Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model

Effects of acetylcholine on survival of Galleria mellonella larvae after Candida albicans infection and the ability of Candida albicans to form a biofilm in vivo. (A) Kaplan-Meier plot showing the effect of ACh on the survival of Candida albicans-infected larvae. The data are derived from three independent experiments with groups of 10 larvae (n = 30). ***, P < 0.001, as determined by the log rank test in comparison to larvae inoculated with C. albicans alone. (B) Real-time PCR determination of the effects of ACh on larval fungal burden as determined by colony-forming equivalents (CFE). Data are derived from 3 larvae from each experimental group from 3 independent experiments (n = 9). (C and D) Real-time PCR determination of the effects of ACh on expression of key Candida albicans genes involved in dimorphic switching in vivo: hyphal cell wall protein 1 gene HWP1 (C) and agglutinin-like sequence 3 gene ALS3 (D). Data are derived from 3 larvae from each experimental group from 3 independent experiments (n = 9). *, P < 0.05.

Effects of acetylcholine on Candida albicans biofilm formation and host immunity in vivo. Histological analysis of larvae was performed using periodic acid-Schiff (PAS) staining (A to D) and Giemsa staining (E to H) at 48 and 72 h postinoculation. The control groups are larvae inoculated with PBS (A and E) or ACh (B and F) alone. Black circles highlight Candida albicans biofilm formation in panels C and G and nodule formation, which is representative of hemocyte recruitment and activation, in panels D, G, and H. Representative images are shown from histological analysis of 3 larvae for each condition from 3 independent experiments. Fb, fat body; Ct, cuticle; GI, gastrointestinal tract; T, trachea; Nd, nodule. Scale bars, 100 μm.

Effects of acetylcholine on Candida albicans-induced hemocyte recruitment and activation. (A) Effect of ACh on hemolymph hemocyte counts. Data are expressed as cells per milliliter of hemolymph. The bars represent the mean and standard deviations for at least 3 larvae from 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Effect of ACh on C. albicans-induced hemocyte activation as determined by cytospin analysis. Representative images are shown from cytospin analysis of 3 larvae for each condition from 3 independent experiments. Cytosopin analysis for each condition was performed 24 h (i to iv), 48 h (v to viii), and 72 h (ix to xii) postinoculation. Black arrows highlight small aggregates with melanin deposition. A white arrow highlights pronounced aggregation, melanization, and merger of hemocytes into nodules with tissue-like structures. Asterisks highlight homogenous distribution of polymorphic hemocytes immersed in an eosinophilic extracellular matrix. Scale bars, 100 μm.

Effects of acetylcholine on Candida albicans-induced hemocyte recruitment into Galleria mellonella tissues. Histological analysis of hemocyte recruitment into larval tissues was performed using PAS staining 24, 48, and 72 h postinoculation. The control groups are larvae inoculated with PBS (A, E, and I) or ACh (B, F, and J) alone. Larvae inoculated with C. albicans alone (C, G, and K) and C. albicans plus ACh (D, H, and L) are also represented. Extensive hemocyte recruitment is highlighted with black arrows. Hemocyte nodule formation is highlighted with a black asterisk. Representative images are shown from histological analysis of >3 larvae for each condition from 3 independent experiments. Fb, fat body; GI, gastrointestinal tract; T, trachea. Scale bars, 100 μm.