Innate immunity, innate immune memory and malaria

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1 Innate immunity, innate immune memory and malaria Douglas Golenbock, MD UMass Medical School Malaria remains a significant public health problem 2015: 212 million new cases, >400,000 deaths WHO, World Malaria Report (2016) 1

2 The paroxysm of malaria 2

3 CamilloGolgi first proposed that the paroxysm of malaria coincided with schizont rupture Golgi C. Sulle febbri malariche estivoautunnalidi Roma. Letteraal Prof. Guido Baccelli GazzettaMedicadi Pavia. 2: ; ; Golgi hypothesized that when schizontsburst, they released a malaria toxin that was responsible for the paroxysm. Golgi C. Sulle febbri malariche estivoautunnalidi Roma. Letteraal Prof. Guido Baccelli GazzettaMedicadi Pavia. 2: ; ;

4 What is the origin of this cytokine storm that causes the malaria paroxsym? What is the toxin and what are its receptors? Hemozoinis the breakdown product of hemoglobin metabolism by plasmodium Parasites metabolize Hgbto a toxic metabolite hemin, which is detoxified by generating a non-toxic (from the parasite s standpoint) crystalline pigment 4

5 14/11/61 Synthetic Hz Coban, Akira et al. Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin. J Exp Med. 2005;201(1): Natural Hz Natural Hemozoin Activates Cytokine Production via TLR9 and its adapter protein, MyD88 Mouse dendritic cells 5

6 The stimulatory activity of hemozoin was destroyed by DNase. Mouse dendritic cells Thus, the TLR9 ligand is parasite DNA that coats the surface of hemozoin 6

7 Plasmodial DNA is thus a malaria toxin, and it is especially potent when complexed to hemozoin Hemozoin/DNA is an important immune activator: 1. Hz acts as signal 2 for NLRP3 (both invitro and in cells from malaria patients) 2. Hz can cause phagolysosomal instability, allowing nucleic acids access to the cytosol 3. DNA can directly activate AIM2 4. Immune complexes containing DNA can be internalized via Fc to activate cytokines 7

8 Problem: TLR9 preferentially binds CpG-rich islands of DNA and P falciparum is highly AT-rich Plasmodial DNA contains multiple copies of an ATrich stem-loop forming motif: ATTTTT(T)AC Daniele Bartholomeu 8

9 The AT-r stem-loop motif is found is common Daniele Bartholomeu Shruti Sharma 9

10 One can study the AT-r stem-loop motif using synthetic ODNs Hmmmmm. I wonder what DNA does when it gets into a cell? 10

11 Microarray analysis of 14 patients with febrile and smear positive P. falciparum showed an Type I IFN signature IFN alpha/beta receptor (IFNAR) knockouts were resistant to cerebral malaria Rosane DeOliveira, PhD 11

12 TRIF/MyD88 DKO have no TLR function MyD88-dependent MyD88-independent TLR1/2 TLR2/6 TLR5 TLR7, 9 TLR4 TLR3 Mal TRAM Mal MyD88 MyD88 TRIF TRIF MyD88 MyD88 IRAKs IRAKs IRAKs IRAKsX X X X X X AT-rich ODN activate type I interferons independently of TLRs WT TRIF/MyD88-/ (-) CpG-B (-) AT2 12

13 What is the receptor for AT-rich DNA that induces Type I interferons? Jacob Schrum Carolina Gallego-Marin 13

14 Could the type I interferon response be generated through adaptor STING (STimulator of INterferon Genes)? Interferon-stimulatory DNA (ISD): double-stranded 45-mer AT5 ODN: single-stranded 20-mer STING??? TBK1 IRF3 IRF3 Ishikawa et al, Nature 461: (2009) Sharma et al, Immunity 35: (2011) Type I IFNs Cyclic GuanosylAdenosylSynthase (cgas) is a cytosolic DNA sensor upstream from STING that generates via a secondary messenger: 2 3 -cgamp P. falciparum genomic DNA (Pf gdna) AT-rich DNA (AT5, AT5 3x) cgas STING 2 3 -cgamp TBK1 IRF3 IRF3 Sun et al, Science 339: (2013) Wu et al, Science 339: (2013) Ablasser et al, Nature 498: (2013) Zhang et al, Mol Cell 51: (2013) Type I IFNs 14

15 Hypothesis: cgas is the sensor for cytosolic P. falciparum genomic DNA and AT-rich DNA ligands cgamp drives the STING pathway Stimulation of IFNBexpression by Hz and Pf gdna is dependent on cgas Pf gdna (±Hz) Fold Induction of IFNB (log10) Medium * (-) THP-1 cells p=0.06 p=0.05 * Hz 10 µm Hz 50 µm Hz 100 µm * Pf gdna cgas +/+ cgas -/- cgas 2 3 -cgamp STING TBK1 IRF3 IRF3 +Lipo Type I IFNs cgas KO THP-1 cells from Veit Hornung 15

16 Stimulation of IFNβ secretion by malarial DNA ligands is dependent on cgasand StiNG Pf gdna (±Hz) AT-rich DNA Mouse bone marrow-derived macrophages (BMDMs) 1500 WT ns cgas -/ STING -/- *** IFNβ (IU/mL) Medium * PBS ** AT5 * AT5 3x +Lipo *** Pf gdna ISD *** p(da:dt) SeV cgas 2 3 -cgamp STING TBK1 IRF3 IRF3 Type I IFNs Quadruplicate stimulations of BMDMs, 1 mouse/genotype. *p<0.05, **p<0.01, ***p<0.001, unpaired t-test. Representative of 3 expts. Stimulation with PfgDNA induces production of the metabolite 2 3 -cgamp 16

17 Summary: 1. The malaria toxin is primarily DNA 2. Many of the effects of DNA are mediated through TLR9 inside the endolysosome(tnf, IL1b, IL6, NF-kB) 3. The most abundant DNA in malaria is AT-rich and turns on type I interferons 4. AT-rich DNA activates the cytosolic DNA sensor cgas, resulting in the production of cgamp, which then turns on the STING pathway Gazzinelli et al, Nat Rev Immunol 14: (2014) BCG the only vaccine against tuberculosis 17

18 Introducing BCG in Norrbotten, Sweden, : Mortality at 0-4 years - 20,000 children Coverage highest in families with TB Reduction was in infancy, but TB deaths occur later This made little sense One could evidently be tempted to find an explanation for this much lower mortality among vaccinated children in the idea that BCG provokes a non-specific immunity... Carl Naeslund 1932 Innate versus specific immunity Innate immunity: -rapid -effective - not-specific, indiscriminate - lacks immunological memory Adaptive immunity: -needs days -a specific activation against a particular microorganism, enhancing the effectivity of the response - builds immunological memory 18

19 Memory: the ability of a system to store and recall information on previously encountered characteristics Molluscs Annelids Insects 95% Innate Fishes 5% Innate and Adaptive Echinoderms Holometabolous Heterometabolous Amphibians Reptiles Birds Gnathostomes -450 My Mammals Placoderms Agnathans Urochordates Plants -800 My -1 By 38 Increased response to secondary infection Netea et al: Cell Host and Microbe

20 Jacob Schrum Jacob Schrum Is there innate immune memory in malaria? 20

21 What is the clinical importance of epigenetic changes due to malaria? 1.Individuals may have cyclical exposure to malaria, and acquired immunity is short lived 2.There may be a negative effect: innate immune memory may contribute to bad outcomes when there is co infection (eg, cerebral malaria and nontyphoidal Salmonella) 3.Epigenetics may explain asymptomatic chronic malaria, ie, the millions of people living in hyperendemicareas who are tolerant to Hypothesis: P. falciparum induces trained innate immunity In vitro assay Add first stimulus Add Cytokine second analysis Wash stimulus (protein, cells (or ChIP) mrna) 24 hr 3 day rest (RPMI + serum) 4-24 hr D0 D1 D4 D5 21

22 Training with irbcs(and Hz) induces increased proinflammatory cytokine secretion after 2 nd stim TNFα (pg/ml) ** *** 0 First stimulus RPMI irbc 10 6 urbc 10 5 irbc 10 5 urbc Second stimulus Pam3CSK4 (24 hr) Pam3 24 hr *p<0.05, **p<0.01, ***p < 0.001, paired t-test vs RPMI+ Training with irbcs(and Hz) induces decreased anti-inflammatory cytokine secretion after 2 nd stim First stimulus Second stimulus Pam3CSK4 (24 hr) Pam3 24 hr *p<0.05, paired t-test vs RPMI+ 22

23 CCL8 CXCL10 APOBEC3A HIST1H3F AC TNFSF15 IFI27 SEPP1 CXCL9 HIST1H3C ANKRD22 NIPAL4 SERPINE1 OCSTAMP SERPING1 SPINK1 LIF HBA2 HIST1H3B C1S C11orf96 TNFAIP6 IL23A EDN1 HIST1H2AL CSF3 HCAR3 GDF15 C1R 14/11/61 Juliet Crabtree irbc 4 irbc 12 irbc irbc 0 rpmi 4 rpmi 12 rpmi RPMI 0 urbc 4 urbc 12 urbc urbc IDO1 CCL8 CXCL10 APOBEC3A SEPP1 CXCL9 ANKRD22 SERPING1 C1S TNFAIP6 HIT1H3B IFI27 HCAR3 C1R CDKN1A WARS IL3RA HCAR2 RNASE1 RSAD2 MCEMP1 HSD11B1 CHI3L1 HBA2 GBP1P1 SPINK1 IL27 RP11-10J5.1 APOL4 SNX10 RNAseqconfirms that trained PBMCs have a robust alteration in gene expression, primarily in NF-kBand IFNa/b target genes Fold increase (TNF) RPMI+ ** 2x10 6 irbc 2x10 6 urbc p = 0.07 Hz 100 µm 23

24 Trained immunity mediated by epigenetic remodeling and metabolic alterations Netea et al, Nat Immunol 16: (2015) Arts et al, Cell Reports 17: (2016) Trained immunity is mediated by epigenetic remodeling and metabolic alterations -Innate immune memory can involve modification of histone 3 at lysine 4 (H3K4me3) at the promoters of proinflammatory genes (e.g., TNFa, IL6). - In addition, there are chromatin modifications at the promoters of key metabolic regulators including mtor (mammalian target of rapamycin). Netea et al, Nat Immunol 16: (2015) Arts et al, Cell Reports 17: (2016) 24

25 ChIP analysis indicates increased H3K4me3 at immunometabolic promoters after irbc training Fold increase (% input TNF) First stimulus RPMI+ TNF H3K4me3 * 5x10 6 irbc 5x10 6 urbc Hz 100 µm Fold increase (% input IL6) RPMI+ IL6 H3K4me3 * 5x10 6 irbc 5x10 6 urbc Hz 100 µm Fold increase (% input MTOR) First stimulus MTOR H3K4me3 p = 0.08 RPMI+ 5x10 6 irbc 5x10 6 urbc ** Hz 100 µm Fold increase (% input PTGS2) PTGS2 H3K4me3 ** RPMI+ 5x10 6 irbc 5x10 6 urbc Hz 100 µm ChIP *p<0.05, paired t-test vs RPMI+ Kate Dobbs and James Kazura (Case Western Reserve) 25

26 Increased H3K4me3 at immunometabolic promoters in monocytes from Kenyan children with malaria and 6 weeks po treatment % input TNF TNF H3K4me3 IL6 H3K4me3 MTOR H3K4me3 * 5 * 4 % input IL * p = 0.08 % input MTOR 8 p = 0.07 * AM 6wk NAM 0 AM 6wk NAM 0 AM 6wk NAM AM: Acute (uncomplicated) malaria 6wk: 6 weeks after curative treatment NAM: North American adult controls CyTOF: Panel of targets, and tsne plot Marker CD3 CD19 HLA-DR CD14 CD16 CD11c CD56 IL-6 TNFa Metal Label 170Er 142Nd 174Yb 148Nd 209Bi 147Sm 163Dy 156Gd 175Lu tsne (t-distributed Stochastic Neighbor Embedding) PBMC were trained for 24hr, rested 3 days, and restimulated for 6 hours for Intracellular Cytokine Staining 26

27 TNFα (IL6 and GM-CSF) are predominantly produced by myeloid cells in irbc-trained PBMC RPMI RPMI LPS Pam3CSK4 urbc irbc Development of newdc and Monocyte populations in irbc-trained PBMC RPMI LPS Pam3CSK4 RPMI urbc irbc 27

28 Summary: P. falciparum induced trained innate immunity Exposure to malaria results in increased proinflammatory/decreased antiinflammatory cytokines (both mrna and protein) after secondary challenge Increased epigenetic markers at immunometabolic promoters in vitro and in patients Potential role for metabolic signaling As a result of training, proinflammatory cytokines are produced by an expanded population of DCs and monocytes Wonderful stuff but what is the clinical significance of innate immune training? 28

29 KATE FITZGERALD & RICARDO GAZZINELLI Contributors Mihai Netea(Center for Trained Immunity, Nijmegen) Katherine Dobbs (Case Western Reserve with Jim Kazura) Eicke Latz: TLR9 confocal microscopy, and much, much more TerjeEspevik: taught us about fluorescent tags in confocal microscopy Peggy Parroche: TLR9/Hz/DNA studies Rosane DeOliveira: P berghei ANKA/IFNαβ KO mouse studies Shruti Sharma: AT-rich DNA motif studies Bernardo Franklin: PBMC microarrays ParisaKalantari: Hz and DNA trafficking; mouse inflammasome studies Luis Hildebrando Peireirada Silva; DhelioPereira; Mauro Tada: Porto Velho human studies Marco Antônio Ataíde Silva: human inflammasome studies 29

30 Contributors DaniellaC. Bartholomeu: bioinformatics Yolanda Corbett : hemozoin studies Kristen Halmen, Brian Monks, NelsyDepaulaTamburro: technical support Gail Germain: transgenic mouse husbandry Shizuo Akira, Tadagatsu Taniguchi. Glen Barber, Hidde Ploegh: provided transgenic & KO mice 30