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Interplay between proinflammatory cytokines, miRNA, and tissue lesions in Anisakis-infected Sprague-Dawley rats

Background Anisakiasis is an emerging public health problem, caused by Anisakis spp. nematode larvae. Anisakiasis presents as variable and unspecific gastrointestinal and/or allergic clinical symptoms, which accounts for the high rate of misdiagnosed cases. Methodology/Principal findings The aim of this study was to characterize the early cellular (6-72 h p.i.) and molecular (6 h p.i.) immune response and general underlying regulatory mechanism in Anisakis infected rats. Each Sprague-Dawley rat was infected with 10 Anisakis spp. larvae by gastric intubation. Tissues with visible lesions were processed for: i) classic histopathology (HE), immunofluorescence (CD3, iNOS, S100A8/A9), and transmission electron microscopy (TEM) ; ii) target genes (Il1b, Il6, Il18, Ccl3, Icam1, Mmp9) and microRNA (Rat Immunopathology MIRN-104ZF plate, Quiagen) expression analysis ; and iii) global DNA methylation. Histopathology revealed that Anisakis larval migration caused moderate to extensive hemorrhages in submucosal and epimysial/perimysial connective tissue. In stomach and muscle, moderate to abundant mixed inflammatory infiltrate was present, dominated by neutrophils and macrophages, while only mild infiltration was seen in intestine. Lesions were characterized by the presence of CD3(+), iNOS(+), and S100A8/A9(+) cells. The greatest number of iNOS(+) and S100A8/A9(+) cells was seen in muscle. Il6, Il1b, and Ccl3 showed particularly strong expression in stomach and visceral adipose tissues, but the order of expression differed between tissues. In total, three miRNAs were differentially expressed, two in stomach (miRNA-451 and miRNA-223) and two in intestine (miRNA-451 and miRNA-672). No changes in global DNA methylation were observed in infected tissues relative to controls. Conclusions/Significance Anisakis infection induces strong immune responses in infected rats with marked induction of specific proinflammatory cytokines and miRNA expression. Deciphering the functional role of these cytokines and miRNAs will help in understanding the anisakiasis pathology and controversies surrounding Anisakis infection in humans. Author summary Anisakiasis is a zoonotic disease (infection transmitted between animals and humans) contracted by consumption of raw or undercooked seafood contaminated with Anisakis spp. nematode larvae. Anisakiasis usually presents with variable and unspecific gastrointestinal and/or allergic symptoms, which accounts for the high rate of misdiagnosed cases. Due to changes in dietary habits, such as eating raw or undercooked seafood, anisakiasis is considered an emerging public health problem. Despite the increase in number of reported cases worldwide, mechanisms of immune response to this unspecific human pathogen are poorly known. We have shown that in experimentally infected rats, Anisakis larvae cause severe hemorrhages and necrotic changes of affected tissues in the early phase of infections. Neutrophils and macrophages were abundantly present in tissue lesions, while eosinophils, hallmark of helminth infections, were scarcely present. We have also demonstrated particularly strong expression of several inflammatory genes. Moreover, we give for the first-time insight into putative regulatory mechanism mediated via a distinct class of RNA molecules. Our study may provide new opportunities for better understanding of cellular and molecular response to Anisakis spp., aiming at development of more specific therapeutics and alleviation of pathologies associated with Anisakis spp. infection.

Anisakis ; anisakiasis ; accidental host ; histopathology ; inflammation ; miRNA

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