Fig 1.
Infection-induced changes in DC protein abundance and localization.
Influenza virus strain A/PuertoRico/34 (IAV) was added to DC for 2 hours to allow entry. Then, the medium was replaced, and the infection proceeded for 17 hours followed by cell lysis and protein extraction. Proteins were labeled with either SIL or iTRAQ and subjected to LC-MS/MS. Both proteomes were combined, redundancies removed and confidently identified peptides with abundance changes of 2-fold or greater linked to protein identifiers. The lists of upregulated and downregulated proteins were submitted to the Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.7 and mapped to KEGG pathways. (A-B) Significantly enriched KEGG pathways were grouped by KEGG Orthology (KO) Class for the increased (A) and decreased (B) proteomes. (C) In the network summary of metabolic protein dynamics, the soluble and insoluble proteomes were subdivided by abundance change. Proteins with relative expression changes of two-fold or greater are connected to their location in the soluble or insoluble fraction by red (top) or blue (bottom) edges indicating increased or decreased abundance change, respectively. Each protein node size was held static and color coded by KEGG subclass with the corresponding metabolic pathway of the protein mapped and delineated in the legend. The nodes of each KEGG subclass were arranged into circles that are proportional to the number of proteins with abundance changes in each significantly enriched metabolic pathway.
Fig 2.
Metabolic changes in infected DC.
DC were infected at MOI of 1 for 17 hours. (A-E) Use of isotopically labeled substrates, as indicated on the y axis, were used to monitor the generation of traceable products (3H2O or 14CO2) two hours after DC treatments substrates were quenched and allowed to accumulate overnight. Substrates were indicative of the following metabolic pathways: (A) [3-3H]glucose (glycolysis), (B) [1-14C]glucose (pentose phosphate pathway), (C) [2-14C]pyruvate (TCA cycle), (D) [U-14C]glutamine (glutaminolysis), and (E) [9,10-3H]palmitic acid (fatty acid oxidation). (F) The concentration of fatty acids (≥C8) in DC was determined by a coupled enzyme assay releasing fluorometric product proportional to the fatty acids present and quantifiable with standards after treatments. (G-I) The rates of pyruvate, glutamine, or long chain fatty acids oxidation for respiration were calculated as the percentage of inhibition of oxygen consumption by UK5099, BPTES, or etomoxir, which are specific inhibitors of mitochondrial pyruvate carrier, glutaminase, and carnitine palmitoyltransferase 1A, respectively. Capacity for a specific substrate to drive respiratory OCR was tested by determining baseline OCR, inhibiting the 2 off-target substrates determining OCR, and inhibiting import of the target metabolite. Percent capacity is one minus the baseline OCR less the off-target OCR divided by the baseline OCR less the OCR after all targets inhibited times 100. Dependency on a specific substrate was tested as above reversing the inhibitor sequence and the percent dependence was calculated by deducting the target OCR from the baseline and dividing by the baseline OCR less the OCR after all targets inhibited times 100. Fuel flexibility was calculated as the difference between capacity and dependency. (G) The average capacity of uninfected or infected DC to use pyruvate, glutamine, or long chain fatty acids was determined. (H) The average dependence of uninfected or infected DC on the oxidation of pyruvate, glutamine, or long chain fatty acids was determined. (I) The average flexibility of DC to alternate oxidation of pyruvate, glutamine, or long chain fatty acids was determined for uninfected or infected. The graphs represent the values of two (E-F), three (B-D), or four (A, G-I) independent experiments (3 ≥ technical replicates) and are presented as the experimental mean +/- SD. The normality of these data was tested followed by the appropriate parametric (t-test) or nonparametric (Wilcoxon signed rank test) for normal distributions (A, B, E-H)) or non-normal distributions (C,D,I), respectively. Asterisks correspond to p-values <0.05 (*) and a p-value < 0.01 (**).
Fig 3.
Compared to TLR agonists, IAV infection induces distinct bioenergetics primarily through aerobic glycolysis.
(A-D) DC were infected or treated with TLA agonists lipopolysaccharide (LPS), polyinosinic polycytidylic acid (PolyIC), or Resiquimod (R848) for 17 hours followed by metabolic analysis with a Seahorse Xfe96 Flux Analyzer. (A) Glycolytic function was tested with the Glycolysis Stress Test while monitoring real-time extracellular acidification rate (ECAR) with the Xfe96 metabolic analyzer during sequential injections of glucose, oligomycin (Oligo), and 2-Deoxy-D-glucose (2-DG) indicated by arrows. (B) Glucose uptake was monitored from the medium using a standard blood glucometer with glucose standard calibration curves. (C) Mitochondrial respiration was tested with the Mitochondrial Stress Test while monitoring oxygen consumption rates (OCR) in real-time with the Xfe96 metabolic analyzer during sequential injections of oligomycin (Oligo), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and a mixture of rotenone and antimycin A (Rot/AntA) indicated by arrows. (D) DC maximal mitochondrial ATP changes induced by oligomycin plotted against maximal ATP changes upon glucose depletion determined by respirometry using Xfe96. Each dataset represents the mean of 3 experiments +/- SD for bar graphs or +/- SEM for energetic traces. Significant differences among means were found with ANOVA followed by Tukey and validated with Dunnett’s multiple comparison tests. Tukey test derived p values are symbolized by asterisks indicating adjusted p-values (* p≤0.05, ** p≤0.001, *** p≤0.0001, and **** p<0.0001).
Fig 4.
c-Myc inhibition blocks IAV-induced glycolysis.
(A) Up to 17 hours PI, qPCR and immunoblotting of RNA and lysates were performed and quantification of target gene expression normalized to β-actin using the 2−ΔΔCt method. One representative blot is presented in the inset. (B-E) DC were seeded in Seahorse XFe-96 plates and pretreated with 2 μM cMyc inhibitor (MI) for 4 hours then IAV infected for 17 hours. (B-C) Glycolytic function was tested with the Glycolysis Stress Test while monitoring real-time extracellular acidification rate (ECAR) with the Xfe96 metabolic analyzer during sequential injections of glucose, oligomycin (Oligo), and 2-Deoxy-D-glucose (2-DG). Data represent means ± SD of 4 experiments. (D-E) Mitochondrial respiration was tested with the Mitochondrial Stress Test while monitoring oxygen consumption rates (OCRs) in real-time with the Xfe96 metabolic analyzer during sequential injections of oligomycin (Oligo), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and a mixture of rotenone and antimycin A (Rot/AntA). These graphs represent the mean values of 3–4 independent experiments +/- SD. Significant differences among means were found with ANOVA followed by Tukey's multiple comparisons test with asterisks indicating adjusted p-values (* p≤0.05 and ** p≤0.001).
Fig 5.
Limiting glycolysis or mitochondrial oxidation of pyruvate, glutamine or long chain fatty acids impairs infected DC function.
(A) DC were seeded on precoated Oris 96-well motility plate and allowed to adhere for 18+ hours followed by 4 hours of inhibitor treatment (2 μM c-Myc inhibitor, 3 μM BPTES, 4 μM etomoxir or 2 μM UK5099), plug removal and infection (MOI = 5) for 17 hours. Cells were stained with Calcein-AM (2 μM), and viability and motility were determined with fluorescence at 485/528 nm. The graph represents the mean values of 3 experiments +/- SD. (B-C) CD8+ T cells were isolated by negative depletion from fresh splenocytes of female Ot1 mice and co-cultured with DC at 5:1 ratio T cells to DC for 24 hours. Basal ECAR was established, and T cell activation by anti-CD3/CD28-coated DynaBeads was monitored for 2 hours and maximal ECAR determined. (B) One representative graph is shown from 3 independent experiments. (C) The graph represents the mean values of 3 experiments +/- SD. (D-F) CD8+ T cells were isolated by negative depletion from fresh splenocytes derived from C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT1) and co-cultured with DC at 5:1 ratio of T cells to DC for 24 hours. T cell were then stained for CD8, CD45.1, CD25, and CD44 and enumerated with flow cytometry. We selected CD8 and CD45.1 positive (D) and then gated on CD25 (E) and CD44 (F). The graph represent the mean values of 3 experiments +/- SD. Significant differences among means were found with ANOVA followed by Tukey's multiple comparisons test with asterisks indicating adjusted p-values (* p≤0.05, ** p≤0.001, *** p≤0.0001, and **** p<0.0001).
Fig 6.
TipDC reprogram metabolism after accumulating in the lungs of IAV-infected mice.
At zero or nine days following intranasal infection of mice with IAV (PR8), lungs were homogenized, and cells extracted. Cells were antibody stained for TipDC surface markers CD11b, Ly6c, GR-1, and MHCII. TipDC controls (Ctl) from day 0 were sorted based on high levels of CD11b, Ly6c, and GR-1. TipDC from day 9 of the IAV infection (IAV) were sorted based on high levels of CD11b, Ly6c, GR-1, and MHCII. cDNA libraries were generated from RNA and sequenced. (A) Confidentially identified transcripts were k-means clustered followed by ascendant hierarchical clustering, and genes with standard deviation threshold < 50% were removed. The data values were replaced by corresponding color intensities of blue to red through white based on interquartile range. (B-D) Significant gene expression differences were determined using Tukey’s honest significant difference test for multiple comparisons with a Benjamini-Hochberg post-hoc false discovery rate correction. TipDC genes that were either upregulated (red) or downregulated (blue) following IAV infection were mapped to KEGG pathways and significantly enriched pathways identified and sorted by KEGG Class (B-C). All isoforms of the central enzymes in glycolysis from the TipDC transcriptome are presented in color, including those that were unchanged (gray), increased (red) and decreased (blue).
Fig 7.
Dendritic cells globally reprogram metabolism in response to IAV infection.
Proteins were extracted from in vitro differentiated uninfected and infected DC, separated into insoluble and soluble fractions, and subjected to mass spectrometry. RNA was extracted from lung TipDC on day 0 or 9 of IAV infection and subjected to RNA-Seq. The log2 ratio (IAV/Ctl) of transcripts and peptides were determined. Identifiers were converted to gene symbols and official protein names, each dataset were manually searched for all NCBI listed genes and synonyms corresponding to the major regulatory enzymes of glycolysis, pyruvate metabolism, and TCA cycle. (A) All isoforms of each enzyme were grouped (y-axis) and log2 ratios plotted. (B) Peptides, from the in vitro DC proteome, of enzymes were grouped into glycolysis, pyruvate metabolism, and TCA cycle. (C)TipDC transcripts were grouped into glycolysis, pyruvate metabolism, and TCA cycle.