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Immune evasion by fungi

Dr Ruth Ashbee

Principal Clinical Scientist

Mycology Reference Centre

Leeds General Infirmary

Lecture overview

• Fungal pathogens introduction

• Immune defences against fungi

• Immune evasion by fungi

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Fungal pathogens

• Fungi are eukaryotes

• Most fungi can only cause systemic disease in people who are immunocompromised (opportunistic); a few fungi can cause systemic disease in healthy people (true pathogens)

• Antifungal drugs are often toxic due to non-specificity of their targets (i.e. humans often have the same/similar targets)

• Yeasts: Candida, Cryptococcus• Moulds: Aspergillus, Fusarium• Dimorphic: Histoplasma, Coccidioides, Blastomyces,

Paracoccidioides

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PATHOGENIC FUNGI

Moulds Yeasts

Pneumocystis jiroveci

Dimorphic

Fungal pathogens

Fungal pathogens

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Innate host defences

• Skin and mucosal surfaces

– skin integrity, antimicrobial peptides, secretions

– mucociliary escalator

– urinary tract

• Commensal population– established populations in niches

• pH– skin pH (sweat & sebaceous secretions); vaginal pH, gastric pH

• Complement cascade– classical pathway (immune complexes)

– lectin pathway (terminal mannose residues)

– alterative pathway (LPS, yeast cell wall)

• Phagocytes– neutrophils

– macrophages

– dendritic cells

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Draining lymph node

Innate immune responses

Pathogen associated molecular patterns (PAMP),

e.g. β-glucans, galactomannan, mannanFungus invading tissue

Macro

Immature

dendritic cell

Neutro

Pattern recognition receptors (PRR), e.g.

Toll-like receptors, dectins, pentraxin 3

Dendritic cell

T-cell receptor

INFLAMMATORY

RESPONSE

TH1

Treg

TH2

TNF-α, IFN-γ, IL-1, IL-6, IL-12

IL-10, IL-4, IL-5

B-cell

Antibody

Plasma cell

MacroNeutro

Innate immune responses

Pathogen associated molecular patterns (PAMP),

e.g. β-glucans, galactomannan, mannan

Pattern recognition receptors (PRR), e.g.

Toll-like receptors, dectins, pentraxin 3

T-cell receptorAntibody

Respiratory burst Superoxide produced by

NADPH

oxidase

complex

H2O2 HOCl

Granule releaseincluding defensins,

elastase,

myeloperoxidase and

reactive oxygen

species

MacroNeutro

Superoxide

IFN-γ

Fungus invading tissue

Adaptive host defences

• T lymphocytes (CD4, CD8)– cytokine production

– some killing

– regulation of immune response

• B lymphocytes– antibody production

Role of the phagocyte in fungal infection

• Neutropenia is the main predisposing factor for invasive fungal infections

• Functional problems with neutrophils (e.g. Chronic granulomatous disease) predisposes to fungal infections

• Neutropenic patients who are treated with antifungals often don’t respond until their neutrophil count recovers

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Pattern recognition receptors (PRR)

• PRR’s (TOLL-like receptors, C-type lectins e.g. Dectin 1) on host cells interact with polysaccharides (e.g. chitin, glucans) and mannosylated proteins of the fungal cell wall.

• Dectin 1 recognises β-1-3-glucan, which triggers multiple signalling pathways via Syk kinase and NF-κB.

• Results in – initiation of respiratory burst and

enhanced phagocytosis

– production of IL-12 and TNFα

– induction of humoral immunity

– stimulation of cytotoxic T cells

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Modulation of inflammatory signals

• TLR 2 activation leads to Th2 response, production of IL4, IL5 and IL10, a humoral immune response and lack of effective antifungal response.

• TLR4 activation leads to a Th1 response, production of pro-inflammatory cytokines and a good antifungal response

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Modulation of inflammatory signals

• C. albicans and A. fumigatus activate the TLR2 and hence bias the response to Th2

• Polysaccharide capsule of Cryptococcusinduces IL10 and biases towards Th2 response

• Blastomyces dermatitidis has a surface adhesin which limits release of TNF-α by macrophages

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Masking of PAMPS (β-glucans)

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Organism Masking strategy

C. albicans Mannoprotein*

Aspergillus fumigatus Hydrophobin layer on conidia

Cryptococcus neoformans Polysaccharide capsule

Histoplasma capsulatumSwitch to α-linked glucans in yeast phase

Paracoccidioides brasiliensis

Blastomyces dermatitidis

* Antifungal treatment may reverse this by unmasking β-1-3-glucan

Phagocytosis

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Phagocytosis of fungal spores

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Antiphagocytic mechanisms

• Cell size: too big to be taken up

– Hyphal forms of Candida albicans and Aspergillus fumigatus aren’t internalised efficiently

– Polysaccharide capsule of Cryptococcus

– “Titan” or “Giant” cells of Cryptococcus(10x size of normal yeasts)

• Metabolites produced by C. albicans inhibit uptake by macrophages by interfering with cytoskeleton

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Capsule of Cryptococcus

• Blocks opsonic effect of complement and antibodies

• Negative charge of capsule repels negatively charged host cells

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Titan/Giant cells of Cryptococcus

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Intraphagocytic survival

• Histoplasma capsulatum can survive within phagocytes and replicate in macrophages – modulates pH to minimise activity of enzymes

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• C. glabrata recently been shown to survive macrophage phagocytosis and replicate intracellularly– Doesn’t cause apoptosis

– Inhibits proinflammatory cytokine production

– Suppresses ROS production

– Alters phagosome maturation

– Replicates in non-acidic phagosome

– Escapes from macrophages by uncertain mechanisms

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Outgrowth from phagocytes

• C. albicans can transform to hyphal phase, under the influence of carbon dioxide, pierce and kill the macrophage

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Expulsion from phagocytes

• Cryptococcus can permeabilise the phagosomal membrane

• Ejected from the macrophage causing no damage to fungus or host during “vomocytosis”

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Modulation of phagocytic killing mechanisms

• Phagocytes undergo respiratory burst and produce toxic metabolites:– Nitric oxide

– Hydrogen peroxide

– Superoxide anion

– Hypochlorous acid

• Various fungi have ways to modulate the ability of these toxic metabolites to kill

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Inhibition of nitric oxide production

• Occurs by several fungi – mechanisms unclear for some

• C. albicans produces detoxification enzymes (e.g. catalases, superoxide dismutase (SOD))

• C. posadasii* secretes an uncharacterisedfactor which may mediate by down-regulation of inducible NO synthase mRNA in macrophages

• C. neoformans produces many enzymes (4 catalases, 2 SOD’s and many others) and can also enlarge capsule

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Melanin

• High MW, hydrophobic pigments

• Produced by organisms in all biological kingdoms

• Extremely resistant – need boiling in acid to dissolve!

• Provide pigment in many organisms – humans, insects, fungi

• In fungi, produced via phenoloxidases

• Associated with virulence in fungi

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Melanin in Cryptococcus

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Melanin: protection against host defences

Effect Organism Magnitude (%)*

Phagocytosis C. neoformans 7 (in vivo)27 (in vitro)

P. brasiliensis 9

S. schenckii 50

F. pedrosoi 36

Killing by host cell C. neoformans 31

A. fumigatus 18

P. brasiliensis >30

S. schenckii 27

F. pedrosoi 22

Oxidants C. neoformans 22

Aspergillus spp 75

S. schenckii 20

Microbial peptides C. neoformans 22

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* Maximum protection for organisms with melanin cf those without

Complement cascade

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Classical Pathway:

Ag-Ab complexes

Lectin Pathway:

Carbohydrates

Alternative

Pathway e.g LPS

C1q, C1r, C1s

C4 and C2

MBL, MASPs,

C4 and C2

C3, FB

FD

C3 convertase

C3b

C3a

C3

C5b

C5

C5a

Amplification

step

C5 convertase

C5b-9

Mast cell activation

Chemotaxis

Phagocyte activation

Generation of oxygen radicals

T-cell activation and survival

Opsonin

Membrane attack

complex

Lysis of some

pathogens and cells

Potent anaphylatoxin

Chemotaxis

Phagocyte activation

Generation of oxygen radicals

T-cell activation and survival

Inhibition of the complement cascade

Binding of complement components

• Secretion of antiphagocytic protein 1 (App1) protein by Cryptococcus which binds to complement receptors 2 and 3, so inhibiting complement-mediated uptake of Cryptococcus

• C. albicans binds several complement regulatory factors, including Factor H and C4b-binding protein (C4BP) thus inhibiting complement activity.

• C. albicans produces Pra1 (pH-regulated antigen) which binds plasminogen, Factor H and Factor H-like protein. This mediates complement evasion and extracellular degradation

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Inhibition of the complement cascade

Binding of complement components

• Other fungi also bind complement regulators:

– Aspergillus fumigatus conidia

– Paracoccidioides brasiliensis– Cryptococcus– Pneumocystis jiroveci

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Inhibition of the complement cascade

• Degradation of complement components

• C. albicans produces Secreted Aspartyl Proteases (SAP’s) which can degrade complement proteins (C3b, C4b and C5) and extracellular matrix proteins.

• Asp fumigatus also produces a secreted serine protease (Alp1) which degrades C3b, C4b and C5

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Summary

• Mechanisms used by fungi to evade host defences:

– Shielding of PAMPs

– Ability to live and multiply in phagocytes

– Outgrowth from phagocytes

– Modulation of phagocytic killing mechanisms

– Role of melanin in immune protection

– Inhibition of complement cascade

– Degradation of complement proteins

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References

• GD Brown (2011) Innate antifungal immunity: the key role of phagocytes. Ann Rev Immunol 29:1-21

• LA Chai et al (2009) Fungal strategies for overcoming host innate immune response. Med Mycol 47:227-236

• JR Collette & MC Lorenz (2011) Mechanisms of immune evasion in fungal pathogens Curr Opin Micro (in press)

• L Romani (2004) Immunity to fungal infections Nature Rev Immunol 4:1-13

• K Seider et al (2010) Interaction of pathogenic yeasts with phagocytes: survival, persistence and escape. Curr Opin Microbiol (2010) 13: 392-400

• K Seider et al (2011) The facultative intracellular pathogen Candida glabrata subverts macrophage cytokine production and phagolysosomematuration. J Immunol (in press)

• PF Zipfel et al (2011) Immune escape of the human facultatitvepathogenic yeast Candida albicans: The many faces of the Candida Pra1 protein. Int J Med Microbiol 301:423-430

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