1. Immunity to fungal infections

Question 1

What are the four main phyla of fungi and which most commonly causes human fungal infection?

Answer 1

Ascomycota – MAIN ONE
Basidiomycota
Chytridiomycota
Zygomycota

Question 2

Give examples of how the morphogesis of fungi contributes to its ability to cause disease in the host.

Answer 2

- Candida albicans exist as single spores but they can become hyphae, which allows tissue invasion – dimorphism allows tissue invasion.

- Cryptococcus forms a capsule to evade phagocytosis

- Aspergillus sp. are inhaled as conidia and invade tissues as hyphae

Question 3

What is Toll and what is its role?

Answer 3

Toll (TLR) is an innate PRR (Pattern Recognition Receptor) required for fungal immunity

Dectin 1 is a major fungal pattern recognition receptor

Question 4

What acts as a physical barrier in defense against pathogens?

Answer 4

The intrinsic immunity is a physical barrier – pulmonary mucosa for airborne pathogens and skin, oral, GIT mucosa for other pathogens.

Question 5

What is the first line of dense of the body?

What are the defenses that occur?

Answer 5

The first line of defence is the innate immune system:

• sIgA and complement leads to opsonisation by pentraxin 3 and mannose-binding lectin
• Phagocytosis by alveolar macrophages – phagocytes are a critical first line of defence
• NK cells provide early IFN gamma
• A failure of innate immunity leads to adaptive responses

Question 6

What are fungi opsonised by?

Answer 6

• Fungi are opsonised by pentraxin-3 and mannose-binding lectin (MBL)

Question 7

What is activated if there is a lesion in the barrier/mucus?

What occurs as a result?

Answer 7

If there is a lesion in the barrier/mucus, then second line of defence is activated:

• Killing and phagocytosis leads to antigen release – these are taken up by APCs, leading to cytokine release – activates T cells
• Dendritic cells influence T cell differentiation
• Th1 and Th17 play a role
• A Th1 response (IL-2 and IFN-gamma) leads to protection whereas a Th2 response (IL-4, IL-5) leads to allergy

Question 8

What do Th1 vs. Th2 responses lead to?

Answer 8

- Th1 response (IL-2 and IFN-gamma) leads to protection

- Th2 response (IL-4, IL-5) leads to allergy

Question 9

Which host cells are important for defence against fungal infection?

Answer 9

Innate immunity:

• • Phagocytes – are the first line of defence
• • NK cells – provide early INF-gamma
• • Neutrophils recruited by damaged epithelial cells and activated phagocytes via cytokine release
• • APC cells are macrophages, dendritic cells, B cells – process antigens and present them via MHC which activates T cells
• • Dendritic cells – influence T-cell differentiation

Adaptive immunity:

• • Th1, Th17 (Th2 in allergic disease play a role)

Question 10

When do the following effector cells play an especially important role:

- neutrophils

- alveolar macrophages

Answer 10

NB: some effector cells are especially important for certain infections:

- Neutrophil mediated killing of Aspergillus fumigatus via neutrophil NETs of extracellular fibres derived from chromatin and coated with granule proteins

- Alveolar macrophages play an essential role in clearing Cryptococcus neoformans infection (however, they can multiply inside the macrophage phagolysosome and escape)

Question 11

How do innate immunity innate immune responses protect against Aspergillus fumigatus infection?

Answer 11

• • Ciliary clearance and pulmonary mucosal barrier
• • Pulmonary polymorphonuclear leukocytes (neutrophils)
• • Neutrophils are of primary importance (more so than macrophage phagocytes) – neutrophils produce neutrophil extracellular nets (NETs)

Question 12

How do innate immune responses protect against Candida albicans infection?

Answer 12

• • Skin and epithelial barrier.
• • In addition, candida Albicans experience nutrient starvation inside macrophage phagolysosome promoting germination and escape.

Question 13

How do innate immune responses protect against Cryptococcus neoformans infection?

Answer 13

Alveolar macrophages play an essential role → internalisation and chemokine and cytokine secretion → recruitment of other immune cells → killing of pathogen and antigen presentation.

Question 14

What risk factors promote susceptibility to infection?

Answer 14

• Immunocompromised
• Surgery
• Hereditary defects

Question 15

How does being immunocompromised promote susceptibility to infection?

Answer 15

Reduced numbers of the key effector cells: dendritic cells, macrophages, and neutrophils so they cannot execute the immune response against the pathogens → they can replicate and survive

Question 16

How does surgery promote susceptibility to infection?

Answer 16

Allows direct inoculation deep into the body tissues, which bypasses the intrinsic and innate defense mechanisms → the pathogen enters the systemic bloodstream very quickly

Question 17

What different hereditary/genetic defects can occur?

How do each promote susceptibility to infection?

Answer 17

Plasminogen alleles

• • Plasminogen directly binds aspergillus fumigatus conidia
• • Increased susceptibility to aspergillosis in stem cell transplantation

Mutation in dectin-1

- important C type leptin receptor, activate killing, inflammation, phagocytosis

• • Fungal PRR → deficiency leads to mucocutaneous infections with candida Albicans due to impaired IL-6 production by macrophages and impaired macrophage binding
• • Increased susceptibility to invasive aspergillosis in stem cell transplantation

CARD9 deficiency

• • Chronic mucocutaneous candidiasis because no TNF alpha is produced in response to beta-glucan stimulation
• • No T cell Th17 differentiation in humans

TLR4 polymorphisms (loss of function mutation)

• • Increased risk of invasive aspergillosis in a BM transplantation
• • Important in sensing LPS (gram –ve cell wall component)

Question 18

What antifungal functions are performed by macrophages?

Answer 18

Major functions:

• • Phagocytose opsonised (by ab and complement – pentraxin 3 and mannose binding lectin) and unopsonised fungi
• • Pro-inflammatory cytokine production
• • Killing of fungi
• • Granuloma formation
• • Maintenance of latency

Other functions: antigen presentation

Question 19

What antifungal functions are performed by neutrophils?

Answer 19

Major functions

• • Phagocytose opsonised (by ab and complement – pentraxin 3 and mannose binding lectin) and unopsonised fungi
• • Oxidative and non-oxidative fungal killing

Other functions: produce cytokines

Question 20

What antifungal functions are performed by dendritic cells?

Answer 20

Major functions:

• • Antigen presentation to T cells
• • Th1/2 polarisation
• Other functions:*
• • Phagocytosis and kill fungi*
• • Produce cytokines*

Question 21

How is adaptive immunity to fungal pathogens orchestrated?

How does adaptive immunity impact the host-commensal relationship?

Answer 21

see previous answer

Question 22

What is the result of Dectin 1 deficiency?

Answer 22

Dectin 1 (a fungal PRR) deficiency leads to mucocutaneous fungal infections – e.g. vulvovaginitis & onychomycosis.

• • This leads to impaired macrophage IL-6 production and binding in response to fungal infections.
• • It also leads to increased susceptibility to invasive aspergillosis in stem cell transplants.

Question 23

What is CARD-9 required for?

What is the result of CARD-9 deficiency?

Answer 23

CARD-9 deficiency leads to chronic mucocutaneous candidiasis.

CARD-9 is required for:

• • TNFa production in response to b-glucan stimulation.
• • T-cell Th17 differentiation.

Question 24

What is the result of TLR4 polymorphisms?

Which SNPs are associated with increased susceptibility to invasive fungal infections and disease?

Answer 24

- TLR4 polymorphisms: lead to an increased risk of Invasive Aspergillosis (IA) in transplantation (i.e. haematopoietic stem cell transplants).

SNPs (single nucleotide polymorphisms): i.e. CXCL10, IL1-R, IL-23R, TLR-2/4/6/9.

Conclusion: mutations on Dectin-1, TLR4, and plasminogen confer increased susceptibility to fungal disease

Question 25

Name 3 factors that are associated with increased risk of Aspergillosis in transplantation. Which PRR is actively recruited to Aspergillus fumigatus phagolysosomes?

Answer 25

TLR4 S4 – loss of function Dectin 1 Plasminogen alleles TLR9 activated recruited to Aspergillus fumigatus phagolysosomes

Question 26

What can plasminogen directly bind to?

Answer 26

Aspergillus fumigatus conidia

Question 27

What are examples of cellular and innate defences? What is the role of neutrophils and fungal morphogenesis?

Answer 27

**_Cellular defences:_** * **- Neutrophils** are very important in fungal defence: * For Aspergillus, neutrophils are of a primary importance (able to be trapped via NETs) * - Neutrophil NETS – neutrophils throw out chromatin “nets” to capture pathogens. * - These chromatin molecules outside the nucleus act as “danger signals” and recruit’s effector cells to the area as well. * **- Fungal morphogenesis** – fungi can transition between yeast, candida and hyphae forms (multicellular) and this can drive modulation of Dendritic cell response and can be bad for the immune response (as it gets confused) **_Innate defenses:_** * - Mucosal immunity governs fungal tolerance and resistance. * Dendritic cells modulate adaptive immune responses* * Adaptive T-cell INF-gamma responses augment host immunity to fungi*

Question 28

Describe how fungal morphogenesis governs the dendritic cell modulation of adaptive immunity.

Answer 28

Hyphal forms = Th2 response Conidium = Th1 response

Question 29

What is the role of adoptive immunotherapy and gene therapy in defense against fungi? What new treatments are being used?

Answer 29

**- Adoptive immunotherapy** – generate lots of antifungal T-cells in a sample and then give these to the patients that need to fight a fungal infection. **- Gene therapy** – e.g. restore gp91 function (make reactive oxidative species to fight fungal spores) to treat chronic granulomatous disorder. E.g. restore neutrophil NET formation. **_New treatments:_** - INF-gamma or adoptive T-cell therapy could be new treatments. - Gene therapy for primary immunodeficiencies could be a new treatment.

Question 30

What are the host responses to the inhalation of fungal spores? What is the main driver and what other fungi contribute?

Answer 30

*Many fungal spores are inhaled daily.* **Host response may be normal, ineffective or exaggerated (allergy)** * - This leads to either an allergic or invasive fungal disease. **Aspergillus is a primary driver – other fungi may contribute.** * - Aspergilli – Aspergillus niger, Aspergillus fumigatus. * - Other supporting fungi – Alternaria, Cladosporium, Penicillum.

Question 31

What are the features of each hypersensitivity reaction? What types of hypersensitivity reaction are associated with fungal allergies?

Answer 31

**Important fungal reactions include type 1, 3, 4 hypersensitivity reactions.** * **- T1** – IgE-driven, involves histamine and leukotrienes, in minutes. * **- T2** – IgG-, IgM-driven, involves complement, in 1-24 hours. * **- T3** – IgG-, IgM-driven, involves complement, in 1-24 hours. * **- T4** – T-cell-driven, involves lymphokines, in 2-3 days.

Question 32

What are the criteria for diagnosing ABPA?

Answer 32

_ABPA – Allergic Bronchopulmonary Aspergillosis:_ _Criteria for diagnosis:_ * **- Predisposing condition** – asthma or cystic fibrosis. * **- Obligatory criteria** – high baseline serum IgE, +ve T1 hypersensitivity (immediate response) skin test OR Aspergillus-specific IgE. * **- Supportive criteria (more than 2)** – eosinophilia, IgG AB to Aspergillus fumigatus, consistent radiologic abnormalities.

Question 33

What radiological abnormalities may be seen in ABPA? What might be seen in a CT scan of a patient with ABPA?

Answer 33

**Radiologic abnormalities – also have hyper-dense mucus:** * - Dilated bronchi, thick walls * - Upper/central predilection * - Lobar collapse * - Ring or linear opacities * - Proximal bronchiectasis * - Fibrotic scarring * **Hyper dense mucous sign - see in CT scan**

Question 34

How is ABPA managed?

Answer 34

* **- Corticosteroids.** * **- Itraconazole (steroid-sparing agent)** – benefit past 16 weeks is unclear however. * - Indicated if not responding to steroids or if steroid-dependency. * **- Omalizumab** – recombinant IgE monoclonal ABs may be useful.

Question 35

What are other examples of pulmonary allergies to fungi and how are they diagnosed?

Answer 35

***Diagnosis is via immediate hypersensitivity skin tests,*** **_- ABPA_** - Allergic bronchopulmonary aspergillosis (treatment: corticosteroids and itraconazole) - total baseline serum of IgE \> 1000, hyper dense mucus production and bronchi dilation **_- Aspergillus rhinosinusitis_** (can be allergic or invasive) - treatment: oral corticosteroids, surgical removal of obstructing nasal tissue **_- Severe asthma and fungal sensitisation_** **_- Hypersensitivity pneumonitis_** - allergen-specific precipitins usually present

Question 36

What test is used to diagnose fungal allergies?

Answer 36

Skin prick testing

Question 37

Which type of hypersensitivity is each of the previously mentioned fungal allergies?

Answer 37

ABPA – type 1 or 4 Asthma – type 1 Rhinitis – type 1 Hypersensitivity pneumonitis – type 4