The INFLAMMASOMES

Guardians of the BodyFabio Martinion, Annick Mayor, Jurg Tschopp

Annual Reviews Immunology 2009

Marcin Cebula

Innate immunity Toll like receptors – extracellular sensing (PAMPs, DAMPs)

RIG-like helicases (RIG-I, MDA5 viral sensors) – intracelular sensing

NOD - like receptors – intracellular sensing- microbial products- danger signals- metabolic stress

Some NLR form large cytoplasmatic complexes

INFLAMMASOMES

Link between sensing of microbial products with proteolytic activation of proinflammatory cytokines

IL-1 and IL-18

Structure of NLR family (I)

Multidomain proteins

1) C-terminal region (Leucin rich repeats 20-30 - LRR)

2 ) NACHT – belongs to STAND family of NTPases

3) N-terminal effector domain

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

Structures of NLR

Two sub familiesI PYD containing NALPs (14 in human)II 5 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 – baculovirys IAP repeat AD- activation dmain

No experimental data have convincingly demonstrated direct interaction between LRRs of NLRs and their respective activators, suggesting that sensing of patogens by NLRs may be indirect.

It is believed that the crucial step in NLRs activation lies in the oligomerization of the NACHT domains

Formation of active high molecular wieght complexes - INFLAMMASOMES

NALPs

NALP1, NALP2, NALP3 are central scafold of caspase-1-activating complex known as INFLAMMASOMES

Have PYD domain NALP1 posses additionaly CADR

domain

IPAF, NAIP

Evolutionary seperates from other NLRs

IPAF – CARD domain NAIP – BIR damain (often

found in proteins involved in apaptosis

Both form INFLAMMASOMES alone or in combination of both

CARD-containing NLRs

NOD1,2,4 and CIITA and separated NOD3, NOD5

NOD1 and NOD2 once activated recruits kinase RIP2 through CARD-CARD interaction. Oligomerization of RIP2 in NOD signalosomes activate NFBb

NOD1, NOD2 detects PNG NOD2 detects MDP

NLRs expression pattern and gene regulation NLRs are expressed in cell and tissues that have role in

immunity such as phagocytes

Epitelial cells - the first barrier

NAIP, IPAF – brain, spleen, lung, liver

Some like NALP5, 8, 4, 7, 10, 11 have restricted expression – germ cells and preimplantation embryos

Regulation – TLR stimulation increases the expression of NLRs (NOD1, NOD2 NALP3)

In Plants…

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

Contain: LRR, oligomerization domain NB-ARC, and TIR domain (recruitment domain involved in the TLR and IL-R1 family of immune mediators)

Convergent evolution

No NLR-like proteins in insects

Prototypical inflammasomes (II)

Biochemistry and diversity if inflammasomes is poorly understood, three prototypes

NALP1 NALP3 IPAF

Prototypical inflammasomes NALP3

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

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

NALP1 has C-terminal extension with CARD that rectuits caspase-5 or second caspase-1 to inflammasomes

Prototypical inflammasomes IPAF

Direct way of recruitment of caspase-1

Both IPAF and NALP3 bind ATP/dATP what is necessary for oligomerization of NACHT domain. Signal for this comes from LRR that are proposed to sense activating signal

Both IPAF and NALP3 bind SGT1 and HSP90 and activity of HSP90-SGT1 complex is essential for NALP3 activation (by keeping inflammasomes inactive but competent for activation)

Heterocomplexes – diversity?

Sensors of Danger signals (III)

How innate immune system discriminate between pathogenic and self nonpathogenic microbes

Matzinger suggest DANGER hypothesis Presentation of an antigen in the context of

danger signal triggrs efficient immune response - not only the foreignnes of antigen

Signals released by damaged or stressed tissues

First evidence found in plansMSU – monosoduim urate crystalsCPPD – calcium pyrophosphate dihydrate crystals

Sensing extracellular ATP

Extracellular ATP released by damage or cellular stress hydrostatic pressure, hypotonic shock

Danger signal binds to purinoreceptor P2X7 thereby activating NALP3 and caspase-1

Extracellular ATP in vivo my be rapidly hydrolyzed. Other ATP sourses

- insulin containing granules from pancreatic cells- microbial flora and pathogenes

ASC deficient mice demonstrated that ATP mediated caspase-1 activation requires ASC and is therefore dependent on activation of NALP

Uric Acid – a danger signal involved in gout (Arthritismus)

Uric acid form supernatant of dying cells tiggers adjuvanticity. Uric acid with free sodium in extracellular enviroment form

monosodium urate MSD crystals MSU adjuvanticity depends on NALP3 inflammasome

activation → IL-1 ASC, NALP3 deficient mice have reduced crystal induced IL-1

Examples- Erytrocytes infected with Plasmodium contain high levels of hipoxanthine which is released from damaged cells and converted to uric acid – results in inlamation- DC incubated with alum also release uric acid

Aluminium particles act as adjuvant by augumented production of IL-1 Alum-induced caspase -1 is dependent on NALP3 inflammasome

activation

Silica and Asbestos Inflamation in the lung

Alveolar macrophages reside on barrier between body and the external enviroment

This phagocytes are important defence against microorganisms, dust particles

Silica, asbestos dust are strong inflamation inducers in the lungs.

This compounds act as activators of NALP3

and skin inflamations

UV activate NALP3 in keratinocytes Inflammasomes play a role in contact hypersensitivity an inflammatory

disease caused by irritant chemicals penetrating skin and inducing T cell response

Two phases- sensitization (chamicals as adjuvants and foreign hapten)- activation (after reexpousure)

SENSITIZATION phase depends on functional:– caspase-1 IL-1, IL-18

– confirmed role of ASC, NALP3 inflammasome

DNFB (dinitrofluorobenzene) shown to promote release IL-1 in caspase-1 dependent manner in DC and keratinocytes.

Suggestion that inflammasomes may detect such a compound directy or recognise the danger signals produced by irritants

Reactive oxygen species

ROS production occurs upon expousure of macrophages to:

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

ROS production is signal involved in stress and damage sensing

Knockdown of NADPH oxidaze subunits or use of antioxidants inhibit inflammasome activation by mentioned above compound

It is proposed that ROS is directly sensed by NALP3 or indirectly sensed by cytoplasmic modulators of inflammasome activity

Sensors of Pathohens (IV)

Extracelular 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)

Additionaly to PAMPs inflammasomes detects toxins and signals restricted to certain pathogens

PAMPs & toxins

Mainly bacterial PGNs and nucleic acids (indirect).

PGN is degraded to MDP that is sensed by NLR- NOD2 results in activation of NFB - NALP3 results IL-1 activation via caspase-1 (but NOD2 is required)

Sugesstion that NOD2 and NALP3 can cooperate directly or indirectly as part of the same complex

PORE-FORMING bacterial toxins antivate NALP3 inflammasome- -toxin Staphylococcus aureus- aerolysin Aeromonas hydrophila- listeriolysin O Listeria monocytogenes

-potassium efflux, calcium influx (danger signals)

Antrax lethal toxin activates NALP1

MDP - Muramyl dipeptide

IPAF inflamasomme activation by injected

virulent factors Gram negative pathogens that activate IPAF require

type III or type IV secretion system for injection of virulent factors activating IPAF

Mainly flagellin activates IPAF but not only.

INFLAMMASOME regulators (V)

What are the mechanisms silenting the inflamation iduced by inflammasomes

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

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

•Targeted disruption of Pyrin in mice causes increased endotoxin sensitivity and enhanced caspase-1 activation

•But Pyrin overexpression can be proinflamatory

•vPYD defficient poxoviruses – enhanced activation of caspase-1

POPs – poxoviral gene product vPYDs – viral PYD

Pi9 – serpin protease inhibitorvCrmA – cowpox virus-encoded inhibitor of caspase-1

& inflamatory diseases

Autoinflammatory but not autoimmune disorders

Inflammasomes & Adjuvanticity

Adjuvants: alum, oil based emulsions TLR agonist ? IL-1 has adjuvant properties. Mice immunization

together with IL-1 results in higher antibody production Inflammasome activators have adjuvant properties

(MDP, MSU) MSU, alum (ASC,NALP3,caspase-1) biases immune

response towards Th2 type via IL-1, IL-18, IL-33

Inflammasomes are important in linking innate immunity to adaptive immunity

Pyroptosis Apoptosis – silent death

Pyroptosis – „Pyro” Fire Cell death dependent on caspase-1, associated with high inflammatory state

Shigella f. infects colon epitelium, evades phagosome to enter cytosol where tigers death requires caspase-1 but not apoptotic caspase-3

Salmonella induced pyroptosis in infected macrophages (ASC, IPAF) is blocked in IPAF defficient mice

Bacillus anthracis induces pyroptosis dependent on NALP1

Emerging inflammasomes functions

Research on IL-1 and inflammatory caspases revealed its role as mediators in neurodegenerative disorders, cancer and fertility-associated conditions

Amyloid- in Alzheimer’s disease similary to phagocytosed uric acid crystals, activate NALP3 inflammasome, might be important for inflamation and tissue damage

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

Thank you

PAMPs & DAMPs

The Caspase-1 Inflammasome: Apilotof innate immune responseB. Brett Finlay et al. Cell Host & Microbe 2008

General mechanisms of Inflammasome activation