www.sciencetranslationalmedicine.org/cgi/content/full/6/245/245ra95/DC1

Supplementary Materials for

An annexin A1–FPR1 interaction contributes to necroptosis of keratinocytes in severe cutaneous adverse drug reactions

Nao Saito, Hongjiang Qiao, Teruki Yanagi, Satoru Shinkuma, Keiko Nishimura,

Asuka Suto, Yasuyuki Fujita, Shotaro Suzuki, Toshifumi Nomura, Hideki Nakamura, Koji Nagao, Chikashi Obuse, Hiroshi Shimizu,* Riichiro Abe*

*Corresponding author. E-mail: aberi@med.hokudai.ac.jp (R.A.); shimizu@med.hokudai.ac.jp (H.S.)

Published 16 July 2014, Sci. Transl. Med. 6, 245ra95 (2014) DOI: 10.1126/scitranslmed.3008227

The PDF file includes:

Fig. S1. Causative drug–specific lymphocytes in patients’ peripheral blood. Fig. S2. The cytotoxicity of supernatant from DIHS/DRESS PBMCs. Fig. S3. The cytotoxicity of supernatant from irrelevant drug–exposed SJS/TEN PBMCs. Fig. S4. The cytotoxicity of keratinocytes from normal-appearing postlesional skin and nonlesional skin. Fig. S5. Protein levels of necroptosis signaling molecules in keratinocytes from SJS/TEN patients, ODSR patients, or healthy controls. Fig. S6. Effect of poly(I:C), TNF-α, and granulysin on SJS/TEN keratinocyte cytotoxicity. Fig. S7. The cytotoxicity of CD14bright CD16– and CD14dim CD16+ cells on SJS/TEN keratinocytes. Fig. S8. FPR1 stimulation does not induce phosphorylation of JNK, p38, or ERK. Fig. S9. Cytotoxicity in SJS/TEN keratinocytes, ODSR keratinocytes, or healthy control keratinocytes induced by PBMC supernatant, as measured by LDH assay. Table S1. Mass spectrometry result of the proteins in SJS/TEN supernatant. Table S2. Patient and healthy control information. Data File S1. The promoter region of FPR1 has no pathogenic mutations. Reference (31)

Supplementary Materials

Fig. S1. Causative drug–specific lymphocytes in patients’ peripheral blood. Causative drug--

specific cells were detected by human IFN-γ ELISPOT. SJS/TEN PBMC, PBMCs from patients

who had recovered from SJS/TEN; Re-stimulated SJS/TEN PBMC, PBMCs from patients who

had recovered from SJS/TEN that had been cultured with causative drugs for 5 days to

proliferate drug-specific T cells. PBMCs were obtained from patient No. 2 and healthy control

No. 2.

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Fig. S2. The cytotoxicity of supernatant from DIHS/DRESS PBMCs. We analyzed the

cytotoxicity of supernatants from five DRESS/DIHS patients. The causative drugs were

lamotrigine (#1), carpamazepin (#2 and #5), sulfamethoxazole and trimethoprim (#3 and #4).

Keratinocytes were obtained from patients No. 5 (SJS/TEN) and No. 22 (DRESS/DIHS) and

healthy control No.1. PBMCs were obtained from patients No. 22 (#1), No. 23 (#2), No. 24 (#3),

No. 25 (#4) and No. 26 (#5) in Table S2.

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Fig. S3. The cytotoxicity of supernatant from irrelevant drug–exposed SJS/TEN PBMCs.

The cytotoxicity of supernatants from PBMCs stimulated with an irrelevant drug (amoxicillin)

were tested. *P < 0.01. Keratinocytes were obtained from patient No. 5 (non-lesional skin) and

healthy control No.9. PBMCs were obtained from patient No. 5.

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A

Post-lesional skin

Non-lesional skin

Detachment phase Healing phase

B

Fig. S4. The cytotoxicity of keratinocytes from normal-appearing postlesional skin and

nonlesional skin. (A) Photographs of lesional and nonlesional skin. Skin biopsies from SJS/TEN

patients were obtained from normal-appearing skin that was lesional during the acute detachment

phase but that had returned to normal (postlesional skin), and from normal-appearing non-

lesional skin (nonlesional skin). (B) Cytotoxicity induced by supernatant from PBMCs of

SJS/TEN PBMCs in cultured keratinocytes from normal-appearing postlesional skin and

nonlesional skin. Postlesional skin (n=3, patients No. 1, No. 3, No. 10); normal-appearing skin

that was nonlesional (nonlesional skin)(n=4, patients No. 4, No. 5, No. 8, No. 9) . Each point

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represents the average cytotoxicity for each patient. All the experiments were performed at least

three times.

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Fig. S5. Protein levels of necroptosis signaling molecules in keratinocytes from SJS/TEN

patients, ODSR patients, or healthy controls. The expression of RIP1, RIP3, FADD and

CYLD in keratinocytes was analyzed by immunoblotting. Keratinocytes were obtained from

patients No. 3, 4, 5, 18, 19 and 20 and healthy controls No. 3, 4, 5 and 7.

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Fig. S6. Effect of poly(I:C), TNF-α, and granulysin on SJS/TEN keratinocyte cytotoxicity.

Poly: IC (10, 20, 50 µg/ml), TNF-α (100, 200, 500 ng/ml) and granulysin (15-kD form) (0.01,

0.1, 1.0 µg/ml). Keratinocytes were obtained from patient No. 3 (post-lesional skin) and healthy

control No. 5.

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Fig S7. The cytotoxicity of CD14bright CD16− and CD14dim CD16+ cells on SJS/TEN

keratinocytes. Purified CD14bright CD16- and CD14dim CD16+ cells were exposed to supernatant

from CD14-depleted PBMCs and were cultured in RPMI-1640 medium supplemented with 2

mM L-glutamine and 25 mM HEPES buffer for 1 day. The supernatant was collected, and

cytotoxicity was assessed. *P < 0.05 vs. medium. SJS/TEN#1 and #2 keratinocytes were

obtained from patients No. 3 (post-lesional skin) and No. 5 (non-lesional skin), respectively.

Healthy control No.3. PBMCs were obtained from patient No. 3.

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Fig. S8. FPR1 stimulation does not induce phosphorylation of JNK, p38, or Erk. Erk 1/2, p-

38 and JNK phosphorylation were analyzed in FPR1-HeLa and mock-transfected cells treated

with fMLP (5 nM). Cell lysates were analyzed by immunoblotting with anti-pErk 1/2, p-p38 and

p-JNK antibodies.

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Fig. S9. Cytotoxicity in SJS/TEN keratinocytes, ODSR keratinocytes, or healthy control

keratinocytes induced by PBMC supernatant, as measured by LDH assay. *P < 0.01 vs.

ODSR and healthy control keratinocytes. Keratinocytes were obtained from patients No. 10

(post-lesional skin) and No. 19 (non-lesional skin) and healthy control No. 5. PBMCs were

obtained from patients No. 3 and No. 14. *P < 0.01 vs. ODSR and healthy control keratinocytes.

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Table S1. Mass spectrometry result of the proteins in SJS/TEN supernatant.

# Identified Protein (126) Accession # SJS/TEN ODSR#1 ODSR#2 medium

1 talin gi|119578757

(+4)

53 0 0 0

2 myosin-9 gi|12667788

(+1)

43 0 0 0

3 filamin-A isoform 1 gi|116063573

(+5)

23 1 0 0

4 unnamed protein product gi|21752646

(+2)

18 0 0 0

5 Chain A, Crystal Structure Of Human Enolase gi|203282367

(+10)

13 2 3 0

6 fibrinogen alpha chain isoform alpha preproprotein gi|11761629

(+5)

11 0 0 0

7 vinculin gi|119574932

(+13)

10 0 0 0

8 78-kDa glucose-regulated protein precursor gi|16507237

(+4)

9 0 0 0

9 unnamed protein product gi|158256710

(+6)

8 0 0 0

10 Alpha-actinin-1 gi|122146006

(+27)

8 1 0 0

11 hemoglobin gi|109893891

(+56)

8 0 2 0

12 glyceraldehyde-3-phosphate dehydrogenase gi|31645 (+3) 8 0 0 0

13 moesin gi|119625804

(+3)

7 0 0 0

14 Heat shock cognate 71-kDa protein gi|123647

(+31)

7 0 0 0

15 tubulin beta-1 chain gi|13562114 6 0 0 0

16 aldolase A, fructose-bisphosphate gi|119600342

(+5)

6 0 0 0

17 calelectrin gi|179976 (+8) 5 0 0 0

18 ATP synthase, H+ transporting, mitochondrial F1 gi|127798841 5 0 0 0

12

complex, alpha subunit 1, cardiac muscle (+9)

19 CAP, adenylate cyclase-associated protein 1 gi|119627645

(+6)

4 0 0 0

20 unnamed protein product gi|193787540

(+14)

4 0 0 0

21 unnamed protein product gi|189055002

(+5)

4 0 0 0

22 keratin 1 gi|11935049

(+2)

4 22 6 19

23 Chain A, Three-Dimensional Structure Of A

Transglutaminase: Human Blood Coagulation

Factor Xiii

gi|1127268

(+15)

3 0 0 0

24 annexin A1 gi|119582950

(+7)

3 0 0 0

25 Chain A, Human Annexin A2 With Heparin

Tetrasaccharide Bound

gi|114794644

(+16)

3 0 0 0

26 Putative HLA class I histocompatibility antigen,

alpha chain H

gi|308153595

(+1)

3 0 0 0

27 GDP dissociation inhibitor 2 gi|119606836

(+9)

3 0 0 0

28 Chain A, Crystal Structure Of Human

Phosphoglycerate Kinase Bound To D-Adp

gi|193506632

(+12)

2 0 1 0

29 albumin gi|11493459

(+19)

2 3 0 0

30 coronin-like protein gi|1002923

(+6)

2 0 0 0

31 malate dehydrogenase 1, NAD (soluble) gi|119620368

(+3)

2 0 0 0

32 ribonuclease/angiogenin inhibitor 1 gi|119622735

(+6)

2 0 0 0

33 Chain R, Twinning In Crystals Of Human Skeletal

Muscle D- Glyceraldehyde-3-Phosphate

Dehydrogenase

gi|230867 2 0 0 0

34 peptidyl-prolyl cis-trans isomerase A gi|10863927 2 0 0 0

13

(+53)

35 hemoglobin beta gi|229149 2 0 0 0

36 clathrin, heavy polypeptide (Hc) gi|119614801

(+36)

2 0 0 0

37 Chain A, Glutathione Transferase P1-1 gi|11514451

(+23)

2 0 0 0

38 hCG2016942 gi|119594944

(+15)

2 0 0 0

39 Chain A, Structure Of The Aflatoxin Aldehyde

Reductase In Complex With Nadph

gi|157881389

(+6)

2 0 0 0

40 ubiquitin-like modifier-activating enzyme gi|23510338

(+4)

2 0 0 0

41 Chain A, Human Muscle L-Lactate Dehydrogenase

M Chain

gi|13786849

(+6)

1 2 0 0

42 fibrinogen beta chain gi|119625338

(+21)

1 0 0 0

43 acetyl-Coenzyme A acyltransferase 2 gi|119583356

(+6)

1 0 0 0

44 Chain A, Crystallographic And Kinetic Studies Of

Human Mitochondrial Acetoacetyl-Coa Thiolase

(T2)

gi|145579602

(+5)

1 0 0 0

45 Protein disulfide-isomerase gi|110815912

(+42)

1 0 0 0

46 Parvin, beta gi|127801538

(+5)

1 0 0 0

47 hypothetical protein DKFZp762H157.1 gi|11276938

(+31)

1 0 0 0

48 SAM domain and HD domain 1 gi|22209036

(+4)

1 0 0 0

49 septin 6 gi|119610263

(+19)

1 0 0 0

50 92 kDa type IV collagenase gi|177205 (+7) 0 1 1 0

51 Chain A, Solution Structure Of Human Normal

Adult Hemoglobin

gi|157883730

(+17)

0 0 1 0

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52 Chain A, Structural Changes Of The Active Site

Cleft And Different Saccharide Binding Modes In

Lysozyme

gi|1065033

(+173)

0 0 1 0

53 epidermal cytokeratin 2 gi|181402 (+1) 0 0 2 1

54 Keratin 14 gi|12803709

(+7)

0 0 0 1

55 keratin, type I cytoskeletal 9 gi|55956899 0 10 3 8

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Table S2. Patient and healthy control information.

Case Age/sex Causative drug Type of cutaneous adverse reaction

1 56/F Excemide TEN 2 55/M Acetaminophen TEN 3 37/F Amoxicillin SJS 4 70/M Ambroxol Hydrochloride SJS

5 27/F Lamotrigine SJS

6 49/F Acetaminophen SJS 7 49/M Benzbromarone SJS 8 71/M Phenytoin SJS 9 68/M Acetaminophen SJS

10 79/F Lansoprazole SJS

11 50/F Phenytoin Maculopapular

12 30/M Lamotrigine Maculopapular

13 37/F Amoxicillin Maculopapular

14 64/M Levothyroxine Sodium Hydrate Maculopapular

15 65/M Sulfamethoxazole, Trimethoprim Maculopapular

16 70/M Carbamazepine Maculopapular

17 24/F Carbamazepine Maculopapular

18 75/F Celecoxib Maculopapular

19 63/F Isoniazid sodium methanesulfonate hydrate Maculopapular

20 61/F Zonisamide Maculopapular

21 8/M Acetaminophen Maculopapular 22 17/F Lamotrigine DRESS/DIHS 23 70/M Carbamazepine DRESS/DIHS 24 61/F Sulfamethoxazole, Trimethoprim DRESS/DIHS 25 65/M Sulfamethoxazole, Trimethoprim DRESS/DIHS 26 80/M Carbamazepine DRESS/DIHS

Case Age/sex 1 6/M Healthy control 2 33/F Healthy control 3 35/M Healthy control 4 40/F Healthy control

5 47/M Healthy control 6 56/M Healthy control 7 56/F Healthy control 8 62/M Healthy control 9 68/F Healthy control 10 80/F Healthy control

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Data File S1. The promoter region of FPR1 has no pathogenic mutations.

Nucleotides from the –88 to the –72 position (–88/–72) of FPR1 were shown to influence

its transcriptional activity (31), if the nucleotide number is designated as its position relative to

the guanidine (+1) at the transcriptional start site. Therefore, we hypothesized that mutations

within the promoter region of FPR1, especially the –88/v72 region, could increase FPR1

production and might be involved in the pathogenesis of SJS/TEN. To address this hypothesis

Japanese patients with SJS/TEN and ODSR were recruited (8 patients and 7 patient,

respectively), and genomic DNA was isolated from their peripheral blood. Participants gave

written informed consent in compliance with the Declaration of Helsinki Principles. This study

was approved by the Medical Ethics Committee of Hokkaido University, Sapporo, Japan. For

mutation analysis of the promoter region of FPR1, nucleotides from the –630 to the +117

position (–630/+117) were amplified and sequenced as described previously (31), but no

mutation except c.-533G>A (rs59390147) was identified in the –630/+117 region in any of the

patients with either SJS or ODSR. The mutation c.-533G>A (rs59390147) was carried by two

and five patients with SJS and ODSR, respectively. The lack of statistically significant difference

between the two groups with respect to the SNP highly suggests that mutations in the promoter

region of FPR1 are not associated with the pathogenesis of SJS, at least in the Japanese

population.