#5607 EFFECTS OF COMMON OSMOLYTES ON THE EXPRESSION OF FOXP3 SPLICE VARIANTS IN HUMAN PBMCS
Authors: Luo Zhidan , Saleh Qais , Tepel Martin 
Published: June 2023

Abstract

Background and Aims
Regulatory T cells modulate autoimmune and allogenic responses in glomerulonephritis and transplantation. The two most abundant fork-head box P3 (FOXP3) splice variants, full-length FOXP3 (FOXP3-FL) and FOXP3 lacking exon 2 (FOXP3-Δ 2), confer suppressive ability to regulatory T cells. Some studies showed a weaker suppressive ability of FOXP3-Δ 2. Renal organic osmolytes, urea and Trimethylamine N-oxide (TMAO), play critical roles in the hyperosmotic renal environment with high concentrations of Na+ and glucose. However, the effects of these osmolytes on FOXP3 splice variants in peripheral blood mononuclear cells (PBMCs) remain unclear. The aim of this study was to evaluate the effects of common osmolytes on the expression of FOXP3 splice variants in human PBMCs.

Method
PBMCs were isolated from the blood of healthy donors using Ficoll density gradients and cultured in RPMI-1640 with or without additional osmolytes at 37°C. A part of PBMCs were activated with 25ng/ml Phorbol-12-myristate-13-acetate (PMA) and 1ug/ml ionomycin (PMA+IONO). FOXP3-FL and FOXP3-Δ 2 mRNA were detected by qPCR after 4hrs’ incubation, using specific primers. The relative expression value was calculated by the formula of (1+E0)∧ Ct0(β-actin)/(1+E)∧ Ct (target gene). Values are given as the mean±S.E.M. Statistical significance was determined by One-Way ANOVA with Post Hoc Test, using Graphpad Prism 9. A P-value &lt;0.05 was considered significant.

Results
The cell viabilities after 4 hours’ incubation were all above 90%. PMA+IONO significantly increased the relative expression of both FOXP3-Δ 2 (from 0.001051 to 0.001609, P = 0.0289, n = 5) and FOXP3-FL (from 0.001024 to 0.001684, P = 0.0101, n = 5) (Figure 1). However, increased concentrations of urea (from 5mM to 50mM) had no effect on either of the two FOXP3 splice variants (P &gt; 0.05, n = 5, Figure 1). TMAO of 4um significantly reduced FOXP3-FL in the PMA+IONO-activated PBMCs from 0.001605 to 0.0007208 (P = 0.0051, Figure 2). However, TMAO didn’t affect FOXP3-FL expression in non-activated PBMCs and had little effect on FOXP3-Δ 2 expression either (P &gt; 0.05, n = 4). On the other hand, compared with control group, the high concentration of glucose (25mM) significantly increased the expression of FOXP3-Δ 2 from 0.002815 to 0.003461 (P = 0.0375, n = 3) in non-activated PBMCs (Figure 2). The high concentration of NaCl (147mM) had no significant effect on either of the FOXP3 splice variants (P &gt; 0.05, n = 3, Figure 2). Furthermore, the equal osmolarity of mannitol didn’t affect the mRNA expressions of FOXP3 splice variants, either (P &gt; 0.05, n = 3, Figure 2). But compared with glucose group (0.003461), the expression of FOXP3-Δ 2 was significantly lower in mannitol group (0.002785) (P = 0.0479, n = 3, Figure 2). It suggested that the effect of high glucose in non-activated PBMCs was independent of the osmolarity change.

Conclusion
In isolated human PBMCs, PMA+IONO increases both FOXP3-Δ 2 and FOXP3-FL expressions. PMA+IONO-induced FOXP3-FL increase can be inhibited by TMAO. Urea and NaCl had little effect on FOXP3 splice expressions. High concentration of glucose increases FOXP3-Δ 2 mRNA in non-activated PBMCs, which was independent of the osmolarity change.