Distinct Roles for Dectin-1 and TLR4 in the Pathogenesis of Aspergillus fumigatus Keratitis

Abstract

Aspergillus species are a major worldwide cause of corneal ulcers, resulting in visual impairment and blindness in immunocompetent individuals. To enhance our understanding of the pathogenesis of Aspergillus keratitis, we developed a murine model in which red fluorescent protein (RFP)-expressing A. fumigatus (Af293.1RFP) conidia are injected into the corneal stroma, and disease progression and fungal survival are tracked over time. Using Mafia mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, we demonstrated that the presence of resident corneal macrophages is essential for production of IL-1β and CXCL1/KC, and for recruitment of neutrophils and mononuclear cells into the corneal stroma. We found that β-glucan was highly expressed on germinating conidia and hyphae in the cornea stroma, and that both Dectin-1 and phospho-Syk were up-regulated in infected corneas. Additionally, we show that infected Dectin-1 ^−/− corneas have impaired IL-1β and CXCL1/KC production, resulting in diminished cellular infiltration and fungal clearance compared with control mice, especially during infection with clinical isolates expressing high β-glucan. In contrast to Dectin 1 ^−/− mice, cellular infiltration into infected TLR2 ^−/−, TLR4 ^−/−, and MD-2 ^−/− mice corneas was unimpaired, indicating no role for these receptors in cell recruitment; however, fungal killing was significantly reduced in TLR4 ^−/− mice, but not TLR2 ^−/− or MD-2 ^−/− mice. We also found that TRIF ^−/− and TIRAP ^−/− mice exhibited no fungal-killing defects, but that MyD88 ^−/− and IL-1R1 ^−/− mice were unable to regulate fungal growth. In conclusion, these data are consistent with a model in which β-glucan on A.fumigatus germinating conidia activates Dectin-1 on corneal macrophages to produce IL-1β, and CXCL1, which together with IL-1R1/MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4-dependent fungal killing.

Author Summary

Corneal infection with filamentous fungi, including Aspergillus species, is a common cause of visual impairment and blindness in the southern USA and worldwide. The incidence in India and China greatly increases during harvest season when infection occurs after traumatic injury with fungal spores (conidia). In contrast to pulmonary aspergillosis, keratitis occurs in immunocompetent individuals. To characterize the host response, we injected Aspergillus fumigatus conidia expressing red fluorescence into the transparent mouse cornea, and showed that cytokine production, neutrophil and monocyte recruitment to the corneal stroma and fungal killing is dependent on the presence of macrophages and dendritic cells, and on expression of the β-glucan receptor Dectin-1. We also found that fungal killing, but not cellular infiltration, is dependent on expression of the LPS receptor TLR4. In addition, we demonstrate that IL-1R1 and MyD88 regulate neutrophil recruitment and fungal killing in Aspergillus keratitis, whereas TLR4 associated adaptor molecules TRIF and TIRAP have no role. Together, these findings identify specific mediators of the innate immune response to these organisms that regulate disease severity and survival of Aspergillus that may have potential application as targets for therapeutic intervention.