Recognition of DHN-melanin by the C-type lectin, MelLec, is required for protective immunity to Aspergillus fumigatus

Objectives
C-type lectin receptors (CLRs) recognize microbial invasion, induce protective immune responses and play a central role in antifungal immunity. Most CLRs recognize conserved carbohydrate structures of the fungal cell wall. Recognition of other fungal virulence factors, including melanin, remain largely undefined. Here, we identify a CLR, Melanin sensing C-type Lectin receptor (MelLec), involved in the recognition of fungal DHN-melanin and describe its role in antifungal immunity to Aspergillus fumigatus. 

Methods
Soluble protein chimeras consisting of the C-type lectin-like domain of murine or human MelLec fused to the Fc-region of human IgG1 (Fc-MelLec) were generated as probes to screen for recognition of fungi. To characterize the expression of MelLec in mice, we screened various tissues by PCR and generated antibodies against murine MelLec. To explore the role of MelLec in vivo, we generated MelLec-deficient mice (MelLec-/-) and performed intravenous infection models with A. fumigatus conidia. In humans, associations between susceptibility to the risk of invasive aspergillosis and a common missense single nucleotide polymorphism (SNP) in MelLec were studied in a cohort of stem-cell transplant patients. 

Results
Using Fc-MelLec, we were able to show that MelLec recognizes melanin of A. fumigatus conidia and other DHN-melanised fungi, but not A. fumigatus mutant conidia deficient in DHN-melanin or melanins produced via other pathways. In mice, MelLec RNA was detected in various tissues, with highest expression in the lung. Surprisingly, unlike most other CLRs, presence of MelLec could not be detected on myeloid cell populations by flow cytometry. Instead, ubiquitous expression of MelLec was detected on CD31+ endothelial cells in the lung and various other tissues. To understand the role of MelLec in antifungal immunity, we performed in vivo infection models. MelLec-/- mice were significantly more susceptible to systemic infection with A. fumigatus conidia compared to wildtype counterparts, and this was associated with significantly increased tissue fungal burdens and inflammatory cytokines. 
In humans, we could demonstrate that human MelLec also recognizes DHN-melanised A. fumigatus conidia. To understand the role of MelLec in human fungal infections, we determined the presence of a SNP in MelLec in stem-cell transplant patients. We found a highly significant association between this SNP and the risk of invasive aspergillosis, with increased risk of disease when the variant was carried by the donor, but not when carried by the recipient. This suggests that in humans, the protective functions of MelLec are primarily mediated by myeloid cells and we could observe that myeloid cells of individuals carrying this SNP produced significantly less pro-inflammatory cytokines following in vitro stimulation with A. fumigatus conidia. 

Conclusion
We have shown that fungal DHN-melanin is sensed through MelLec, a CLR that plays a crucial role in controlling systemic A. fumigatus infection in both mice and humans. Our data indicates that identifying genetic variants of MelLec in donors could help reduce the incidence of invasive aspergillosis in stem-cell transplant recipients. Furthermore, our data suggests that MelLec could also play an important role in immunity to other DHN-melanised fungi, such as those that cause phaeohyphomycosis, mycetoma, and chromoblastomycosis.