Understanding the Role of the Inflammasome in Inflammation

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1 Understanding the Role of the Inflammasome in Inflammation Martha O Brien, Ph.D. Assay Design Group 215

2 Innate Immune Response in Inflammation LPS flagellin TLR-1/2/6 TLR-4 TLR-5 Adaptor proteins PAMPs, DAMPs Viral nucleic acids endolysosome TLR-3/7/9 NLR inflammasome PYD LRR NBD ASC-CARD Pro-caspase-1 Pro-IL-1 expression nucleus Pro-IL-1 Caspase-1 IL-1, IL-18 Inflammation

3 Inflammasome Multiprotein Complex Inflammasome: GFP-labeled ASC Santos, G. et al. (212) Lung Cellular and Molecular Physiology 33: L

4 Different Stimuli Induce Different Inflammasome Complexes Naip/NLRC4 NLRC

5 Inflammasome Engagement Activates Caspase-1 LPS T3SS needle protein T3SS rod protein flagellin Gasdermin D modified from Kanneganti, T-D. (215) The inflammasome: firing up innate immunity. Immunological Reviews 265:

6 Current Methods for Monitoring Inflammasome Activation Western Blots for processed IL-1β, IL-18 or Caspase-1 Cumbersome, time consuming Need to make lysates or use serum-free supernatants Variable Ab quality Activity can be missed because oligomerization but not cleavage of caspase-1 required for activity (Broz, et al., 21) ELISAs for Released IL-1β, IL-18, or Caspase-1 Cumbersome, time consuming Ab exhibit some cross-reactivity with pro-il-1β Pro-IL-1β can also be released complicating analysis IL-1β can be processed extracellularly by other proteases (van de Veerdonk, et al. 211) Dynamic Range is limited FLICA (fluorescently-labeled inhibitor of caspase, e.g., FAM-YVAD-FMK) Cells need to be washed, very problematic since caspase-1 is released from cells FLICA is not blocked by unlabeled inhibitor leading to questions of specificity HTRF (IL-1β), AlphaLISA (IL-1β) Similar to the ELISA, Abs exhibit some cross-reactivity with pro-il-1β Requires special instrumentation

7 S/N Bioluminescent Caspase-1 Substrates H Z-WEHD- N S N COOH N S Optimal caspase-1 tetrapeptide substrate determined from recombinant peptide library H Z-YVAD- N S COOH N S Known tetrapeptide caspase-1 substrate similar to YVHD endogenous IL-1ß cleavage site N 1,, 1, 1, Z-WEHD-aminoluciferin Z-YVAD-aminoluciferin Ac-WEHD-AMC Ac-YVAD-AMC 1, Caspase-1 (U/ml) 1 1 7

8 RLU Substrate Performance in Cell Model for Inflammasome Activation 5, 45, 4, 35, 3, 25, 2, 15, 1, 5, Z-WEHD-aminoluciferin No cell Vehicle a-hemolysin No MG132 Cell Model -hemolysin-treated THP-1 cells -hemolysin is a poreforming toxin virulence factor from Staphylococcus aureus that elicits inflammasome activation and pyroptosis in THP-1 monocytic leukemia cells

9 RLU Substrate Performance in Cell Model for Inflammasome Activation 1, 9, 8, 7, 6, 5, 4, 3, 2, 1, Z-WEHD-aminoluciferin +MG-132 No cell Vehicle a-hemolysin MG132+ added Cell Model -hemolysin-treated THP-1 cells -hemolysin is a poreforming toxin virulence factor from Staphylococcus aureus that elicits inflammasome activation and pyroptosis in THP-1 monocytic leukemia cells

10 RLU RLU Substrate Performance in Cell Model for Inflammasome Activation 1, 9, 8, 7, 6, 5, 4, 3, 2, Z-WEHD-aminoluciferin +MG-132 No cell Vehicle a-hemolysin Cell Model -hemolysin-treated THP-1 cells -hemolysin is a poreforming toxin virulence factor from Staphylococcus aureus that elicits inflammasome activation and pyroptosis in THP-1 monocytic leukemia cells 2, 18, 16, 14, 12, 1, 8, 6, 4, Z-YVAD-aminoluciferin +MG-132 No cell vehicle a-hemolysin 1, 2, MG132+ added YVAD Inhibition of proteasome with MG-132 required to see inflammasome signal Z-WEHD-aminoluciferin is the better substrate

11 RLU Coupled-Enzyme Assay System nigericin 12, 1, 8, THP-1 cells K+ ionophore triggers inflammasome No cell Vehicle Nigericin 6, 4, 2, Time after reagent addition (min)

12 % Inhibition Ac-YVAD-CHO is Caspase-1 Selective Inhibitor Enzyme Aldehyde K i YVAD (nm) Group I Caspase-1.76 Caspase Caspase Group II Caspase-3 >1, Caspase-7 >1, Caspase-2 >1, Group III Caspase-6 >1, Caspase Caspase-9 97 Caspase-1 48 Garcia-Calvo et al. (1998) J. Biol. Chem 273: % Inhibition of -hemolysin THP-1 signal: YVAD-CHO Titration 6' read YVAD-CHO (um)

13 RLU Ac-YVAD-CHO (1µM) Completely Inhibits Cell Signal 1, 9, 8, 7, 6, a-hemolysin-treated a-hemolysin-treated + YVAD-CHO Vehicle R² = , 4, 3, 2, 1, R² =.9997 R² = , 2, 3, 4, 5, 6, 7, 8, 9, Cell Number/well Assay is linear

14 RLU Ac-YVAD-CHO can Distinguish Caspase-1 from Cross-reacting Caspases 45, 4, 35, 3, 25, 2, 15, 1, Ac-YVAD-CHO inhibitor at 1µM in the reagent knocks down 99% of the caspase-1 activity. Caspase-Glo 1 Caspase-Glo 1 +YVAD-CHO (1 um) 5% Ac-YVAD-CHO does not substantially inhibit crossreacting caspases 5, 99% 15% Control inflammation caspases Caspase (125 pm) apoptotic caspases

15 RLU Ac-YVAD-CHO Confirms that the Luminescent Signal is Due to Caspase-1 in THP-1 cells 7, 6, 5, 4, Caspase-Glo 1 Caspase-Glo 1+YVAD-CHO Caspase-Glo 1+VEID-CHO 2h treatment 18h treatment 3, 2, 1, 215. Signal inhibited by YVAD-CHO= caspase-1! Signal not inhibited by YVAD-CHO is not caspase-1! 15

16 RLU Apoptosis Activity in THP-1 cells is Inhibited by VEID-CHO 5, 45, 4, 35, 3, 25, 2, 15, 1, 5, THP-1 Caspase-Glo 1 Caspase-Glo 1+YVAD-CHO Caspase-Glo 1+XIAP Caspase-Glo 1+VEID-CHO No cell Vehicle Doxorubicin Paclitaxel XIAP (endogenous inhibitor of caspases 3,7, and 9) partially inhibits signal VEID-CHO completely inhibits the apoptotic signal Although VEID-CHO is not completely selective for caspase-6, it is consistent with our in vitro data that caspase-6 contributes to the apoptotic signal YVAD-CHO does not inhibit the apoptosis signal providing the assay specificity

17 Caspase-Glo 1 Assay Cell-based Protocol

18 RLU Caspase-1 Activation Detected with Several Inflammasome Inducers in THP-1 cells THP-1 cells PMA-differentiated and treated in 384-well plates Caspase-Glo 1 Caspase-Glo 1+YVAD-CHO

19 RLU Assay Enables High Throughput Screening for Inflammasome Activation 24, 2, 16, 12, 384-well plate with automated dispensing Testing high, medium, and low inducer Nigericin LPS Flagellin Nigericin +YVAD LPS +YVAD Flagellin +YVAD Unstim. Nigericin LPS Flagellin S/B= Z' , 4, Stable signal in 384-well Time (min)

20 RLU RLU Macrophages Require Two Signals for Caspase-1 Activation Unprimed cells LPS-primed J774A.1 cells 4, 35, 3, Caspase-Glo 1 Caspase-Glo 1 +YVAD-CHO 25, 2, Caspase-Glo 1 Caspase-Glo 1+YVAD-CHO 25, 15, 2, 15, 1, 1, 5, 5, No cell Vehicle Nigericin Vehicle Nigericin No cell Vehicle Nigericin THP-1 J774A

21 Two Signal Hypothesis for NLRP3 Inflammasome Activation and Cytokine Processing Macrophages require two signals for processing and release of IL-1 and IL-18 mediated through the NLRP3 inflammasome Signal 1: Pathogen associated molecular patterns (PAMPs) bind to TLRs and upregulate pro-il- 1 /IL-18 and NLRP3 via the NF-kB pathway Yang et al. (212) Int. Neurourology J. 16: Signal 2: ATP, crystals, and other damage associated molecular patterns (DAMPs) trigger inflammasome formation, caspase-1 activation, and IL-1, IL-18 processing through a common mechanism thought to be K+ efflux, ROS, or lysosomal damage 21

22 RLU Caspase-1 Activated by One Inflammasome Stimulus in THP-1 Cells In THP-1 cells, Signal 2 (nigericin, -hemolysin) alone can activate caspase-1 via K+ efflux 4, 35, 3, 25, Caspase-Glo 1 Caspase-Glo 1 +YVAD-CHO 2, 15, 1, 5, No cell Vehicle Nigericin Vehicle Nigericin THP-1 J774A

23 No cell Unstim. LPS Pam3CSK4 Unstim. LPS Pam3CSK4 RLU Caspase-1 Activated by One Inflammasome Stimulus in THP-1 Cells In THP-1 cells, Signal 2 (nigericin, -hemolysin) alone can activate caspase-1 via K+ efflux Signal 1 (TLR agonists) alone can activate caspase-1 in differentiated THP-1 cells 3, 25, 2, 15, 1, 5, Undifferentiated PMA-differentiated

24 RLU Caspase-1 Activated by Two Inflammasome Signals in J774A.1 Macrophages J774A.1 macrophages require both Signal 1 and 2 for NLRP3 inflammasome engagement and caspase-1 activation 6 5 Caspase-Glo 1 J774A.1 Caspase-Glo 1+YVAD-CHO No cell LPS Nigericin ATP LPS+ LPS+ATP Nigericin

25 RLU RLU Caspase-1 Activation is Rapid and Transient 25, PMA-differentiated THP-1 cells 18, 16, LPS-primed J774A.1 cells 2, 15, 1, No cell Vehicle LPS LPS + YVAD-CHO 14, 12, 1, 8, 6, No cell Vehicle Nigericin Nigericin+YVAD-CHO 5, 4, 2, Time of treatment (h) Time of treatment (h)

26 No cell Unstim. Pam3CSK4 R848 Unstim. Pam3CSK4 R848 RLU Caspase-1 is Released into Culture Medium 3, 25, 2, Caspase-Glo 1 Caspase-Glo 1 +YVAD-CHO Caspase-1 is released with its substrates, IL-1 and IL-18 A simple transfer of half the culture medium to a 2 nd plate can be done to detect released caspase-1 15, 1, 5, Advantages Although the RLUs are lower, better S/B (greater sensitivity) is achieved due to lower background Leaves the cells available for multiplexing 215. Cells Culture medium S/B

27 S/B Culture Medium can be Frozen at -2 C Before Monitoring for Caspase-1 Activity fresh culture medium culture medium -2oC 2 days h 2h 3h 1h 2h 3h Flagellin YVAD-CHO

28 RLU Released Caspase-1 Activity from Mouse Primary BMDMs and Human Primary PBMCs Culture medium from mouse bone marrow-derived macrophages 18, 16, 14, 12, 1, 8, 6, 4, 2, Culture medium from human peripheral blood mononuclear cells No caspase-1 inhibitor Ac-YVAD-CHO Drs. Sivapriya Kailasan Vanaja and Vijay Rathinam University of Connecticut Assay validated in human PBMCs and mouse BMDMs Carlene Petes and Dr. Katrina Gee Queen s University

29 Caspase-1 Induced Pyroptosis is Protective Pathogen replicating Pathogen evading caspase-1 Pyroptosis (in contrast to apoptosis) is lytic and inflammatory releasing IL-1, IL-18 and other cytokines. Macrophage Pathogen detected by caspase-1 Neutrophil Recent research demonstrates that pyroptosis can also limit pathogen replication by releasing the pathogen before replication and exposing them to neutrophil uptake and clearance caspase-1 Clearance of pathogen pyroptosis

30 RLU RFU Multiplexing to Monitor Caspase-1 Activity and Pyroptosis 7, 6, 5, 4, 3, Undifferentiated THP-1 cells Caspase-Glo 1 Caspase-Glo 1 +YVAD-CHO 4, 35, 3, 25, 2, 15, CellTox Green Cytotoxicity 2, 1, 1, 5, Cell death with caspase-1 activation= pyroptosis Cell death, no caspase-1 activation = no pyroptosis

31 RLU RFU RLU RLU Multiplexing to Monitor Caspase-1 Activity and Cell Viability or Cell Death 25, 2, 15, Caspase-Glo 1 Culture Medium Caspase-Glo 1 Caspase-Glo 1 +YVAD-CHO 5, 45, 4, 35, 3, CellTox Green Cytotoxicity 3,5, 3,, 2,5, 2,, RealTime-Glo Viability 6,, 5,, 4,, CellTiter-Glo Viability 1, 25, 2, 15, 1,5, 1,, 3,, 2,, 5, 1, 5, 1,, 5, Cell permeability Reducing capacity decrease ATP decrease

32 No cell Vehicle LPS Nigericin Vehicle LPS Nigericin No cell Vehicle LPS Nigericin Vehicle LPS Nigericin RLU pg/ml Multiplexing to Monitor Caspase-1 Activity, IL-1 Release, and Pyroptosis 25, Caspase-1 Activity in Culture Medium Caspase-Glo 1 7, IL-1 Release (ELISA on Culture Medium) 2, Caspase-Glo 1+YVAD-CHO 6, 5, 15, 4, 1, 3, 5, 2, 1, PMA 2nM 2 days Undifferentiated PMA 2nM 2 days Undifferentiated

33 No cell Vehicle LPS Nigericin Vehicle LPS Nigericin No cell Vehicle LPS Nigericin Vehicle LPS Nigericin RLU RFU Multiplexing to Monitor Caspase-1 Activity, IL-1 Release, and Pyroptosis 25, 2, Caspase-1 Activity in Culture Medium Caspase-Glo 1 Caspase-Glo 1+YVAD-CHO 2, 18, 16, CellTox Green Cytotoxicity (Pyroptosis) 14, 15, 12, 1, 1, 8, 6, 5, 4, 2, PMA 2nM 2 days Undifferentiated PMA 2nM 2 days Undifferentiated

34 Multiplexing Summary in THP-1 Cells In differentiated THP-1 cells, nigericin induces caspase-1 activation, pyroptosis, and IL-1 release. In undifferentiated THP-1 cells, nigericin induces caspase-1 activation and pyroptosis, but not IL-1 release. The lack of IL-1 release in undifferentiated THP-1 cells is expected since differentiation or priming is required for pro-il-1 up-regulation. The caspase-1 assay has enabled a demonstration of caspase-1 activation and pyroptosis separate from IL-1 /IL-18 release in THP-1 cells. This led to the question: Is caspase-1 activation directly driving pyroptosis in the IL-1 independent cell model?

35 RFU Abs 54nM-56nM RFU Inhibition of Pyroptosis with Cell Permeable Caspase Inhibitors CellTox Green Cytotoxicity PMA-differentiated THP-1 IL-1 ELISA PMA-differentiated THP-1 CellTox Green Cytotoxicity Undifferentiated THP-1 35, 3, No inhibitor z-yvad-fmk (2 um) z-vad-fmk (2 um) no inhibitor z-yvad-fmk (2 um) z-vad-fmk (2uM) 6, 5, no inhibitor z-yvad-fmk (4 um) z-vad-fmk (2 um) 25,.8 4, 2,.6 3, 15, 1,.4 2, 5,.2 1, Vehicle Nigericin. Vehicle Nigericin Vehicle Nigericin Our results suggest that caspase-1 is driving pyroptosis in these cell models, but without complete inhibition with YVAD-FMK, further work will be needed to confirm this

36 Understanding the Outcomes of Inflammasome Activation LPS Gasdermin D Kanneganti, T-D. (215) The inflammasome: firing up innate immunity. Immunological Reviews 265:

37 Summary We have developed a bioluminescent assay to directly monitor activated caspase-1 for assessing inflammasome activation. Caspase-1 activity can be measured directly in cells in the lytic assay or from culture medium when caspase-1 is released from cells. The assay has enabled determining the kinetics of caspase-1 activation and we have shown that it is typically rapid and transient in our cell models. The assay can be multiplexed with other assays to monitor IL-1 /IL-18 release or cell death to assess pyroptosis. The assay demonstrated that two signals are not required for caspase-1 activation in THP-1 cells, whereas two signals are required in macrophages. In the THP-1 model we were able to separate the distinct outcomes of caspase-1 activation namely IL-1 /IL-18 release and pyroptosis by testing undifferentiated and PMA-differentiated cells. The high throughput capability of this convenient caspase-1 assay will enable screening for modulators of inflammasome activation

38 Acknowledgments Danielle Moehring Gedi Vidugiris Dan Lazar Jim Cali Troy Good Laurent Bernad Lauren Hongo Tenaya Noce Jeri Culp Jackie Jackson Stuart Forsyth Hélène Benink Shikha Gupta Pam Guthmiller Sally Floyd Terry Riss Thank you for your attention! Collaborators Justin Callaway and Jenny Ting (U. North Carolina) Raúl Muñoz-Planillo and Gabriel Núñez (U. Michigan) Carlene Petes and Katarina Gee (Queen s University) Sivapriya Kailasan Vanaja and Vijay Rathinam (U. Connecticut)

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