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Methylation on RNA: A Potential Mechanism Related to Immune Priming within But Not across Generations - PubMed

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Methylation on RNA: A Potential Mechanism Related to Immune Priming within But Not across Generations

Cynthia Castro-Vargas et al. Front Microbiol. 2017.

Abstract

Invertebrate immune priming is a growing field in immunology. This phenomenon refers to the ability of invertebrates to generate a more vigorous immune response to a second encounter with a specific pathogen and can occur within and across generations. Although the precise mechanism has not been elucidated, it has been suggested that methylation of DNA is a cornerstone for this phenomenon. Here, using a novel method of analytical chemistry (a reversed-phase liquid chromatography procedure) and the beetle Tenebrio molitor as a model system, we did not find evidence to support this hypothesis taking into account the percentage of methylated cytosine entities in DNA (5mdC) within or across generations. However, we found a lower percentage of methylated cytosine entities in RNA (5mC) within but not across generations in immune priming experiments with adults against the bacteria Micrococcus lysodeikticus and larvae against the fungus Metarhizium anisopliae. To our knowledge, this is the first report suggesting a role of differential methylation on RNA during immune priming within generations.

Keywords: DNA; RNA; Tenebrio molitor; epigenetics; high-performance liquid chromatography; immune priming; innate immune memory; methylation.

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Figures

FIGURE 1
FIGURE 1

Schematic representation of the experimental design. Image courtesy of Ángela Arita.

FIGURE 2
FIGURE 2

Larvae of T. molitor infected with Metarhizium anisopliae strains Ma10mCherry (A) or Ma10GFP (B). Figure shows the larvae cuticle covered by the fungus. Both strains contain the same genotype except for the overexpression of mCherry or Green Fluorescent Protein (GFP) under the GPDa promoter, respectively. Images were acquired using a Nikon Optiphot-2 microscope with a SPOT RT- Color camera and SPOT 4–6 software (Diagnostic instruments). GFP and mCherry fluorescence was detected using the recommended filters (Chroma Technology). Larvae of dual challenges did not show evidence of hyphae growing on its body 9 days after the first challenge, just before inoculation with 200 conidia of Ma10GFP.

FIGURE 3
FIGURE 3

Typical chromatograms of immune priming within generations obtained from one Control insect (dashed line) and one Priming insect (solid line). The full-scale mode represents (A) one insect per treatment from the experiment of immune priming within generations; (B) one insect per treatment from the experiment of immune priming across generations. C, cytidine; dC, 2′-deoxycytidine; 5mC, 5-methylcytidine.

FIGURE 4
FIGURE 4

Percentage of females (A) or males (B) that survived according to treatment 24 h after the second challenge. Sample sizes are shown in the text. Taking into account the females (A), after 24 h of the second challenge it was more likely that PBS (live females: 96%, dead females: 4%, n = 92) and Priming group survived (live females: 75%, dead females: 25%, n = 95) than the Control group (live females: 24%, dead females: 76%, n = 92; X2 = 68.34, p < 0.0001). A similar result was found in males (B), after 24 h of the second challenge it was more likely that PBS (live males: 93%, dead males: 7%, n = 95) and Priming group (live males: 67%, dead males: 33%, n = 51) survived than males of the Control group (live males: 44%, dead males: 56%, n = 112; X2 = 43.48, p < 0.0001).

FIGURE 5
FIGURE 5

Levels of RNA Methylation by treatment within generations in Tenebrio molitor. (A) Adult primed insects (n = 25) had a lower percentage of methylation than did Control animals (n = 20) when challenged with M. lysodeikticus, as measured 24 h after the second challenge. (B) Primed larvae (n = 19) had a lower percentage of methylation than did Control animals (n = 18) when challenged with M. anisopliae, as measured 24 h after the second challenge. The means ± standard errors of the percentages of methylation among treatments are shown.

FIGURE 6
FIGURE 6

RNA methylation for treatments and time periods in larvae against M. anisopliae. Tissue was extracted at 4 (Control n = 20 and Priming = 20), 12 (Control n = 23 and Priming = 19), 48 (Control n = 19 and Priming = 20) and 96 h (Control n = 20 and Priming = 20). The means ± standard errors of the percentage methylation are shown.

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