Deciphering inflammation and immunomodulation in host-parasite interface: caco-2 cells and human intestinal organoids response to Anisakis’ messengers of pathogenicity

Background: Anisakiasis is a zoonosis caused by consumption of raw fish parasitized with Anisakis spp third stage larvae (L3). Even if humans are accidental hosts, only one L3 is sufficient to induce mild to severe symptoms. In fact, larval migration through the gastrointestinal tract, the excreted/secreted products and extracellular vesicles (EVs) can progressively determine allergic reactions, erosive ulcerous lesions, granulomas and chronic inflammation (Audicana et al., 2008 Clin Microbiol Rev. 21,360). Furthermore, reports regarding patients affected by gastric and intestinal tumors in co-occurrence with anisakiasis are increasing from endemic countries, suggesting exposure as a risk factor (Sonoda et al., 2015 Surg Today. 45, 1321; Murata et al., 2018 BMC Infect Dis. 4;619; Kita et al., 2019 Int J Surg Case Rep. 60, 209). Despite its relevance for public health, investigations on host inflammatory and immune response during anisakiasis are still very scarce. To date, studies aimed to understanding the messengers of pathogenicity, based on in vitro models able to recount the microenvironment in which the parasite acts during infection in humans, are at their infancy (Speciale et al., 2017 Parasitol Res. 116, 2471; Carballeda-Sangiao et al., 2020 PLoS Negl Trop Dis. 14, e0008462). Objective: the general aim of this PhD thesis is to investigate Anisakis-human interactions, focusing on human host inflammatory response and recreating the microenvironment in which Anisakis act. To achieve this goal, the project has been structured in two sections: i) the gene expression and the amounts of the pro-inflammatory cytokines (IL-6, IL-8) were analyzed to investigate the inflammatory pathway in in-vitro human epithelial colorectal adenocarcinoma cells (Caco-2) exposed to the live L3, the crude extract (CE) and the extracellular vesicles (EVs), as representative of the mechanical action of larval motility, whole body of senescent larvae and inflammation silencing, respectively. ii) expanding the knowledge on Anisakis EVs effects on human interface, using a bi or three-dimensional cutting-edge model capable of exhibiting the architecture and functionality of the organ of origin: the human intestinal organoids (HIO). In particular, the gene expression of three cytokines of interest was analyzed (Il33, Il1β, Il8). Methods: a total of 855 L3 were collected from fishes from selected areas (FAO37) and species identification was carried out through PCR-RFLP. CE were analyzed using Qubit and EVs were isolated through commercial kit and characterized using western blot and Nanoparticle tracking analyses. The gene expression (Caco-2 cells check-points: 1h, 6h, 24h; HIO checkpoint: 48h) and amounts of the pro-inflammatory cytokines (IL-6, IL-8) were analyzed by qRT-PCR and ELISA, respectively. Results: ELISA tests on Caco-2+L3 showed a progressive decrease of IL-6 (P<0.01) and IL-8 if compared to controls. Interestingly, no signals were obtained for IL-6 in Caco-2+EVs (P<0.01) and IL-8 was downregulated (P<0.01). Contrariwise, CE induced a strongly increased secretion of IL-6 (P<0.01) and a decreasing trend was observed for IL-8. Real-time PCR results on Caco-2+L3, CE and EVs suggested a detectable early effect on cytokines expression (1h). RT-PCR carried out on HIO exposed to EVs revealed a decreasing trend in Il33 gene expression and a slight increase in Il1β, a dynamic possibly linked to helminth infection chronicity, while Il8 gene expression seemed to be not affected. Conclusion: This study represents the first attempt to decipher the Anisakis EVs effects on human host (for both the models used) and results obtained showed an intricate host-parasite interplay, characterized by an early phase where the active L3 and its released EVs modulate the immune response to find a long-lasting niche to survive, and a second phase where L3 senescence may induce host immune response activation, leading to the granuloma formation.