA’s

were analyzed by protein blot for ATG16L1 (top), the ATG5-‐12 conjugate (middle)

and Tubulin (bottom). Right panel: Cells transduced with scrambled or ATG9a

shRNA were probed for the expression of ATG9a (top) or tubulin (bottom) by

protein blot. * indicates a non-‐specific band. (B-‐D) Expression of ATG5 (B),

ATG16L1 (C), and ATG9a (D) mRNA was measured in cells transduced with the

indicated shRNAs (n=3, **, p<0.01 by t test). (E-‐F) Scrambled, ATG5, and ATG16L1

(E) or Scrambled, ATG16L1, and ATG9a (F) knockdown cells were stimulated with

rapamycin (25 µg/mL) for 2 hours. Formation of LC3-‐II was measured by protein

blot (top). Tubulin was used as a loading control (bottom). Blots are representative

of three independent experiments. (G) Representative images of Scrambled, ATG5,

and ATG16L1 knockdown cells transduced with GFP-‐LC3 and infected with RFP

expressing Shigella for 3.5 hours. (H) Quantification of GFP-‐LC3 autophagosomes

targeted to intracellular Shigella in control and knockdown cells (n=3, with 25 cells

counted per condition per experiment, * indicates p<0.05 by t test). (I) Lysates of

MDAMC cells transduced with the indicated shRNA’s were analyzed by protein blot

for expression of ATG16L1 (top panel), and Tubulin (bottom). (J) Expression of

ATG16L1 relative to β-‐actin in MDAMC cells was quantified in control and ATG16L1

knockdown cells (n=3, **, p<0.01 by t test). All values are present as mean+SEM.

Figure S2: The increased cytokine response in ATG16L1-‐deficient cells in response

to cyto-‐invasion is not dependent on increased bacterial load, related to Figure 3.

(A) Scrambled (left), ATG5 (middle) or ATG16L1 (right) knockdown cells were

infected with invasive or non-‐invasive Shigella, or wild type or endosome-‐confined

Listeria. At 16 hour’s post-‐infection secretion of CXCL1 was quantified by ELISA

(n=5-‐8). (B) Representative images of ModeK cells (Blue – DAPI) 3.5 hours after

being infected with RFP-‐Shigella (Red). To block bacterial replication post-‐invasion,

cells were treated with 5µg/mL chloramphenicol 30 minutes post-‐infection. (C)

Production of CXCL1 in control or ATG16L1-‐silenced ModeK cells was measured by

ELISA 16 hours post-‐infection with Shigella infection with or without

chloramphenicol treatment (n = 5). (D) Following infection of control of ATG16L1-‐

knockdown ModeK cells with wild-‐type Salmonella or a ΔphoP mutant, production

of CXCL1 was measured by ELISA at 16 hours post-‐infection (n=3). At 4.5 hours

post-‐infection, there was an average of 83.6% fewer bacteria in wells infected with

ΔphoP mutant compared to wild type. All values are presented as mean+SEM.

*p<0.05, **,p<0.01 by t test.

Figure S3: Knockdown of ATG16L1 but not ATG5 or ATG9a triggers increased Nod

responses in ModeK cells, related to Figure 4. (A) Production of CXCL1 was

measured by ELISA after stimulation of control, ATG5 and ATG16L1-‐knockdown

ModeK cells with C12-‐iE-‐DAP (20 μg/mL) or L18-‐MDP (20 μg/mL) for 16 hours

(n=3). (B) Scrambled (left), ATG5 (middle) and ATG16L1 (right) knockdown cells

were stimulated with C12-‐iE-‐DAP (20 μg/mL), L18-‐MDP (20 μg/mL), TNF-‐

α (10ng/mL) or LPS (100 ng/mL). At 16 hour’s post-‐stimulation, secretion of CXCL1

was quantified by ELISA (n=3-‐8). (C) Production of CXCL1 was measured by ELISA

after stimulation of control, ATG9a and ATG16L1 deficient ModeK cells with C12-‐iE-‐

DAP (10 μg/mL) or L18-‐MDP (10 μg/mL) for 16 hours (n=9). (D) Production of

CXCL1 by ModeK cells in the presence or absence of 3-‐MA (5 mM) and stimulated

for 16 hours with C12-‐iE-‐DAP (20 μg/mL), L18-‐MDP (20 μg/mL), TNF-‐α (10

ng/mL), LPS (100 ng/mL) (n=3 from a representative experiment). (E) Expression

of ATG16L1 (top panel) was examined by protein blot in ModeK cells transduced

with control or ATG16L1 targeting shRNAs. Tubulin was used as a loading control

(bottom panel). (F) Expression of ATG16L1 in cells transduced with control or

ATG16L1 targeting shRNA was quantified by qPCR (n=3). (G) Production of CXCL1

was measured by ELISA after stimulation of control, or ATG16L1-‐knockdown

ModeK cells with L18-‐MDP (20 μg/mL) or LPS (100ng/mL) for 16 hours (n=3). (H)

Production of CXCL1 was measured by ELISA after stimulation of control, or Rip2

inhibitor-‐treated ModeK cells with C12-‐iE-‐DAP (20 μg/mL), TNF-‐α (10 ng/mL),

L18-‐MDP (20 μg/mL) or LPS (100ng/mL) for 16 hours (n=3 from a representative

independent experiment.). All values are presented as mean+SEM. *p<0.05, by t

test.

Figure S4: A truncated ATG16L1 lacking the N-‐terminal binds Nod1 and Nod2 but

not ATG5, related to Figure 6. A) HEK293T cells were transfected with HA-‐Nod1

(left panels) or HA-‐Nod2 (right panels) and Flag-‐mCherry, Flag-‐ATG16L1, or Flag-‐

85−607 and interaction between the Flag-‐tagged proteins, Nod1 or Nod2, and ATG5

was assessed by performing a Flag-‐immunoprecipitation. Input lysates and

immunoprecipitated proteins were probed for Flag (top), ATG5 (middle) and HA

(bottom).

Table S1: shRNA sequences used for ATG5, ATG9a and ATG16L1 knockdown

Target shRNA Sequence

Murine ATG16L1 #1 AACAGACTTTGGAAAGATGGG

Murine ATG16L1 #2 AAAGTTCTCAAGAAAGCTACG

Murine ATG16L1 #3 AAACAATGTCATTGCAGCTGG

Murine ATG16L1 #4 AAGTTCTCAAGAAAGCTACGG

Murine ATG5 TTATCTGGGTAGCTCAGATGC

Murine ATG9a ATGTGTGCAAGAATCACTCGG

Human ATG16L1 Coding TTCAGTGTTATTCTTCCAGGC

Human ATG16L1 3’UTR GCATGAATGTGTCTCATTACT

Table S2: Primers used to generate pLJM1-‐ATG16L1 constructs.

Construct

Flag-‐

mCherry

Forward CCCACCGGTGCCACCATGGATTACAAGGATGACGACGATAAAATGGTGAGCAAGGGCGAG

Reverse CCCGAATTCTTACTTGTACAGCTCGTCCATGC

Flag-‐

ATG16L1

Forward CCCACCGGTGCCACCATGGATTACAAGGATGACGACGATAAAATGTCGTCGGGCCTCCGC

Reverse CCCGAATTCTCAGTACTGTGCCCACAGCACAG

Flag-‐85-‐

607

Forward CCCACCGGTGCCACCATGGATTACAAGGATGACGACGATAAAATGGCCCAACTGAGGATT

Reverse CCCGAATTCTCAGTACTGTGCCCACAGCACAG

Flag-‐ΔCCD Amplifying

1-‐69

For: CCCACCGGTGCCACCATGGATTACAAGGATGACGACGATAAAATGTCGTCGGGCCTCCGC Rev: CCGGGCTTGCCGCCTCCTTATCTCGTGCCTGTTTGGTACGTC

Amplifying

213-‐607

For: GACGTACCAAACAGGCACGAGATAAGGAGGCGGCAAGCCCGG Rev: CCCGAATTCTCAGTACTGTGCCCACAGCACAG

Table S3: Primer sequences for qPCR

Gene Forward Reverse

Murine Rpl19 GCATCCTCATGGAGCACAT CTGGTCAGCCAGGAGCTT

Murine Cxcl1 TGGGATTCACCTCAAGAACA TTTCTGAACCAAGGGGAGC

Murine Cxcl2 CCCCCTGGTTCAGAAAATCA CTCTTTGGTTCTTCCGTTGAGG

Murine

Atg16L1

TGTGAATTCAAGGGCTCCCTGTCT TCATCCACAGTCCAGATTCGGCTT

Murine Atg5 AACCACCTTGAGTCAGGACAACGA TGCATTTCACGAGAAGAGGAGGCT

Murine Atf3 CGAAGACTGGAGCAAAATGATG CAGGTTAGCAAAATCCTCAAATAC

Murine Atg9a GAATTGCTTCTTGTCACCGCTGCT AGGACGTGTTCCACAGCTAACACA

Human Actb CGTTGCACATGCCGGAG

GCACAGAGCCTCGCCTT

Human Il8 ATGACTTCCAAGCTGGCCGTGGCT

TCTCAGCCCTCTTCAAAATCTC

Human

Atg16L1

PCR1

AGACAGGCGTTCGAGGAGATCAT

ATCTCGTGCCTGTTTGGTACGTCA

Human

Atg16L1

PCR2

TAACCAAATGCAGCGGAAGGACAG

TTCAGGGTCTGGTTGGCTCTTTCA

Supplemental Information The Protein ATG16L1 Suppresses ...

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