The natural product phyllanthusmin C enhances IFN-γ production by human NK cells through upregulation of TLR-mediated NF-κB signaling

Abstract

Natural products are a major source for cancer drug development. NK cells are a critical component of innate immunity with the capacity to destroy cancer cells, cancer-initiating cells, and clear viral infections. However, few reports describe a natural product that stimulates NK cell IFN-γ production and unravel a mechanism of action. In this study, through screening, we found that a natural product, phyllanthusmin C (PL-C), alone enhanced IFN-γ production by human NK cells. PL-C also synergized with IL-12, even at the low cytokine concentration of 0.1 ng/ml, and stimulated IFN-γ production in both human CD56(bright) and CD56(dim) NK cell subsets. Mechanistically, TLR1 and/or TLR6 mediated PL-C's activation of the NF-κB p65 subunit that in turn bound to the proximal promoter of IFNG and subsequently resulted in increased IFN-γ production in NK cells. However, IL-12 and IL-15Rs and their related STAT signaling pathways were not responsible for the enhanced IFN-γ secretion by PL-C. PL-C induced little or no T cell IFN-γ production or NK cell cytotoxicity. Collectively, we identify a natural product with the capacity to selectively enhance human NK cell IFN-γ production. Given the role of IFN-γ in immune surveillance, additional studies to understand the role of this natural product in prevention of cancer or infection in select populations are warranted.

Figure 1. PL-C enhances IFN-γ production in human primary NK cells

(A) Chemical structure of PL-C. (B) Healthy donor PBMCs (left) or enriched NK cells (right) were treated with DMSO vehicle control or 10 µM PL-C for 18 hours in the presence of IL-12 (10 ng/ml) or IL-15 (100 ng/ml). The cells were harvested and analyzed by intracellular flow cytometry to determine the frequency of IFN-γ⁺ cells in CD56⁺CD3⁻ NK cells (n = 8 for PBMC and n = 5 for enriched NK). (C) Highly purified (≥ 99.5%) human primary NK cells were treated with 10 µM PL-C for 18 hours to determine the levels of IFN-γ secretion. IFN-γ secretion from treatment with PL-C alone (left) or in combination with IL-12 (10 ng/ml, middle) or IL-15 (100 ng/ml) (right) is shown. (D) Cells were treated as described in (C) and harvested at 12 hours. IFNG mRNA expression were assessed by real-time RT-PCR and the relative IFNG mRNA expression of each treatment was normalized to untreated vehicle control in the same donor. Data are shown as mean ± S.E.M. (n = 6 in each treatment; error bars represent S.E.M.). * and ** indicate P < 0.05 and P < 0.01, respectively, which denote statistical comparison between the two marked treatment groups (B, C and D). (E) Highly purified ((≥ 99.5%) primary human NK cells were treated with 10 µM PL-C in combination with various concentrations of IL-12 (10, 1, 0.1 ng/ml) or IL-15 (100, 10, 1 ng/ml) for 24 hours to determine the levels of IFN-γ secretion. Representative data from 1 out of 3 donors with the similar data are shown. * and ** indicate P < 0.05 and P < 0.01, respectively, which denote statistical comparison between the two marked treatment groups and are calculated from data of all tested donors. (F) NKL cells were treated with 10 µM PL-C in the presence of IL-12 or IL-15 for 18 hours or 12 hours to determine the levels of IFN-γ secretion (left) or IFNG mRNA (right) expression respectively. Data shown represent at least 3 independent experiments. * and ** indicate P < 0.05 and P < 0.01, respectively, compared to vehicle control. Error bars represent S.D.

Figure 2. PL-C activates both CD56^dim and CD56^bright NK cells to secrete IFN-γ

(A) Enriched NK cells were sorted via FACS into CD56^dim and CD56^bright NK cells based on the relative density of CD56 expressed on the cell surface. CD56^dim and CD56^bright NK cells were treated with 10 µM PL-C in the presence of IL-12 (10 ng/ml) for 18 hours and assessed for the levels of IFN-γ secretion. (B) Cells were isolated, treated and analyzed as in (A), but in the presence of IL-15 (100 ng/ml) instead of IL-12. Representative data from 1 out of at least 3 donors with similar results are shown. * and ** indicate P < 0.05 and P < 0.01, respectively, which denote statistical comparison between the two marked treatment groups and are calculated from data of all tested donors (A and B). Error bars represent S.D.

Figure 3. PL-C increases the phosphorylation of p65 in human primary NK and NKL cells

(A) Purified primary human NK cells were treated with 5 and 10 µM PL-C for 18 hours. The cells were harvested and lysed for immunoblotting using p65 and phosphorylated p65 (p-p65) antibodies. β-actin immunoblotting was included as the internal control. Data shown are for treatment with PL-C alone (top) or in combination with IL-12 (10 ng/ml) (middle) or IL-15 (100 ng/ml) (bottom), and are the representative plots of 4 donors with similar results. Numbers under each lane represent quantification of p-p65 or p65 via densitometry, after normalizing to β-actin. (B) NKL cells were treated, and data are presented as described in (A). Data from 1 of 3 independent experiments with similar results are shown. (C) Purified primary human NK (left) or NKL cells (right) were co-treated with 10 µM PL-C and IL-12 (10 ng/ml) in the presence or absence of the NF-κB inhibitor TPCK (10 µM) for 18 hours. Supernatants were assayed for IFN-γ secretion (top) and cells were harvested and lysed for immunoblotting of p-p65 (bottom). Representative data from 1 out of 3 donors with the similar data (left panel) and the summary of three independent experiments with similar results (right panel) are shown. ** indicates P < 0.01, and error bars represent S.D.

Figure 4. PL-C augments the binding of p65 to the IFNG promoter in human NK cells

(A) Schematic of IFNG promoter potential binding sites for p65 (45). (B) NK cells purified from healthy donors were treated with 10 µM PL-C or DMSO vehicle control in the presence of IL-12 (10 ng/ml) for 12 hours. Cell pellets were harvested for nuclear extraction, followed by EMSA with a ³²P-labeled oligonucleotide containing the C3-3P NF-κB p65 binding site of the IFNG promoter. Data shown represent 1 out of 3 donors with similar results. (C) Cells were treated as described in (B), and the cell pellets were harvested to extract protein for ChIP assay of p65 binding to the IFNG promoter locus C3-3P. Mean of relative association of p65 at the IFNG promoter locus C3-3P from three independent experiments is shown. * indicates P < 0.05, compared to cells treated with IL-12 alone. Error bars represent S.D.

Figure 5. TLR1 and/or TLR6 mediate IFN-γ induction by PL-C in human NK cells

(A) Human NK cells were purified and pretreated with a non-specific IgG or anti-TLR1, anti-TLR3, anti-TLR6 or the combination of TLR1 and TLR6 blocking antibodies (α) for 1 hour. Cells were then treated with PL-C and IL-12 (10 ng/ml) for another 18 hours and were assessed for IFN-γ secretion. Data shown are representative of one of 6 different donors with similar results. * and ** indicate P < 0.05 and P < 0.01, respectively, which denote statistical comparison between the two marked treatment groups and are calculated from data of all tested donors. (B) Cell pellets were harvested from the above mentioned samples in (A), followed by p-p65 immunoblotting. Numbers underneath each lane represent quantification of protein by densitometry, normalized to β-actin. Representative data from 1 out of 6 donors with similar results are shown in (A and B). (C) Purified NK cells were treated with Pam3CSK4 (1 µg/ml, TLR1/2 ligand), or FSL-1 (1 µg/ml, TLR6/2 ligand) in the presence of IL-12 (10 ng/ml), with or without PL-C (10 µM) for 18 hours, and then supernatants were harvested to assay for IFN-γ secretion. Data shown are representative of 1 of 6 donors with similar results. * and ** indicate P < 0.05 and P < 0.01, respectively, which denote statistical comparison between the two marked treatment groups and are calculated from data of all tested donors. (D) Purified primary NK cells were treated with various low concentration of Pam3CSK4, or FSL-1 with or without PL-C (10 µM) in the presence of IL-12 (10 ng/ml), and then supernatants were harvested to assay for IFN-γ secretion. Data shown are representative 1 out of 3 donors with similar results. Error bars indicate S.D. (E) 293T cells were transfected with TLR1 (0.5 µg), or TLR6 (0.5 µg) expression plasmid along with pGL-3×κB-luc (1 µg) and pRL-TK renilla-luciferase control plasmids (5 ng, Promega). Cells were then treated with various concentration of PL-C for another 24 hours with fresh medium, and DMSO was included as vehicle control. The ratio of the firefly to the renilla luciferase activities was used to show the relative luciferase activity, which corresponded to NF-κB activation. * and ** indicate P < 0.05 and P < 0.01, respectively, compared to vehicle control. Error bars represent S.D. (F) NKL cells were infected with pSUPER or pSUPER-shTLR1 retroviruses and sorted based on GFP expression. Confirmation of TLR1 mRNA knockdown is shown in the left panel. Both the vector-transduced cells (pSUPER) and the TLR1 knockdown cells (pSUPER-shTLR1) were treated with or without PL-C in the presence or absence of IL-12 or IL-15. Cell pellets were harvested at 12 hours for real-time RT-PCR. The relative IFNG mRNA expression induced by PL-C under IL-12 (10 ng/ml) or IL-15 (100 ng/ml) was shown in middle or right panel, respectively. The summary of three independent experiments with similar results are shown. ** indicates P < 0.01, and error bars represent S.D.