Download Mechanisms of Danger-signal mediated Immune Modulation

Mechanisms of Danger-signal
Mediated Immune Modulation
The Self-Non-Self theory
• Dominant model in immunology since the 1950s
• The body is able to discern between self and non-self
• Thus an immune response is triggered against all foreign entities
• No immune response is triggered against an organism’s endogenous
entities
Danger Theory
• Rooted in Janeway’s work (Infectious non-self
theory)
• Proposed by Polly Matzinger in a 1994 article
title “Tolerance, Danger and the Extended
Family”
• States that the immune response is a result of
the organism reacting to the emission of “danger
signals” by the organism, not “non-self” entities
• Self constituents can trigger an immune
response if they are dangerous (cellular stress)
and non-self constituents can be tolerated
(commensal bacteria)
Predications made by Self-non-self, Infectious
non-self theory and Danger Theory
Burnet 1969
Janeway 1989
Matzinger 1994
Danger Theory
*Alarm Signals = DAMPs,
damage associated
molecular patterns
DAMPs – Damage signal criteria
• Should be active as a highly purified molecule
• Biological activity should not be due to contamination with microbial
molecules (LPS)
• Should be active at concentrations that are actually present in
pathophysiological situations
• Selective elimination or inactivation of a DAMP should ideally inhibit
the biological activity of dead cells (in vitro and vivo)
Molecular identification of Danger Signals
• Cellular stress – when a cell is stressed, even in the absence of any foreign
substance, it emits molecules that activate APCs
• Heat-shock proteins – expression increased with elevated temperature and
other stresses, can bind antigen and activate APCs
• Necrotic cell death – intracellular contents, including damage-associated
molecular patterns (DAMPs). Apoptosis?
• Uric Acid – released by injured cells, dendritic cell maturation, with antigen
it enhances respsonses from CD8+ cells
• High-mobility-group box 1 – signals damage, initiates inflammatory
response/repair
• Inflammasomes?
Inflammasomes
• Component of innate immunity, triggered by danger signals;
stress/infection
• Multiprotein complex
• Expressed in myeloid cells
• Senses damage activate caspase1 production of IL-1β
• Subsets – NLRP1, NLRP3 (codes Nalp3 inflammasome), NLRC4
• Consists of caspase-1, caspase recruitment domain (CARD), NALP and
ASC (adaptor)
• NALP – NOD like receptor that contains NACHT (nucleotide binding
domain), LRR and Pyrin domain
NLRP3 Inflammasome Structure
Malaria
• Infects 300-500 million
• Kills over 1 million children annually
• Causative agent is a parasitic protozoan;
Plasmodium species
• Complex life cycle involving a mosquito vector
and a human host
• Erythrocyte (RBC) lysis resulting in fever, anemia
and death
• 1-2% cases develop Cerebral Malaria (deadly)
Life Cycle of the Malaria Parasite
Immune response and Plasmodium
infection
Adaptive:
• Induces an immune response characterized by IFNγ producing T cells
• Production of antibodies against infected RBCs
Innate:
• Several molecular conserved structures of Plasmodium act as pathogenassociated molecular patterns (PAMPs)
• PAMPs activate Toll-like receptors (TLRs) on macrophages and dendritic
cells
• Hemozoin activation of a Nalp3 inflammasome
Hemozoin
•
•
•
•
Heme crystal formed by Plasmodium
During the intraerythrocytic cycle hemoglobin is digested
Results in free heme
Parasite is able to convert free heme into insoluble hemozoin crystals as a means of
detoxification
• RBC lysis results in hemozoin entering the blood stream
Studies about the immuno-modulatory capacity are conflicting
• Activation of TLR9 signaling
• Dependence upon the presence of malarial DNA complexed to hemozoin
• Inflammasome
Hypothesis
Hemozoin acts as a Nalp3 inflammasome
activating danger signal resulting in IL-1β
production.
Hemozoin induces IL-1β secretion
in myeloid cells
• Experiments used synthetic hemozin;
β-hematin
• Bone marrow-derived macrophages
(BMDM) produced low levels of TNF,
IL-6 and MIP-1α with hemozoin,
relative to CpG (TLR9 activator).
• BMDM robustly secreted IL-1β and IL18 when primed and stimulated with
HZ.
IL-1β HZ induction in THP1 cells and Murine
BMDCs
THP1 cells: human macrophage
like cell line
BMDCs: murine bone marrow-derived
Dendritic cells
Hemozoin IL-1β secretion is NALP3
inflammasome dependent
Hemozoin IL-1β secretion is independent
from P2X7 activation
HZ induced IL-1B not mediated
by ATP released from dying
cells.
Uric acid crystals have no effect
on hemozoin IL-1B, uric acid itself
can act as danger signal
Toxic heme cannot activate
caspase1, but is toxic (PARP
cleavage)
Hemozoin IL-1β secretion is independent
from MyD88-mediated signaling pathways
Chloroquine – anti-malarial drug
Performed in order to remove any
implication of DNA-mediated TLR9
signaling
Bafilomycin – shown to inhibit inflammasome activation, no effect seen in
these experiments
Phagocytosis, K+ Efflux and activation of a NADPH
oxidase are all essential for Hz-mediated
inflammasome
Phagocytosis, K+ Efflux and activation of a NADPH
oxidase are all essential for Hz-mediated
inflammasome
Phagocytosis, K+ Efflux and activation of a NADPH
oxidase are all essential for Hz-mediated
inflammasome
Role of the Inflammasome in a mouse model
of Hz-induced peritonitis
Role of the Inflammasome in a mouse model
of Hz-induced peritonitis
Role of the Inflammasome in a mouse model
of Hz-induced peritonitis
Role of Nalp3 in a mouse model of
Cerebral Malaria
Role of Nalp3 in a mouse model of Cerebral
Malaria
H & E Stain
CD45 Stain
Conclusion
• Were able to show that Malarial Hemozoin is a Nalp3 inflammasome
activating signal, therefore enhancing the pro-inflammatory activity
along with TLRs
• May lead to novel more efficient anti-malaria drugs
• Investigate the mechanism for inflammasome activation by agonists
and therefore the exact role of hemozoin
• Self-non-self vs Danger? Maybe a combination of both.