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Immune response of sea bass peripheral blood leukocytes to Anisakis pegreffii crude extract: what does RNA-seq reveal?

Introduction: Nematodes of the genus Anisakis Dujardin, 1845 have an indirect life cycle and their life-stages are propagated through trophic webs of their marine hosts. About 200 marine fish species and 25 cephalopod species are confirmed as Anisakis spp. paratenic or secondary intermediate hosts. Previous research suggested that excretory/secretory Anisakis simplex products could have immunomodulatory role in fish hosts. The aim of this study was to assess the immunogenic potential of Anisakis pegreffii crude extract (CE) in European sea bass (Dicentrarchus labrax) peripheral blood leukocytes (PBLs). Methodology: The experiment was performed on in vitro stimulated PBLs of sea bass, prepared using 51% iso-osmotic Percoll solution, seeded in 6-well plates with L-15 medium, supplemented with 15% FCS and 1% penicillin/streptomycin. Two hours (h) after settlement of PBLs (final concentration of 107 cells/ml), 5 μg/ml of previously prepared A. pegreffii CE was added to test wells. In total, 18 replicates were collected 1 and 12 h post-stimulation and preserved for RNA isolation, including control wells. Illumina NextSeq 500 was used for paired-end sequencing of total of 7 pooled PBLs samples prepared from test and control treatments. Results: After quality control, filtering and mapping, differential expression (DE) analysis of sea bass PBLs revealed 195 (60 down and 135 up- regulated) DE transcripts 1 h post-stimulation and 968 (206 down and 762 up-regulated) DE transcripts 12 h post-stimulation with A. pegreffii CE. Strong and significant fold changes (LogFC >> 1), with steady up-regulation as the experiment progressed, was especially noted for transcripts of pro-inflammatory cytokines (interleukin-1 beta and tumor necrosis factor alpha), as well as interleukin-27 subunit beta, known for its diverse roles in innate immunity. Conclusion: Overall, the most frequent gene ontology terms associated with DE transcripts were positive regulation of cell proliferation, response to stimulus, immune response, and chemokine activity. These results help explain the nature of interaction between sea bass PBLs and A. pegreffii CE.

European sea bass, in vitro, PBLs, total protein, nematode

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