The Toll-like receptor gene family is integrated into human DNA damage and p53 networks

Abstract

In recent years the functions that the p53 tumor suppressor plays in human biology have been greatly extended beyond "guardian of the genome." Our studies of promoter response element sequences targeted by the p53 master regulatory transcription factor suggest a general role for this DNA damage and stress-responsive regulator in the control of human Toll-like receptor (TLR) gene expression. The TLR gene family mediates innate immunity to a wide variety of pathogenic threats through recognition of conserved pathogen-associated molecular motifs. Using primary human immune cells, we have examined expression of the entire TLR gene family following exposure to anti-cancer agents that induce the p53 network. Expression of all TLR genes, TLR1 to TLR10, in blood lymphocytes and alveolar macrophages from healthy volunteers can be induced by DNA metabolic stressors. However, there is considerable inter-individual variability. Most of the TLR genes respond to p53 via canonical as well as noncanonical promoter binding sites. Importantly, the integration of the TLR gene family into the p53 network is unique to primates, a recurrent theme raised for other gene families in our previous studies. Furthermore, a polymorphism in a TLR8 response element provides the first human example of a p53 target sequence specifically responsible for endogenous gene induction. These findings-demonstrating that the human innate immune system, including downstream induction of cytokines, can be modulated by DNA metabolic stress-have many implications for health and disease, as well as for understanding the evolution of damage and p53 responsive networks.

Figure 1. 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 p21^WAF1 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.

Figure 2. Inhibition of p53 activity by pifithrin-alpha dramatically reduces p53-dependent TLR induction by DNA damage and p53 activation.

The p53 inhibitor pifithrin-alpha (PFTα, 40 µM) or DMSO (control) were added to PHA-stimulated T-lymphocytes 2 h prior to Doxo (0.3 µg/mL), 5FU (300 µM) or nutlin (10 µM) exposure. Following 24 h of exposure, gene expression was assessed by qPCR. Presented is the mRNA fold-change compared to untreated cells for subjects BS#7 and BS#19. Each bar represents an average of 3 PCR replicates with its standard deviation.

Figure 3. Induced TLR expression in primary human T-lymphocytes obtained from the same subjects on different days.

TLR gene expression assessed by real time-PCR was measured in PHA-stimulated lymphocytes from 2 volunteers who were sampled twice, separated by 4 months, in a blind study as indicated in the figures (i.e., 12-2009 and 04-2010). The cells were exposed to DNA stressors or nutlin. Presented is the mRNA fold-change compared to untreated cells for patients BS#19 (A) and BS#20 (B).

Figure 4. p53 drives expression of TLR family through direct binding to regulatory regions.

(A) Functionality of presumptive p53 RE sequences associated with TLRs (sequences are described further in Table 2). H1299 cells were transfected with RE::luciferase reporter constructs in the presence (solid bars) or absence (open bars) of a vector expressing wild type p53. At 48 h post-transfection, induction of the luciferase reporter was compared with cells containing the pGL4.26 plasmid lacking p53. Presented are the average and standard deviations of 3 independent experiments. p53REs corresponding to TLRs 5, 6 and 8 contain SNPs as described in Table 2. (B) Occupancy of p53 at promoters of p21 and TLRs 2, 4, 5, 6, 8, 9 and 10 assessed by ChIP analysis of PHA-stimulated T-lymphocytes from subject BS#4 following 24 h of doxorubicin (0.3 µg/ml) treatment.

Figure 5. Nutlin induces TLR2 and TLR5 mRNA expression as well as proteins.

(A) Induction of TLR2 and 5 protein expression in stimulated lymphocytes cells from subjects BS25 and BS26 by nutlin (10 µM) after 24 hr post-treatment. Proteins were determined by western blot analysis using antibodies specific to TLR2 and TLR5 in the membrane fractions. Actin protein provided a loading control. (While there is little change in the amount of actin protein detected, there may be differences in the nonspecific bands.) (B) Induction of TLR2 and 5 mRNA expression in stimulated lymphocytes from subjects BS25 and BS26 (data obtained from Figure 1) by nutlin.

Figure 6. Induced expression of TLR gene family in human alveolar macrophages by DNA stressors and activation of the p53 pathway.

Alveolar macrophages obtained by bronchoalveolar lavage of normal, healthy human subjects were incubated for 24 h in the presence of (A) nutlin or (B) Doxo. TLR gene expression was analyzed by qPCR and is presented as fold-change compared to untreated cells. Horizontal bars correspond to the median. (C) Activation of p53 pathway (p53 and p21) in alveolar macrophages following 24 h of incubation with nutlin or Doxo as analyzed by Western blots.

Figure 7. Induction of p53 sensitizes freshly isolated CD3+ cells to PAMP stimulation.

(A) TLR2 and cytokine (IL-1 and IL-8) expression in CD3+ lymphocytes incubated for 20 h with nutlin (10 µM) or DMSO and then exposed for 4 h to the TLR2 ligand PAM3CSK4 (1 µg/ml). Presented are representative results for subject BS#37. Gene expression was analyzed by qPCR and presented as fold-change compared to untreated cells. (B) Activation of p53 pathway by 24 h of nutlin treatment in CD3+ cells isolated from peripheral blood as determined by Western blot analysis.

Figure 8. Ability of p53 to drive TLR8 expression in primary human cells depends on SNP in p53 response element of TLR8 promoter.

The regulatory region of TLR8 was amplified by PCR and the product was analyzed for the presence of the A and/or the G version of the p53 RE. Presented are SNP genotypes and the expression levels of TLR8 following 24 h of treatment with (A) nutlin, (B) IR, (C) Doxo and (D) 5FU. Since the gene is X-linked, males have one copy and females have two copies of the allele. Presented are results with stimulated lymphocytes (black symbols) or alveolar macrophages (red symbols).

Figure 9. Model describing expression loop between p53 and the TLR gene family in response to chromosome and inflammation stresses.

DNA metabolic stress is induced by both environmental factors and endogenous sources, such as host-derived reactive oxygen species (ROS) generated downstream of Toll-like Receptors and pro-inflammatory cytokines. DNA damage/stress activates p53-dependent and independent (“X”) pathways that in turn induce expression of TLRs. Increased TLR expression sensitizes the cell to both exogenous, pathogen-associated molecular patterns (PAMPs) and endogenous, damage-associated molecular patterns (DAMPs) released during tissue injury which, in this loop, lead to further ROS.