"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"

2. “The poetry of earth is never dead ”
-John Keats

3. 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

4. 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)

5. 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”

6. ……Mastitis for Instance
( Nayan et al., 2012)

7. 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)

8. 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)

9. 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)

10. 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”

11. 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

12. “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”

13. 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

14. 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)

15. 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)

16. (Adapted from Chen et al., 2009)

17. (Adapted from Chen et al., 2009)

18. (Adapted from Chen et al., 2009)
(Adapted from Martinon et al., 2009)

19. 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)

20. 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)

21. 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)

22. 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)

23. In Plants…
NLRs genes have similarities to plant genes involved in
immune defenses (R-genes)
Convergent evolution
No NLR-like proteins in insects

24. 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

25. 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”

26. 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)

27. 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)

28. 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

29. Chronological Discoveries for Danger Signals
(Pelegrin, 2011)

30. Sensors of Danger signals
MSU – monosoduim urate crystals
CPPD – calcium pyrophosphate dihydrate crystals
(Martinon et al., 2009)

31. Inflammasome activation by Danger Signals
(Pelegrín, 2011)

32. 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

33. 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

34. 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

35. ……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”

36. 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

37. 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

38. (Martinon et al., 2009)

39. 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

40. ( of NLRP3
inflammasome)
( Upregulation of
NLRP3 & NF-kB)
(Cholera
Toxin)
Kinase
Activation of
Caspase-1
through
NLRP3
(Franchi et al., 2012)

41. 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)

42. NLRC4
inflammasome
activation
(Franchi et al., 2012)

43. Modulation by Microbial Pathogens
HMGB1: High Mobility Group Box 1
(Walle and Lamkanfi, 2011)

44. Inflammasome Activating Pathways
(Szabo and Csak, 2012)

45. 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)

46. PKR stirs up inflammasomes ??
(Stunden and Latz, 2012)

47. …Disease Associations
Autoinflammatory but not autoimmune
disorders
“ Evidence for adaptive immunity components such as
autoreactive T cells or Igs to self- Ags are lacking”

48. Inflammasomes & Adjuvanticity
APCs can sense Ags (contained within
adjuvants) through inflammasome
(Martinon et al., 2009)

49. Inflammasomes
&
Pyroptosis

50. Cell Death…….
Programmed
Passive
(Labbe and Saleh, 2011)

51. Pyroptosis: A Caspase-1 Dependent
Programmed Cell Death
“Pyro”: Fire
high inflammatory state
(Labbe and Saleh, 2011)

52. Pyroptosis and Apoptosis
(Lamkanfi, 2011)

53. 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???

54. 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

56. Questions ??

58. 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)

59. IPAF/NLRC4 Inflammasome
Direct way of recruitment
of caspase-1
(Martinon et al., 2009)