Immunomodulatory properties of heat-inactivated biomasses by selected bacterial and fungal species included in the Qualified Presumption of Safety list of the European Food Safety Authority

Introduction: The use of microbial lysates has been described to prevent recurrent respiratory infections, to avoid the flare-up of respiratory chronic infections and as a defence against urinary infections by modulating host responses with partially understood mechanisms. Recently, we have demonstrated that a Cutibacterium acnes bacterial lysate (BL) enhances cellular responses against Candida albicans, Escherichia coli, and Gardnerella vaginalis in an in vitro model of vaginal infection, through mechanisms associated with trained immunity. The present work aims to identify new microbial lysates with immunomodulatory capacity comparable to or better than BL, to be tested in an in vitro model of E. coli-induced urinary tract and gastrointestinal infection. Materials and Methods: Ten microorganisms (6 fungi, 3 Gram-positive bacteria and 1 Gram-negative bacterium), included in the Qualified Presumption of Safety (QPS) list published by the European Food Safety Authority (EFSA), were selected. Microorganisms were cultured in broth and incubated at 30 °C for at least 48 h. Microbial growth was monitored by measuring optical density (OD) and colony-forming units (CFU). Subsequently, the cultures were heat-inactivated at 75 °C for 3 h. The resulting heat-inactivated microbial biomasses (Hi-MBs) were harvested by centrifugation and dried overnight using a speed-vacuum system. The murine macrophage cell line J774A.1 was then primed with the obtained Hi-MBs for 24 h at 37 °C and 5% CO2. Then the phagocytic activity versus E. coli fluorescent bioparticles, was assessed after 30 min or 2 h, using the Vybrant™ Phagocytosis Assay Kit according to the manufacturer’s instructions. Results: In a first series of experiments, macrophages primed with Hi-MB obtained by the yeast Hanseniaspora uvarum (H. uvarum) and the Gram negative bacterium Xanthomonas campestris (X. campestris) exhibited a significant increase in phagocytic activity with respect to un-primed controls. Such activity was comparable to the phagocytic activity observed in cells primed with either BL or lipopolysaccharide (LPS), indicating that H. uvarum and X. campestris Hi-MBs could be effective in triggering macrophages activation. Discussion and Conclusion: Our results suggest that H. uvarum and X. campestris Hi-MBs could effectively train innate immune cells, thus supporting the possibility to be employed for the development of new microbial lysates with immunomodulatory properties. Future studies will evaluate whether these lysates can also enhance mitochondrial activity and microbicidal efficacy and if they are able to improve cellular responses to E. coli in an in vitro model of urothelial and intestinal infection.