Deep dermatophytosis and CARD9 deficiency

D’un patient à une thèse

Fanny Lanternier

Service de maladies infectieuses et tropicales

Hôpital Necker Enfants malades

Laboratory of Human Genetics of Infectious Diseases, INSERM U980

Institut Imagine

Ne jamais se décourager!!!

Tu pourras regarder pour lundi le

dossier de ce patient avec une

maladie dermatophytique (???!!!)

qui vient de Belgique?

Un premier patient

Mr R, 37 ans

• Antécédents:

– Pityriasis versicolor

• Mode de vie:

– Origine Marocaine (région de Tanger)

– Né en Belgique, Vit à Bruxelles

• 14 ans: lésions dermatophytiques granulomateuses des pieds.

• 36 ans: aggravation avec atteinte de la cuisse gauche et aggravation des lésions des pieds, lyse osseuse

• 37 ans: Amputation de la première phalange du 2ème orteil gauche

• Aggravation malgré:

– Des traitements antifongiques par lamisil, voriconazole puis posaconazole.

– Des traitements antibiotiques par Dalacine, ciprofloxacine, ceftazidime.

Examens biologiques

• GB: 10 400, PNN 3200, PNE

1800, L: 2600, Monocytes: 900

• Trichophyton rubrum en culture

sur les biopsies cutanées

Histologie

• Hyperkératose, épiderme hyperplasique avec micro-abcès

• Derme:

– nombreux granulomes épithélioïdes et gigantocellulaires,

– inflammation lymphohistiocytaire et nombreux PNE

•Filaments mycéliens dans les granulomes (coloration PAS)

Traitement

• Traitement par:– Interferon γ

– Posaconazole et Lamisil

– Antibiothérapie

• Absence d’amélioration, persistance de T. rubrum en culture

• Reprise d’un traitement antibiotique optimal

• En cas d’échec: amputation

Susceptibilité particulière aux infections à dermatophytes?

Transmission autosomique récessive?

Déficit immunitaire de transmission autosomique récessive?

Une bibliographie

Dermatophytosis physiopathology

Keratinized tissues: epidermis, hair, nails

Infection limited to stratum corneum

Startum corneum

Epidermis

Dermis 3. Conidia ingestion (resident macrophage)

Dermatophyte conidie

1. Keratinocyte adherence (2-6h)

2. Keratinocyte invasion (protease) (6-10h)

4. Hyphae

differenciation

in macrophage

Rupture of macrophage membrane

Vermout S, 2008, Baldo A, 2011, Campos M 2006

Dermatophytosis

Superficial

Stratum corneum

Superficial

Benign

Tinea pedis,

corporis, cruris

and capitis

No risk factor

Invasive

Dermal invasion

Majocchi

granuloma

Perifolliculargranuloma

Risk factors

Deep dermatophytosis

Extensive dermal infection

Lymph node or organ invasion

Riskfactors

Patients without known risk factors

“Maladiedermatophytique”

North Africa

Dermatophytosis

Bernard Liautaud, thèse de

médecine, Alger, 1977

Deep dermatophytosis in otherwise healthy

individuals

Deep dermatophytosis

Elsewhere

Non consanguineous*

18

Sporadic

North Africa

45

Consanguineous

24

Sporadic

5

Familial

19

Non consanguineous*

21

Sporadic

14

Familial

7

*or not reported consanguinity

First reported by Hadida and Schousboe in 1959

Un projet

La prédisposition génétique aux

maladies infectieuses: quelques

exemples

Infections à mycobactéries

• Défaut axe IFN-IL12

• ISG15

Encéphalite herpétique

• TLR3

• UNC93B

• TRIF

Infections à bactéries pyogènes

• IRAK4

• MyD88

• NEMO

• STAT3

Invasive fungal diseases: risk factors

Acute leukemia

Allo HSCT

SolidOrgan

Transplant

Intensive Care Unit

Neonates, Elderly

Surgery

HIV

PIDs

?????

U 980: Unité de Génétique Humaine des maladies infectieuses

Anne Puel, Capucine Picard

Laurent Abel, Jean-Laurent Casanova

Postulat:

Pas d’infections fongiques invasives chez le sujet immunocompétent

Postulat:

Pas d’infections fongiques invasives chez le sujet immunocompétent

Exploration immunogénétique des patients ayant des infections fongiques invasives sans

déficit immunitaire sous-jacent.

Exploration immunogénétique des patients ayant des infections fongiques invasives sans

déficit immunitaire sous-jacent.

Primary immunodeficienciesInvasive fungal diseases

• T cell deficiencies

Severe combined immunodeficiencies, Combined immunodeficiencies, CD4

lymphopenia

• Phagocytic cell deficiency

– Chronic granulomatous disease, severe congenital neutropenia and leukocyte

adhesion deficiency: Invasive aspergillosis and candidiasis

• IL-12/IFN-γ axis deficiency

– Dimorphic fungi infections

• AD STAT1 gain of function mutations

– Dimorphic fungi, chronic mucocutaneous candidiasis +++

• AR CARD9 deficiency

– Central nervous system candidiasis+++, chronic mucocutaneous candidiasis+,

dermatophytosis

Comprehension of antifungal immunity

TLR2

Zymozan

C. albicans

Dermatophyte

Conidia

β-glucans

Zymozan

Curdlan

DECTIN-1DECTIN-2

C. albicans

Dermatophyte

hyphae

Mannose

MINCLE

FcRγFcRγ

F. pedrosoi

Dermatophyte

C. albicans

C. albicans

Fungi and cell wall

components

(natural and

synthetic)

Receptors

Innate antifungal immunity

Fungi cell wall

componentsβ-glucans Chitin Mannans

TLR4

Mannans

Phagocytosis and fungal killing

Monocytes, macrophages, neutrophils

Bridge to adaptive immunity

Dendritic cells

DC-SIGN

DC-HIL

Invasive fungal diseases

Patients without risk factors

Deep dermatophytosis

Multiplex and/or consanguineous families

Des collaborations

Deep dermatophytosis recruitment

• 9 families, 18 patients

– Algeria: 5 families

– Morocco: 1 family

– Tunisia: 2 families

– Egypt: 1 family

• 8 consanguineous families

Dermatophytic disease: clinical presentation

Atteinte des phanères

• Extensive skin lesions:• Palmo-plantar keratodermia• Nodules (fistulizing)• Squamous erythematous

lesions• Ulcero-vegetative lesions

with local extension

Dermatophytic disease: clinical presentation

Thanks to Pr Aomar Ammar-Khodja, Dr Lynda Taibi, Pr Omar Boudghene Stambouli, Dr Boumediene Guellil

P

• Disseminated forms:

– Bone

– Lymph nodes with fistulization

– Brain abcesses

Dermatophytic disease: clinical presentation

Deep dermatophytosis:

clinical characteristics of the 18 patients

Male 12 (70)

Median age at first symptoms [years] 8 [2-21]

First symptoms

Severe or recurrent tinea capitis 14

Severe or recurrent tinea corporis 10

Onychomycosis 6

Presentations in adulthood

Lymph node involvement 10

Central nervous system invasion 1

Local organ invasion (bone, digestive tract) 2

Associated infection: thrush 6

Death 5

Median age at death [years] 34 [28-91]

Histological analysis

Granulome

Nécrose

T. Rubrum

T. violaceum

Traitement

• Itraconazole, posaconazole

• Rechute et rebond à l’arrêt du traitement

• Pas de données sur la sensibilité des souches

Candidate geneapproach

Genome-wideapproach

Candidate genesequencing

Exomesequencing

Mutation identification

Strategy

Functional studies

Linkage

analysis

Autosomal recessive CARD9 deficiency

- One large consanguineous Iranian family:

7 members affected

CMC, dermatophytosis, Candida spp. CNS infection

CARD9: Q295X/Q295X

- One Korean girl:

Candida dubliniensis meningitis

CARD9: G72S/R373P

Blood 2013NEJM 2009

R373PG72S

CARD CC

NH2 COOH1

6 98 140 420

536Q295X

Lanternier, NEJM, 2013

Deep dermatophytosisHomozygous mutations in CARD9

R373PG72S

CARD Coil Coiled

NH2 COOH1

6 98 140 420

536R101C Q289XQ295X

Founder effect

(Algerian and Tunisian patients)

Haplotype common to the 7 unrelated patients carrying HMZ CARD9 Q289X mutation

Most common ancestor around 975 years with 95%CI [575-1750]

CARD9 locus9q34

Distance (Mb)

1.61.1 0.9 1.41.2 0.4

qtermpterm

0.30.8 0.1

α-CARD9

α-GAPDH

P17 P15

Controls

(WT/WT)

Patients

(Q289X/Q289X)

C1 C2

Control

WT/WT

P12

R101C/R101C

P17

Q289X/Q289X

Ce

ll c

ou

nts

Deep dermatophytosis: CARD9 expression

CARD9

Reduced or abolished protein expression

Patients’ whole blood cytokine production

Reduced cytokine production upon fungal ligand stimulation

Ex vivo IL-17 producing T cells

Deep dermatophytosis

(Q289X, R101C)

Summary

CARD9 deficiency:

• Main genetic etiology of deep dermatophytosis:

– All patients studied: HMZ CARD9 mutation

– Founder effect: Tunisia, Algeria

• Reduced or abolished CARD9 protein expression

• Impairment of pro-inflammatory cytokine (IL-6, TNF-α) secretion

upon fungal ligand stimulation: whole blood

• Ex vivo IL-17 producing T cells reduction

Human CARD9 deficiency

Geographic origin

of patients

Patients’ number

(families)CARD9 mutations

References

Algeria 11 (5) Q289X/Q289X Lanternier, NEJM, 2013

Tunisia 4 (2) Q289X/Q289X Lanternier, NEJM, 2013

Morocco 3 (2) Q289X/Q289X

R101C/R101C

Lanternier, NEJM, 2013

Egypt 1 (1) Q289X/Q289X Lanternier, in preparation

Turkey 1 (1) R70W/R70W Lanternier, in preparation

Iran 9 (3) R35Q/R35Q, E322Del/

E322Del, Q295X/Q295X

Lanternier, in preparation

Glocker, NEJM 2009

Angola 1(1) R18W/R18W Lanternier, in preparation

Korea 1(1) G72S/R373P Drewniak, Blood, 2013

Human CARD9 deficiency

Clinical phenotype Number

(%)

CARD9 mutations

Age at first symptoms, median 9 [2-42]

Age at study date 39 [6-91]

Gender, male 17 (55)

Death 7 (22)

Dermatophyte infection 22 (71) Q289X/Q289X, R101C/R101C, Q295X/Q295X

Deep dermatophytosis 18 (58) Q289X/Q289X, R101C/R101C

CNS infection 9 (29)

Candida 7 (22) R35Q/R35Q, R70W/R70W, Q289X/Q289X, Q295X/Q295X,

G72S/R373P

Exophiala dermatitidis 1 R18W/R18W

Dermatophyte (?) 1 Q289X/Q289X

CMC 14 (45) R70W/R70W, R101C/R101C, Q289X/Q289X, Q295X/Q295X

Not associated with other severe common (bacterial, viral..) infections

Discussion: CARD9 central role in antifungal

immunity

Mice

• Increased susceptibility to:

– C. albicans

– Listeria monocytogenes

– Mycobacterium tuberculosis

Humans

• Susceptibility to:

– Fungi: Candida spp.,

dermatophytes, E. dermatitidis

• No susceptibility to:

– Bacteria, virus, mycobacteria

CARD9 non-redondant for: Anti-fungal and

anti-intracellular bacterial

immunities

CARD9 non-redondant for: CARD9 non-redondant for:

Anti-fungal immunity

CARD9 redondant for:

Anti-intracellular bacterial

immunity

Susceptibility of CARD9-/- mice

Hsu, Nature Immunol 2007, Gross Nature 2006 , Dorhoi, JEM, 2010

Candida

Listeria monocytogenes

Mycobacterium

tuberculosis

CARD9 role in ROS production Evidenced from mouse studies

C. albicans

stimulation

LyGDI

CARD9 Phagolysosome

Mice macrophage

C. albicansLyGDI

CARD9

ROS production

Nature Immunol, 2009

TLR2

DECTIN-1

MyD88

NFkB

CARD9Bcl10

Malt1

IL-6, IL-10,

TNF-a, IL-23

T IL17+

RORγtTGF-β

IL17, IFNγ

Dendritic

cells

IL23R

DECTIN-2

PkC-δ

MINCLE

T CD4+

CARD9 role in IL-17 antifungal immunityEvidenced from mouse studies

Syk Syk

Syk

CARD9 role in fungal killing in human

Blood, 2013

CARD9 role in fungal killing in human

*Opsonized C. albicans killing: CARD9

independant, NADPH oxydase dependant

Unopsonised C. albicans killing: CARD9

dependant, NADPH oxydase independant

CARD9 deficiency = Susceptibility to

unopsonied Candida

*Blood brain barrier: reduction of

complement and immunoglobulins:

Unopsonised Candida in CNS

CNS tropism of fungal infection in CARD9

deficient patient

Blood, 2013

CARD9 role in antifungal defense: human

IL6, TNF-α

Roth, Trends in immunology, 2013

Heat killed Candida albicans, heat killed Saccharomyces cerevsiae, Curdlan

X

CARD9 role in antifungal defense: human

IL6, TNF-α

B cell lympho-proliferation

Autoimmunity

No fungal infectionSalzer, Blood 2013, Kuehn, Blood 2013, Belot A, Arthritis

and Rheumatism 2013

B and T cell response defect

Bacterial infections and

Oesophagitis

+/- lungJabara H, JACI 2013

Young age of patients

Roth, Trends in immunology, 2013

Heat killed Candida albicans, heat killed Saccharomyces cerevsiae, Curdlan

X

X

IL-17 T cells and human CARD9 deficiency

• IL-17 producing T cells:

– Differentiation: CARD9 dependent in mice

– Defective in most of CARD9 deficient patients

– Differentiation driven by dendritic cell upon fungal activation

In CARD9 deficient patient:

Dendritic cells fail to drive IL-17 T cell differentiation upon fungal ligand

• IL-17 T cells variation in CARD9 deficient patients:

– Low during infection (deep dermatophytosis: chronic), trapping atinfection site

– Normal after infection resolution (Th17 from CNS infection patients: drawn at distance from infection)

Perspectives

Further characterization of the mutation impact

-CARD9 CoIP with Bcl10, Malt1

-Luciferase assay

What is the role of CARD9 in antifungal defense?

-Identify the receptors responsible for CARD9 activation in different cell types

Phagocytosis

-Identify CARD9 role in fungal killing (different fungi and cell types)

Role of IL-17 T cells?

-Do IL-17 T cells vary with infection?

-Is differentiation of naïve T cells into IL-17 producing T cells after phagocyte

stimulation by fungal pathogens impaired in CARD9 deficient patients?

Innate lymphoid cells?

-Are they impaired in CARD9 deficient patients

Perspectives

Acute leukemia

Allo HSCT

SOT

ICU

Neonates, Elderly

Surgery

HIV

PIDs

CARD9

?????

DD

CNS candidiasis

AcknowledgmentsJean-Laurent Casanova Laurent Abel

Anne Puel

Capucine Picard

Quentin Vincent, Vincent Pedergnana, Luyan

Liu, Mélanie Migaud, Malik Bensifi, Carolina

Prando, Marjorie Hubeau, Sophie Cypowyj,

Laura Israel, Maya Chrabieh, Erika Della Mina,

Ling Yun, Bertrand Boisson, Alexandre Bolze,

Necker and Rockefeller branches

Acknowledgments

Patients and their families

Fabrice Chretien

Gregory Jouvion,

Institut Pasteur (France)

Roel Gazendam, Taco Kuijpers,

Amsterdam (Netherlands)

Infectious disease unit, Necker

Enfants maladesOlivier Lortholary

NCRMA, Institut Pasteur: Françoise Dromer,

Dea Garcia-Hermoso (France)

Lynda Taibi, Aomar Ammar-Khodja,

Omar Boudghene Stambouli, Guellil

Boumediene, Merad Boudia (Algeria)

Lubna Boussofara, Mohamed Denguezli,

Molka Larif (Tunisia)

Frederique Jacobs, Jean-Christophe

Goffard, Kinda Shepers (Belgium)

Alireza Mahdaviani, Davood Mansouri

(Iran)

Marie-Elisabeth Bougnoux, Hervé

Bachelez, Jean-David Bouaziz, Hélène

Chaussade, Jacques Reynes, Anne-Sophie

Bruneel, Laurence Michel, Adela

Angoulvant, Ulrich Meinzer, Marianne

Debre, Louis Bernard, Gérard Lefranc,

Serge Romana (France)

Saad Patan, Bodo Grimbacher (UK)

Objectifs

• Meilleure compréhension immunité

antifongique

• Développement de

– Vaccins

– Nouvelles armes thérapeutiques

La publication