The INFLAMMASOMES Guardians of the Body

1. The INFLAMMASOMESGuardians of the Body Fabio Martinion, Annick Mayor, Jurg Tschopp Annual Reviews Immunology 2009 Marcin Cebula

2. 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 complexesINFLAMMASOMES • Link between sensing of microbial products with proteolytic activation of proinflammatory cytokines IL-1 and IL-18

3. Structure of NLR family (I) • Multidomain proteins1) 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

4. Ligand sensing domain Responsible for NLR oligomerization Effector domain Structures of NLR Two sub families I PYD containing NALPs (14 in human) II 5 members of NODs + CIITA IPAF, and BIR containing NAIP form remaining NLR members PYD – pyrin domain CARD – caspase recruitment domain FIIND – function to find BIR – baculovirys IAP repeat AD- activation dmain

6. 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

7. NALPs • NALP1, NALP2, NALP3 are central scafold of caspase-1-activating complex known as INFLAMMASOMES • Have PYD domain • NALP1 posses additionaly CADR domain

8. 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

9. 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 NFBb • NOD1, NOD2 detects PNG • NOD2 detects MDP

10. 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)

11. 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

12. Prototypical inflammasomes (II) • Biochemistry and diversity if inflammasomes is poorly understood, three prototypes • NALP1 • NALP3 • IPAF

13. 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

14. Prototypical inflammasomes IPAF • Direct way of recruitment of caspase-1

15. 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?

16. 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 plans MSU – monosoduim urate crystals CPPD – calcium pyrophosphate dihydrate crystals

17. 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

18. 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 containhigh 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

19. 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

20. 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

21. 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

22. 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

23. PAMPs & toxins • Mainly bacterial PGNs and nucleic acids (indirect). • PGN is degraded to MDP that is sensed by NLR- NOD2 results in activation of NFB - 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

24. MDP - Muramyl dipeptide

25. 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.

26. POPs – poxoviral gene product vPYDs – viral PYD Pi9 – serpin protease inhibitor vCrmA – cowpox virus-encoded inhibitor of caspase-1 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

27. & inflamatory diseases • Autoinflammatory but not autoimmune disorders

28. 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

29. 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

30. 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

31. Thank you

32. PAMPs & DAMPs The Caspase-1 Inflammasome: Apilotof innate immune response B. Brett Finlay et al. Cell Host & Microbe 2008

33. General mechanisms of Inflammasome activation