Involvement of the P2X7-NLRP3 axis in leukemic cell proliferation and death

Abstract

Lymphocyte growth and differentiation are modulated by extracellular nucleotides and P2 receptors. We previously showed that the P2X7 receptor (P2X7R or P2RX7) is overexpressed in circulating lymphocytes from chronic lymphocytic leukemia (CLL) patients. In the present study we investigated the P2X7R/NLRP3 inflammasome axis in lymphocytes from a cohort of 23 CLL patients. P2X7R, ASC and NLRP3 were investigated by Western blot, PCR and transfection techniques. P2X7R was overexpressed and correlated with chromosome 12 trisomy in CLL patients. ASC mRNA and protein were also overexpressed. On the contrary, NLRP3 was dramatically down-modulated in CLL lymphocytes relative to lymphocytes from healthy donors. To further investigate the correlation between P2X7R, NLRP3 and cell growth, NLRP3 was silenced in THP-1 cells, a leukemic cell line that natively expresses both NLRP3 and P2X7R. NLRP3 silencing enhanced P2X7R expression and promoted growth. On the contrary, NLRP3 overexpression caused accelerated apoptosis. The P2X7R was also up-modulated in hematopoietic cells from NLRP3-KO mice. In conclusion, we show that NLRP3 down-modulation stimulates P2X7R expression and promotes growth, while NLRP3 overexpression inhibits cell proliferation and stimulates apoptosis. These findings suggest that NLRP3 is a negative regulator of growth and point to a role of the P2X7R/NLRP3 axis in CLL.

Figure 1. P2X7R expression in CLL lymphocytes.

(a) P2X7R mRNA level was evaluated by Real-Time PCR as described in Methods. P2X7R expression was normalized on G3PDH internal control and displayed as fold increase. Data are shown as mean ± SEM, n = 3, ***p < 0.001. (b) Representative Western blot of P2X7R from three subjects representative of each population. (c) Densitometry of P2X7R normalized on actin level (AU, arbitrary Units). N = 3, ***p < 0.001. (d) P2X7R mRNA expression in lymphocytes from CLL patients with or without chromosome 12 trisomy; **p < 0.01. (e) IL-1β release (see Methods) was evaluated by ELISA from CLL lymphocytes and HD PBMCs. Cells were overnight (12 h) incubated in the presence of LPS. Data are averages ± SEM, *p < 0.05; ***p < 0.001.

Figure 2. ASC and NLRP3 expression in CLL lymphocytes.

Peripheral CLL lymphocytes and B lymphocytes and PBMCs from healthy donors were isolated and qRT-PCR and Western blot analysis performed as described in Methods. ASC (a–c) and NLRP3 (d–f) protein (b,c,e,f) and mRNA expression (a,d) from lymphocytes from 23 CLL patients (see Table 1), PBMCs from 12 HDs, and B lymphocytes from 5 HDs is shown. Representative Western blot (b,e)and densitometry (c,f) of CLL lymphocytes, HD PBMC and HD peripheral blood B lymphocytes from three subjects (determinations from each subject in triplicate) representative of each population are shown. Data are averages ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3. NLRP3 silencing in THP-1 cells triggers P2X7R up-regulation and stimulates growth.

THP-1 cells were cultured and analyzed for NLRP3 mRNA (a) and protein (b,c) expression as described in Methods. NLRP3 (a–c) was silenced by HuSH-29 transfection and selected with puromycin as described in Methods. Densitometry of NLRP3 protein bands is shown in panel (c) (n = 3). Up-regulation of P2X7R protein in NLRP3-silenced THP-1 cells (d–e). Densitometry of P2X7R protein bands is shown in panel (e) (n = 3). For cell proliferation, 5 × 10⁴ cells were plated in 6-well plates in complete RPMI 1640 medium in a 5% CO₂ incubator (37 °C) and counted (f) or analyzed by MTT assay (g) at the indicated time points. Data from triplicate determinations are shown in panels (a,c,e–g). Data are averages ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4. NLRP3 overexpression in THP-1 cells triggers cell death.

THP-1 cells were transfected with a plasmid encoding NLRP3 as described in Methods, and then analyzed for NLRP3 mRNA and protein expression (a,b). Empty vector (mock)-transfected cells and a HEK293 clone stably transfected with NLRP3 (HEK293 NLRP3) are shown as control. Apoptotic cell number (annexin V-positive cells) is shown in panel (c). Caspase-3 and -9 expression is shown in panels (d,e). Effect of NLRP3 transfection on caspase-3 activation (cleavage) is shown in panels (f,g). Down-modulation of P2X7R protein by NLRP3 overexpression is shown in panels (h,i). Data are averages ± SEM from triplicate independent determinations. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5. NLRP3 overexpression in Ramos cells triggers cell death.

Ramos cells were transfected with a plasmid encoding NLRP3 as described in Methods, and then analyzed for NLRP3 mRNA and protein expression (a,b) Empty vector (mock)-transfected cells and a HEK293 clone stably transfected with NLRP3(HEK293NLRP3) are shown as control. Effect of NLRP3 over- expression on caspase-3 mRNA (c) and protein (e) expression and caspase-3 activation (d) is shown. Effect of NLRP3 transfection on caspase-9 expression (f) and apoptotic cell number (annexin V-positive cells) is shown in panel (g). Data are averages ± SEM from 3 to 4 independent determinations. *p < 0.05; ***p < 0.001.

Figure 6. NLRP3 overexpression inhibits proliferation of HEK293 cells.

HEK293 cells were transfected with a NLRP3-encoding plasmid as described in Methods, cultured in complete DMEM-F12 medium and assayed for proliferation. Panels (a,b) show level of mRNA and protein expression, respectively, in mock- and NLRP3-transfected cells. THP-1 cells (a) are shown as a control for baseline NLRP3 expression. Panel (c) shows growth kinetics of NLRP3- and mock-transfected HEK293 cells, respectively. **p < 0.01. Morphology of mock- (d) and NLRP3-transfected (e) HEK293 cells. **p < 0.01 Bar = 40 μm.

Figure 7. P2X7R expression in tissues and cells from Nlrp3^−/− mouse.

NLRP3 mRNA expression in bone marrow-derived macrophages from wt and Nlrp3^−/− mice (a). P2X7R mRNA expression in MEF (b), liver (c), macrophages (d), bone marrow (e) and spleen (f) from wt and Nlrp3^−/− mice. P2X7R protein expression in spleen (g) and MEF (h) from wt and Nlrp3^−/− mice. Growth rate of MEFs from wt and Nlrp3^−/− mice (i). Cell were isolated and processed as described in Methods. Data are averages ± SEM from triplicate independent determinations. *p < 0.05; **p < 0.01; ***p < 0.001.