Nucleotide-Binding Oligomerization Domain 1/Toll-Like Receptor 4 Co-Engagement Promotes Non-Specific Immune Response Against K562 Cancer Cells

Abstract

Nucleotide-binding oligomerization domain 1 (NOD1) receptor and Toll-like receptor 4 (TLR4) belong to the family of pattern recognition receptors. Interactions between these receptors profoundly shape the innate immune responses. We previously demonstrated that co-stimulation of peripheral blood mononuclear cells (PBMCs) with D-glutamyl-meso-diaminopimelic acid (iE-DAP)-based NOD1 agonists and lipopolysaccharide (LPS), a TLR4 agonist, synergistically increased the cytokine production. Herein, we postulate that stimulation of NOD1 alone or a combined stimulation of NOD1 and TLR4 could also strengthen PBMC-mediated cytotoxicity against cancer cells. Initially, an in-house library of iE-DAP analogs was screened for NOD1 agonist activity to establish their potency in HEK-Blue NOD1 cells. Next, we showed that our most potent NOD1 agonist SZZ-38 markedly enhanced the LPS-induced cytokine secretion from PBMCs, in addition to PBMC- and natural killer (NK) cell-mediated killing of K562 cancer cells. Activation marker analysis revealed that the frequencies of CD69⁺, CD107a⁺, and IFN-γ⁺ NK cells are significantly upregulated following NOD1/TLR4 co-stimulation. Of note, SZZ-38 also enhanced the IFN-γ-induced PBMC cytotoxicity. Overall, our findings provide further insight into how co-engagement of two pathways boosts the non-specific immune response and attest to the importance of such interplay between NOD1 and TLR4.

Keywords: K562; LPS; NK cells; NOD1 agonist; PBMC; TLR4 agonist; cytolytic activity; synergy.

FIGURE 1

Structural modifications of the parent D-Glu-meso-DAP (iE-DAP) resulting in conformationally constrained analogs.

FIGURE 2

Proliferation rates of NOD1-specific HEK-Blue cells following 20 h incubation with C12-iE-DAP (1 µM) and constrained iE-DAP derivatives SZZ-38, SZZ-39, SZZ-40, and SZZ-41 (all at 1 µM). The rates, expressed as means of duplicates ± SEM of two independent experiments, are shown relative to that of the control (NT).

FIGURE 3

Synergistic effect of SZZ-38 and LPS on the induction of cytokine production from PBMCs. Cytokine concentrations were measured after 18 h stimulation with SZZ-38 (1 µM), LPS (2 ng/mL), both, or the corresponding vehicle (0.1% DMSO; NT). Data are expressed as means ± SEM of three independent experiments. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparisons test; ***, p < 0.001.

FIGURE 4

Effect of NOD1 agonists and LPS on the cytolytic activity of PBMCs against K562 cells. (A) PBMCs were treated for 18 h with C12-iE-DAP (1 µM) or NOD1 agonists (1 and 10 µM), before the addition of K562 cells. Cytotoxicity was determined after 4 h of co-incubation. (B) The concentration-dependent effect of NOD1/TLR4 agonist combinations on the induction of PBMC cytotoxicity. Data are shown relative to the negative control (0.1 % DMSO) and expressed as means ± SEM of three independent experiments. Statistical significance was determined by two-way ANOVA followed by Dunnets multiple comparisons test; *, p < 0.05, **, p < 0.01, ***, p < 0.001.

FIGURE 5

Effect of NOD1 and TLR4 antagonist pre-treatment on the SZZ-38- and LPS-induced PBMC cytolytic activity against K562 cells. PBMCs were pre-treated with the NOD1 antagonist Nodinitib-1 (10 µM), TLR4 antagonist TLR4-IN-C34 (100 µM), or both for 1 h, before the addition of SZZ-38 (1 µM), LPS (10 ng/mL), or IL-2 (200 IU/mL). Following 18 h activation, K562 cells were added. Cytotoxicity was determined after 4 h of co-incubation. Data are shown relative to the negative control (0.1 % DMSO) and expressed as means ± SEM of three independent experiments.

FIGURE 6

Effect of SZZ-38 and LPS on the cytolytic activity of NK cells against K562 cells. Isolated NK cells were treated for 18 h with SZZ-38 (1 µM), LPS (1 µg/mL), their combination, or IL-2 (200 IU/mL) before the addition of K562 cells. Cytotoxicity was determined after 4 h of co-incubation. Data are shown relative to the negative control (0.1% DMSO) and expressed as means ± SEM of three independent experiments. Statistical significance was determined by Dunnet’s multiple comparisons test; **, p < 0.01, ***, p < 0.001.

FIGURE 7

NK cell activation, degranulation, and IFN-γ production in response to NOD1 (SZZ-38, 1 µM) and TLR4 co-stimulation (LPS, 1 µg/mL). Live CD3⁻/CD56⁺ cells were gated as NK cells. Cell surface expression of CD69 (A) and CD107a (B), and the percentage of IFN-γ producing (C) NK cells were assessed by cell surface and intracellular staining, followed by flow cytometric analysis. The results are expressed as percentages of CD69⁺ (A), CD107a⁺ (B), or IFN-γ⁺ (C) CD3⁻ CD56⁺ cells. The data represent the means ± SEM of three independent experiments. Statistical significance was determined by Dunnet’s multiple comparisons test; *, p < 0.05, ***, p < 0.001.

FIGURE 8

The concentration-dependent effect of NOD1 agonist/IFN-γ combinations on the induction of PBMC cytotoxicity against K562 cells. PBMCs were treated for 18 h with SZZ-38 (1 and 10 µM), IFN-γ (10 and 100 ng/mL), their combinations, or IL-2 (200 IU/mL) before the addition of K562 cells. Cytotoxicity was determined after 4 h of co-incubation. Data are shown relative to the negative control (0.1% DMSO) and expressed as means ± SEM of three independent experiments. Statistical significance was determined by two-way ANOVA followed by Dunnet’s multiple comparisons test; **, p < 0.01, ***, p < 0.001.