host response to fungal infections - molekularediagnostik.ch · • the neutrophil response and the...

Host Response to

March 9th, 2016Molecular Diagnostics 2017, Zürich

Salomé [email protected]

- immunology- immunogenetics- fungal pathogenicity

Fungal Infections

1 2 3

4

Mycosis# life-threateningcases/year

Aspergillosis >200,000

Candidiasis >400,000

Cryptococcosis >1,000,000

Mucormycosis >10,000

Pneumocystosis >400,000

Dimorphic (endemic) mycoses

~65,000

Systemic fungal infections

mortality rate: 30-70%

mainly nosocomial

~25 % ~1.7 bn

Superficial fungal infections

The spectrum of fungal diseases

Underhill and Iliev, Nat.Rev.Immunol., 2014

The organisms causing diseaseare mainly opportunistic pathogens

~ 105 fungal species(only a few hundreds are pathogenic)

yeast (C. albicans)mold (Aspergillus)

Predisposing conditions for opportunistic fungal infections

Candidiasis(thrush)

CD4+ T cells

HIV

time

• barrier defects e.g. severe burn wounds, cathethers

• dysbiosis e.g. vulvovaginal candidiasis, systemic candidiasis

• chemotherapy e.g. in leukemia patients

• immunosuppression e.g. in transplant patients

• immune defects - neutropenia e.g. systemic candidiasis

- functional neutrophil defects e.g. aspergillosis in CGD patients

- T cell defects e.g. superficial candidiasis

Despite similar clinical risk factors:

some patients rapidly develop fungal infections,

while others seem to be protected and never do so.

Genetic Differences ?

1. Immunogenetics of systemic candidiasis and aspergillosis

2. Congenic defects associated with superficial fungal infections

3. Genetic diversity of the fungus

SNP array

limited to the SNPs on the array

screen of 200’000 SNPs in a large candidemia cohort

identify novel genetic risk factors for candidaemia

Kumar et al., Nat.Communications (2014)

significance treshold

SNP array whole exome sequencingwhole genome sequencing

limited to the SNPs on the array

the majority (85%) of all disease-causing mutations occur in the exome

the exome is 1-2% of the entiregenome

WES = cost effective

Candidate gene polymorphisms associated with invasiveAspergillus and/or Candida infections

PRRscytokines

chemokinescytokine receptors

cytokine antagonists

Wójtowicz and Bochud, Semin Immunopathol (2015)

Caveats: • sample size• study design, controls• statistical testing• confirmation by independent cohorts• functional evidence supporting the association

Khanna et al, Swiss Med Wkly (2016)

Caveats: • sample size• study design, controls• statistical testing• confirmation by independent cohorts• functional evidence supporting the association

Is the identified mutation the disease-causing mutation?

Validation!

- synonymous vs. non-synonymous SNPs

- exclude SNPs that are frequent gene variants

- analyse the transcriptome and proteome to find the relevance of the mutation

Future perspectives:

- assign each patient an individual risk score based on his genetic background (‘genetic prediction’)

- apply personalized prophylaxis and treatment schemes (as in modern oncology)

- Whole-Exon-Sequencing will likely be the method of choice.

Khanna et al., Swiss Med Wkly (2016)

protective mechanisms against systemic fungal infection

Identification of novel mutations also promotes the understanding of

Mendelian defects in the IL-17 pathway

Chronic Mucocutaneous Candidiasis (CMC)

STAT3 (AD-HIES)STAT1 GOFRORcCard9

AIRE (APECED)

induction of IL-17/Th17

5

NFkB

target genes

a-IL-17

IL-17 plays a non-redundant role in protection from superficial Candida infection

(Ling et al., JEM, 2015)e.g. inherited IL-17RC-deficiency

• oroal mucosa• skin• nails• vaginal mucosa

The IL-17 cytokine family

Pappu et al., Trends Immunol (2012)

Monte et al., Infect. Immun. (2013)

IL-17 in autoinflammatory disease

Lowes et al., TRIMM, 2013

FDA-aproved in 2015

Kirkham et al., Immunology, 2013

high moderatemoderate-low

response rate:

targetting IL-17 as a therapeutical appraoch

side effects...

“...localized mucosal or cutaneous candidiasis, primarily oral candidiasis. These infections were mild to moderate in severity and were clinically manageable/treatable with standard therapy without discontinuation. “

Mendelian defects in the IL-17 pathway

(Ling et al., JEM, 2015)

e.g. inherited IL-17RC-deficiency

immortalized fibroblasts frompatients (P1, P2) & controls (C1, C3)

response to IL-17 stimulation transfectants

5

NFkB

target genes

epithelium

?

IL-17A/F?

• Regulation of IL-17 induction in response to C. albicans

• IL-17-mediated effector mechanisms

• IL-17-independent antifungal defense

Candida albicans

C57BL/6 (WT)

C. albicans(strain SC5314)

WT

IL-17RA-/-

0 1 2 3 5 760

70

80

90

100

110

days post infection

%body

weig

ht

IL-17RC-/-

WT IL-17RA-/-

day 7 p.i.

IL-17RC-/-

IL-17RA-/-IL-17RC-/-

Experimental model of oropharyngeal candidiasis (OPC)

WT, day 1naive

acute inflammatory response

Trautwein-Kirchner et al., Mucosal Immunology 2015

lumen

epithelium

innate lymphocytes

IL-17

Th17

DC

T cell priming in lymph nodes Complementary and redundant cellular sources

for the IL-17 response during murine OPC

?

Multiple sources of IL-17 during acute OPC infection

Gloadiator et al., J.Immunol (2013)Trautwein-Weidner et al., PLoS Pathogens (2015)Sparber et al, in preparation (in humans?)

FSCIL

-17

ATCRγδ

TCR

β

Viable CD45+ CD90+ tongue cells, 1 day post-inf.

5

NFkBMAPK

target genes

control neutrophil-depleted

neutrophils

chemokines, cytokines

IL-17 & neutrophils

Trautwein-Weidner, Gladiator et al., Mucosal Immunology (2015)

epithelium

day 1 p.i.

Neutrophil recruitment during OPC is IL-17-independent

Trautwein-Weidner, Gladiator et al., Mucosal Immunology (2015)

epithelium

C. albicans

IL-17 immunity and neutrophil response are uncoupled and act complementary in the oral mucosa

confinement of the fungus

IL-17R

IL-17-producing cells

IL-17

neutrophils

epithelium

C. albicans

IL-17 immunity and neutrophil response are uncoupled and act complementary in the oral mucosa

confinement of the fungus

IL-17R

IL-17-producing cells

IL-17

?antimicrobial

factors

neutrophils

8h 1d 3d 7d

fold induction relative to naive (log2)

tongue, strain SC5314

IL-17-dependent expressionlimited to the epithelium

Identification of genes/pathways that contribute to disease susceptibility

Generation of new hypothesis

antifungal host defense research

genetic studiesexperimental studies

(human PBMCs, mouse models)

Dissection of protective mechanismsat a cellular and molecular level

new therapeutic approaches, vaccines

The host immune status determines susceptibility to disease

What about the fungus?

Impact of the intraspecies diversity on the outcome of infection?

• Colonizing population of C. albicans is monoclonal

• Vertical transmission

• Large intraspecies diversity (10’000 – 100’000 SNPs)

C. d’Enfert and ME. Bougnoux (Pasteur)

isolate 101

isolate SC5314

Very distinct outcomes of infection with different fungal isolates

S C 5 3 1 4 1 0 1

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

1 0 6

1 0 7

d ay 1 p .i

cfu

/g t

on

gu

e

101SC5314 S C 5 3 1 4 1 0 1

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

1 0 6

1 0 7

d ay 7 p .i

cfu

/g t

on

gu

e101SC5314

Schönherr et al., Mucosal Immunology, 2017

0 1 2 3 4 5 6 7

7 0

8 0

9 0

1 0 0

1 1 0

t im e (d a y s )

we

igh

t (%

)

S C 5 3 1 4

1 0 1

day 1 p.i.

Pathogenicity correlates with the invasion depth into the epithelium

SC5314 101

day 1 post-inf.

stratum granulosumstratum spinosum

stratum corneum

stratum basale

12h post-inf.


The epithelium senses the variation in C. alicans andtranslates it in various degrees of inflammation

e

SC5314 101


The epithelium senses the variation in C. alicans andtranslates it in various degrees of inflammation

IL-1αDAPI

SC5314

101

in vivo (1 day p.i.)

SC5314 101

IL-1 signaling controls theneutrophil response

Are persistent strains ‘unseen’ by the immune system?


1 0 1 3 5

0

2

4

6

8

1 0

se

ru

m G

-CS

F (

ng

/ml)

SC5314 101

1d 3d 5d1dpost-inf.

Persistent C. albicans isolates do not induce neutrophils

Ly6G (Neutrophils)

101SC5314

d1

d3

d5


1 0 1 3 5

1 0 -6

1 0 -5

1 0 -4

1 0 -3

IL -1 7 k in e tic s

re

l. m

RN

A e

xp

re

ss

ion

(Il

17

a /

Ac

tb)

SC5314 101

1d 3d 5d1d post-inf.

Delayed induction of the host response with non-pathogenic isolates

05

00

10

00

15

00

20

00

dif

fere

nti

ally

exp

ress

ed

ge

ne

s

8h 1d 3d 7d

SC5341

101

p.i.

kinetic & qualitative differences in the response to different C. albicans isolates

Schönherr et al., Mucosal Immunology, 2017Altmeier et al., unpublished

β-defensins mediate IL-17-dependent fungal control

SC5314 101

0.3 1 3 7 0.3 1 3 7 days p.i.

Defb1

Defb3

Defb4

Defb6

Defb14

re

l. m

RN

Ae

xp

re

ss

ion

(D

efb

3/A

ctb

)n

aiv

eS

C101

0

5

1 0

1 5

2 0

W T

IL 1 7 R C - /-

101SC5314naive

Altmeier, Mertens and LeibundGut-Landmann., unpublished

SC5314 101

B l/6 I l1 7 ra -/- B l/6 I l1 7 ra -/-

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

1 0 6

1 0 7

cfu

/g t

on

gu

e

day 7 p.i.

IL-17 signaling is essential for fungal control –irrespective of the degree of fungal pathogenicity

101

101


IL-17R

epithelium

neutrophil response

C. albicans

IL-17

bone marrow

circulation

IL-1α

IL-17 pathway

chemokinesG-CSF

• Natural variations in C. albicans determine theoutcome of fungal-host interactions.

• The epithelium translates differences in the fungus into qualitatively distinct host responses.

• The neutrophil response and the IL-17 pathway are uncoupled.

• The key role of the IL-17 pathway for fungal control isconserved, irrespective of the fungal pathogenicity.

predictive value ?

Alumni: Kerstin Trautwein-WeidnerAndré GladiatorAlbulena ToskaLaura LauenerPatrizia DiethelmAna Chavez-SteenbockIsabelle Zenklusen

Eva

Sarah

Jesper

Florian K.Selim Florian S. Simon

Carina

FranziSchönherr

Natalia

Thank you!

Promedica Stiftung

Fonds zur Förderung des

akademischen Nachwuchses

des Zürcher Universitätsvereins

Dominique Sanglard

Cristina Fragoso

Christophe d’Enfert

David Sancho

Federica Sallusto

Thomas Rülicke

Norman Pavelka

Cornelia Halin

Anja Kipar, Udo Hetzel

host response to fungal infections - molekularediagnostik.ch · • the neutrophil response and the...

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