Induced expression of the TLR gene family in primary human T-lymphocytes by DNA stressors and activation of the p53 pathway in cells from healthy subjects.
Human peripheral blood mononuclear cells (PBMC) freshly isolated from healthy subjects (n = 17–18) were incubated with PHA to stimulate T-lymphocyte expansion. After 48 h incubation, cells were exposed to (A) ionizing radiation (IR); (B) 5-fluorouracil (5FU); (C) doxorubicin or (D) nutlin. Cells were harvested following 24 h treatment in the presence of PHA. In the case of IR, they were harvested 24 h after exposure to 4 Gy. (Robust responses were observed under these conditions; however, further studies might reveal more optimum times and dose for primary cells.) Presented in panels A to D are mRNA expression levels of TLRs and p21WAF1 as compared to expression in untreated cells for each subject (the dashed red line corresponds to no change with treatment, i.e., a value of 1; horizontal bars correspond to the median). Gene expression was analyzed by qPCR and normalized to 18S ribosomal RNA. Statistical analysis in A–D. Unless noted, the TLR genes of the population as a whole exhibited statistically significant change in expression induced by an agent (vs. untreated) at the p<0.006 level (see text). Black arrows indicate changes that are not significantly different from the control for specific TLR genes in the population (p≥0.05). (E) Changes in TLR expression after IR of cells from all subjects (BS#) presented as a heat map. The subjects are grouped according to IR responsiveness (i.e., purple, for nearly all TLRs being inducible and blue for people with only a few TLRs induced by IR). (F) p53 and p21 activation analyzed by Western blot in a representative experiment for subject BS#7 with actin as a loading control. (G) Inhibition of IR-induced TLR gene expression by the p53 inhibitor pifithrin-α (PFTα, 40 µM) added to cells 2 h prior to IR and kept there after exposure. Values are relative to cells treated with DMSO represented as the dash red line, i.e., a value of 1. Presented is a representative experiment with T-lymphocytes from subject BS#19.
