Fungal-derived cues promote ocular autoimmunity through a Dectin-2/Card9-mediated mechanism

Abstract

Uveitis (intraocular inflammation) is a leading cause of loss of vision. Although its aetiology is largely speculative, it is thought to arise from complex genetic-environmental interactions that break immune tolerance to generate eye-specific autoreactive T cells. Experimental autoimmune uveitis (EAU), induced by immunization with the ocular antigen, interphotoreceptor retinoid binding protein (IRBP), in combination with mycobacteria-containing complete Freund's adjuvant (CFA), has many clinical and histopathological features of human posterior uveitis. Studies in EAU have focused on defining pathogenic CD4⁺ T cell effector responses, such as those of T helper type 17 (Th17) cells, but the innate receptor pathways precipitating development of autoreactive, eye-specific T cells remain poorly defined. In this study, we found that fungal-derived antigens possess autoimmune uveitis-promoting function akin to CFA in conventional EAU. The capacity of commensal fungi such as Candida albicans or Saccharomyces cerevisae to promote IRBP-triggered EAU was mediated by Card9. Because Card9 is an essential signalling molecule of a subgroup of C-type lectin receptors (CLRs) important in host defence, we evaluated further the proximal Card9-activating CLRs. Using single receptor-deficient mice we identified Dectin-2, but not Mincle or Dectin-1, as a predominant mediator of fungal-promoted uveitis. Conversely, Dectin-2 activation by α-mannan reproduced the uveitic phenotype of EAU sufficiently, in a process mediated by the Card9-coupled signalling axis and interleukin (IL)-17 production. Taken together, this report relates the potential of the Dectin-2/Card9-coupled pathway in ocular autoimmunity. Not only does it contribute to understanding of how innate immune receptors orchestrate T cell-mediated autoimmunity, it also reveals a previously unappreciated ability of fungal-derived signals to promote autoimmunity.

Keywords: autoimmunity; eye; inflammation; rodent; uveitis.

Figure 1

Candida. albicans‐derived signals promote autoimmune uveitis. Uveitis was evaluated in mice immunized with interphotoreceptor retinoid binding protein (IRBP) by both clinical examination (a,b) and histopathology scoring (c,d). (b) Representative fundus images depicting uveitis; black arrow denotes the optic nerve head. (d) Representative histological images depicting pathological features; green asterisk indicates vasculitis. L = lens; V = vitreous; R = retina. Ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), retinal pigment epithelium (RPE). (e) Enumeration of cells within the aqueous of anterior segment (AS) or vitreous cavity of posterior segment (PS). Asterisk (*) indicates significance in IRBP‐immunized mice compared to adjuvant‐immunized (n = 10–20 mice/group/time from two to three experiments).

Figure 2

Fungal‐derived uveitis requires Card9 but is independent of Mincle. To assess the role of Mincle (a,c) or Card9 (b,c) in fungal‐promoted uveitis, wild‐type (WT), Card9 knock‐out (KO) and Mincle KO mice were immunized with interphotoreceptor retinoid binding protein (IRBP) in an emulsion containing Candisa albicans and uveitis was evaluated by clinical examination and histology on day 21. (c) Clinical and histological images of the fundus and posterior eye are shown. Asterisk (*) indicates significance compared to WT mice (n = 12 mice/group combined from two independently performed experiments).

Figure 3

Dectin‐2 contributes to pathogenesis of fungal‐promoted uveitis. Representative immunoblotting of indicated ocular tissues (pooled from both eyes of three mice) from naive wild‐type (WT) mice showing C‐type lectin receptors (CLR) expression (a). Uveitis was induced in WT or Dectin‐1 knock‐out (KO) (b,c) versus Dectin‐2 KO (d,e) mice by immunization with interphotoreceptor retinoid binding protein (IRBP) in an emulsion containing Candida albicans and evaluated by clinical and histological examination. Fundus and histological images of the posterior eye d21 post‐immunization (green asterisk indicates vasculitis). No significant differences between WT and Dectin‐1 KO mice were observed (n = 10–12 mice/group combined from two independently performed experiments). Asterisk (*) indicates significance compared to WT mice (n = 12 mice/group combined from two independently performed experiments).

Figure 4

Activation of the Dectin‐2/Card9 coupled signalling axis promotes autoimmune uveitis. B10.RIII mice were immunized with interphotoreceptor retinoid binding protein (IRBP) emulsified in incomplete Freund's adjuvant (IFA) supplemented with α‐mannan and uveitis was scored from histological sections (a). For comparison, mice were also immunized with heat‐killed Candida albicans (HKCA), complete Freund's adjuvant (CFA) or the mycobacterial‐derived products trehalose‐6–6‐dibehenate (TDB), peptidoglycan (PGN) or muramyl dipeptide (MDP). An asterisk (*) indicates significance compared to CFA‐immunized mice (n = 10 mice/group combined from two independent experiments). To determine the specific role(s) for Dectin‐2 or Card9 in α‐mannan‐induced uveitis, disease severity was evaluated clinically and histopathologically in knock‐out (KO) mice (b,c). Asterisk (*) indicates significance compared to wild‐type (WT) mice while # indicates significance between Dectin‐2 and Card9 KO mice (n = 24 mice/group combined from three independently performed experiments). (c) Fundus and histological images of the posterior eye d21 post‐immunization. (d) Heat map showing retinal gene expression at d10 post‐immunization as evaluated by multiplex quantitative polymerase chain reaction analysis. Fold‐change in gene expression is relative to naive control animals (n = 6 mice pooled/group; array results are representative of four independently performed experiments). (e) Cytokine concentration in culture supernatants was determined at 24 h post‐IRBP stimulation by enzyme‐linked immunosorbent assay (n = 5 mice/group, performed in triplicate). Asterisk (*) indicates significance compared to media‐stimulated control cells.