Galectin-9 induces IL-1β production as a key inflammatory cytokine in the acute myeloid leukemia cell line (U937)

Abstract

Background and purpose: T-cell immunoglobulin and mucin-domain containing protein-3 (TIM-3)/ galectin-9 (Gal-9)/ autocrine loop in myeloid leukemia stem cells provokes inflammation through the NF-κB signaling pathway, which is influential in the expression of inflammatory factors. Interleukin 1β (IL-1β) is a vital inflammatory cytokine that plays an important role in the proliferation and therapy resistance of acute myeloid leukemia (AML) cells. This study aimed to assess the effect of Gal-9 on IL-1β in the human leukemic U937 cell line.

Experimental approach: The U937 cells were cultured in different concentrations of Gal-9. Cell counting kit-8 was used to assess the effect of Gal-9 on human leukemic U937 cell proliferation. Also, its impact on the expression of TIM-3, Gal-9, IL-1β, IL-1βR, IL-1βRAP, and NLRP3 genes and IL-1β protein was studied by RT-PCR and ELISA, respectively. Moreover, the effect of Gal-9 on the NF-κB signaling pathway was evaluated by western blotting.

Findings/results: U937 cells were expanded in the presence of Gal-9 in a concentration-dependent manner. Following treatment of U937 cells with Gal-9, the gene expression of Gal-9, IL-1B, IL-1BR, and IL-1BRAP were significantly upregulated compared to the control group. The IL-1β concentration increased following Gal-9 treatment in a concentration-dependent manner, while following time-pass its level significantly decreased. Furthermore, Gal-9 slightly increased NF-κB phosphorylation.

Conclusion and implications: Gal-9 increased IL-1β level as a critical inflammatory cytokine in the proliferation and resistance of AML cells to therapy. According to this finding, targeting and blocking the TIM-3/Gal-9 autocrine loop can suppress IL-1β production and facilitate AML treatment.

Keywords: AML; GAL-9; IL-1β; Inflammation; NF-κB.

Fig. 1

The effect of Gal-9 on the proliferation of U937 cells. U937 cells were treated with various concentrations of rhGal-9 and the proliferation of the cells was measured by CCK-8 after 24 h. The data were achieved through two independent experiments; each was performed in triplicate. Data are represented as the mean ± SD. ^**P < 0.01 indicates significant differences compared to the control group. rhGal-9, recombinant human galectin-9; CCK-8, Cell-counting Kit-8.

Fig. 2

The effect of (A and B) Gal-9 alone or (C) in combination with LPS on TIM-3 and Gal-9 gene expression of U937 cells. Different concentrations of rhGal-9 alone or in combination with LPS were added to U937 cells and after 24 h incubation, the TIM-3 and Gal-9 gene expression were studied by RT-PCR. The data were achieved through two independent experiments, each performed in triplicate. Data are represented as the mean ± SD. ^**P < 0.01 and ^***P < 0.001 indicate significant differences compared to the control group and ^###P < 0.001 between the designated groups. Gal-9, Galectin-9; LPS, lipopolysaccharide; TIM-3, T cell immunoglobulin domain and mucin domain-3.

Fig. 3

The effect of Gal-9 on IL-1β gene and protein expression of U937 cells. U937 cells were treated with different concentrations of rhGal-9 or a determined concentration of rhGal-9 and then IL-1β gene and protein expression levels were measured by RT-PCR and ELISA methods, respectively. In the concentration-dependent study, the effect of various concentrations of rhGal-9 on IL-1β (A) gene and (C) protein expression was studied after 24 h treatment, and in the time-dependent study, the effect of a determined concentration of rhGal-9 on IL-1β (B) gene and (D) protein expression was studied following 6, 12, 24, and 48 h treatment of the cells. The data were obtained through two independent experiments, each performed in triplicate. Data are represented as the mean ± SD. ^**P < 0.01 and ^***P < 0.001 indicate significant differences compared to the control group and ^#P < 0.05 and ^##P < 0.01 versus the 6-h treatment groups. Gal-9, Galectin-9; LPS, lipopolysaccharide; IL, interleukin.

Fig. 4

The effect of Gal-9 in combination with LPS on (A) gene expression and (B) protein level of IL-1β. The specific concentration of rhGal-9 in combination with LPS was used for the treatment of U937 cells and following 24 h incubation the IL-1β gene and protein expression was measured by RT-PCR and ELISA methods, respectively. The data were achieved through two independent experiments, each performed in triplicate. Data are represented as the mean ± SD. ^***P < 0.001 indicates significant differences between the defined groups. Gal-9, Galectin-9; LPS, lipopolysaccharide; IL, interleukin.

Fig. 5

The effect of Gal-9 alone or in combination with LPS on (A and C) IL-1βR and (B and C) lL-1βRAP gene expression. Different concentrations of rhGal-9 alone or in combination with LPS were added on U937 cells and after 24 h incubation, the IL-1βR and IL-1βRAP gene expression were studied by RT-PCR. The data were achieved through two independent experiments, each performed in triplicate. Data are represented as the mean ± SD. ^**P < 0.01 and ^***P < 0.001 indicate significant differences compared to the control group; ^###P < 0.001 between the defined groups. Gal-9, Galectin-9; LPS, lipopolysaccharide; IL, interleukin.

Fig. 6

The effect of Gal-9 (A) alone or (B) in combination with LPS on NLRP3 gene expression. Different concentrations of rhGal-9 alone or in combination with LPS were added to U937 cells and after 24 h incubation, NLRP3 gene expression was measured by RT-PCR. The data were achieved through two independent experiments, each performed in triplicate. Data are represented as the mean ± SD. ^*p < 0.05 indicates a significant difference compared to the control group. Gal-9, Galectin-9; LPS, lipopolysaccharide; NLRP3, NLR family pyrin domain containing 3.

Fig. 7

The effect of Gal-9 alone or in combination with LPS on (A) NF-κB expression and (B) phosphorylation. rhGal-9 alone or in combination with LPS was added to U937 cells and after 24 h incubation, NF-κB expression and its phosphorylation were studied by western blotting, while β actin was considered for normalization (bands at the lower boxes). Data represent the median ± IQR of normalized protein expression with β-actin from experiments, which were performed in a triplicate manner. Statistical analysis was performed on protein expression using the Kruskal-Wallis, Dunn test, and Benjamini-Hochberg. ^**P < 0.01 indicates a significant difference in comparison with the control group. Gal-9, Galectin-9; LPS, lipopolysaccharide; NF-κB, nuclear factor-κB.