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 Outline

Part 1: Using an ‘arrayed’ shRNA library to identify receptors important in fungal recognition (2009 Means et al. JEM 206(3):637-653)

Part 2: Using inducible shRNA delivery in vivo to test candidate in mouse models of fungal pathogenesis PartPart 1:1: RNAiRNAi Identifies Identifies anan ImportantImportant RoleRole forfor ScavengerScavenger ReceptorsReceptors inin anti-Fungalanti-Fungal ImmunityImmunity Discovery of Toll and Toll-like Receptors in Innate Immunity Awarded Nobel Prize 2011

Jules Hoffmann Bruce Beutler

Discovered the function of Identified the mammalian the fruit fly Toll in Toll homolog, TLR4 as the innate immunity sensor for LPS 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 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. MD2 triacyl- diacyl- flagellin profilin lipopeptides lipopeptides LPS

TLR1 TLR2 TLR6 TLR4 TLR5 TLR11 CD14

TRIF MyD88 MyD88 MyD88 Mal MyD88 MyD88 MyD88 intracellular TRAM Mal dsRNA TLR3 IRAK4 IRF3 TR ssRNA TLR7 IF TRAF6

M ssRNA yD NF-κB TLR8 88

M IRF7 CpG-DNA y cytokines TLR9 D 88

M y D IFN-α IFN-β 88 Scavenger Receptor Specificity

mLDL mLDL mLDL mLDL mLDL mLDL mLDL THP Endogenous (self) β-amyloid β-amyloid gp96

S. aureus Pathogens (nonself) S. aureus CpG DNA OmpA OmpA E. coli E. coli diacylglycerides PorB

C C C N N

C C C N

C

N N N NC C C C N N N 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) Scavenger Receptors Function as TLR co-Receptors

Endogenous apoptotic cells AGE products HSP60/70? self DNA? “self” oxLDL oxLDL mmLDL ligands Amyloid β Amyloid β GP96

S. aureus E. coli LPS Bacterial outer Microbial S. aureus CpG-DNA ligands membrane diacyl-lipopeptides Mycobacterial Listeria lipopeptides

LOX-1 TLR2/TLR6 CD36 TLR2/TLR6 TLR4 SR-A TLR? CD14 TLR4 TLR2/TLR1 CXCL16 & MARCO C

MyD88 MyD88 MyD88 ? N Lyn IRAK IRAK IRAK intracellular endosome TRAF6 Fyn TRAF6 TRAF6 NF-κB NF-κB NF-κB TLR9 MAPK p44/p42

MyD88 IRAK TRAF6 NF-κB 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 Cryptococcus neoformans

• Encapsulated pathogenic yeast

• Cause of severe disease in immuno-suppressed patients

• Outgrowth in the 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) 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 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, 15675-15680

Copyright ©2002 by the National Academy of Sciences 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 Y49E10.20 Y76A2B.6 F11C1.3 R07B1.3 F07A5.3 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. CED-1 and C03F11.3 are required for C. neoformans-induced production of the anti-fungal peptides ABF-1 and ABF-2

Nematode

CED-1 C. neoformans N C. albicans

C03F11.3 Tol-1

? ? ?

N C ? C ? TIR-1

Abu

ABF-1 ABF-2 shRNA silencing of Scavenger Receptors in mammalian macrophages shRNA Library RNAi Consortium at Broad Institute High-throughput 96-well Lentiviral Production

Lentiviral shRNA construct

U6 hPGK 5’LTR shRNA puroR 3’LTR

Cotransfect with Viral supernatants in packaging vector Transfection 96-individual wells and VSV-G shRNA viruses

gag/pol env HEK293 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

Cryptococcus add virus puromycin neoformans selection stimulation TLRs and SRs Expression in the Murine RAW macrophage cell line

0.25

0.20 H D P

A 0.15 G / s

e 0.10 opi c 0.05

0.00 1 2 3 4 5 6 7 8 9 1 1 2 O 6 8 1 1 2 6 1 R R R R R R R R R 1 A A 3 6 F B B 1 - L L L L L L L L L R - - C D D R R R L X T T T T T T T T T L R R R C C A A A C O T S S A C C C X L M S S S C

C. neoformans per cell Fold induction Primary Screen: shRNA knockdown of Scavenger Receptors in C. neoformans stimulated RAW macrophages

siControl 10 0/0 Seconday Screen:Validation of Knock-downs mRNA expression expression

shTLR2 #4 shControl

* * **

TLR2

shCD36 #4 shControl

* * * **

CD36 shSCARF1 #3 shControl

* * **

SCARF1 Tertiary Screen: Cytokine response to C. neoformans in RAW macrophages silenced for TLR2, CD36, or SCARF1

* * *

(shRNA #4) (shRNA #4) (shRNA #3) Characterization: SCARF1 and CD36 mediate binding and internalization of C. neoformans C. neoformans activates TLR2/6 signaling via SCARF1 and CD36 Generation of anti-CD36 and anti-SCARF1 antibodies

BM-derived macrophages C-terminus of SCARF1 is required for TLR2/6 signaling and C. neoformans internalization Tyrosine 463 in the C-terminus of CD36 is required for TLR2/6 signaling and C. neoformans internalization. CD36 expression mediates macrophage recognition of C. neoformans

cytokine expression C. neoformans binding C. neoformans uptake Increased fungal burden and mortality in C. neoformans infected CD36-KO mice

Fungal burden lung

Cytokine expression lung Creation of SCARF1-knockout mice SCARF1-ko macrophages are hyper-responsive to Cryptococcus stimulation

* * Copies IL-1/GAPDH Copies IL-1/GAPDH Copies CD36\GAPDH SCARF1-ko macrophages express10-fold * higher CD36

Copies IL-1/GAPDH WT +C. neoformans * SCARF1-ko * Summary Part 1 Mammalian Nematode

SCARF1 CED-1 N C. Neoformans N C. Neoformans C. albicans C. albicans

CD36 C03F11.3 TLR2 TLR6 ?

? ?

C N C N C Mal MyD88 C IRAK4 ? TRAF6

NF-κB ABF-1 ABF-2 cytokines ?

C. elegans CED-1 and C03F11.3 and their mammalian orthologues SCARF1 and CD36 are components of an evolutionarily conserved pathway for fungal recognition Part 2: Using inducible shRNA delivery in vivo to test candidate ‘hit’ genes in mouse models of fungal pathogenesis 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 anti- fungal immune responses in macrophages

How can we test these candidates quickly in vivo for their role in mediating innate and adaptive immune responses? 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 Development of inducible lentiviral shRNA gene silencing for use in vitro and in vivo

PAC: Puro resistance gene IPTG LacI: Lac Repressor LacO: Lac Repressor binding site 2A: self-processing polyprotein cleavage sequence for multicistronic

U6 hPGK 2A 5’LTR shRNA puroR LacI 3’LTR

3x LacO Evaluation of inducible shGFP in bone marrow-derived macrophages from actin-GFP mice

100 no IP TG e c

n 80 plus IP TG e c s

e 60 uor

Fl 40 P

F *

G 20 % 0 e l p F P ro F P ty G t G - - o n h ild tg C s h O W s c O L a a c x L 3 3 x Tg-GFP BM-derived macrophages

hPGK U6 2A 5’LTR shRNA puroR LacI 3’LTR

3x LacO Optimization of the performance of inducible shGFP in vitro

Days

IPTG 1mM IPTG induction removal Figure 2. Inducible lentiviral shRNA gene silencing in vivo

Step 1: Step 2: transduce BM cells with inducible shGFP lentivirus 7 Isolate bone 10 Bone hPGK marrow cells U6 marrow 5’LTR shGFP puroR LacI 3’LTR (Tg-GFPpos cells mice) 3x LacO

Step 3: Step 4: Step 5: Step 6: WT recipient mouse Inject syngeneic bone allow for add IPTG to mice bone marrow aplasia marrow transduced with reconstitution drinking H2O to induce (total body inducible shGFP (~ 6 weeks) shRNA (~ 1 week) irradiation) lentivirus inducible shGFP inducible shGFP without IPTG with IPTG Performance of inducible shGFP in vivo

Week 0 Week 6 Week 7 Week 8 BM transplant BM reconstitution IPTG induction IPTG removal without IPTG 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 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 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 Collaborators

Joseph El Khoury Lab Nir Hacohen Lab Means Lab Center for Immunology Broad Institute Massachusetts General Hospital Massachusetts General Hospital Melissa Tai Robert Friday Eleftherios Mylonakis Lab Andrew Luster Lab Division of Infectious Diseases Lindsay Puckett Center for Immunology and Massachusetts General Hospital Richard Colvin Inflammatory Diseases Massachusetts General Hospital Edward Seung Kathryn Moore Lab Douglas Golenbock Lab Lipid Metabolism Division of Infectious Diseases Massachusetts General Hospital UMASS Medical School RNAi Consortium

This work was supported by grants from the NIH, Irvington Institute and Dana Foundation, Massachusetts Life Sciences Center, and Lupus Research Institute Outline

Part 1: Using an ‘arrayed’ shRNA library to identify receptors important in fungal recognition (2009 Means et al. JEM 206(3):637-653)

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 Part 2: Using genome-wide “pooled” shRNA screens to identify genes that mediate innate immune cell responses to fungal pathogens Cytokine and Co-Stimulatory Reporter System for shRNA Pooled Screen

Control shRNA shTLR2

untreated C. neoformans untreated C. neoformans

TNF/CD80 TNF/CD80 Pooled Viral supernatants

Contains ~90,000 shRNAs 7 targeting 18,000 genes 10 RAW macrophages

Lentivirus encoding shRNA

Select with puromycin macrophage Stimulate cell sorting on FACSAria

Day 0 1 2 3 4 56789 10 11

e e te a say +puro s infect passaglls passaglls imul a e e t c c s FACS sort Pooled shRNA assay Screen 90,000 shRNAs for 18,000 genes in each experiment

(+ C. neoformans) 90k FACSor shRNA t viruses 107 cells CD80+\TNF+ infect expand Isolate gDNA

puromycin TNF PCR hairpins CD80-\TNF- selection (sort bottom Sequence shRNAs 108 macrophages5%) Measure shRNA abundance Fungal stimulation Enrichment/depletion of shRNAs CD80 equally expressed shRNAs TNF sort enriched in low Top 100shRNAs TNF sort enriched in high Top 100shRNAs Decreased shRNA expressi Decreased shRNA express Increased shRNA Pooled IRF7 TRAF6 IRAK2 TLR2 MyD88 Nfkbib Tollip PIAS1

screen

ion (>8-fold, *p<0.05) Low sort #1 on (>8-fold, *p<0.05) Low sort #2

Hi sort #1 primary Hi sort #2 Unsorted #1 Unsorted #2

hits 349. STAT1 256. RelA 227. STAT4 200. IRF8 149. IRAK4 61. NF genes known Additional κ B (p50)