engleski

Leukocyte nucleolus and Anisakis pegreffii: when falling apart actually is falling in place

Introduction: Ribosome biosynthesis (RB) is orchestrated by nucleolus whose main function is rapid production of small and large ribosome subunits. Nucleolus is also essential in the sensing of stress stimuli that target ribosome biogenesis ; a condition known as nucleolar or ribosomal stress. It is exhibited through expression of several p53-dependent or p53- independent response pathways that maintain cell homeostasis. Our aim was to characterise molecular mechanisms and architectural changes in the nucleolus of the Anisakis-stimulated rat peripheral blood leukocytes (PBLs). Methodology: In vitro A. pegreffii crude extract-stimulated Sprague-Dawley rat PBLs were harvested 1 and 12 h post-stimulation for RNA isolation. Illumina NextSeq 500 was used for paired-end sequencing of 7 pooled PBLs. Markers of ribosomal biogenesis (Tp53, CDKN1, CCND1, mTOR, RPL5, RPL11, RPL23, NFE2L2) and inflammation (Il4, Il6, Il7, TNF-α) were measured by qPCR, while nucleolar architecture was assessed by localisation of nucleophosmin, NOP58 and coilin by confocal microscopy. Results: Differential expression (DE) analysis of rat PBLs revealed 53 (22 down and 31 up-regulated) DE transcripts 1 h post- stimulation, whose strong and significant fold changes (LogFC >> 1) were especially noted for transcripts involved in regulation of protein complex assembly, membrane polarisation, mitotic cell cycle and calcium ion binding, to name only the few. In contrast, 39 (5 down and 34 up- regulated) DE transcripts were observed 12 h post-stimulation, mainly involved in proinflammatory functions. Strong scattering of nucleophosmin from nucleolus into the nucleus was evidenced, while nucleolus granular component and Cajal bodies remained mostly unaffected. Conclusion: Transcriptomics, targeted gene expression and confocal microscopy support the hypothesis that A. pegreffii CE in PBLs trigger the onset of nucleolus functional and morphological cues toward increased ribosomal biogenesis already 1 h post-stimulation, enriched in transcripts of 40S and 60S ribosomal proteins. This suggests that nucleolar rearrangements occur early and are soon balanced in order to provide an efficient proinflammatory response to the nematode.

Anisakis pegreffii, nucleolus, ribosomal proteins, inflammation

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