Phd in ADVANCES IN INFECTIOUS DISEASES, MICROBIOLOGY, LEGAL
MEDICINE AND PUBLIC HEALTH SCIENCES

Thesis title: Impact of Anisakis Extracellular Vesicles on Human Intestinal Organoids: a Characterization and Functional study

Background. Anisakis spp. are zoonotic nematodes whose infectious third-stage larvae (L3) are acquired by humans through raw or undercooked seafood and cause anisakiasis. Clinical manifestations range from acute gastrointestinal symptpms (epigastric pain, nausea, vomiting, bleeding) to allergic reactions, including urticaria and anaphylaxis. Increasing reports and experimental observations worldwide have raised concerns about the possible tumorigenic potential of Anisakis in humans, however mechanisms and causality remain unresolved. Like other helminths that exert immunomodulation strategies, the extent and mechanisms of this phenomenon in Anisakis remain to be clarified. In particular, among the ES, the extracellular vesicles (EVs) are emerging mediators of host-parasite communication, but their effects on human intestinal epithelium remain unclear. This thesis was aimed at (i) isolating and characterizing EVs secreted by Anisakis L3 larvae through a multimodal approach and (ii) testing their impact on human intestinal organoids (HIOs) to define epithelial signaling and structural responses. Methods. L3 larvae were incubated and EVs were purified from incubation media by polymer precipitation or size-exclusion chromatography (SEC) with one or two steps of ultrafiltration. EVs were characterized by NTA/iNTA, DLS, TRPS, TEM/SEM, and AFM. Surface biomolecules were probed by proteinase K and PNGase F treatments. Polarized HIOs monolayers were exposed to EVs apically and responses were profiled by RNA-seq (mRNA/miRNome), qRT-PCR, multiplex cytokine assays, immunofluorescence and western blot of junction/differentiation markers (ZO-1, Villin-1, MUC-2). Results. SEC with ultrafiltration yielded higher-purity EVs than precipitation. Preliminary results on EVs characterization returned intact, spherical vesicles by EM and a “shaving” effect following enzymatic treatment. In HIOs, EVs induced alterations in seven FDR-significant genes: upregulation of NUPR1 and H2BC5 (stress/transcriptional control) and downregulation of LEFTY1, MKI67, MYBL2, TACC1, and EPHB2 (cell-cycle, adhesion/architecture). Parasite miRNAs packed into EVs were predicted to target human TACC1 and EPHB2. At the protein level, EVs reduced IL-33R, CD40, CEACAM-1, IL-1β, GM-CSF, IL-15, and IL-23, consistent with dampened type-2/type-3 axes. No disruption of ZO-1, Villin-1, or MUC-2 was detectable in treated HIOs. Conclusions. Anisakis EVs reshape epithelial programs and attenuate cytokine output, possibly promoting a tolerogenic, hypo-inflammatory environment that may, alongside suppressed proliferation/repair pathways, create conditions permissive to tumorigenesis. These data establish a novel human organoid platform to dissect Anisakis–host interactions and underscores the importance of EVs as key players of host–parasite communication at the interface of immunity, pathology, and cancer risk.