TNF-α Regulates Human Plasmacytoid Dendritic Cells by Suppressing IFN-α Production and Enhancing T Cell Activation

Abstract

Human plasmacytoid dendritic cells (pDCs) play a vital role in modulating immune responses. They can produce massive amounts of type I IFNs in response to nucleic acids via TLRs, but they are also known to possess weak Ag-presenting properties inducing CD4⁺ T cell activation. Previous studies showed a cross-regulation between TNF-α and IFN-α, but many questions remain about the effect of TNF-α in regulating human pDCs. In this study, we showed that TNF-α significantly inhibited the secretion of IFN-α and TNF-α of TLR-stimulated pDCs. Instead, exogenous TNF-α promoted pDC maturation by upregulating costimulatory molecules and chemokine receptors such as CD80, CD86, HLA-DR, and CCR7. Additionally, RNA sequencing analysis showed that TNF-α inhibited IFN-α and TNF-α production by downregulating IRF7 and NF-κB pathways, while it promoted Ag processing and presentation pathways as well as T cell activation and differentiation. Indeed, TNF-α-treated pDCs induced in vitro higher CD4⁺ T cell proliferation and activation, enhancing the production of Th1 and Th17 cytokines. In conclusion, TNF-α favors pDC maturation by switching their main role as IFN-α-producing cells to a more conventional dendritic cell phenotype. The functional status of pDCs might therefore be strongly influenced by their overall inflammatory environment, and TNF-α might regulate IFN-α-mediated aspects of a range of autoimmune and inflammatory diseases.

FIGURE 1.

Human pDCs produce both IFN-α and TNF-α in response to TLR9 and TLR7 agonists. (A) Gating strategy for human pDCs; pDCs are characterized by the lack of lineage markers (CD3, CD19, CD14, CD56, and CD11c), intermediate to high expression of MHC-II (HLA-DR), and high expression of CD123 and CD303 (BDCA-2). Freshly isolated PBMCs were cultured and stimulated with TLR9 (ODN 2216) or TLR7 (ORN R-2336) agonists for 6 h, and then IFN-α and TNF-α production was detected using intracellular staining. (B) Unstimulated pDCs produced no IFN-α and/or TNF-α. (C and D) Upon stimulation with TLR9 or TLR7 agonists, there were three major pDC populations: 1) nonproducers, 2) TNF-α producers, 3) IFN-α and TNF-α producers. Results shown are representative of three independent experiments.

FIGURE 2.

TNF-α regulates IFN-α and TNF-α production in TLR-stimulated pDCs. (A) Freshly isolated PBMCs were cultured in the absence or presence of recombinant human TNF-α. After 24 h, PMBCs were washed twice and stimulated with TLR9 (ODN 2216) or TLR7 (ORN R-2336) agonists for 6 h, and then IFN-α and TNF-α production by pDCs was measured using intracellular staining. Results shown are representative of three independent experiments. (B and C) PBMCs were cultured according to (A) with different concentrations of exogenous TNF-α (0–50 ng/ml). After TLR9 or TLR7 stimulation for 6 h, both IFN-α and TNF-α production by pDCs was measured using intracellular staining. (D) Purified pDCs were stimulated with TLR9 or TLR7 agonists in the absence or presence of anti-TNF Ab or isotype control. After 24 h, the supernatants were collected, and IFN-α production was measured by ELISA (0–24 h). (E) pDCs were washed twice and restimulated according to (D), and the supernatants were collected after an additional 24 h. IFN-α production was measured by ELISA (24–48 h). Results shown are representative of three independent experiments. Bars represent median value with 95% confidence interval. *p < 0.05, **p < 0.01. ns, not significant.

FIGURE 3.

TNF-α promotes transcriptional changes associated with Ag processing and presentation. (A) Enriched Reactome pathways in DEGs upregulated by TNF-α in pDCs compared with untreated pDCs. (B–D) Heat maps showing that TNF-α promotes *DEGs associated with Ag processing and presentation, T cell proliferation, and T cell activation pathways in pDCs.

FIGURE 4.

TNF-α promotes transcriptional changes associated with T cell differentiation. (A) Enriched KEGG pathways in DEGs upregulated by TNF-α in pDCs. (B–D) Heat maps showing that TNF-α promotes DEGs associated with T cell differentiation toward Th17, Th1, and Th2 pathways in pDCs.

FIGURE 5.

TNF-α inhibits TLR cascade signaling pathways. (A) Enriched Reactome pathways in DEGs downregulated by TNF-α in pDCs. (B) Heat maps showing that TNF-α promotes DEGs associated with negative regulation of TLR cascade signaling pathway in pDCs. (C) DEGs in TNF-treated versus untreated pDCs associated with negative regulation of TLR-mediated type I IFN production.

FIGURE 6.

TNF-α upregulates costimulatory molecules and maturation markers on pDCs. pDCs were purified from freshly isolated PBMCs and cultured in the absence or presence of recombinant human TNF-α. After 24 h, pDCs were analyzed by flow cytometry. Fluorescence intensity is shown on the x-axis. Results shown are representative of three independent experiments. (A) TNF-α upregulates HLA-DR (MHC-II), costimulatory molecules such as CD80 and CD86, and CCR7 but downregulates pDC-specific markers such as BDCA-2 (CD303). TNF-α also upregulates receptors related to type I IFN regulation such as ILT7 (CD85g) and CD317 (BST2; tetherin). Results shown are representative of three independent experiments.

FIGURE 7.

TNF-α enhances Ag uptake and processing by pDCs. (A) pDCs were purified by freshly isolated PBMCs and cultured alone or with 10 μg/ml DQ OVA in the presence or absence of 50 ng/ml TNF-α for 18 h. Ag uptake and processing was measured by flow cytometry based on mean fluorescence intensity (MFI). One representative experiment is shown out of three independent experiments. (B) Summary of the DQ OVA uptake of untreated pDCs, DQ OVA–treated pDCs, and DQ OVA/TNF–treated pDCs. Data are presented as means ± SEM for three independent experiments. **p < 0.005.

FIGURE 8.

TNF-α–treated pDCs enhance T cell proliferation and activation. (A) Allogeneic naive CD4⁺ T cells were labeled with CellTrace Violet and cultured alone or with pDCs or TNF-α–treated pDCs for 5 d. T cell proliferation was analyzed by flow cytometry based on CellTrace Violet dilution. One representative experiment is shown out of three independent experiments. (B) Average percentage of proliferated CD4⁺ T cells cocultured with pDCs or TNF-α–treated pDCs (n = 3). (C) Expression of CD69 on CD4⁺ T cells from the cultures shown in (A). One representative experiment is shown out of three independent experiments. (D) Average expression of CD69 on CD4⁺ T cells cocultured with pDCs or TNF-α–treated pDCs (n = 3). (E–J) Allogeneic naive CD4⁺ T cells were cultured alone or with pDCs or TNF-α–treated pDCs for 5 d. Percentage of TNF-α (E), IFN-γ (G), and IL-17A (I) production by CD4⁺ T cells was measured by intracellular staining. One representative experiment is shown out of three independent experiments. Average percentage of TNF-α (F), IFN-γ (H), and IL-17A (J) produced CD4⁺ T cells cocultured with pDCs or TNF-α–treated pDCs (n = 3). *p < 0.05, **p < 0.001, ***p < 0.001.