deepdermatophytosisand card9 deficiency d’un patient à une … · 2015-09-30 · severe or...
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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