Innate Immunity to Fungi

Most fungal exposure ends at a barrier. Inhaled conidia, commensal yeasts and skin dwelling fungi are cleared, tolerated or contained without clinical disease, and invasive infection usually requires a breach of a barrier or a defect in the cells behind it.

Epithelial cells are active sensors and not passive walls. Yeast cells of Candida albicans are tolerated by oral epithelium, whereas hyphae that damage cells are not. Candidalysin and hyphal invasion activate epidermal growth factor receptor and MAP kinase signalling, with release of interleukin 1 alpha, interleukin 1 beta, interleukin 36 and GM-CSF. These signals recruit neutrophils and shape the later T cell response. The epithelium therefore responds to damage and not to the mere presence of the fungus, which is why colonisation is usually silent.

Two additional points have clinical consequences:

• Dermatophytes are restricted to dead keratin and are opposed by desquamation, sebum and inflammatory signals from keratinocytes. Medium chain fatty acids in post pubertal sebum are fungistatic, which contributes to the prepubertal predominance of tinea capitis caused by anthropophilic species.
• Mucosal and cutaneous Candida control depends on interleukin 17 and interleukin 22 acting on epithelial cells. This is a link between innate sensing and the T cell response, and its failure produces mucocutaneous candidiasis without bloodstream disease.

Fungal cell wall components are detected by pattern recognition receptors on epithelial cells, macrophages, neutrophils and dendritic cells. Signalling through the adaptor CARD9 is the common hub for the C type lectin receptors.

Recognition depends on exposure of the ligand. Resting conidia of Aspergillus fumigatus are covered by melanin and a hydrophobin rodlet layer and are largely invisible to Dectin 1. Swelling and germination expose beta glucan and trigger recognition.

Candida albicans conceals glucan beneath outer mannan and exposes it at bud scars and after echinocandin exposure. Capsule shields Cryptococcus, and alpha 1,3 glucan covers the yeast phase of Histoplasma and Blastomyces.

Pathogens therefore regulate their visibility, and the immune system reads the morphology as well as the species.

Interleukin 1 signalling downstream of the inflammasome is required for neutrophil recruitment and Th17 priming. Receptor deficiencies are mostly weak, redundant and organism specific, whereas loss of the shared adaptor CARD9 is severe.

• The alternative pathway is activated directly on fungal walls. The lectin pathway is activated by mannose binding lectin and ficolins binding mannan and N acetylglucosamine, and the classical pathway by antibody.
• C3b and iC3b opsonise fungi for uptake through complement receptor 3 (CD11b/CD18), which also binds beta glucan. C5a recruits and primes neutrophils.
• The membrane attack complex does not lyse fungi, because the cell wall is too thick. Deficiency of terminal components is therefore not a recognised cause of fungal susceptibility, in contrast to Neisseria infection.
• Cryptococcus capsule activates the alternative pathway, but C3 is deposited on the outer capsule, away from the wall, and opsonisation is inefficient.
• Candida albicans recruits complement factor H through Pra1 and degrades C3b and C5 with secreted aspartyl proteases.
• Pentraxin 3 binds Aspergillus fumigatus conidia and enhances phagocytic recognition. Donor pentraxin 3 gene variants have been associated with invasive aspergillosis after haematopoietic stem cell transplantation.
• Surfactant proteins A and D bind and agglutinate Aspergillus conidia and opsonise Pneumocystis and Cryptococcus.
• Mannose binding lectin deficiency has been associated with recurrent vulvovaginal candidiasis and chronic cavitary pulmonary aspergillosis. The effect is modest.

Neutrophils are the central defence against Candida and against the hyphal phase of moulds. Hyphae are too large to ingest, so killing is extracellular.

Recruitment follows interleukin 8 and CXCL1, C5a, leukotriene B4 and the interleukin 1 and interleukin 17 axis. Killing mechanisms:

• NADPH oxidase (gp91phox, p22phox, p47phox, p67phox, p40phox) generating superoxide and hydrogen peroxide.
• Myeloperoxidase converting hydrogen peroxide to hypochlorous acid.
• Granule contents: defensins, elastase, cathepsin G, lactoferrin.
• Neutrophil extracellular traps carrying calprotectin, which chelates zinc and manganese and restrains hyphal growth.
• Neutrophil swarming that concentrates cells at the site of a growing hypha.

Both quantity and function matter. The risk of invasive mould infection rises sharply when the absolute neutrophil count falls below 500 per microlitre, and rises further with duration beyond about 10 days and with profound neutropenia below 100 per microlitre. Qualitative defects include chronic granulomatous disease, uncontrolled hyperglycaemia and ketoacidosis, corticosteroid exposure, myelodysplasia, uraemia and burns.

The most informative contrast for clinical reasoning is between lung phagocytes and neutrophils. Alveolar macrophages kill inhaled conidia. Neutrophils kill hyphae, so hyphal disease implies a neutrophil failure, whereas persistent conidial survival implies macrophage failure, as with corticosteroids.

Neutrophil recovery is also a clinical event. Neutrophil return in a patient with invasive aspergillosis can worsen the radiological picture transiently, with cavitation and the air crescent sign, as the necrotic lung sequesters the fungus. This is a sign of host recovery and not of treatment failure.

• Alveolar macrophages are the first cells to meet inhaled spores. They ingest conidia and kill them in the phagolysosome through acidification and reactive oxygen species. Melanin on conidia interferes with LC3 associated phagocytosis, which depends on NADPH oxidase and normally accelerates conidial killing. Corticosteroids impair macrophage conidial killing and cytokine release, which is why invasive aspergillosis arises on corticosteroids without neutropenia.
• Inflammatory monocytes are recruited from the bone marrow through CCR2 and differentiate into macrophages and dendritic cells, contributing to control of Candida, Aspergillus and Cryptococcus.
• Nutritional immunity is a macrophage function. Iron is withheld through ferroportin downregulation and hepcidin, zinc through metallothionein and zinc transporter changes, and manganese through NRAMP1. Macrophages activated by interferon gamma and GM-CSF restrict Histoplasma by withholding zinc and iron.
• Classical activation by interferon gamma produces fungicidal macrophages. Alternative activation by interleukin 4 and interleukin 13 produces macrophages that tolerate intracellular fungal growth, which is one reason Th2 skewing worsens cryptococcosis.

Several fungi use the macrophage as a niche: Histoplasma blocks phagolysosome acidification, Cryptococcus replicates and is expelled by vomocytosis, Candida glabrata survives in the phagosome, and Talaromyces marneffei divides by fission within the cell. These organisms are controlled only when T cell derived signals activate the macrophage, which links innate function to the adaptive response.

Dendritic cells capture fungal antigen in the lung, skin and gut, migrate to lymph nodes and prime T cells. The pattern of receptor engagement sets the cytokine output:

• Dectin 1 together with Toll like receptor 2 drives interleukin 1 beta, interleukin 6 and interleukin 23, which promote Th17 cells.
• Interleukin 12 production promotes Th1 cells.
• Interleukin 10 and transforming growth factor beta promote regulatory T cells.

Morphotype shapes the outcome. In experimental systems, Aspergillus conidia favour Th1 priming, and hyphae favour Th2 and regulatory responses. Langerhans cells in epidermis sense Candida hyphae and promote cutaneous Th17 responses. Corticosteroids and HIV impair dendritic cell maturation and function.

• Natural killer cells kill Cryptococcus directly by perforin release after recognition through NKp30, and have activity against Aspergillus hyphae and Candida. They also supply interferon gamma and GM-CSF to activate macrophages. Natural killer cells are depleted and dysfunctional in advanced HIV, but their independent clinical role is difficult to isolate.
• Type 3 innate lymphoid cells, gamma delta T cells and mucosal associated invariant T cells produce interleukin 17 and interleukin 22 rapidly at epithelial surfaces, before antigen specific T cells expand. They provide the early interleukin 17 signal in the oral mucosa and skin.

Innate responses also injure tissue. Patients with chronic granulomatous disease who inhale large quantities of fungal spores, for example from mulch, hay or compost, can develop an acute fungal pneumonitis with intense inflammation and few organisms. Treatment is antifungal therapy with adjunctive corticosteroids, because the lung injury is largely inflammatory.

Excess interleukin 1 beta signalling and neutrophil recruitment also contribute to tissue damage in severe candidiasis and aspergillosis. The same system that protects at low fungal burden can damage the host at high burden, and this is the reason immune modulation is sometimes part of treatment.