1. The role of p110β class I PI3K in microcrystal uptake and inflammasome activation in phagocytes Student: Maarten Verdonckt Mentor: Dr. Ezra Aksoy Promoter: Stefanie Salliau Work placement: Barts Cancer Institute, department of Cell Signalling 1

2. Table of contents 1. Introduction - PI3K - Gout 2. Experimental design 3. Results 4. Conclusion 5. Future perpectives 2

3. 1. Introduction 3

4. Phosphoinositide 3-kinase (PI3K) Three classes Class I phosphorylate PIP 2 -> PIP 3 PIP 3 is second messenger in lots of pathways 4 PIP 2 PIP 3 p110 p85 Kinase signalling e.g. Akt

5. Gout Auto-inflammatory disease Caused by deposition of monosodium urate (MSU) crystals in the joints 5

6. MSU crystals Receptors & lipid sorting Tyr kinase PI3K 6 Phagocytosis? Inlammasome activation?

7. MSU Inflammasome Caspase-1 Pro-IL-1β IL-1β Inflammation Phagocytosis “Frustrated phagocytosis” (Phagolysosome rupture) 7

8. 2. Experimental design Aim: Determine the role of p110β in crystal-uptake and inflammasome activation by phagocytes Design: -In vitro: Dendritic cells & macrophages from bone marrow (BMDC & BMMF) - Ex vivo: Peritoneal macrophages 8 Cell cultures derived from WT and kinase dead (KI) mice - Inflammasome pathways activation - Phagocytosis activity Experimental procedures - Western blotting - ELISA - Confocal microscopy - Flow cytometry PIP 2 PIP 3 p110 p85 DADA protein KI allele and function WTp110β Knock-in (KI)

9. 3. Results 9

10. 10 Analysis of Caspase-1 processing Caspase 1 full length long exposure Caspase1 full length short exposure MSU (min) Gapdh Cleaved Caspase 1 p10 01060010600 WTp110β KI 10 p110β KI BMMFs

11. 10 75 Caspase1 long exposure Caspase1 short exposure MSU (min) 010 Gapdh 60010600 p10 WT p110β KI 10 p110β KI 20 50 37 25 50 37 kDa 37 BMMFs 11 Analysis of Caspase-1 processing (continued)

12. 12 Total Akt P Akt T308 MSU (min) 010 Gapdh 600 WTp110β KI 10 P Akt S473 Analysis of Akt phosphorylation ex-vivo MFs

13. Total Akt P Akt T308 MSU (min) 010 Gapdh 600 WTp110β KI 10 P Akt S473 50 37 kDa ex-vivo MFs 13 Analysis of Akt phosphorylation (continued)

14. 14 Total Akt P Akt T308 MSU (min) 010 Gapdh 60010600 WTp110β KI 10 WT + Tgx 21 P Akt S473 50 37 kDa BMDCs Analysis of Akt phosphorylation (continued)

15. 1. IL-1β IL-6 concentration (ng/ml) 2. IL-6 Cytokine Production (ELISAs) 15 ex-vivo MFs

16. Microtubule structure – βeta-Tubulin staining 16 Confocal Microscopy BMDCs

17. Confocal Microscopy Actin cytoskeleton – Phalloidin (F-actin)staining 17 p110β KI WT + Medium (basal condition) BMDCs

18. 18 p110β KI 10μm WT + LPS (10 min)

19. WT 19 Analysis of bead uptake by confocal microscopy in BMDCs Green: Phalloidin (F-actin) Red: 0.5mM polystyrene beads

20. p110β KI 20

21. 060 time (min) Mean fluorescent intensity (%) F-actin dynamics time (min) 03060 WT p110β -/- a. G-actin dynamics time (min) 03060 Mean fluorescent intensity (%) WT p110β -/- b. n=3 Flow cytometry analysis of actin dynamics ex-vivo MFs 21 c.

22. 4. Conclusions 1.Loss of p110β leads to a defect in actin depolymerization and alters actin dynamics in phagocytes. 2. The defects in actin dynamics hinders the phagocyte ability to internalize MSU crystals. 3.Diminished MSU crystal uptake results in the inhibition of inflammasome activation and IL-1β secretion. 22

23. 23 5. Future Perspectives Study cell biological and molecular mechanism of p110β action in more detail Investigate whether a similar mechanism is active in other similar disease models (asbestosis with asbestos, silicosis with silica or Alzheimer’s Disease with amyloid beta) Translational aspects of the study and use of p110β inhibitors as potential therapeutic targets

24. Mentor: Dr. Ezra Aksoy Co-mentor: Prof Bart Vanhaesebroeck Promoter: Stefanie Salliau Work placement: Barts Cancer Institute, department of Cell Signalling 24 Acknowledgements Stay abroad was partially funded by Erasmus