Chapter 4: Does activation of PBMCs change transcriptional regulation of the genes encoding
4.3.1 Relationship between CD69 expression and mRNA expression
The relationship between the proportion of CD69 positive events measured in the PBMC gate by flow cytometry and relative mRNA expression were analysed by Spearman’s rank-order correlation for the total EPUFA population (Figure 4.3). There were significant positive correlations between CD69 expression and the mRNA expression of FADS2 (rs = 0.354, P < 0.01), FADS1 (rs = 0.470, P = < 0.0001) and ELOVL5 (rs = 0.348, P < 0.01).
Figure 4.3. Relationship between CD69 expression and FADS2, FADS1 and ELOVL5 mRNA expression.
The relationships between the proportion of CD69 positive events (% CD69+) measured within a PBMC gate and the relative mRNA expression of FADS2 (n = 67), FADS1 (n = 66) and ELOVL5 (n = 66) were assessed by Spearman’s rank-order correlation, where rs is the Spearman’s Correlation
4.4 Discussion
Activation of PBMCs by Con A significantly increased the mRNA expression of three genes (FADS2,
FADS1 and ELOVL5) encoding enzymes involved in conversion of 18:3n-3 to longer chain
metabolites, compared with untreated PBMCs. In Chapter 3, it was shown that the synthesis of 20:3n-3, 20:4n-3, 20:5n-3 and 22:5n-3 was also significantly increased in Con A activated cells. These findings suggest that changes in the mRNA expression of these genes affects the capacity of cells for n-3 PUFA synthesis. This is in agreement with other studies that have shown that increased activity of this PUFA biosynthesis pathway is associated with increased mRNA expression of the genes encoding the enzymes that operate in the pathway (105, 213). Analysis of the proportion of cells within the PBMC gate expressing the cell surface activation marker CD69 revealed significant positive correlations between CD69 expression and the mRNA expression of FADS2, FADS1, and
ELOVL5. This suggests that the level of transcription relates to the level of cell activation and
complements the positive relationships observed between the proportion of cells expressing CD69 and the levels of synthesised n-3 PUFAs. Taken together, the present findings suggest that the effects of Con A activation on n-3 PUFA synthesis are mediated at the transcriptional level. Consistent with the current findings, upregulation of FADS2 mRNA expression in activated lymphocytes has been demonstrated previously. Antibody induced activation of cultured CD4+ T- cells increased FADS2 mRNA expression compared with naïve CD4+ T-cells, which was shown to be controlled by direct binding of the transcription factor SREBP1 activated through TCR stimulation and MTOR-mediated signalling (146). However, in contrast to the present study, Angela et al., 2016 found no increase in ELOVL5 mRNA expression. It is presumed that the present analysis should be most representative of T-lymphocyte activation since the majority of PBMCs are T-lymphocytes and Con A primarily activates this lymphocyte subset. However, the difference in findings could be explained by ELOVL5 mRNA expression changing in a different cell type or lymphocyte sub- population present in PBMCs. PBMCs are a heterogeneous population of cells and therefore measured gene expression profiles are not resolved to an individual cell population; a limitation when interpreting gene expression results as it is not known which cell types are the source of detected differences.
Chapter 3 reported no measurable fatty acid peak for 18:4n-3, the delta-6 desaturase conversion product of 18:3n-3 and the most highly synthesised n-3 PUFA was 20:3n-3, which is the immediate elongation product of 18:3n-3. Together, this suggests that PBMCs may use the alternative delta-8 desaturation pathway as the primary route to longer chain n-3 PUFA synthesis. However, FADS2 mRNA was expressed in PBMCs and increased in activated cells. This suggests conversion of 18:3n- 3 to 18:4n-3 could have been operating but the presence of FADS2 protein would need to be
verified by Western blotting. Inspection of raw Ct values suggested that ELOVL5 (mean Ct 23.53), was more highly expressed than FADS2 (mean Ct 26.86); based on the principle that the lower the RT-PCR template amount, the more amplification cycles are needed to reach the threshold fluorescence. However, this should be interpreted with caution as the amplification efficiency was not identical for the FADS2 (96%) and ELOVL5 (98%). In a previous study, expression of human
ELOVL5 in S. cervisiae revealed that it is capable of elongating 18:3n-3 to 20:3n-3 (26). Therefore,
since in the present study both ELOVL5 expression and 20:3n-3 synthesis were elevated, it is speculated that elongation of 18:3n-3 to 20:3n-3 in PBMCs is catalysed by elongase-5. Furthermore, potential competition between elongase-5 and delta-6 desaturase for the substrate 18:3n-3 may explain the predominance of the alternative delta-8 desaturation pathway. However, the involvement of elongase-5 in 20:3n-3 synthesis in PBMCs would need confirming, which would be possible by using siRNA to knock down ELOVL5 expression.
ELOVL2 mRNA expression was below the quantitation limit of the assay for all the unstimulated
samples and was only quantifiable in four of the 34 stimulated samples. Furthermore, no amplification of ELOVL2 could be detected in 21 of the unstimulated compared with six of the Con A stimulated PBMC samples. This indicates that the mRNA expression of ELOVL2 is either absent or very low in both unstimulated and stimulated PBMCs. The observation that amplification of ELOVL2 was detectable in a greater number of stimulated compared with unstimulated samples suggests some upregulation of ELOVL2 in stimulated cells may occur. However, the biological importance of very low copy numbers of ELOVL2 mRNA is not known. Elongase-2 has been shown to be involved in the conversion of 22:5n-3 to 22:6n-3 by elongation of 22:5n-3 to the 24 carbon intermediate 24:5n-3 (27). Consequently, the minimal expression of ELOVL2 mRNA could explain the lack of 22:6n-3 synthesis observed in PBMCs. However, conversion of 22:5n-3 to 22:6n-3 involves multiple steps including desaturation at the delta-6 position, translocation to peroxisomes and one cycle of β-oxidation, after which 22:6n-3 may be further degraded or moved back to the endoplasmic reticulum for membrane biosynthesis (4). Therefore, there are additional steps that could act as control points in the synthesis of 22:6n-3 and incubation of PBMCs with the pathway intermediates downstream of elongation (24:5n-3) and desaturation (24:6n-3) could help to verify the mechanism responsible. Finally, it has been shown that conversion of 20:5n-3 to 22:5n-3 can be catalysed by both elongase-2 and elongase-5 enzymes (29) but the minimal expression of ELOVL2 mRNA suggests that ELOVL5 may be more important for the conversion of 20:5n-3 to 22:5n-3 in activated PBMCs.
In summary, Con A mediated activation of PBMCs significantly increased the mRNA expression of
with the data in Chapter 3, the findings from this chapter suggest that Con A mediated upregulation of n-3 PUFA synthesis is controlled at the transcriptional level. Elucidation of the molecular mechanisms controlling the transcription of these genes would be important for understanding the regulation of n-3 PUFA synthesis in activated PBMCs.