Inflammasomes: Guardian Angels of

the BodyVarij Nayan1 and Anuradha

Bhardwaj21 Scientist, CIRB, Hisar-125001, Haryana, INDIA2 Scientist, NRCE, Hisar-125001, Haryana, INDIA

“The poetry of earth is never dead ”

-John Keats

The Invaders . . .

Bacteria Viruses Fungi Protista Worms

worm trichura.jpg

(http://www.cdc.gov/mrsa/mrsa_initiative/skin_infection/

mrsa_photo_9994.html)

MRSA

HIV

(http://www.scientificamerican.com/article.cfm?id=experts-where-did-

viruses-come-fr)

(http://www.medicine.cmu.ac.th/dept/parasite/nematode/wbmf.htm)

W. bancroftiT. brucei

(© 2011, American Society for Microbiology)

Immune system

“The body system in humans and other animals that protects the organism by distinguishing that is foreign and neutralizing potentially pathogenic organisms or substances”

(The American Heritage® Science Dictionary, 2010)

Cardinal Signs of Inflammation

“ Inflammation is the coordinated immune response to harmful stimuli such as infectious agents, tissue

damage, or cellular irritants like toxins”

(adapted from Lawrence et al., Nat. Rev. Immunol. ,2002)

……Mastitis for Instance

( Nayan et al., 2012)

Innate and Adaptive Immunity

Skin - “Walls and Moats”

Immune System - “Sentries”

Cellular Counterattack - “Roaming Patrols” ( Dranoff, 2004)

Innate Immunity

(rapid response)

Adaptive Immunity

(slow response)

Traditionally, Innate immunity – “First line of Defense”Now, Innate immunity emerged as:

Sophisticated system for sensing signals of ‘danger’

(Schroder and Tschopp, 2010)

Activated by engagement of germ-line encoded PRRs (Pattern Recognition Receptors)

( Kumar et al., 2011)

PRR recognize the presence of – Microbial PAMPs (Pathogen-Associated Molecular Patterns) Endogenous DAMPs (Danger-Associated Molecular Patterns)

(Chen and Nunez, 2010)

Innate Immunity

Such Detection System Includes…….

C-Type Lectin Receptors

Toll like receptors (TLRs)

Pentraxins

RNA-sensing RIG-like helicases

(RLHs): RIG-I, MDA5 viral sensors

DNA- sensors (DAI, AIM2)

NOD - like receptors (NLRs)microbial products

Host-derived danger signalsmetabolic stress

Survey the extracellular milieu & Endosomal Compartments

{extracellular sensing (PAMPs)}

Survey the Cytoplasm

{intracellular sensing orCytosolic

surveillance}

(Schroder and Tschopp, 2010; Pétrilli and Martinon, 2011)

Recognize PAMPs as well as DAMPs

PRRs → Signalling Cascade → Inflammatory Response

Activation of Adaptive Immunity

Members of NLR family form large cytoplasmic complexes

“INFLAMMASOMES”

“Molecular platforms that link between sensing of microbial/ cellular products following infection or stress with proteolytic activation of proinflammatory cytokinesIL-1β and IL-18”

Inflammasomes

Multiprotein

Complexes

Cytoplasmic

localization

Activate CASP-1

Processes :Pro-IL-1β → IL-1βPro-IL-18 → IL-18

First Inflammasome complex - reported in 2002

Assembly of PRR NLRP1, adaptor ASC, CASP-

1 & 5

“Generally speaking, the inflammasome depends on the assembly of a sensor (e.g. NLRP), with an adaptor, ASC (apoptosis-associated Speck-like protein containing a CARD), allowing the recruitment and activation of an inflammatory caspase, Caspase-1”

Structure of NLR family “NLRs form central molecular platforms that organize signalling complexes such as inflammasomes & NOD Signalosomes”

Multidomain proteins – Tripartite Architecture

1) C-terminal region (Leucine rich repeats - LRRs)

2) Central NACHT domain (NOD Domain) – belongs to STAND family of NTPases

3) N-terminal effector domain- can be a Pyrin domain (PYD),

CARD or BIR domain

LRR – ligand sensing, autoregulation of NLR signaling LRR domains are formed by tandem repeats

NB-ARC domain of apoptotic mediator APAF1

/ NLRPs

Structure of NLRs

2 Large Sub familiesI PYD containing NALPs / NLRPs(14 in human)II 6 members of NODs + CIITA

IPAF, and BIR containing NAIP form remaining NLR members

Ligand sensing domain

Responsible for NLR oligomerization

Effector domain

PYD – pyrin domainCARD – caspase recruitment domainFIIND – function to findBIR – baculovirus IAP repeat AD- activation domainAPAF1- Apoptotic protease activating factor-1 (Martinon et al., 2009)

Number of NBDs in Multicellular Organisms

Subsuming the NB-ARC domain and the NACHT domain under the term NBD (NOD-binding Domain)

(Lange et al., 2010)

(Adapted from Chen et al., 2009)

(Adapted from Chen et al., 2009)

(Adapted from Chen et al., 2009)

(Adapted from Martinon et al., 2009)

NALPs/ NLRPs 1-14

NALP ( or NLRP) 1, 2 & 3 are central scafold of caspase-1-activating complex known as INFLAMMASOMESHave PYD domainNALP1 possess additional CARD domain

(Martinon et al., 2009)

IPAF, NAIP

Evolutionary seperated from other NLRsIPAF – CARD domainNAIP – BIR domain (often found in proteins involved in apoptosisBoth form INFLAMMASOMES alone or in combination of both

(Martinon et al., 2009)

CARD-containing NLRs

NOD 1, 3, 4 and CIITA and separated NOD2, NOD5NOD1 and NOD2 activate NFBCIITA- in transcriptional regulation of genes encoding MHC II

(Martinon et al., 2009)

NLRs expression pattern and gene regulation

NLRs are expressed in cell and tissues that have role in immunity such as phagocytes

Epitelial cells - the Physical barrier

NAIP, IPAF – brain, spleen, lung, liver

Some like NLRP5, 8, 4, 7, 10, 11 have restricted expression – germ cells and preimplantation embryos

Regulation – TLR stimulation increases the expression of NLRs (NOD1, NOD2, NLRP3)

In Plants…

NLRs genes have similarities to plant genes involved in immune defenses (R-genes)

Convergent evolution

No NLR-like proteins in insects

Caspases- Cysteine Proteases, initiate/execute events leading to inflammation or cell deathSynthesized as catalytically inactive zymogens and undergo proteolytic activationExecutioner caspases- Cleave substrates in apoptosis (CASP-3, 6, 7)Initiator capases- activate EC (CASP-2, 8,9, 10)Inflammatory Caspases (CASP-1, 11 &12 in mouse; CASP-1, 4 & 5 in humans) – have CARD domain + a domain having catalytic CysteineCASP-1 : fully characterized Catalytic activity regulated by signal-dependent autoactivation

within inflammasomes

Caspases

Prototypical inflammasomesBiochemistry and diversity of inflammasomes is poorly understood, four prototypes

NLRP1 Inflammasome

NLRP3 Inflammasome

IPAF Inflammasome

AIM2 Inflammasome

“ Inflammasomes are assembled by self-oligomerizing scaffold proteins”

NLRP1, NLRP3 & IPAF are danger sentinels that self-oligomerize via homotypic NACHT domain interactions to form high MW complexes (hexamers/heptamers)HIN-200 family member, Cytosolic dsDNA sensor, also mediates inflammasome assembly Oligomerization by clustering on multiple binding sites in dsDNA via C-terminal HIN domain of AIM2

(Schroder and Tschopp, 2010)

NLRP1 have C-terminal extension with CARD domain – interact directly with procaspase-1; bypass requirement of ASC

Consists of NLRP3 scaffold, ASC (PYCARD) adaptor & caspase-1

Contains CARD domain – interact directly with procaspase-1; As no PYD domain, role of ASC unclear, collaboration of IPAF with NLRP (PYD containing protein) ????

First non-NLR family member; PYD domain of AIM2 interacts with ASC via homotypic PYD-PYD interactions, allowing ASC CARDDomain to recruit procaspase-1

IPAF, NLRP3 bind ATP/dATP - for oligomerization of NACHT domain

IPAF, NLRP3 bind HSP90-SGT1 chaperone: essential for NALP3 activation

Heterocomplexes – diversity?

(Martinon et al., 2009)

How innate immune system discriminate between pathogenic and self non-pathogenic microbes and commensals?“DANGER Model” (Matzinger, 1994)

Presentation of an antigen in the context of danger signal triggers efficient immune response - not only the foreignness of antigenSignals released by damaged or stressed tissues First evidence found in plants

Both the self-from-nonself model & the danger model may synergize to determine quality & extent of innate immune responseInflammasomes: Key piece in this puzzle ?

Key players in inflammatory & immune responseSensors of danger signals

Inflammasomes as sensors of Danger

Chronological Discoveries for Danger Signals

(Pelegrin, 2011)

Sensors of Danger signals

MSU – monosoduim urate crystalsCPPD – calcium pyrophosphate dihydrate crystals

(Martinon et al., 2009)

Inflammasome activation by Danger Signals

(Pelegrín, 2011)

Sensing extracellular ATP

Extracellular ATP released by cell damage/ cellular stress hydrostatic pressure changes, hypotonic shockDanger signal binds to purinoreceptor P2X7 thereby activating NLRP3 and caspase-1 & IL-1b maturationOtherATPsources - insulin containing granules from pancreatic cells- microbial flora and pathogensATP mediated caspase-1 activation requires ASC and is therefore dependent on activation of NLRP

Uric Acid – a danger signal involved in gout Uric acid from supernatant of dying cells tiggers adjuvanticityUric acid with free sodium in extracellular enviroment form monosodium urate (MSU) crystalsMSU adjuvanticity depends on NLRP3 inflammasome activation → IL-1Erytrocytes infected with Plasmodium : high hypoxanthine - released from damaged cells → to uric acid → → inflammationAlum-induced caspase -1 → dependent on NLRP3 inflammasome activation Inflammatory response in gout is dependent on inflammasomes

Silica and Asbestos and Inflammation in lung

Silica, asbestos dust are strong inflammation inducers in the lungs

These compounds act as activators of NALP3/ NLRP3

……and skin inflammations

UV irradiation activate NALP3/ NLRP3 in keratinocytes Inflammasomes → role in contact hypersensitivityTwo phases:

Sensitization phase (chemical act both as adjuvant and foreign hapten)Elicitation phase (after reexpousure)

SENSITIZATION phase depends on functional:caspase-1, IL-1, IL-18 confirmed role of ASC, NALP3 inflammasome

“Inflammasomes may detect such a compound directy or recognise the danger signals produced by irritants”

ROS, the common NLRP3 Activator?

ROS production occurs upon expousure of macrophages to:

silica, asbestos, MSU, alum, ATP, toxin nigericin, UV

ROS production is signal involved in stress and damage sensing

ROS sensing: Direct → NALP3 Indirect → cytoplasmic modulators of inflammasomes

Sensors of Pathogens

Extracellular PAMPs and danger signals – TLR,

RAGE (receptor for advanced glycation end product)

NLRs samples PAMPs reaching cellular compartments (invasion, degradation products from phagocytosed bacteria, viruses)

In addition to PAMPs, inflammasomes detects toxins and signals that are restricted to certain pathogens

(Martinon et al., 2009)

PAMPs & toxinsInflammasomes respond to bacterial PGNs (peptidoglycans) and nucleic acids PGN → degraded to MDP (muramyl dipeptide) → sensed by NLR NOD2 → activation of NFB NLRP3 → additional MDP sensor → IL-1 activation via caspase-1 NOD2 and NLRP3 can cooperate directly or indirectly as part of the same complexPORE-FORMING bacterial toxins activate NLRP3 inflammasome

-toxin Staphylococcus aureusaerolysin Aeromonas hydrophilalisteriolysin O Listeria monocytogenes

ionic imbalance, potassium efflux, calcium influx

Antrax lethal toxin (LeTx) → activates NLRP1

(Franchi et al., 2012)

Activation of Caspase-1

throughNLRP3

(Cholera Toxin)

Kinase

( Upregulation of NLRP3 & NF-

kB)

( of NLRP3 inflammaso

me)

IPAF/NLRC4 inflamasomme activation

by injected Virulence Factors Gram negative pathogens that activate IPAF require type III or type IV secretion system for injection of virulent factors activating IPAFMainly flagellin activates IPAF

(Martinon et al., 2009)

NLRC4 inflammasome activation

(Franchi et al., 2012)

Modulation by Microbial Pathogens

(Walle and Lamkanfi, 2011)

HMGB1: High Mobility Group Box 1

Inflammasome Activating Pathways

(Szabo and Csak, 2012)

What are the mechanisms silencing the inflammation iduced by inflammasomes

Factors: proteins that interfere with inflammasome assembly and inflammatory caspase activation

MAIN inflammasome regulators are those containing CARD domain, and those with PYD domain

POPs – poxoviral gene product vPYDs – viral PYD

Pi9 – serpin protease inhibitorvCrmA – cowpox virus-encoded inhibitor of caspase-1 (Martinon et al., 2009)

INFLAMMASOME regulators

PKR stirs up inflammasomes ??

(Stunden and Latz, 2012)

…Disease Associations

Autoinflammatory but not autoimmune disorders

“ Evidence for adaptive immunity components such as autoreactive T cells

or Igs to self- Ags are lacking”

Inflammasomes & Adjuvanticity

APCs can sense Ags (contained within adjuvants) through inflammasome

(Martinon et al., 2009)

Inflammasomes &

Pyroptosis

Cell Death…….Programmed Passive

(Labbe and Saleh, 2011)

Pyroptosis: A Caspase-1 Dependent Programmed Cell Death

(Labbe and Saleh, 2011)

“Pyro”: Firehigh inflammatory state

Pyroptosis and Apoptosis

(Lamkanfi, 2011)

Defects in inflammasome cause rare auto-inflammatory syndromes

variants in NLR genes may predispose to common inflammatory or autoimmune disease

The discovery of inflammasome involvement in common pathologies such as gout lead to new therapy strategies

Alum adjuvant stimulates inflammation through NALP3 inflammasome. Design of new adjuvant???

Why it is interesting to study inflammasome?

Design and dicovery of effective and specific drugs that alter inflammasome function

Translating basic research into development of novel targeted strategies using pharmacological manipulation of NLRs

IL-1β perfusion rabbit ovary blocks embryo development. Inflammasomes may link innate immunity to reproductive biology

Establishing role as mediators in neurodegenerative disorders, cancer and fertility-associated conditions

Future Perspective

“Inflammasomes are emerging as Guardians

of Body”

Questions ??

NALP3/ NLRP3 Inflammasomes

Assumed that PYD of NLRPs recruits adaptor ASC (apoptosis assiciated spec-like protein containing caspase recruitment domain)

The CARD within ASC binds and recruits caspase-1 to the inflammasomes

(Menu and Vince, 2011)

(Martinon et al., 2009)

IPAF/NLRC4 Inflammasome

Direct way of recruitment of caspase-1

(Martinon et al., 2009)