TOLL 2018 • • • • 247--P mice lethality 2: Figure activation causes 1: Figure • E0 Timeline of mouse development (days) E16.5 small intestine 06 (C362A). expressing catalytically development, we have engineered activation of RIPK1 and RIPK3 during To better understand how caspase MLKL. which can be rescued by deletion of RIPK3 or Caspase its substrate MLKL by an ill suppresses mediated by RIPK3 and In addition to promoting , caspase termed and RIPK3 Receptor Casp8 Mlkl • atrophy (R). intestinal lumen (L normal but show increased E18.5 Placenta Heart - development. Caspase - / - NLR/ NLR/ now +/+ Phospho embryos E10.5 Casp8 Casp8 embryonic WT Mlkl H&E H&E necroptosis. due E11 Loss - - INTRODUCTION 8 loss causes embryonic lethality, interacting protein kinase 1 (RIPK1) die at die Casp8 Mlkl - trigger a C362A - - / / - Casp8 8 catalytic activity is required for to * Katherine Wickliffe 06 -NLR/Inflammasome - RIPK3 - of MLKL / - caspase C362A Casp8 birth birth - C362A 8 C362A8 – ) and severe intestinal villous lethality – pro IHC C362A inactive caspase - embryos are inflammatory rescues - 8 C362A8 Casp8 Mlkl - defined mechanism. E18.5 intestine neutrophils in the mutation and RIPK3 and RIPK3 Casp8 Casp8 - / - and Catalytic E12.5 +/+ adult Casp8 H&E H&E embryonic - - - mice / / 8 inhibits - - but Mlkl Ripk3 grossly WT embryos Casp8 Mlkl Department Department of Physiological Chemistry Roose WickliffeKatherine - / C362A the - - / 8 - - / - – C362A - 8 - Girma 37 50 50 25 37 50 50 75 75 50 cytokines of andelevated levels deathincreased cell Figure3: ripoptosome Figure5: deletion of Caspase Figure4 C362A

• Serum Level (pg/ml) Casp8

• C 10 10 10 10 •

c Inactive Percent survival l2 -/- -/- 1 2 3 4 -/- -/- embryos were measured by Levels of circulating cytokines in the serumofE17.5 C / 100 M Mlkl Mlkl c E17.5 small intestine

Mlkl Casp8 adulthood (L) and show no intestinal atrophy (R). Mlkl Western blot. intestines. Co Caspase c 20 40 60 80 l3 p C /M 1 -/- -/- 0

Mlkl Ripk1 c i IHC andWestern blots l p 3 0 4 p C / 1 M a * Input -/- ,

c - Mlkl - C l i 5 p RIPK3 / IHC : / 1 - c R b * Casp8 l -/- C362A 1 a Søren Mlkl Mlkl Casp8 inactive caspase Catalytic Mlkl 1 n

Casp8 Mlkl /E t e ** 50 -/- Age (d) o s C ta Ripk1 x x - C c in

components l -/- 8 was -/- x 1 / -

Fadd - c K 1 l / /

2 C - -

C362A C 100 / Casp8 , Maltzman Allie C362A M

-/- Casp8 Casp1/11 x i cl p

b Caspase 1 F TRADD RIPK3 RIPK1 p-RIPK1 2 Casp1 Mlkl 9 p - /M Casp3 IHC -actin purifying proteins were detected by ADD - I R G G During * - Cleaved

immunoprecipitated C Warming I G -

P Casp1/11 150 M S / F - - Casp8 K

- C Mlkl C362A

/11 S 1/11

3

C362A F IF – N Caspase DOI: 10.3252/pso.eu.TOLL2018.2018 - -g - / 1 - / IL - -1 C362A Casp1/11 in vivo (n=20) 100 a

I mice 37 50 75 50 L 1

lethality lethality is rescued by -1 , Department of Pathology Luminex b * -

I 100 / L 25 75 75 50 50 75 50 - -/- -/- -2 50 37 20 Mlkl Casp8 I

mice survive to L

Caspase -

3 ofshow signs

-/- -/- 1 2 IL 3

Mlkl Ripk1 -

/ -

5 M , -/- - Mlkl (n=12) IL lk - . - - 6 l /- Vishva 8 interacts with interacts 8 I E18.5 intestine I L -/- C362A L - Development Mlkl Casp8 -1 1 0 * Casp8 Mlkl 2 M from E16.5

- I -/- L p l 1 8 IP k - 4 ** C Ripk1 1 0 l -/ 2 a - Debra Dugger , p s -/- -/- 7 RIPK3 p-RIPK3 Caspase 8 0 FADD FLIP Caspase 3 Cleaved Casp3 RIPK1 p-RIPK1 p Fadd H&E I L 8 C - 3 -/- C362A 1 6 3 2 Casp1/11 IL A -1 - Caspase 8 TRADD RIPK3 RIPK1 IL 5 -1 8

7 Dixit A I presented L M -1 - 8

Poster C – S F T Promotes N F 1 at: , KimNewton 2 , Department of • • Casp8 Casp8 Survival of • • • • • • Mlkl components partially rescued Figure7 perinatal lethality of Figure6 birth • weeks of age, but the mice are severely • required for the pathology of determine which We are performing additional rescue crosses to components Caspase not perinatal lethality. phenotypes Deletion of FADD or RIPK1 rescues some Casp1/Casp11. can be rescued to by ablation of RIPK3 or The embryonic lethality of pro atrophy, increased levels of Casp8 Casp8 Unexpectedly, deletion of MLKL only rescues suppressing necroptosis during embryogenesis. Caspase Deletion of RIPK3 rescues Deletion of FADD has no effect on survival. mice RIPK1 loss delays the - / - Casp8 atrophy and neutrophil transmigration seen in RIPK3, RIPK1, or FADD deletion reduces Casp8 C362A

deleted. reduced when RIPK1, Casp1/11, or RIPK3 are Circulating levels of IL Serum level (pg/mL) C362A 1 - 10,000 20,000 30,000 40,000 inflammatory cytokines , Joshua Webster, Joshua to P4 Mlkl Mlkl C362A C362A 0 : : C362A C362A Casp8 P RIPK3 and RIPK1 contribute to the - - - - / / 8 C362A 8 catalytic activity is critical for - - Fadd athology athology in - Mlkl P6, but none survive to mice to birth. associated with CONCLUSIONS in vivo mice. Casp8 - / - C362A - 1 E18.5 intestine / Ripk3 IL - Casp8 inflammasome - embryos by deletionof 1 Casp8 interacts with 8 C362A and in cells. rescue crosses - / * P4 - C362A death of Casp8 - - 6 Mlkl C362A 18 in E18.5 serum are Mlkl Casp8 exhibit severe intestinal . cell death, Casp8 - * Casp8 - / / Ripk1 Casp8 - - Casp8 C362A + Ripk1 –

Casp8 mice C362A H&E H&E Mlkl 2 components are ripoptosome ripoptosome C362A , - - / 3 / - C362A Mlkl Ripk3 Mlkl Mlkl Casp8 Mlkl Ripk3 Mlkl - C362A C362A weaning. C362A Merone - / embryos - Casp8 Mlkl -/- -/- -/- -/- -/- Casp8 and elevated Mlkl Mlkl mice to ~6 Fadd Casp8 Ripk1 Casp1/1 -/- -/- runted Mlkl -/- - / Mlkl - - / C362A - Fadd - C362A Casp8 adult mice - / villous - -/- / - -/- but -/- C362A mice. . Ripk3 is 1 - / - -/- C362A - / -