Immunity to pathogenic fungi in the eye

Abstract

Fusarium, Aspergillus and Candida are important fungal pathogens that cause visual impairment and blindness in the USA and worldwide. This review will summarize the epidemiology and clinical features of corneal infections and discuss the immune and inflammatory responses that play an important role in clinical disease. In addition, we describe fungal virulence factors that are required for survival in infected corneas, and the activities of neutrophils in fungal killing, tissue damage and cytokine production.

Keywords: Aspergillus; Candida; Cornea; Cytokines; Fungal infection; Fusarium; IL-1 alpha; IL-1 beta; Innate immunity; Keratitis; NADPH oxidase; Neutrophil; Nutritional immunity; Ocular infection; Reactive oxygen species.

Fig. 1.

(A) Diagram of the human eye; (B) H&E stain of a human corneal section showing the major layers. (C) Aspergillus flavus infected human cornea (GMS stain).

Fig. 2.

Percent of cases of microbial keratitis caused by fungi. Reproduced with permission from Brown et al. [1].

Fig. 3.

Clinical features of fungal keratitis in England. (A,B) contact lens related Fusarium keratitis (A) with multifocal infiltrates (green and red arrows); (B) Later stage fungal keratitis with hypopyon (neutrophil infiltrate to the anterior chamber, yellow arrow). (A–C) slit lamp images; C (inset)-optical coherence tomography of the eye in C showing endothelial plaque (yellow arrow). (D) In vivo confocal microscopy of a fungal infected cornea showing hyphae in the stroma. (A–C) From Ting et al. [10]. (d) From Wang et al. [12].

Fig. 4.

Immune response in patients with corneal ulcers caused by Aspergillus and Fusarium. (A) Fungal keratitis caused by Aspergillus flavus (* neutrophil rich hypopyon in the anterior chamber). (B,C) A. flavus hyphae and cellular infiltrate in a corneal ulcer showing >90% of the cells were neutrophils. (D) Gene expression of cytokines in corneal ulcers caused by Aspergillus and Fusarium compared with uninfected (donor) cornea. Data points represent individual patients. From Karthikeyan et al. [14].

Fig. 5.

RodA hydrophobin proteins mask conidia cell wall carbohydrates and impair A. fumigatus hyphal killing in infected corneas. (A,B) β-glucan and α-mannose on parent G10 A. fumigatus, ΔrodA mutants and hydrofluoric acid (HF) treated Aspergillus and Fusarium conidia. (C) CFU 24 h post-corneal infection with G10 or ΔrodA conidia. Note that mutants are more rapidly cleared, which is dependent on Dectin-1 and Dectin-2. Data points represent corneas from infected mice. From Carrion Sde et al. [24].

Fig. 6.

Neutrophil ROS mediated regulation of Aspergillus fumigatus corneal infection (keratitis). (A,B) Red fluorescent (RFP) expressing A. fumigatus spores were injected into the corneal stroma of mice expressing GFP neutrophils. Brightfield: Corneal opacification, RFP and GFP were examined by fluorescence microscopy. Neutrophils were depleted by intra-peritoneal injection of Ly6G antibody NIMP-R14 (corneas were examined 48 h post infection). (C) Infection of mice with a deletion in the NADPH oxidase subunit GP91^PHOX (original magnification: corneas x20; histology (PASH) x 200). From Leal et al. [25].

Fig. 7.

Calprotectin (CP) is required for Aspergillus fumigatus hyphal growth in infected corneas. (A, B) Exogenous Zn⁺⁺ and Mn⁺⁺ rescue hyphal CP inhibition of hyphal growth in vitro. (C, D) Increased hyphal growth in corneas of calprotectin deficient S100A9^−/− mice infected with RFP expressing A. fumigatus AF293. Representative images (C) and quantification by imaging analysis. From Clark et al. [35].

Fig. 8.

NETosis induced by curdlan is dependent on PAD4. (A–C) Neutrophil elastase (NE) and citrullinated histone 3 (H3cit) in bone marrow neutrophils from C57BL/6 or PAD4^−/− mice. (D–F) highlighted areas showing NE in the nucleus of neutrophils from C57BL/6 but not PAD4^−/− mice and extracellular H3cit associated with NETs. Original magnification for A-C: x200, for D-F x400. From Clark et al. [45].

Fig. 9.

Optical maps of the human systemic fusariosis strain NRRL 32931 and the tomato wilt pathogen Fol4287. The core genome comprises 11 homologous chromosomes (gray) that are conserved within the Fusarium oxysporum complex. However, the four unique and smaller lineage-specific chromosomes of human-infecting strain NRRL 32931 in red are clearly distinct from the tomato pathogen. From Zhang et al. [54].

Fig. 10.

Aspergillus fumigatus keratitis is regulated by IL-1β and caspase 11. Hyphal mass of Red fluorescent (RFP) expressing A. fumigatus in the corneal stroma of C57BL/6 and IL-1β^−/− mice at d1 and d2 post infection (A, B) or caspase1/11^−/− or caspase-11^−/− mice at d2 post-infection. RFP was examined. (A,C) representative RFP expressing corneas; (B,D) RFP quantification by image analysis. Data points represent individual infected corneas. From Sun et al. [21].

Fig. 11.

Co-localization of IL-1α with the tetraspanin CD63 marker for exosomes. Neutrophils from the peritoneal cavity of C57BL/6 mice (following induction of sterile inflammation) incubated 6 h with LPS or curdlan, and immunostained with antibodies to CD63 and IL-1α and examined by confocal microscopy. Reproduced with permission from Ratitong et al. [69].