Abstract
Phagocytosis by innate immune cells is an important process for protection against multiple pathologies and is particularly important for resistance to infection. However, phagocytosis has also been implicated in the progression of some diseases, including the dissemination of the human fungal pathogen, Cryptococcus neoformans. Previously, we identified Dectin-1 as a likely phagocytic receptor for C. neoformans spores through the use of soluble components in receptor-ligand blocking experiments. In this study, we used gain-of-function and loss-of-function assays with intact cells to evaluate the in vivo role of Dectin-1 and other C-type lectins in interactions with C. neoformans spores and discovered stark differences in outcome when compared with previous assays. First, we found that non-phagocytic cells expressing Dectin-1 were unable to bind spores and that highly sensitive reporter cells expressing Dectin-1 were not stimulated by spores. Second, we determined that some phagocytes from Dectin-1-/- mice interacted with spores differently than wild type (WT) cells, but the effects varied among assays and were modest overall. Third, while we detected small but statistically significant reductions in phagocytosis by primary alveolar macrophages from Dectin-1-/- mice compared to WT, we found no differences in survival between WT and Dectin-1-/- mice challenged with spores. Further analyses to assess the roles of other C-type lectins and their adapters revealed very weak stimulation of Dectin-2 reporter cells by spores and modest differences in binding and phagocytosis by Dectin-2-/- bone marrow-derived phagocytes. There were no discernable defects in binding or phagocytosis by phagocytes lacking Mannose Receptor, Mincle, Card-9, or FcRγ. Taken together, these results lead to the conclusion that Dectin-1 and other C-type lectins do not individually play a major roles in phagocytosis and innate defense by phagocytes against C. neoformans spores and highlight challenges in using soluble receptor/ligand blocking experiments to recapitulate biologically relevant interactions.
Highlights
Therapeutic approaches in treating human disease have directly targeted the causative agents of disease
Spores pre-treated with Fc-Dectin or alveolar macrophages pretreated with an antibody against Dectin-1 prior to co-culture of spores and macrophages led to a 66% and 60% reduction in phagocytosis of spores, respectively [15]
Because Dectin-1 has been shown previously to be sufficient for binding C. albicans, we used C. albicans as a positive control for Dectin-1 binding
Summary
Therapeutic approaches in treating human disease have directly targeted the causative agents of disease (e.g. anticancer drugs, antimicrobials, etc.). Immunomodulatory biologics that instead target and alter the host immune response have shown promise [1]. Developing similar methods to treat fungal diseases is appealing because current antifungal drugs often lack efficacy, cause off-target toxicity in the host, or are not readily available worldwide [5]. Phagocytosis of microbes by host immune cells is mediated by receptors that bind to immunoreactive moieties on microbes (Reviewed in [6]). The most well characterized of these interactions among fungi is the binding between the C-type lectin receptor Dectin-1 and the predominantly fungal carbohydrate β-(1,3)-glucan. Dectin-1 was discovered to be the major receptor responsible for phagocytosis of the pathogenic yeast Candida albicans, being both necessary and sufficient for uptake [7]. The absence of Dectin-1 or blocking it through the addition of soluble ligand causes a profound loss of C. albicans phagocytosis [8]
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