NICOLA SMITHNATIONAL ASPERGILLOSIS CENTRE

UHSMMANCHESTER

Support Meeting for Aspergillosis Patients

Fungal Research Trust

Nicola Smith

The University of Manchester, Manchester Academic Health Science Centre, University Hospital of South

Manchester, UK

Genetic Susceptibility to Aspergillosis

Introduction: Aspergillosis (1)

• Disease caused by fungus – Usually A. fumigatus

• Causes multiple types of lung disease– Allergic (ABPA, SAFS)

– Chronic (CCPA, CNPA, CFPA)

– Invasive (IPA)

• Project focuses on chronic and allergic diseases

Genetic Susceptibility in Aspergillosis

• ABPA occurs in families – Shah et al., 1990; Shah et al., 2008

• Genetic associations found in literature

My Project• Investigating possible genetic associations • Find genes underlying susceptibility to

aspergillosis

DNA, Genes & Proteins

• Genes are made of DNA• Genes are the blueprint

for proteins • Proteins are the building

blocks of our bodies

Fig: http://www.bioteach.ubc.ca/Bioinformatics/humangenome

• Genes (and proteins) vary between people– We look different (eye, hair colour etc)

• Differences in genes can also affect susceptibility to disease

• When cells in the body make proteins we say that the gene is being expressed

• Some genes are expressed all the time• Others are turned on and off as required

• Gene expression can be turned on or off by various factors

Gene Expression (1)

Example: Person gets an infection • Cells see germs – gene expression of anti-germ

genes is turned on• Cells produce proteins to fight of the germ• Germ is killed by proteins• Cells no longer see germs – gene expression is

turned off• Cells stop making anti-germ protein

Gene Expression (2)

Immune cell

Protein

Germ

Gene Expression:

Do the cells of aspergillosis patients respond differently to those from healthy volunteers?

• Which genes are expressed in aspergillosis?

• Macrophages: cells in blood, fight germs• Aspergillus: fungus (germ) causing aspergillosis

• Grow macrophages and Aspergillus together and look at gene expression

Gene Expression and Aspergillosis (1)

Aspergillus

Macrophage

• Changes in expression after exposure to fungus = genes are important in fighting fungus

• Look at cells from healthy volunteers and patients

• Differences in expression between the groups = may be important in disease susceptibility

• Differences may be at before or after exposure to fungus

Gene Expression and Aspergillosis (2)

Gene expression is measured at 30min, 1hr, 3hr, 6hr and 9hr – how does this relate to the fungus?

Fungal growth: resting conidia (RC) germinating conidia (GC) germtube (GT) hyphae (H)

Counted the amount of fungus at each stage over time

Timepoints for gene expression (1)

Gene expression is measured at 30min, 1hr, 3hr, 6hr and 9hr – how does this relate to the fungus?

3hr – mostly resting conidia

6hr – mostly germtubes

9hr – mostly hyphae

Timepoints for gene expression (2)

Method

Ficoll prep

PBMCs stored in liquid nitrogen

Cultured for 12-15 days to differentiate to macrophages (~2x105)

24well plate – one plate per timepoint

0hr timepoint – investigate gene expression

Aspergillus spores added (4x105)

30min, 1hr, 3hr, 6hr, 9hr timepoints - Investigate gene expression

Macrophages and Aspergillus cultured together

Heparinised Blood

Macrophages

PBMCs

Ficoll prepped blood

Plasma

Ficoll

Granulocytes

PBMCs thawed and plated out (2x106)

Gene Expression Results

I studied the expression of 84 genes!

Fold regulation in ABPA (1)A

DO

RA

2A

C5 C8A

CA

MP

CA

SP

1

CC

R3

CD

14 CD

55

CH

UK

CR

P

CY

BB

DE

FB

4

DM

BT

1

FN

1

HM

OX

1

IFN

A1

IFN

B1

CC

L2

CD

1DCA

SP

4

CO

LEC

12 CX

CR

4

-40

-35

-30

-25

-20

-15

-10

-5

0

5

10

Gene

Fo

ld r

egu

latio

n (c

om

par

ing

to 0

hr)

30min

1hr

3hr

6hr

9hr

Fold regulation in ABPA (2)

IKB

KB

IL1

2R

B2

IL1

A

IL1

B

IL1

F1

0

IL1

F5

IL1

F6

IL1

F7

IL1

F8

IL1

F9

IL1

R1

IL1

RA

P

IL1

RA

PL

2

IL1

RL

2 IL1

RN IL6

IRA

K2

IRA

K1

IL1

R2

IL1

0

IFN

GR

2

IFN

GR

1

-15

-10

-5

0

5

10

Gene

Fo

ld r

egu

latio

n (c

om

par

ing

to 0

hr)

30min

1hr

3hr

6hr

9hr

IRF1LALBA

LBPLTF

LY96 LYZMAPK14 MAPK8

MIFMYD88 NCF4

NFKB1NFKB2

NFKBIANLRC4 NOS2

PGLYRP1PGLYRP2

PGLYRP3PPBP

PROCPTAFR

-15

-10

-5

0

5

10

15

Fold regulation (comparing to 0hr)

Gene

Fold regulation in ABPA (3)

30min

1hr

3hr

6hr

9hr

Fold regulation in ABPA (4)

SE

RP

INE

1

TLR

8

TLR

9

TN

F

TR

AF

6

RP

L13

A

SE

RP

INA

1

SF

TP

D

TG

FB

1

TLR

1

TL

R1

0

TL

R2

TLR

4

TL

R6

HP

RT

1

B2

M

TN

FRS

F1

A

TO

LLIP

TR

EM

1

S1

00A

12

TLR

3

-70

-60

-50

-40

-30

-20

-10

0

10

20

Gene

Fo

ld r

egu

latio

n (c

om

par

ing

to 0

hr)

30min

1hr

3hr

6hr

9hr

RED: more expression in ABPA (2)

GREEN: less expression in ABPA (12)

ABPA 0hr vs Asthma 0hr

>2-fold higher expression in ABPA

>2-fold lower expression in ABPA

Expression before Exposure: ABPA

RED: more expression in CCPA (4)

GREEN: less expression in CCPA (5)

CCPA 0hr vs Healthy 0hr

>2-fold higher expression in CCPA

>2-fold lower expression in CCPA

Expression before Exposure: CCPA

Less expression in ABPA:• 9 genes involved in IL1 pathway• TREM1: receptor

Less expression in CCPA:• TLR10, TLR3, TLR1, TLR8: receptors

Expression before Exposure:ABPA and CCPA

Less expression in CCPA than controls:

• TLR10, TLR3, TLR1, TLR8

• TLRs:

• are receptors on the cell surface

• are important in recognising germs

• tell the cell to respond to the germ (to fight it off)

• Less expression fewer receptors harder for cell to recognise fungus fungus grows get disease

Expression before Exposure: TLRs

Less expression in ABPA than in controls:

• 9 genes involved in IL1 pathway

• IL1 pathway:

• Proinflammatory – leads to inflammation

• Inflammation is often useful in fighting off fungus

• Uncontrolled inflammation can be a problem

• What happens in this pathway over time?

Expression before Exposure: IL1 pathway

• Asthma (control) - expression alters in response to fungus• ABPA - expression remains low

• ABPA cells don’t respond to presence of fungus less inflammation less fungal death disease

Expression over time: IL1 pathway

IL6 IL1A IL1B

• TNF expression increases over time in all groups (CCPA, ABPA and controls)

• TNF is proinflammatory – is important in fighting fungus

Expression over time

Genetic Variation: Are there similar differences in the

genes (DNA)?

• DNA is like a string of letters (A, T, G, C)• The letters in the string tell the body how and

when to make protein

• Small changes in the letters (SNPs) can change:• When a protein is made• How much of a protein is made• Whether a protein works or not (or how well)

Genetic Variation

ATGGGTAATGCCATGTCAC = normal proteinATGGGTAACGCCATGTCAC = more proteinATGGATAATGCCATGTCAC = non-functional protein

Example: STAT6

Example: MBL

Protein Function• Binds to germs, helps cells see them

SNPs cause:• Low/High expression levels

– Low expression = less protein to fight infection

• Functional/Non-functional protein– Non-functional protein = can’t fight infection

• Associated with CPA (Vaid et al., 2007; Crosdale et al., 2001)

• Most SNPs have 2 possible letters (e.g. C and G)

• Count how many patients and how many controls and each letter:• More C in patients than in controls• Statistics prove it’s not due to chance• SNP is associated with disease (C in particular)

Genetic Variation and Disease

Genetic Variation Results (1)

CCPA 0hr vs Healthy 0hr

>2-fold higher expression in ABPA

>2-fold lower expression in CCPA

• TLR1: SNP associated with CCPA• TLR10: SNP associated with ABPA+CPA

• Both show low expression in CCPA• Do the SNPs cause this?

Genetic Variation Results (2)

• IL6: 2 SNPs associated with ABPA

• Differences are seen in IL6 expression by ABPA and control cells

• Do the SNPs cause this difference?IL6

Genetic Variation Results (3)

ABPA 0hr vs Asthma 0hr

>2-fold higher expression in ABPA

>2-fold lower expression in ABPA

• TREM1: 1 SNP associated with SAFS

• Lower expression by ABPA than control cells – before and after exposure to fungus

• Does the SNP cause this difference?

Genetic Variation Results (4)IL1RN: • 1 SNP associated with CCPA• 2 SNPs associated with ABPA+CPA• 1 SNP associated with ABPA

• Expression increases over time: must be important in response – do the SNPs affect the function?

Genetic Variation Results (5)• Dectin-1: receptor that recognises A. fumigatus

• 2 SNPs associated with CCPA• 1 SNP associated with ABPA+CPA• 1 SNP associated with ABPA• 1 SNP associated with SAFS

• SNPs may affect the ability of the cells to recognise and respond to the fungus

• Expression of Dectin-1 was not investigated

So what?• We can find differences between patients and

controls – but so what?

• Differences in expression over time: tells us which genes are important in fighting the fungus (and preventing the disease)

• Differences between patients and controls: suggests genes that may be involved in disease susceptibilty

Knowledge of these helps us know which proteins may be useful drug targets/therapies…

How?Knowledge of these helps us know which proteins may

be useful drug targets/therapies… how?

This is a long way in the future, but…

• If a protein is shown to be important and is reduced in a patient, it may be possible to replace this protein as a therapy

• If a receptor is shown to be important in recognition of the fungus, it may be possible to boost the response of this receptor with drugs

Summary• DNA Genes Proteins• Small changes in DNA (SNPs) can affect protein

expression or function• Differences in expression can be seen between ABPA

and CCPA patients and controls• SNPs can be found in these and other genes

Future work:• Discover the function of these SNPs• Use this information to further understanding

aspergillosis

AcknowlegementsPatients

Supervisors:

• Dr Paul Bowyer

• Dr Angela Simpson

• Dr David Denning

Advisor:

• John Curtin

Blood Processing:

• Liz Hadley

• Lucy Yates

Recruitment / clinics:

• Dr David Denning

• Dr Robin Gore

• Dr Rob Niven

• NWLC nurses

• NAC nurses (Georgina & Debbie)

• Marie Kirwan

• Marian Denson

Funding / Resources:

• MRC

• NHS