In vitro Study of Fungus-Like Organisms Peronospora ficariae and Wilsoniana bliti as Potential Inhalant Allergens

Monika Sztandera-Tymoczek,1 Sylwia Wdowiak-Wróbel,2 Urszula Świderska,3 Marta Palusińska-Szysz,2 Agnieszka Szuster-Ciesielska1 1Department of Virology and Immunology, Institute of Biological Sciences, Maria Curie-Skłodowska University, Lublin, Poland; 2Department of Genetics and Microbiology, Institute of Biological Sciences, Maria Curie-Skłodowska University, Lublin, Poland; 3Department of Botany, Mycology and Ecology, Institute of Biological Sciences, Maria Curie-Skłodowska University, Lublin, PolandCorrespondence: Agnieszka Szuster-Ciesielska, Department of Virology and Immunology, Institute of Biological Sciences, Maria Curie-Skłodowska University, Akademicka 19, Lublin, 20-033, Poland, Tel +48 81 537 59 43, Email agnieszka.szuster-ciesielska@mail.umcs.plPurpose: Allergic conditions have surged to epidemic proportions globally, affecting almost 30% of people worldwide. Fungi are a significant source of allergens, contributing to up to 6% of respiratory illnesses in the general population. Nonetheless, the exact cause of respiratory allergies often remains elusive. This research sought to explore the potential of two fungus-like species from the Peronosporales (Peronospora ficariae) and Albuginales (Wilsoniana bliti) to induce a proinflammatory response in vitro models of the upper and lower respiratory tract.Materials and Methods: BEAS-2B and A549 cell lines were used to mimic upper and lower respiratory epithelial cells. The cytotoxicity of fungal extracts was assessed using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) and flow cytometry assays. Reactive oxygen species (ROS) production in the cells was measured through flow cytometry, while ELISA (enzyme-linked immunosorbent assay) tests were used to quantify the production of proinflammatory cytokines and metalloproteinases. Immunofluorescence techniques were utilized to evaluate markers of cell integrity.Results: Although W. bliti extracts did not trigger notable inflammatory responses, P. ficariae significantly enhanced the production of proinflammatory cytokines IL-1β and GM-CSF in both cell lines, which are linked to allergic reactions. The rise in these cytokines and increased ROS production were associated with disrupting tight junction proteins, such as E-cadherin and occludin, in epithelial cells.Conclusion: The results indicate that P. ficariae extracts have the potential to collectively compromise the epithelial barrier in the upper and lower respiratory tracts by inducing proinflammatory cytokines and promoting the production of reactive oxygen species and metalloproteinases. While none of these parameters were exceptionally high, their combined effect was observed to disrupt epithelial cell junctions.Plain Language Summary: Microscopic fungi and fungus-like organisms that cause plant diseases often found in human environments are plant parasites. They negatively impact crop production, reduce the quality of agricultural goods, and lessen the visual appeal of decorative plants. Additionally, these fungi can pose a health hazard to humans, as they are significant sources of allergens. The most prominent allergenic fungi include Alternaria, Aspergillus, Cladosporium, Penicillium, and Fusarium. Even fungus-like organisms such as P. ficariae, which infects Ficaria verna, can provoke allergic responses. P. ficariae infest plants extensively, releasing large quantities of spores, especially during spring. This increases the likelihood of human exposure to allergens as the plants and their parasitic fungi proliferate in early spring. Since conventional skin or blood tests do not always identify the underlying causes of allergies, discovering new fungal allergens is essential for more accurate hypersensitivity diagnoses. Our research shows that the parasitic P. ficariae fungi have strong pro-inflammatory properties, which form the basis of allergenic reactions in in vitro models of the upper and lower respiratory tracts.Keywords: phytopathogenic microfungi, Peronospora ficariae, Wilsoniana bliti, airway epithelial cells, inflammatory response