Inflammatory mechanisms in Caco-2 cells stimulated with Anisakis-derived messengers of pathogenicity

“Inflammatory mechanisms in Caco-2 cells stimulated with Anisakis-derived messengers of pathogenicity” I BELLINI, D SCRIBANO, M SARSHAR, C AMBROSI, A PIZZARELLI, AT PALAMARA, S D’AMELIO, S CAVALLERO. Department of Public Health and Infectious Diseases, Sapienza University of Rome Keywords: Anisakis, Caco-2, crude extract, exosomes, inflammation INTRODUCTION: Anisakiasis is a zoonotic disease caused by consumption of raw fish parasitized with Anisakis spp third stage larvae (L3). Larval migration in the gastrointestinal tract, the excreted/secreted products and extracellular vesicles (EVs) can progressively determine allergic symptoms, erosive ulcerous lesions and granulomas (Audicana et al., 2008 Clin Microbiol Rev. 21: 360–379). Reports of tumors co-occurrence with Anisakis L3 are increasing (Sonoda et al., 2015 Surg Today. 45:1321-5; Murata et al., 2018 BMC Infect Dis. 2018 4;18:619) but pathogenic mechanisms, role of host's immune response and potential implications are still unknown. The aim of this study was 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 exosomes enriched fraction, as representative of mechanical action of larval motility, whole body of senescent larvae and inflammation silencing, respectively. In particular, the activation of inflammatory response key molecules (ERK1/2, NF-kB), and the amounts of the pro-inflammatory cytokines (IL-6, IL-8) were analyzed. MATERIAL AND METHODS: L3 collected were used for incubation as live L3 (38), for CE preparation (36) and for EVs isolation (136). Samples were incubated with Caco-2 cells (6h/24h) at 37°C in the presence of 5%CO2. Cell lysates were used for Western Blot (WB) and Real-Time PCR analyses and supernatants were used for ELISA tests. RESULTS AND CONCLUSION: Caco-2+L3 showed a progressive decrease of IL-6 (P<0,01) and IL-8 if compared to non-treated cells. Differently, in Caco-2+EVs, IL-6 was not detected (P<0,01) and IL-8 was strongly downregulated (P<0,01). On the contrary, CE induced a strongly increased secretion of IL-6 (P<0,01) and a decreasing trend in IL-8. Accordingly, WB analyses showed an increased phosphorylation level of ERK1/2 in Caco-2+CE (P<0,05) and in Caco-2+L3, with a slight increased level of activated NF-kB. No differences were observed in the phosphorylation levels of ERK1/2 in cells incubated with EVs respect to the controls. Furthermore, real-time PCR preliminary results on Caco-2+L3 suggested an early effect on cytokines expression. The results obtained showed an intricate host-parasite interplay, characterized by an early phase where active L3 and its released EVs modulate the immune response to find a long-lasting niche, and a second phase where L3 senescence may lead to the activation of host immune response leading to the formation of granuloma.