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Inflammasomes: Guardian Angels of the body

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"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"

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Inflammasomes: Guardian Angels of the body

  1. 1. “The poetry of earth is never dead ” -John Keats
  2. 2. The Invaders . . . Bacteria Viruses Fungi Protista Worms MRSA (http://www.cdc.gov/mrsa/mrsa_initiative/skin_i nfection/mrsa_photo_9994.html) HIV (http://www.scientificamerican.com/article.cfm?i d=experts-where-did-viruses-come-fr) T. brucei W. bancrofti (© 2011, American Society for Microbiology) (http://www.medicine.cmu.ac.th/dept/parasite /nematode/wbmf.htm) worm trichura.jpg
  3. 3. 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)
  4. 4. Cardinal Signs of Inflammation (adapted from Lawrence et al., Nat. Rev. Immunol. ,2002) “ Inflammation is the coordinated immune response to harmful stimuli such as infectious agents, tissue damage, or cellular irritants like toxins”
  5. 5. ……Mastitis for Instance ( Nayan et al., 2012)
  6. 6. Innate and Adaptive Immunity Innate Immunity (rapid response) Adaptive Immunity (slow response) Skin - “Walls and Moats” Cellular Counterattack - “Roaming Patrols” Immune System - “Sentries” ( Dranoff, 2004)
  7. 7. Innate Immunity 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)
  8. 8. Such Detection System Includes……. C-Type Lectin Receptors Toll like receptors (TLRs) Pentraxins Survey the extracellular milieu & Endosomal Compartments {extracellular sensing (PAMPs)} RNA-sensing RIG-like helicases (RLHs): RIG-I, MDA5 viral sensors DNA- sensors (DAI, AIM2) Survey the Cytoplasm {intracellular sensing or Cytosolic surveillance} NOD - like receptors (NLRs) microbial products Host-derived danger signals metabolic stress Recognize PAMPs as well as DAMPs (Schroder and Tschopp, 2010; Pétrilli and Martinon, 2011)
  9. 9. 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 cytokines IL-1β and IL-18”
  10. 10. Inflammasomes Multiprotein Complexes Cytoplasmic localization Activate CASP-1 Processes : Pro-IL-1β → IL-1β Pro-IL-18 → IL-18 First Inflammasome complex - reported in 2002 Assembly of PRR NLRP1, adaptor ASC, CASP-1 & 5
  11. 11. “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”
  12. 12. Structure of NLR family “NLRs form central molecular platforms that organize signalling complexes such as inflammasomes & NOD Signalosomes” Multidomain proteins – Tripartite Architecture NB-ARC domain of apoptotic mediator APAF1 1) 2) 3) – - can be a or LRR – ligand sensing, autoregulation of NLR signaling LRR domains are formed by tandem repeats
  13. 13. Structure of NLRs Ligand sensing domain Responsible for NLR oligomerization Effector domain / NLRPs PYD – pyrin domain 2 Large Sub families CARD – caspase recruitment domain I PYD containing NALPs / NLRPs(14 in human) FIIND – function to find II 6 members of NODs + CIITA BIR – baculovirus IAP repeat AD- activation domain IPAF, and BIR containing NAIP form remaining NLR members APAF1- Apoptotic protease activating factor-1 (Martinon et al., 2009)
  14. 14. Number of NBDs in Multicellular Organisms Subsuming the NB-ARC domain and the NACHT domain under the term NBD (NODbinding Domain) (Lange et al., 2010)
  15. 15. (Adapted from Chen et al., 2009)
  16. 16. (Adapted from Chen et al., 2009)
  17. 17. (Adapted from Chen et al., 2009) (Adapted from Martinon et al., 2009)
  18. 18. NALPs/ NLRPs 1-14 NALP ( or NLRP) 1, 2 & 3 are central scafold of caspase-1activating complex known as INFLAMMASOMES Have PYD domain NALP1 possess additional CARD domain (Martinon et al., 2009)
  19. 19. IPAF, NAIP Evolutionary seperated from other NLRs IPAF – CARD domain NAIP – BIR domain (often found in proteins involved in apoptosis Both form INFLAMMASOMES alone or in combination of both (Martinon et al., 2009)
  20. 20. CARD-containing NLRs NOD 1, 3, 4 and CIITA and separated NOD2, NOD5 NOD1 and NOD2 activate NF B CIITA- in transcriptional regulation of genes encoding MHC II (Martinon et al., 2009)
  21. 21. 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)
  22. 22. In Plants… NLRs genes have similarities to plant genes involved in immune defenses (R-genes) Convergent evolution No NLR-like proteins in insects
  23. 23. Caspases Caspases- Cysteine Proteases, initiate/execute events leading to inflammation or cell death Synthesized as catalytically inactive zymogens and undergo proteolytic activation Executioner 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 Cysteine CASP-1 :  fully characterized  Catalytic activity regulated by signal-dependent autoactivation within inflammasomes
  24. 24. Prototypical inflammasomes Biochemistry and diversity of understood, four prototypes inflammasomes is poorly NLRP1 Inflammasome NLRP3 Inflammasome IPAF Inflammasome AIM2 Inflammasome 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 Cterminal HIN domain of AIM2 “ Inflammasomes are assembled by self-oligomerizing scaffold proteins”
  25. 25. 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 CARD Domain to recruit procaspase-1 (Schroder and Tschopp, 2010)
  26. 26. IPAF, NLRP3 bind ATP/dATP for oligomerization of NACHT domain IPAF, NLRP3 bind HSP90SGT1 chaperone: essential for NALP3 activation Heterocomplexes diversity? – (Martinon et al., 2009)
  27. 27. Inflammasomes as sensors of Danger 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 antigen Signals 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 response Inflammasomes: Key piece in this puzzle ? Key players in inflammatory & immune response Sensors of danger signals
  28. 28. Chronological Discoveries for Danger Signals (Pelegrin, 2011)
  29. 29. Sensors of Danger signals MSU – monosoduim urate crystals CPPD – calcium pyrophosphate dihydrate crystals (Martinon et al., 2009)
  30. 30. Inflammasome activation by Danger Signals (Pelegrín, 2011)
  31. 31. Sensing extracellular ATP Extracellular ATP released by cell damage/ cellular stress hydrostatic pressure changes, hypotonic shock Danger signal binds to purinoreceptor P2X7 thereby activating NLRP3 and caspase-1 & IL-1b maturation OtherATPsources - insulin containing granules from pancreatic cells - microbial flora and pathogens ATP mediated caspase-1 activation requires ASC and is therefore dependent on activation of NLRP
  32. 32. Uric Acid – a danger signal involved in gout Uric acid from supernatant of dying cells tiggers adjuvanticity Uric acid with free sodium in extracellular enviroment form monosodium urate (MSU) crystals MSU adjuvanticity depends on NLRP3 inflammasome activation → IL-1 Erytrocytes infected with Plasmodium : high hypoxanthine - released from damaged cells → to uric acid → → inflammation Alum-induced caspase -1 → dependent on NLRP3 inflammasome activation Inflammatory response in gout is dependent on inflammasomes
  33. 33. 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
  34. 34. ……and skin inflammations UV irradiation activate NALP3/ NLRP3 in keratinocytes Inflammasomes → role in contact hypersensitivity Two 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”
  35. 35. 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
  36. 36. 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
  37. 37. (Martinon et al., 2009)
  38. 38. PAMPs & toxins Inflammasomes respond to bacterial PGNs (peptidoglycans) and nucleic acids PGN → degraded to MDP (muramyl dipeptide) → sensed by NLR NOD2 → activation of NF B NLRP3 → additional MDP sensor → IL-1 activation via caspase-1 NOD2 and NLRP3 can cooperate directly or indirectly as part of the same complex PORE-FORMING bacterial toxins activate NLRP3 inflammasome -toxin Staphylococcus aureus aerolysin Aeromonas hydrophila listeriolysin O Listeria monocytogenes ionic imbalance, potassium efflux, calcium influx Antrax lethal toxin (LeTx) → activates NLRP1
  39. 39. ( of NLRP3 inflammasome) ( Upregulation of NLRP3 & NF-kB) (Cholera Toxin) Kinase Activation of Caspase-1 through NLRP3 (Franchi et al., 2012)
  40. 40. 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 IPAF Mainly flagellin activates IPAF (Martinon et al., 2009)
  41. 41. NLRC4 inflammasome activation (Franchi et al., 2012)
  42. 42. Modulation by Microbial Pathogens HMGB1: High Mobility Group Box 1 (Walle and Lamkanfi, 2011)
  43. 43. Inflammasome Activating Pathways (Szabo and Csak, 2012)
  44. 44. INFLAMMASOME regulators POPs – poxoviral gene product vPYDs – viral PYD What are the mechanisms silencing inflammation iduced by inflammasomes the Factors: proteins that interfere with inflammasome assembly and inflammatory caspase activation MAIN inflammasome regulators are those containing CARD domain, and those with PYD domain Pi9 – serpin protease inhibitor vCrmA – cowpox virus-encoded inhibitor of caspase-1 (Martinon et al., 2009)
  45. 45. PKR stirs up inflammasomes ?? (Stunden and Latz, 2012)
  46. 46. …Disease Associations Autoinflammatory but not autoimmune disorders “ Evidence for adaptive immunity components such as autoreactive T cells or Igs to self- Ags are lacking”
  47. 47. Inflammasomes & Adjuvanticity APCs can sense Ags (contained within adjuvants) through inflammasome (Martinon et al., 2009)
  48. 48. Inflammasomes & Pyroptosis
  49. 49. Cell Death……. Programmed Passive (Labbe and Saleh, 2011)
  50. 50. Pyroptosis: A Caspase-1 Dependent Programmed Cell Death “Pyro”: Fire high inflammatory state (Labbe and Saleh, 2011)
  51. 51. Pyroptosis and Apoptosis (Lamkanfi, 2011)
  52. 52. Why it is interesting to study inflammasome? 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???
  53. 53. Future Perspective 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
  54. 54. Questions ??
  55. 55. 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 (Martinon et al., 2009) (Menu and Vince, 2011)
  56. 56. IPAF/NLRC4 Inflammasome Direct way of recruitment of caspase-1 (Martinon et al., 2009)

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