Hypoxia potentiates monocyte-derived dendritic cells for release of tumor necrosis factor α via MAP3K8

Abstract

Dendritic cells (DCs) constantly sample peripheral tissues for antigens, which are subsequently ingested to derive peptides for presentation to T cells in lymph nodes. To do so, DCs have to traverse many different tissues with varying oxygen tensions. Additionally, DCs are often exposed to low oxygen tensions in tumors, where vascularization is lacking, as well as in inflammatory foci, where oxygen is rapidly consumed by inflammatory cells during the respiratory burst. DCs respond to oxygen levels to tailor immune responses to such low-oxygen environments. In the present study, we identified a mechanism of hypoxia-mediated potentiation of release of tumor necrosis factor α (TNF-α), a pro-inflammatory cytokine with important roles in both anti-cancer immunity and autoimmune disease. We show in human monocyte-derived DCs (moDCs) that this potentiation is controlled exclusively via the p38/mitogen-activated protein kinase (MAPK) pathway. We identified MAPK kinase kinase 8 (MAP3K8) as a target gene of hypoxia-induced factor (HIF), a transcription factor controlled by oxygen tension, upstream of the p38/MAPK pathway. Hypoxia increased expression of MAP3K8 concomitant with the potentiation of TNF-α secretion. This potentiation was no longer observed upon siRNA silencing of MAP3K8 or with a small molecule inhibitor of this kinase, and this also decreased p38/MAPK phosphorylation. However, expression of DC maturation markers CD83, CD86, and HLA-DR were not changed by hypoxia. Since DCs play an important role in controlling T-cell activation and differentiation, our results provide novel insight in understanding T-cell responses in inflammation, cancer, autoimmune disease and other diseases where hypoxia is involved.

Keywords: dendritic cells; hypoxia; inflammation; mitogen-activated protein kinases; tumour necrosis factors.

Figure 1. Hypoxia potentiates LPS-induced TNF-α secretion

(A–C) Cell viability of moDCs during atmospheric (20% O₂) and hypoxic (1% O₂) oxygen levels in presence or absence of LPS, determined by Zombie Violet as analyzed by flow cytometry. Representative dot plot (A), histograms (B), and quantitation for three donors ((C); average ± S.E.M.) are shown. (D–I) LPS-induced maturation of moDCs cultured at atmospheric or hypoxic oxygen levels, determined by expression levels of maturation markers CD83 (D,G), CD86 (E,H), and HLA-DR (F,I). Representative histograms showed quantitation for five donors. (J,K) Secretion by ELISA of the pro-inflammatory cytokines TNF-α (J) and IL-6 (K) from moDCs cultured at atmospheric or hypoxic oxygen levels. Quantitation from ten donors (average ± S.E.M.). *P<0.05; **P<0.01; ***P<0.001. Abbreviation: ns, not significant.

Figure 2. Hypoxia-mediated potentiation of TNF-α secretion is controlled by p38/MAPK

(A–F) Effects of inhibitors of p38/MAPK (1 µM SB203580; (A,B,E,F)) and/or IKK (100 nM IKK-16; (C–F)) on LPS-induced TNF-α secretion by moDCs cultured under atmospheric (20% O₂; (A,C,E)) or hypoxic (1% O₂; (B,D,F)) oxygen levels. Individual donors shown. (G,H) Effects of SB203580 and/or IKK-16 on moDC viability, determined by flow cytometry labeling with Zombie Violet as analyzed by flow cytometry. Representative histograms (G) and quantitation for four donors ((H); average ± S.E.M.). *P<0.05; **P<0.01; ***P<0.001. Abbreviaton: ns, not significant.

Figure 3. LPS-induced phosphorylation of p38/MAPK is increased at hypoxia

(A) Representative Western blot of moDCs cultured at atmospheric (20% O₂) or hypoxic (1% O₂) oxygen levels and in presence or absence of LPS. The blot was stained with an antibody specific for p-p38/MAPK (top) and total p38/MAPK (bottom). (B) Quantitation of (A) for five donors. p38/MAPK phosphorylation during hypoxic inflammation, normalized to unstimulated cells at atmospheric oxygen levels (LPS-, 20% O₂). *P<0.05; **P<0.01; ***P<0.001. Abbreviation: ns, not significant.

Figure 4. Hypoxia increases expression of HIF target gene MAP3K8

(A) Positions and sequences of HRE and HIF-binding site (HIF-1) in the promoter region of human MAP3K8. (B) MAP3K8 mRNA expression by moDCs as determined by PCR on cDNA obtained from unstimulated moDCs. Expected band size is 201 bp. (C) mRNA expression levels of MAP3K8 in moDCs cultured at atmospheric or hypoxic conditions in absence or presence of LPS determined by RT-qPCR. Quantitation from six donors. *P<0.05, **P<0.01, ***P<0.001.

Figure 5. MAP3K8 knockdown in moDCs blocks hypoxia-induced potentiation of LPS-induced TNF-α secretion

(A) Representative Western blot of siRNA knockdown of MAP3K8 in moDCs (MAP3K8^siRNA) stained with an antibody specific for MAP3K8 (top) and loading control (GAPDH; bottom). MoDCs were transfected with non-targetting siRNA as negative control. (B) Quantitation of (A) by band intensities normalized to GAPDH and shown relative to non-targetting control. Individual donors are shown. Compared using Wilcoxon’s matched-pairs signed rank test. (C) TNF-α secretion by moDCs with or without MAP3K8^siRNA and cultured at atmospheric (20% O₂) or hypoxic (1% O₂) oxygen levels. Quantitation of 14 donors (average ± S.E.M.). (D) Phosphorylation of p38/MAPK in moDCs with or without MAP3K8^siRNA and cultured at atmospheric (20% O₂) or hypoxic (1% O₂) oxygen levels. Representative Western blot probed for phosphorylated and total p38/MAPK shown. (E) Quantitation of (D) for six donors. Shown are the ratios of p38/MAPK phosphorylation for MAP3K8^siRNA moDCs compared with non-targetting siRNA controls. (F) TNF-α secretion by moDCs following overnight incubation with LPS and Tpl2 kinase inhibitor (Tpl2 KI) normalized to atmospheric oxygen condition (average ± S.E.M.). (G) Representative flow cytometry histogram showing Zombie Violet cell viability staining on moDCs incubated overnight with Tpl2 KI, DMSO negative control, or 2 min 70% ethanol (EtOH) as positive control. *P<0.05. Abbreviation: ns, not significant.

Figure 6. Model of MAP3K8-mediated potentiation of TNF-α secretion in hypoxic inflammation

(1) Hypoxia inhibits PHD activity, leading to stabilization of HIF-1α. (2) HIF-1α dimerizes with HIF-1β and up-regulates MAP3K8 expression, which potentiates the p38/MAPK signaling cascade. (3) LPS-induced dimerization of TLR4 activates the p38/MAPK signaling cascade. (4) c-JUN is phosphorylated by p-p38α and up-regulates TNF-α expression. (5) TNF-α is transported to the plasma membrane and released.