1. Protein Protein Interactions: The Inflammasome
Mark D. Wewers, M.D.
Davis Heart and Lung Research Institute

2. Outline of Presentation
Innate vs. Adaptive Immunity
Focus on TLR and NLRs
Pyrogens (endogenous and exogenous)
Intracellular sensors/inflammasomes
Example of intracellular pathogen
Example of population differences in pathogen responses (caspase-12)

3. Time Line for Innate vs. Adaptive Immunity
Cooper MD and Alder MN. Cell 124:815, 2006.

4. INFECTION
Fever/Inflammation
ARDS
Multiple Organ Failure
IL-1/TNF

5. Shock and Poor Tissue Perfusion May Lead to Distal Gangrene

6. Black Death
25% of population died

8. Illness due to natural causes
Hippocrates
5th century B.C.
Illness due to natural causes

9. Guns Germs and Steel Hypothesis
Jared Diamond

11. Meningococcemia

12. core polysaccharide
bacterial membrane
“O” antigen
lipid A
Components of lipopolysaccharide

13. Acquired host response:
How can our defense repertoire cover the enormous diversity in pathogen challenges?
Innate host response:
Static defense system
Focus on immutable components of pathogens (e.g., cell wall component)
Acquired host response:
Creative ability to change with pathogen
Depends upon the acquisition of recombinase gene (RAG)

14. Innate Pathogen Sensors
NLR
TLR
Extracellular
sensors
Intracellular
TIR NB
LRR

15. Family of TLR’s and Ligands
Beutler et al.

16. TLRs and their ligands
Akira et al. Nature Reviews (Immunology):4:499, 2004.

17. The Signaling Pathway of Toll-like Receptors
Figure 2.
The Signaling Pathway of Toll-like Receptors.
Some of the toll-like receptors (TLRs) function as pattern-recognition
receptors of the innate immune system. Their recognition
of microbial products leads to the activation of the nuclear
factor- k
B (NF-
B) signaling pathway. In this example, the
recognition of lipopolysaccharide is mediated by three different
gene products: CD14, toll-like receptor 4 (TLR4), and MD-2.
The binding of lipopolysaccharide to CD14 presumably leads to
the association of CD14 with the TLR4–MD-2 complex and is
thought to induce the dimerization of TLR4. Once TLR4 is activated,
it recruits the adapter protein MyD88, which is associated
with the serine–threonine protein kinase interleukin-1 receptor
–associated kinase (IRAK). IRAK is then phosphorylated
and associated with the tumor necrosis factor–associated factor
6 (TRAF-6) adapter protein. Oligomerization of TRAF-6 is
thought to activate a member of the mitogen-activated protein
kinase kinase kinase (MAP3K) family, which directly or indirectly
leads to the activation of I
B kinase 1 (IKK1) and I
B kinase
2 (IKK2). These kinases phosphorylate I
B on serine residues,
thus targeting I
B for degradation and releasing NF-
B, which
moves into the nucleus and induces the transcriptional activation
of a wide variety of inflammatory- and immune-response
genes.
Medzhitov R. NEJM 343:338,2000.

18. Exogenous pyrogen induces endogenous pyrogen.

19. Both Endogenous and Exogenous Pyrogens converge at NFkB

20. Innate versus Adaptive Immunity Characteristics
Figure 2-10

21. Bridge to Adaptive Immunity
Figure 3.
The Receptors Involved in the Interplay of the Innate and Adaptive Immune Systems.
Recognition of the pathogen-associated molecular pattern (PAMP) by pattern-recognition receptors, such as the toll-like receptors,
generates signals that activate the adaptive immune system. Endocytic pattern-recognition receptors, such as the macrophage mannose
receptor, bind to components of microbial cell walls and mediate the uptake and phagocytosis of pathogens by antigen-presenting
cells (macrophages and dendritic cells). Proteins derived from the microorganisms are processed in the lysosomes to generate
antigenic peptides, which form a complex with major-histocompatibility-complex (MHC) class II molecules on the surface of
the macrophage. These peptides are recognized by T-cell receptors. In the case of the signaling class of pattern-recognition receptors,
the recognition of pathogen-associated molecular patterns by toll-like receptors leads to the activation of signaling pathways that
induce the expression of cytokines, chemokines, and costimulatory molecules. Therefore, pattern-recognition receptors have a role
in the generation of both the peptide–MHC-molecule complex and the costimulation required for the activation of T cells.
Medzhitov R. NEJM 343:338,2000.

22. News from the Genome Project that has expanded our understanding of the innate host response repertoire.

23. Plant Immunity: Pathogen sensing without Tolls

25. Dangl JL, Jones JDG. Nature 411:826, 2001.

26. Guard Hypothesis NATURE | VOL 411 | 14 JUNE 2001
NB-LRR protein dissociates due to pathogen
NB-LRR protein binds target complex due to pathogen binding to complex
Dangl, J. et al.
NATURE | VOL 411 | 14 JUNE 2001

27. Exogenous pyrogen induces endogenous pyrogen.

28. Caspase-1 (ICE) CARD p45 Active ICE N C p20 p10 p20 p10 p20 p10 p20
QACRG
p45 N
p20
p10 C
p20
p10
Active ICE
p20
p10
p20
p10

29. Caspase Recruitment Domains:
CARDs

30. CATERPILLER: A Large Family of Mammalian Genes Containing CARD, Pyrin, Nucleotide-binding, and Leucine-Rich Repeat Domains
NBD
CARD/PYD
LRR
Harton JA et al. J. Immunol. 169:4088, 2002.

31. Inflammasome Concept caspase-1 NALP1 ASC caspase-5 NBD LRR pyrin CARD
10kD kD
caspase-5
20kD kD
caspase-1

32. Domain structure of CATERPILLERs
Martinon, F et al. Mol.Cell 10: , 2002.

33. Toll like receptors and the Signalosome
gene
expression
proteasome NF k B I a
IKK complex
TLR * g b
100 ’
s of genes
including IL - 1
and TNF
signalosome

34. Toll-like receptors
Cell Death and Differentiation (2006) 13, 816–825

35. Sea Urchin’s Pathogen Sensors
Rast,J.P. et al. Science 314:952, 2006.

36. pyrin
CARD
ASC
NBD
pyrin
CARD
LRR
NALP1
NBD
CARD
LRR
IPAF
NBD
CARD
LRR
NOD1
NBD
CARD
CARD
LRR
NOD2
NBD
pyrin
LRR
NALP3

37. inflammasome TLR CASPASE-1 NOD-2 ASC NALP-1 CARD domain pyrin domain
proIL-1
IL-1
Martinon, F et al. Mol.Cell 10: , 2002.

38. Multiprotein Complex Assembly requires ASC
ASC (apoptosis associated speck forming complex)
ASC

39. ASC Critical to IL-1 Processing
Sarkar A et al. J.Immunol. 176:4979, 2006.

40. Need for Intracellular Pathogen Sensors
Phagosome escape

41. Francisella as Model of Inflammasome Activation
F. novicida
F. n. heat-killed
F. n. + cytochalasin D
E. coli
Gavrilin MA, PNAS 103:141, 2006.

42. Phagosome Escape Induces Inflammasome Assembly
F.novicida
proIL-1
phagosome
IL-1

43. F. novicida induces both mRNA and processing and release of IL-1
Gavrilin MA, PNAS 103:141, 2006.

44. Toll like receptors and the Signalosome
proIL-1

45. inflammasome TLR CASPASE-1 1 2 ASC NALP-1 CARD domain pyrin domain
proIL-1
IL-1

46. Molecular Cell 25, 713–724, March 9, 2007

47. Inflammasome structure

48. What about Septic Shock?

49. Figure 2-45 part 1 of 3

50. Caspase 1 and Sepsis TLR CASPASE-1 NOD-2 ASC NALP-1 Caspase-5 proIL-1

51. Caspase-1: Role in Sepsis Survival and Spleen Apoptosis
Sarkar A et al. AJRCCM 2006

52. Caspase 12 and Sepsis TLR CASPASE-1 Caspase-12 NOD-2 ASC NALP-1
proIL-1
IL-1

53. Caspase-12 Comes in Two Forms
Saleh M. Nature 429:75, 2004.

54. Caspase-12L (active) versus Caspase-12S (inactive) Allelic Distribution
The American Journal of Human Genetics Volume 78 April 2006

55. Caspase-12L Linked to Sepsis
Source
Total
(n)
Genotype Frequency (%)
Allele Frequency (%)
T/T
T/C
C/C T C
Sepsis 38
60.5 29
10.5 75 25
Control
148
81.1
17.6
1.3
89.9
10.1
Saleh M. Nature 429:75, 2004.

56. NLRP3, NLRP1 and Caspase-1 and 12

57. Summary
Innate immune responses are critical to our host defense against pathogens.
Innate mechanisms are probably more critical than previously recognized.
Protein/protein interactions that are modified by pathogens (or their products) are central to pathogen sensing and signaling.