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. 2016 Mar 1;196(5):2004-9.
doi: 10.4049/jimmunol.1501557. Epub 2016 Jan 29.

Cutting Edge: Critical Role of Glycolysis in Human Plasmacytoid Dendritic Cell Antiviral Responses

Gagan Bajwa  1 , Ralph J DeBerardinis  2 , Baomei Shao  3 , Brian Hall  4 , J David Farrar  5 , Michelle A Gill  6
Affiliations

Cutting Edge: Critical Role of Glycolysis in Human Plasmacytoid Dendritic Cell Antiviral Responses

Gagan Bajwa et al. J Immunol. .

Abstract

Plasmacytoid dendritic cells (pDCs) are vital to antiviral defense, directing immune responses via secretion of huge concentrations of IFN-α. These cells are critical in protecting the lung against clinically relevant respiratory viruses, particularly influenza (Flu), a virus responsible for substantial worldwide morbidity and mortality. How pDC responses to such viral pathogens are regulated, however, is poorly understood in humans. Using an unbiased approach of gene chip analysis, we discovered that Flu significantly affects metabolism in primary human pDCs. We demonstrate that Flu and RV, another common respiratory virus, induce glycolysis in pDCs and that this metabolic pathway regulates pDC antiviral functions, including IFN-α production and phenotypic maturation. Intranasal vaccination of human volunteers with live influenza virus also increases glycolysis in circulating pDCs, highlighting a previously unrecognized potential role for metabolism in regulating pDC immune responses to viral infections in humans.

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Figures

FIGURE 1
FIGURE 1
Flu, RV and gardiquimod (Gard) induce glycolysis in human pDCs. (A) pDCs were cultured in cRPMI with no stimulant (Contl), Flu, RV, or Gard for 24h, washed, and reincubated in non-buffered media. Real-time ECAR was measured in supernatants of pDC cultures using a XF-24 analyzer. Vertical lines indicate addition of glucose (glycolysis substrate), oligomycin (ATP synthase inhibitor) and 2-DG (glycolysis inhibitor). One of 3 independent experiments shown. (B) Summary of pDC basal ECAR and (C, left) basal OCR measured simultaneously in the same well. (C, right) Red CMXRos mean fluorescence intensity (MFI) was quantified by flow cytometry in pDCs cultured +/− Flu for 48 h. Data represent mean ± SEM, N=3 for B and C. (D) pDCs were exposed to Flu, RV, or Gard for 6, 24 and 48 h and glucose-derived-13C-lactate measured in supernatants by mass spectrometry. One of 3 experiments shown. (E) Mean ± SEM values of 13C-lactate normalized to control in Flu-, RV- and Gard-treated pDCs after 24 and 48 h; N=4–6. (F) Lactate efflux rate (nmoles/h/105 cells) in pDCs exposed to Flu, RV or Gard, N=4–6. *p ≤ 0.05; **p ≤ 0.01, paired t-test, control versus treatment.
FIGURE 2
FIGURE 2
Inhibiting glycolysis impairs viral-induced pDC antiviral responses without disrupting viability. (A) 13C-lactate and IFN-α in supernatants of pDCs exposed to Flu in media containing 10 mM D-glucose with or without 2-DG (1 mM, 10 mM and 20 mM). One of 3 experiments shown. Arrows indicate 2-DG concentration (10 mM) utilized in all subsequent experiments. (B) Effects of 2-DG on pDC viability. (C) IFN-α (pg/ml) in supernatants of pDCs exposed to Flu, RV or Gard for 24 h +/− 2-DG. (D) Surface expression of HLA-DR, CD80 and CD86 on pDCs cultured for 48 h in conditions shown in (C). (E) Relative expression of IFN-α, HLA-DR, CD80 and CD86 to PPIA in pDCs exposed to Flu +/− 2-DG for 24 h. (F) Total protein and total RNA in pDCs exposed to Flu +/− 2-DG for 24 h. (G) Impact of 2-DG on expression of GUSB, IL3R, CD2AP, CD317 and ACTB. Data represent mean ± SEM, N= 3–8 in B-G, *p ≤ 0.05; ***p ≤ 0.001, paired t-test.
FIGURE 3
FIGURE 3
In vivo viral infection (LAIV) increases glycolysis in human pDCs and correlates with pDC antiviral responses. Blood pDCs were purified from healthy donors before (D0), 1 (D1) and/or 3 (D3) days after LAIV and pDCs (5×104) were cultured ex vivo for 24 h. (A) 13C-lactate quantification and (B) lactate efflux rate in pDC supernatants. (C) Relative pDC mRNA expression of HK2 and LDHA. (D) IFN-α2a secretion (left) and relative IRF7 expression (right) in pDCs cultured ex vivo for 24 h. (E) Effect of LAIV on pDC HLA-DR expression. (F) Nasal and T lymphocyte responses to LAIV. Nasal cell IRF7 expression normalized to PPIA (left); Flu-specific IFN-γ secreting T cells measured by ELISPOT before (D0) and after (day 8–10) LAIV (right). (G) Correlations between pDC glycolysis (lactate production) and HLA-DR expression (upper) and IFN-α secretion (lower). Data from 3 time points (D0, D1 and D3) displayed. Data in histograms represent mean ± SEM. In A and E, lines connect data from individual donors. N= 6–11 *p ≤ 0.05, paired t-test.
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