HMGN1 and R848 Synergistically Activate Dendritic Cells Using Multiple Signaling Pathways

Abstract

High mobility group nucleosome-binding protein 1 (HMGN1 or N1) is a Th1-polarizing alarmin, but alone is insufficient to induce antitumor immunity. We previously showed that combination of N1 and R848, a synthetic TLR7/8 agonist, synergistically activates dendritic cells (DCs) and induces therapeutic antitumor immunity, however, it remained unclear how N1 and R848 synergistically activate DCs. Here, we show that co-stimulation with N1 and R848 of human monocyte-derived DCs (MoDCs) markedly upregulated DC's surface expression of CD80, CD83, CD86, and HLA-DR, as well as synergistic production of pro-inflammatory cytokines including IL-12p70, IL-1β, and TNF-α. This combination also synergistically activated NF-κB and multiple MAPKs that are involved in DC maturation. Moreover, N1 and R848 synergistically increased nuclear translocation of interferon (IFN) regulatory transcription factors (e.g., IRF3 and IRF7) and promoted the expression of type 1 IFNs such as IFN-α2, IFN-α4, and IFN-β1. Similar signaling pathways were also induced in mouse bone marrow-derived DCs (BMDCs). RNA-seq analysis in human MoDCs revealed that N1 plus R848 synergistically upregulated the expression of genes predominantly involved in DC maturation pathway, particularly genes critical for the polarization of Th1 immune responses (e.g., IL12A, IL12B, and IFNB1, etc.). Overall, our findings show that (1) N1 synergizes with R848 in activating human and mouse DCs and (2) the synergistic effect based on various intracellular signaling events culminated in the activation of multiple transcriptional factors. These findings have important implications for future clinical trials since N1 and R848 synergistically promoted optimal Th1 lineage immune responses resulting in tumor rejection in mice.

Keywords: HMGN1; IFN; MAPK; NF-κB; R848; TLR; Th1 polarization; dendritic cell.

Figure 1

N1 and R848 dose dependently induce phenotypic maturation of human MoDCs. (A,C) Human MoDCs were incubated at 5 × 10⁵ cells/ml in the absence (sham) or presence of indicated concentrations of recombinant N1 or R848 for 48 h before they were immunostained and analyzed by flow cytometry for the expression of the indicated surface molecules. Shown are the overlay histograms (gray area = sham-treated) of one experiment, with the average relative fluorescence intensity (geometric mean ± SD) of three independent experiments inscribed. (B,D) Cytokine levels in the culture supernatants of DCs treated with N1 or R848 at indicated concentrations for 48 h were quantitated by cytokine array (mean ± SD; n = 3). *p<0.05, ***p<0.001 and ****p<0.0001 when compared with the sham treatment according to one-way ANOVA followed by Tukey's post hoc test.

Figure 2

N1 and R848 synergistically induce phenotypic maturation of human MoDCs. Human MoDCs were incubated at 5 × 10⁵ cells/ml in the absence (sham) or presence of various concentrations of N1 and/or R848 for 48 h before they were analyzed for surface marker expression and cytokine production. (A,C) The expression of indicated surface molecules is shown as overlay histograms (gray area = sham-treated) of one donor, with the average relative fluorescence intensity (geometric mean ± SD) of three independent donors inscribed. (B,D) Cytokine levels in the culture supernatants were quantitated by cytokine array (mean ± SD; n = 3). ***p<0.001, and ****p<0.0001 according to one-way ANOVA followed by Tukey's post hoc test.

Figure 3

N1 and R848 synergistically induce functional maturation of human MoDCs. (A–E) Human MoDCs cultured in the presence or absence of N1 (250 ng/ml) and/or R848 (250 ng/ml) for 48 h were incubated in triplicate with allogenic human peripheral blood CD4+ T lymphocytes (L) (10⁵ cells/well). (A) On day 4, 1 μCi of [³H]TdR was added to each well to pulse the cultures for another 18 h. The proliferation was measured as the average (mean ± SD) of [³H]TdR incorporation (CPM) of triplicate wells. (B) IFN-γ production by CD4+ T cells co-cultured with DCs (DCs:L = 1:250) for 3 days (mean ± SD, n = 3). (C–E) The expression of T-bet and Gata-3 by CD4+ T cells co-cultured with DCs (DCs:L = 1:250) for 3 days were measured by flow cytometry. (C) Representative dot plots. (D,E) The average (mean ± SD) of triplicate wells. Shown is the results of one representative of three independent experiments obtained from different donors. **p<0.01 according to one-way ANOVA followed by Tukey's post hoc test.

Figure 4

N1 and R848 synergistically activate NF-κB and MAPK signaling in human MoDCs. Serum-starved human MoDCs (2 × 10⁶ cells/ml) were treated for 0–90 min with N1 (250 ng/ml) and R848 (250 ng/ml) before they were solubilized in lysis buffer (10⁶ MoDCs/0.1 ml) to obtain whole cell lysates or in buffer (2 × 10⁶ MoDCs/0.1 ml) for analysis of the translocation of proteins from the cytoplasmic to nuclear cell fractions. An identical amount of protein was loaded and separated by electrophoresis to detect the levels of phospho-I-κBα, I-κBα, phospho-p65, phospho-p38, p38, phospho-CREB, CREB, phospho-JNK, JNK, phospho-c-Jun, c-Jun, and GAPDH in whole cell lysates (A,C) or (B) cytoplasmic fraction of phospho-p65, GAPDH and nuclear fraction of phospho-p65, lamin B1. Quantitation of band intensities from three donors using ImageJ software were normalized against GAPDH or lamin B1. **p<0.01, ***p<0.001, and ****p<0.0001 according to one-way ANOVA followed by Tukey's post hoc test.

Figure 5

N1 and R848 synergistically activate IRF signaling as well as type 1 IFN responses in human MoDCs. (A,B) Serum-starved human MoDCs (2 × 10⁶ cells/ml) were treated for 0–90 min with N1 (500 ng/ml) and R848 (500 ng/ml) before they were solubilized in buffer (2 × 10⁶ MoDCs/0.1 ml) for analysis of the translocation of proteins from cytoplasmic to nuclear cell fractions. An identical amount of protein was loaded and separated by electrophoresis to detect the levels of (A) cytoplasmic fraction of IRF3, GAPDH, and nuclear fraction of IRF3, lamin B1 or (B) cytoplasmic fraction of IRF7, GAPDH, and nuclear fraction of IRF7, lamin B1. Band intensities from three donors was quantified and normalized with GAPDH or lamin B1 using ImageJ software. (C–E) Human MoDCs were treated with N1 (500 ng/ml) and/or R848 (500 ng/ml) for 6 h before extraction of total RNA. The levels of IFN-α2, IFN-α4, and IFN-β1 mRNA were quantitated by qPCR and shown as a fold increase over the sham treated DCs. Data are shown as the average (mean ± SD) of triplicates of one experiment representative of three. *p<0.05, and **p<0.01 according to one-way ANOVA followed by Tukey's post hoc test.

Figure 6

N1 and R848 synergistically induce phenotypic maturation of mouse BMDCs. (A,C) Mouse BMDCs were incubated at 5 × 10⁵ cells/ml in the absence (sham) or presence of N1 (125, 250 ng/ml) and R848 (125, 250 ng/ml) for 48 h before they were immuno-stained and analyzed by flow cytometry for the expression of the indicated surface molecules [inscribed numbers are the average (geometric mean fluorescence intensity ± SD) of three batches of male mice; gray = sham-treated]. (B,D) Cytokine levels in the culture supernatants for 48 h were quantitated by cytokine array (mean ± SD; n = 3). Shown are the results of one experiment representative of three from three different batches of male mice (n = 6). *p<0.05, **p<0.01, and ****p<0.0001 according to one-way ANOVA followed by Tukey's post hoc test.

Figure 7

N1 and R848 synergistically activate NF-κB, IRF3, and IRF7 signaling as well as type 1 IFN responses in mouse BMDCs. (A–C) Mouse BMDCs (2 × 10⁶ cells/ml) were treated for 0–90 min with N1 (500 ng/ml) and R848 (500 ng/ml) before they were solubilized in the buffer for analysis of the translocation of proteins from cytoplasmic to nuclear cell fractions. An identical amount of proteins was loaded and separated by electrophoresis to detect the levels of cytoplasmic fraction of IRF3, IRF7, phospho-p65, and GAPDH, as well as nuclear fraction of IRF3, IRF7, phospho-p65, and lamin B1. Quantitation of band intensities from three different batches of male mice (n = 6) were normalized against GAPDH or lamin B1 using ImageJ software. (D) Mouse BMDCs were treated with N1 (500 ng/ml) and/or R848 (500 ng/ml) for 6 h before being used for the extraction of total RNA. The levels of IFN-β1 mRNA was quantitated by qPCR and shown as fold increase over the sham treated DCs. Data are shown as the average (mean ± SD) of triplicates of one experiment representative of three. *p<0.05 according to one-way ANOVA followed by Tukey's post hoc test.

Figure 8

Synergy of N1 and R848 as revealed by RNA-seq analysis. Human MoDCs from three donors were stimulated with N1 (250 ng/ml) and/or R848 (250 ng/ml) for 4 h before extraction of total RNA. (A) Venn diagram of 2355 genes out of 14,284 genes were found to be variable (adjusted p<0.05 and absolute fold change relative to sham ≥2.0). (B) Heat map showing pathways identified as significantly (absolute activation z-score ≥2.0) de/activated in all three experimental contrasts vs. sham. Cell values are activation z-scores and row centering has been applied. (C) Heat map showing the expression of 642 genes only found to be variable relative to sham when treated with N1 plus R848. (D) Results of gene ontology (GO) term analysis of the 642 genes exclusively variable in the N1 plus R848 treatment. Significant (p<0.05) GO terms for the up- and down-regulated subsets of these genes are given; the bar graph visualizes log₁₀-transformed, Bonferonni-corrected p-values for each term. (E) Heat map showing the expression of 32 selected genes with relevance to DCs maturation. All genes in this subset were included in the set of 2,355 unique variable genes as well as the significantly de/activated pathway analysis. (C,E) Expression values in these heat maps are log₂-transformed, TMM-normalized counts-per-million (CPM). (F) Shown are the fold expression of CPM of 4 genes selected from 28 synergistic upregulated genes obtained from three donors. Orange shows relatively high expression while purple shows relatively low expression. Row centering and unit variance scaling have been applied. Rows are clustered using Euclidean distances and complete linkage.