P38 MAPK/AKT signalling is involved in IL-33-mediated anti-apoptosis in childhood acute lymphoblastic leukaemia blast cells

Abstract

Background: Acute lymphoblastic leukaemia (ALL) is often characterized by broad clinical and biological heterogeneity, as well as recurrent genetic aberrations. Despite remarkable improvements in the treatment outcome in paediatric ALL over the past several decades, it remains a leading cause of morbidity and mortality among children. Cytokines have been extensively studied in haematologic diseases; however, the mechanisms by which cytokines contribute to ALL pathogenesis remain poorly understood.

Methods: IL-33 levels were measured by enzyme-linked immunosorbent assay (ELISA). IL1RL1 expression on ALL cell surface was accessed by flow cytometry. Expression of phosphorylated p38 MAPK, p38, pAKT, AKT and GAPDH were quantified by western blot. Cell survival signals were evaluated by apoptosis using flow cytometry.

Results: BM samples from ALL patients at diagnosis upregulated their cell surface expression of IL1RL1, and a higher interleukin (IL)-33 level in the serum was observed as compared to the healthy individuals. Moreover, exogenous IL-33 treatment significantly inhibited apoptosis by activating p38 mitogen-activated protein kinase (MAPK) and AKT pathway, while the inhibitor for p38 MAPK, SB203580, counteracted IL-33-induced anti-apoptosis via inactivation of p38 MAPK and AKT. Furthermore, IL-33 negatively regulates cyclin B1 protein level while increasing the expression of CDK1, with SB203580 inhibiting the effect.

Conclusion: Our study reveals an important role for IL-33/IL1RL1 axis in supporting ALL which may represent a novel treatment for paediatric patients.KEY MESSAGESBoth IL-33 and IL1RL1 levels are upregulated in primary ALL samples.IL-33 increased both p38 MAPK and AKT activation in ALL.IL-33 promotes survival and cell cycle progression of ALL cells via activating p38 MAPK.

Keywords: AKT; ALL; IL-33; IL1RL1; P38 MAPK; anti-apoptosis.

Figure 1.

Primary ALL cells upregulate IL1RL1 cell-surface expression and serum IL-33 level. (A-B) IL1RL1 expression is increased on the cell surface of BM cells from ALL cohorts. BM cells from patients with ALL and HD were analysed for IL1RL1 expression by flow cytometry. (A) Histograms show two representative samples for each group of ALL patients (n = 10) or HD (n = 5). (B) IL1RL1 expression on primary ALL cells expressed as relative mean fluorescence intensity (MFI stained/MFI unstained). Each symbol represents one HD or patient with ALL. Paired t test. (C) ELISA assay was used to measure IL-33 levels in serum from ALL patients at diagnosis (n = 15) and HD (n = 5). Each symbol represents one HD or patient with ALL. Paired t test. *p < .05; **p < .01.

Figure 2.

IL-33 promotes cell survival by activating p38 MAPK. (A) BM cells from ALL patients at initial diagnosis were incubated for 72 h with IL-33 (100 ng/mL) or SB203580 (SB, 20 uM) alone, or in combination. Cell lysates from each treatment group were prepared. P-p38, p38, or GAPDH protein expression was probed by western blot analysis. (B) The bar graph shows the relative quantification of p-p38 protein in all groups as compared to the untreated cells. One-way ANOVA with Tukey post hoc test. (C) Cellular viability was measured by DAPI staining. Bar graph shows the relative cell viability as compared to the untreated cells. One-way ANOVA with Tukey post hoc test. (D) Apoptosis was measured by Annexin V staining. Bar graph shows the relative Annexin V staining of leukaemia cells as compared to the untreated cells. One-way ANOVA with Tukey post hoc test. n = 4; *p < .05; **p < .01.

Figure 3.

IL-33 regulates cell cycle progression via p38 MAPK signalling. BM cells from ALL patients were cultured with IL-33 (100 ng/mL), or combined with SB (20 uM) for 72 h and analysed for cell cycle status by flow cytometry. (A) Representative histograms show the cell cycle analysis of primary ALL cells from each treatment. (B) Bar graph shows the relative percentage of leukaemia cells in the indicated phase of the cell cycle. One-way ANOVA with Tukey post hoc test. n = 4; *p < .05.

Figure 4.

IL-33 modulates protein expression related to cell cycle via p38 MAPK/AKT. (A) BM cells from ALL patients were cultured with IL-33 (100 ng/mL), or combined with SB (20 uM) for 72 h. Cell lysates from each treatment group were prepared. CDK1, cyclin B1, AKT, pAKT (Ser473) or GAPDH protein expression was probed by western blot analysis. (B) The bar graphs show the quantification of pAKT, CDK1, and cyclin B1 protein in all groups. One-way ANOVA with Tukey post hoc test. n = 3; *p < .05; **p < .01.

Figure 5.

Proposed mechanism of IL33/IL1RL1 axis in mediating ALL cell survival by activating p38 MAPK.