Scavenger Receptors and Innate Immunity to Fungal Pathogens

Transcription

1 Scavenger Receptors and Innate Immunity to Fungal Pathogens Terry K. Means Ph.D. Assistant Professor in Medicine Center for Immunology and Inflammatory Diseases Massachusetts General Hospital and Harvard Medical School

2 Outline Part 1: Using an arrayed shrna library to identify receptors important in fungal recognition (2009 Means et al. JEM 206(3): ) Part 2: Using inducible shrna delivery in vivo to test candidate genes in mouse models of fungal pathogenesis

3 Part 1: RNAi Identifies an Important Role for Scavenger Receptors in anti-fungal Immunity

4 Discovery of Toll and Toll-like Receptors in Innate Immunity Awarded Nobel Prize 2011 Jules Hoffmann Bruce Beutler Discovered the function of the fruit fly Toll gene in innate immunity Identified the mammalian Toll homolog, TLR4 as the sensor for LPS

5 Drosophila Toll Null mutation of Toll in Drosophila results in the lack of anti-fungal immunity due to loss of induction of antifungal peptide, drosomycin (1996, Cell J. Hoffmann) Toll mutant flies consumed by A. Fumigatus

6 Mammalian TLR4 Macrophages from the mouse strains (C3H/HeJ) are hypo-responsive to LPS from gram-negative E. coli Bruce Beutler, using a positional cloning strategy identified TLR4 as the gene responsible for recognizing LPS Later, TLR transfection studies and TLR knockout mice revealed that each TLR recognized a specific microbial pathogen or modified self danger molecules.

7 diacyllipopeptides triacyllipopeptides LPS MD2 flagellin profilin TLR1 TLR2 TLR6 TLR4 TLR5 TLR11 CD14 intracellular dsrna TLR3 ssrna TLR7 TRIF MyD88 MyD88 MyD88 Mal MyD88 MyD88 MyD88 TRAM Mal IRAK4 TRIF IRF3 TRAF6 ssrna TLR8 MyD88 NF-κB CpG-DNA TLR9 MyD88 IRF7 cytokines MyD88 IFN-α IFN-β

8 Scavenger Receptor Specificity Endogenous (self) mldl β-amyloid mldl mldl β-amyloid mldl mldl mldl mldl THP gp96 Pathogens (nonself) S. aureus E. coli CpG DNA E. coli S. aureus diacylglycerides OmpA OmpA PorB C C C N N C C C N C N N N N C C C N N N C N SR-AI/II MARCO CD36 SR-C1 CD68 LOX-1 SCARF1/SREC (SR-A) (SR-B) (SR-C) (SR-D) (SR-E) (SR-F)

9 Scavenger Receptors Function as TLR co-receptors Endogenous self ligands apoptotic cells oxldl Amyloid β AGE products oxldl Amyloid β HSP60/70? mmldl GP96 self DNA? Microbial ligands Bacterial outer membrane proteins S. aureus E. coli LPS S. aureus diacyl-lipopeptides Mycobacterial Listeria lipopeptides CpG-DNA LOX-1 TLR2/TLR6 CD36 TLR2/TLR6 TLR4 SR-A TLR? CD14 TLR4 TLR2/TLR1 CXCL16 & MARCO C N MyD88 IRAK TRAF6 NF-κB Lyn Fyn MAPK p44/p42 MyD88 IRAK TRAF6 NF-κB MyD88 IRAK TRAF6 NF-κB intracellular endosome? TLR9 MyD88 IRAK TRAF6 NF-κB

10 Hypothesis: Scavenger receptors work in cooperation with TLRs by binding, concentrating, and internalizing exogenous and endogenous TLR ligands Goal: Determine the functional relationship between scavenger receptors and TLRs for uptake, binding, and signaling to various microbes and endogenous self ligands

11 Cryptococcus neoformans Encapsulated pathogenic yeast Cause of severe disease in immuno-suppressed patients Outgrowth in the lung leads to dissemination to the CNS Cryptococcus stimulation of macrophages leads to the production of pro-inflammatory cytokines and chemokines To date only an indirect role for the innate sensing of fungal pathogens by scavenger receptors has been shown. Modified LDL (classic SRs ligand) inhibited beta-glucan (major carbohydrate found in the fungal cell wall) binding to monocytes. (Rice et al. J. Leukoc. Biol. 2002)

12 Question: Are Scavenger Receptors involved in the innate recognition of fungal pathogens? Experimental Design: We performed two RNAi screens in parallel to test the involvement of Scavenger Receptors in the innate sensing of fungal pathogens. 1. RNAi screen in the nematode C. elegans (colloboration with the Mylonakis Laboratory) 2. shrna screen in the murine macrophage cell line RAW 264.7

13 Killing of Caenorhabditis elegans by Cryptococcus neoformans as a model of yeast pathogenesis non-pathogenic C. Laurentii Pathogenic C. neoformans clinical isolates Mylonakis, Eleftherios et al. (2002) Proc. Natl. Acad. Sci. USA 99, Copyright 2002 by the National Academy of Sciences

14 Scavenger receptors in C. elegans Identified 7 potential Scavenger Receptors in the C. elegans genome by BLAST analysis using Wormbase database WS196 Human/Mouse gene: SCARF1 CD36 SCARB1 SCARB2 C. elegans orthologue: CED-1 C03F11.3 F11C1.3 R07B1.3 F07A5.3 Y49E10.20 Y76A2B.6

15 CED-1 (SCARF1 orthologue) and C03F11.3 (CD36 orthologue) mediate recognition of C. neoformans in C. elegans Together these data indicate that CED-1 and C03F11.3 have evolved to mediate the host response to pathogenic fungi.

16 CED-1 and C03F11.3 are required for C. neoformans-induced production of the anti-fungal peptides ABF-1 and ABF-2 Nematode C. neoformans C. albicans CED-1 N C03F11.3 Tol-1??? N C? C? TIR-1 Abu ABF-1 ABF-2

17 shrna silencing of Scavenger Receptors in mammalian macrophages

18 Lentiviral shrna construct shrna Library RNAi Consortium at Broad Institute High-throughput 96-well Lentiviral Production U6 hpgk 5 LTR shrna puro R 3 LTR Cotransfect with packaging vector and VSV-G Transfection Viral supernatants in 96-individual wells shrna viruses gag/pol env HEK293

19 Silencing Scavenger Receptors and TLRs in mouse macrophages shrnas for 9 mouse TLRs and 10 Scavenger Receptors (SR-A1, SRA2, MARCO, SCARF1, CD36, SCARB1, SCARB2, CD68, CXCL16, LOX1) were selected from the genome-wide RNAi library The library contains 5 individual shrnas targeting each gene, subcloned into the lentiviral vector Each shrna viral vector contains puromycin resistance for stable cell selection shrna viruses RAW264.7 cells add virus puromycin selection Cryptococcus neoformans stimulation

20 TLRs and SRs Expression in the Murine RAW macrophage cell line C. neoformans per cell Fold induction copies/gapdh TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR7 TLR8 TLR9 TLR11 SR-A1 SR-A2 MARCO CD36 CD68 SCARF1 SCARB1 SCARB2 CXCL16 LOX

21 Primary Screen: shrna knockdown of Scavenger Receptors in C. neoformans stimulated RAW macrophages sicontrol 10 0/0

22 Seconday Screen:Validation of Knock-downs mrna expression protein expression shtlr2 #4 shcontrol * * ** TLR2 shcd36 #4 shcontrol * * * ** CD36 shscarf1 #3 shcontrol * * ** SCARF1

23 Tertiary Screen: Cytokine response to C. neoformans in RAW macrophages silenced for TLR2, CD36, or SCARF1 * * * (shrna #4) (shrna #4) (shrna #3)

24 Characterization: SCARF1 and CD36 mediate binding and internalization of C. neoformans

25 C. neoformans activates TLR2/6 signaling via SCARF1 and CD36

26 Generation of anti-cd36 and anti-scarf1 antibodies BM-derived macrophages

27 C-terminus of SCARF1 is required for TLR2/6 signaling and C. neoformans internalization

28 Tyrosine 463 in the C-terminus of CD36 is required for TLR2/6 signaling and C. neoformans internalization.

29 CD36 expression mediates macrophage recognition of C. neoformans cytokine expression C. neoformans binding C. neoformans uptake

30 Increased fungal burden and mortality in C. neoformans infected CD36-KO mice Fungal burden lung Cytokine expression lung

31 Creation of SCARF1-knockout mice

32 SCARF1-ko macrophages are hyper-responsive to Cryptococcus stimulation Copies IL-1/GAPDH * * Copies IL-1/GAPDH

33 SCARF1-ko macrophages express 10-fold higher CD36 Copies CD36\GAPDH * Copies IL-1/GAPDH * * WT SCARF1-ko +C. neoformans

34 Summary Part 1 Mammalian Nematode SCARF1 N C. Neoformans C. albicans C. Neoformans C. albicans CED-1 N TLR2 TLR6 CD36 C03F11.3??? C Mal N MyD88 C N C C IRAK4 TRAF6? NF-κB cytokines ABF-1 ABF-2? C. elegans CED-1 and C03F11.3 and their mammalian orthologues SCARF1 and CD36 are components of an evolutionarily conserved pathway for fungal recognition

35 Part 2: Using inducible shrna delivery in vivo to test candidate hit genes in mouse models of fungal pathogenesis

36 Identification of Candidate Genes Using arrayed shrna screens targeting TLRs and SRs and genome-scale pooled RNAi screens we have identified >100 new hit genes that mediate antifungal immune responses in macrophages How can we test these candidates quickly in vivo for their role in mediating innate and adaptive immune responses?

37 Inducible lentiviral shrna gene silencing in vivo in mouse models fungal pathogenesis Key Advantages: 1.Reduced cost and time compared to generating KO mice 2.Avoid the expensive and time-consuming process of backcrossing 3. Can assess the function of your gene of interest before and after infection or disease has occurred 4.Track the shrna-infected cell in vivo 1.Assess the function of genes that have regulatory and developmental functions and would result in a lethal phenotype in KO mice

38 Development of inducible lentiviral shrna gene silencing for use in vitro and in vivo IPTG PAC: Puro resistance gene LacI: Lac Repressor LacO: Lac Repressor binding site 2A: self-processing polyprotein cleavage sequence for multicistronic gene expression U6 hpgk 2A 5 LTR shrna puro R LacI 3 LTR 3x LacO

39 Evaluation of inducible shgfp in bone marrow-derived macrophages from actin-gfp mice % GFP Fluorescence * no IPTG plus IPTG 0 Wild-type tg-gfp 3x LacO shcontrol 3x LacO shgfp Tg-GFP BM-derived macrophages hpgk U6 2A 5 LTR shrna puro R LacI 3 LTR 3x LacO

40 Optimization of the performance of inducible shgfp in vitro Days IPTG 1mM induction IPTG removal

41 Figure 2. Inducible lentiviral shrna gene silencing in vivo Step 1: Step 2: Isolate bone marrow cells (Tg-GFP pos mice) U6 hpgk 5 LTR shgfp puro R LacI 3 LTR 3x LacO transduce BM cells with inducible shgfp lentivirus 10 7 Bone marrow cells Step 3: Step 4: WT recipient mouse bone marrow aplasia (total body irradiation) Inject syngeneic bone marrow transduced with inducible shgfp lentivirus Step 5: allow for reconstitution (~ 6 weeks) inducible shgfp without IPTG Step 6: add IPTG to mice drinking H2O to induce shrna (~ 1 week) inducible shgfp with IPTG

42 Performance of inducible shgfp in vivo Week 0 Week 6 Week 7 Week 8 BM transplant BM reconstitution IPTG induction IPTG removal without IPTG

43 MyD88 expression in hematopoietic cells is required for anti-fungal immunity i.v. C. neoformans infection i.v. C. neoformans infection P<0.003 P<0.001 CFU/g (millions) CFU/g (millions) no IPTG with IPTG

44 Restoration of MyD88 expression in hematopoietic cells post-infection increases survival and anti-fungal immunity i.v. C. neoformans infection CFU/g (millions) +IPTG +IPTG IPTG removal at Day 5

45 Take Home Summary Using arrayed and pooled shrna screens in macrophages to identify novel genes that mediate anti-fungal immunity CED-1 and C03F11.3 and their mammalian orthologues SCARF1 and CD36 mediate host defense against fungal pathogens Create gene knockdown mice using inducible shrna silencing in vivo to test candidate hits in mouse models of fungal pathogenesis MyD88 is essential in circulating hematopoietic cells for anti-fungal immunity in mice

46 Collaborators Joseph El Khoury Lab Center for Immunology Massachusetts General Hospital Eleftherios Mylonakis Lab Division of Infectious Diseases Massachusetts General Hospital Kathryn Moore Lab Lipid Metabolism Massachusetts General Hospital Means Lab Melissa Tai Robert Friday Lindsay Puckett Richard Colvin Edward Seung RNAi Consortium Nir Hacohen Lab Broad Institute Massachusetts General Hospital Andrew Luster Lab Center for Immunology and Inflammatory Diseases Massachusetts General Hospital Douglas Golenbock Lab Division of Infectious Diseases UMASS Medical School This work was supported by grants from the NIH, Irvington Institute and Dana Foundation, Massachusetts Life Sciences Center, and Lupus Research Institute

47 Outline Part 1: Using an arrayed shrna library to identify receptors important in fungal recognition (2009 Means et al. JEM 206(3): ) Part 2: Using genome-wide pooled shrna screens to identify genes that mediate innate immune cell responses to fungal pathogens Part 2: Using inducible shrna delivery in vivo to test candidate genes in mouse models of fungal pathogenesis

48 Part 2: Using genome-wide pooled shrna screens to identify genes that mediate innate immune cell responses to fungal pathogens

49 Cytokine and Co-Stimulatory Reporter System for shrna Pooled Screen Control shrna shtlr2 untreated C. neoformans untreated C. neoformans TNF/CD80 TNF/CD80

50 Pooled Viral supernatants Contains ~90,000 shrnas targeting 18,000 genes 10 7 RAW macrophages Lentivirus encoding shrna macrophage Select with puromycin Stimulate cell sorting on FACSAria Day infect +puro passage cells passage cells stimulate assay FACS sort

51 Pooled shrna assay Screen 90,000 shrnas for 18,000 genes in each experiment 90k shrna viruses 10 7 cells infect expand FACSor t CD80+\TNF+ puromycin CD80-\TNFselection (sort bottom 10 8 macrophages 5%) Fungal stimulation TNF (+ C. neoformans) CD80 Isolate gdna PCR hairpins Sequence shrnas Measure shrna abundance Enrichment/depletion of shrnas

52 Pooled screen primary hits Top 100 shrnas enriched in low TNF sort MyD88 TLR2 IRAK2 TRAF6 IRF7 Low sort #1 Low sort #2 Hi sort #1 Hi sort #2 Unsorted #1 Unsorted #2 Additional known genes 61. NFκB (p50) 149. IRAK IRF8 shrnas expressed equally 227. STAT RelA 349. STAT1 Top 100 shrnas enriched in high TNF sort PIAS1 Tollip Nfkbib Increased shrna expression (>8-fold, *p<0.05) Decreased shrna expression (>8-fold, *p<0.05)