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

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)

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

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

Shock and Poor Tissue Perfusion May Lead to Distal Gangrene

Black Death 25% of population died

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

Guns Germs and Steel Hypothesis Jared Diamond

Meningococcemia

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

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)

Innate Pathogen Sensors NLR TLR Extracellular sensors Intracellular TIR NB LRR

Family of TLR’s and Ligands Beutler et al.

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

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.

Exogenous pyrogen induces endogenous pyrogen.

Both Endogenous and Exogenous Pyrogens converge at NFkB

Innate versus Adaptive Immunity Characteristics Figure 2-10

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.

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

Plant Immunity: Pathogen sensing without Tolls

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

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

Exogenous pyrogen induces endogenous pyrogen.

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

Caspase Recruitment Domains: CARDs

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.

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

Domain structure of CATERPILLERs Martinon, F et al. Mol.Cell 10:417-426, 2002.

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

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

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

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

inflammasome TLR CASPASE-1 NOD-2 ASC NALP-1 CARD domain pyrin domain proIL-1 IL-1 Martinon, F et al. Mol.Cell 10:417-426, 2002.

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

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

Need for Intracellular Pathogen Sensors Phagosome escape

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

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

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

Toll like receptors and the Signalosome proIL-1

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

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

Inflammasome structure

What about Septic Shock?

Figure 2-45 part 1 of 3

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

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

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

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

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

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.

NLRP3, NLRP1 and Caspase-1 and 12

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.