Figure 2: HTLV-1-induced LTB4 secretion depends on Tax-induced cPLA2γ overexpression.

(a) Tax-expressing Jurkat cells secrete significant levels of LTB4. Jurkat cells were transduced with lentiviral vectors encoding GFP alone or together with the viral regulatory proteins Tax and HBZ. Protein expression was detected by western blot 48 h post transduction (right panel). Spontaneous LTB4 secretion levels were determined by ELISA. P value <10⁻³; n=4; mean±s.e.m.; ANOVA, Tukey’s post hoc test. (b) Tax-induced LTB4 secretion is dependent on group 4 phospholipases A2 (PLA2G4). Tax-expressing Jurkat cells were treated with either 1 μM MAFP or 5 μM BEL for 30 min. Spontaneous LTB4 secretion was detected by ELISA. P value=0.019; n=4; mean±s.e.m.; ANOVA, Tukey’s post hoc test. (c) Pla2g4c transcript levels are increased in Tax-expressing Jurkat cells. Expression levels of pla2g4a (encoding cPLA2α) and pla2g4c (encoding cPLA2γ) were determined by RT–qPCR on Jurkat cells transduced with GFP, Tax or HBZ. mRNA levels were normalized to gapdh expression and gene expression in Jurkat cells. P value=2.10⁻³; n=3; mean±s.e.m.; ANOVA test, Tukey’s post hoc test. (d) Pla2g4c mRNA levels are higher on CD4 T cells from HTLV-1-infected individuals when compared to HDs. CD4 T cells from HDs (n=7), HACs (n=5) and HAM/TSP (n=5) were isolated by negative selection. RT–qPCR for pla2g4a and pla2g4c was performed. Values were normalized to gapdh expression and the mean value on CD4 T cells from HDs. P value=0.007; mean±s.e.m.; Mann–Whitney U-test. (e) Pla2g4c KD counters Tax-induced LTB4 secretion. Jurkat cells were transduced with vectors encoding a shRNA-targeting pla2g4c (or a control sh-ctrl). Cells were then transduced with Tax. KD efficiency was determined by RT–qPCR (normalized to gapdh expression and pla2g4c in Jurkat cells) (left panel). LTB4 secretion was detected by ELISA. P value=0.03; n=3; mean±s.e.m.; Friedman test, Dunn’s post hoc test.