Novel Insight into Neutrophil Immune Responses by Dry Mass Determination of Candida albicans Morphotypes

The common fungal pathogen Candida albicans has the ability to grow as a yeast or as a hypha and can alternate between these morphotypes. The overall biomass of both morphotypes increases with growth. However, only yeasts, but not hyphae, exist as discrete cellular entities. Multiplicity of infection (MOI) is a useful parameter to determine the initial inoculum of yeasts for in vitro infection assays. Since the amount of hyphae is difficult to quantify, comparable starting conditions in such assays cannot be determined accurately for yeasts and hyphae using MOI. To circumvent this problem, we have established a set of correlation coefficients to convert fungal metabolic activity and optical density to dry mass. Using these correlations, we were able to accurately compare ROS production and IL-8 release by polymorphonuclear neutrophils upon infection with equal dry mass amounts of yeast and hyphal morphotypes. Neutrophil responses depended on the initial form of infection, irrespective of C. albicans wild-type yeasts transforming to hyphal growth during the assay. Infection with a high mass of live C. albicans yeasts resulted in lower neutrophil ROS and this decrease stems from efficient ROS detoxification by C. albicans without directly affecting the phagocyte ROS machinery. Moreover, we show that dead C. albicans induces significantly less ROS and IL-8 release than live fungi, but thimerosal-killed C. albicans were still able to detoxify neutrophil ROS. Thus, the dry mass approach presented in this study reveals neutrophil responses to different amounts and morphotypes of C. albicans and serves as a template for studies that aim to identify morphotype-specific responses in a variety of immune cells.常见真菌病原体白色念珠菌（Candida albicans）能够以酵母型或菌丝型两种形态生长，并可在这两种形态型之间相互转换。两种形态型的总生物量均随培养进程递增，但仅酵母型以离散的单细胞实体形式存在，菌丝型则不具备这一特征。感染复数（Multiplicity of Infection, MOI）是体外感染实验中确定酵母初始接种量的常用参数，然而由于菌丝的数量难以精准定量，因此无法通过MOI为酵母与菌丝型建立可比的实验起始条件。 为解决这一难题，本研究建立了一组可将真菌代谢活性与光密度转换为干重的相关系数。借助这些系数，我们得以精准比较干重一致的酵母型与菌丝型白色念珠菌感染时，多形核中性粒细胞（polymorphonuclear neutrophils）的活性氧（Reactive Oxygen Species, ROS）生成量与白细胞介素8（IL-8）释放水平。中性粒细胞的应答反应取决于初始感染的形态型，即便实验过程中白色念珠菌野生型酵母可转变为菌丝生长，该规律仍不受影响。 高剂量活的白色念珠菌酵母感染会导致中性粒细胞ROS生成减少，这一现象源于白色念珠菌高效的ROS解毒作用，且并未直接干扰吞噬细胞的ROS产生机制。此外，本研究证实，与活真菌相比，灭活的白色念珠菌诱导的ROS与IL-8释放量显著更低，但经硫柳汞（thimerosal）灭活的白色念珠菌仍可清除中性粒细胞产生的ROS。 综上，本研究提出的干重分析方法可清晰揭示中性粒细胞对不同剂量、不同形态型白色念珠菌的应答特征，同时可为旨在识别多种免疫细胞中形态型特异性应答的研究提供标准化参考范式。