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 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 pathogen- associated 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-1 α with hemozoin, relative to CpG (TLR9 activator). BMDM robustly secreted IL-1β and IL- 18 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 Performed in order to remove any implication of DNA-mediated TLR9 signaling Chloroquine – anti-malarial drug

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

Role of the Inflammasome in a mouse model of Hz-induced peritonitis

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.