the groups. For the RNA sequence analysis, both
wild-type and mHIF1a-KO mice were injected
with PBS or b-glucan i.p. and the total RNA was
extracted from splenocytes at day 4. The total gene
expression profiles were accessed by RNA sequencing. This study was carried out in accordance with
the recommendations of the National Research
Council (46). The protocol was approved by the
Dartmouth IACUC (approval no. cram.ra.2).
Metabolic activity assay
Alveolar macrophages were isolated from 6- to
10-week-old HIF-KO and wild-type mice by flushing lungs 10 times with 1 ml of PBS containing
0.5 mM EDTA. Alveolar macrophages were added
and allowed to adhere for 1 hour to a 96-well
plate at a concentration of 8 × 104 in 200 ml of
CO2-independent media (Leibovitz’s L-15, Life
Technologies) supplemented with 10% FCS,
5 mM HEPES buffer, 1.1 mM L-glutamine, penicillin (0.5 U/ml), and streptomycin (50 mg/ml).
To the media, 10% Resazurin dye (Sigma) was
added and the plate was incubated at 37°C for
24 hours, with readings recorded every 30 min
at 600 nm. A 690-nm reference wavelength was
subtracted from the 600-nm wavelengths and
the data were normalized to wells without cells.
Curdlan (100 mg/ml, Sigma) was used as a stimulator of metabolic activity.
The differences between groups were analyzed
using the Wilcoxon signed-rank test (unless otherwise stated). Statistical significance of the survival
experiment was calculated using the product
limit method of Kaplan and Meier. The level of significance was defined as a P value of <0.05. Cytokine production as well as the band intensity ratio
for Western blot were plotted as a bar chart with
mean T SEM. Replicate numbers of the experiments performed are reported in the figure legends.
REFERENCES AND NOTES
1. C. A. Janeway Jr., R. Medzhitov, Innate immune recognition.
Annu. Rev. Immunol. 20, 197–216 (2002). doi: 10.1146/
annurev.immunol.20.083001.084359; pmid: 11861602
2. Z. Q. Fu, X. Dong, Systemic acquired resistance: Turning local
infection into global defense. Annu. Rev. Plant Biol. 64,
839–863 (2013). doi: 10.1146/annurev-arplant-042811-105606;
3. J. Kurtz, Specific memory within innate immune systems.
Trends Immunol. 26, 186–192 (2005). doi: 10.1016/
j.it.2005.02.001; pmid: 15797508
4. D. M. Bowdish, M. S. Loffredo, S. Mukhopadhyay, A. Mantovani,
S. Gordon, Macrophage receptors implicated in the “adaptive”
form of innate immunity. Microbes Infect. 9, 1680–1687
(2007). doi: 10.1016/j.micinf.2007.09.002; pmid: 18023392
5. J. Kleinnijenhuis et al., Bacille Calmette-Guerin induces
NOD2-dependent nonspecific protection from reinfection via
epigenetic reprogramming of monocytes. Proc. Natl. Acad. Sci.
U.S.A. 109, 17537–17542 (2012). doi: 10.1073/
pnas.1202870109; pmid: 22988082
6. J. C. Sun, J. N. Beilke, L. L. Lanier, Adaptive immune
features of natural killer cells. Nature 457, 557–561 (2009).
doi: 10.1038/nature07665; pmid: 19136945
7. J. Quintin et al., Candida albicans infection affords protection
against reinfection via functional reprogramming of
monocytes. Cell Host Microbe 12, 223–232 (2012).
doi: 10.1016/ j.chom.2012.06.006; pmid: 22901542
8. C. S. Benn, M. G. Netea, L. K. Selin, P. Aaby, A small jab—a big
effect: Nonspecific immunomodulation by vaccines. Trends
Immunol. 34, 431–439 (2013). doi: 10.1016/ j.it.2013.04.004;
9. S. Bekkering, L. A. Joosten, J. W. van der Meer, M. G. Netea,
N. P. Riksen, Trained innate immunity and atherosclerosis.
Curr. Opin. Lipidol. 24, 487–492 (2013). doi: 10.1097/
MOL.0000000000000023; pmid: 24184939
10. M. Jaskiewicz, U. Conrath, C. Peterhänsel, Chromatin
modification acts as a memory for systemic acquired
resistance in the plant stress response. EMBO Rep. 12,
50–55 (2011). doi: 10.1038/embor.2010.186; pmid: 21132017
11. G. D. Brown, S. Gordon, Fungal b-glucans and mammalian
immunity. Immunity 19, 311–315 (2003). doi: 10.1016/S1074-
7613(03)00233-4; pmid: 14499107
12. S. Saeed et al., Epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity.
Science 345, 1251086 (2014).
13. R. Oren, A. E. Farnham, K. Saito, E. Milofsky, M. L. Karnovsky,
Metabolic patterns in three types of phagocytizing cells.
J. Cell Biol. 17, 487–501 (1963). doi: 10.1083/jcb.17.3.487;
14. L. A. O’Neill, D. G. Hardie, Metabolism of inflammation limited
by AMPK and pseudo-starvation. Nature 493, 346–355
(2013). doi: 10.1038/nature11862; pmid: 23325217
15. D. Tello et al., Induction of the mitochondrial NDUFA4L2
protein by HIF-1a decreases oxygen consumption by
inhibiting Complex I activity. Cell Metab. 14, 768–779 (2011).
doi: 10.1016/ j.cmet.2011.10.008; pmid: 22100406
16. T. Klimova, N. S. Chandel, Mitochondrial complex III regulates
hypoxic activation of HIF. Cell Death Differ. 15, 660–666
(2008). doi: 10.1038/ sj.cdd.4402307; pmid: 18219320
17. S. Y. Jang, H. T. Kang, E. S. Hwang, Nicotinamide-induced
mitophagy: Event mediated by high NAD+/NADH ratio and
SIRT1 protein activation. J. Biol. Chem. 287, 19304–19314
(2012). doi: 10.1074/jbc.M112.363747; pmid: 22493485
18. G. M. Tannahill et al., Succinate is an inflammatory signal that
induces IL-1b through HIF-1a. Nature 496, 238–242 (2013).
doi: 10.1038/nature11986; pmid: 23535595
19. T. F. Liu, V. T. Vachharajani, B. K. Yoza, C. E. McCall,
NAD+-dependent sirtuin 1 and 6 proteins coordinate a switch
from glucose to fatty acid oxidation during the acute
inflammatory response. J. Biol. Chem. 287, 25758–25769
(2012). doi: 10.1074/jbc.M112.362343; pmid: 22700961
20. D. R. Donohoe, S. J. Bultman, Metaboloepigenetics:
Interrelationships between energy metabolism and epigenetic
control of gene expression. J. Cell. Physiol. 227, 3169–3177
(2012). doi: 10.1002/jcp.24054; pmid: 22261928
21. E. L. Pearce, M. C. Poffenberger, C. H. Chang, R. G. Jones,
Fueling immunity: Insights into metabolism and lymphocyte
function. Science 342, 1242454 (2013). doi: 10.1126/
science.1242454; pmid: 24115444
22. H. Chi, Regulation and function of m TOR signalling in T cell
fate decisions. Nat. Rev. Immunol. 12, 325–338 (2012).
23. B. Ferwerda et al., Human dectin-1 deficiency and mucocutaneous
fungal infections. N. Engl. J. Med. 361, 1760–1767 (2009).
doi: 10.1056/NEJMoa0901053; pmid: 19864674
24. P. K. Majumder et al., m TOR inhibition reverses Akt-dependent
prostate intraepithelial neoplasia through regulation of
apoptotic and HIF-1-dependent pathways. Nat. Med. 10,
594–601 (2004). doi: 10.1038/nm1052; pmid: 15156201
25. C. C. Hudson et al., Regulation of hypoxia-inducible factor 1a
expression and function by the mammalian target of
rapamycin. Mol. Cell. Biol. 22, 7004–7014 (2002). doi: 10.1128/
MCB.22.20.7004-7014.2002; pmid: 12242281
26. T. W. Kelley et al., Macrophage colony-stimulating factor
promotes cell survival through Akt/protein kinase B.
J. Biol. Chem. 274, 26393–26398 (1999). doi: 10.1074/
jbc.274.37.26393; pmid: 10473597
27. M. J. Marakalala et al., Dectin-1 plays a redundant role in the
immunomodulatory activities of b-glucan-rich ligands in vivo.
Microbes Infect. 15, 511–515 (2013). doi: 10.1016/j.
micinf.2013.03.002; pmid: 23518266
28. D. M. Gwinn et al., AMPK phosphorylation of raptor mediates
a metabolic checkpoint. Mol. Cell 30, 214–226 (2008).
doi: 10.1016/ j.molcel.2008.03.003; pmid: 18439900
29. E. Bosi, Metformin—the gold standard in type 2 diabetes:
What does the evidence tell us? Diabetes Obes. Metab. 11
(suppl. 2), 3–8 (2009). doi: 10.1111/j.1463-1326.2008.01031.x;
30. T. Cramer et al., HIF-1a is essential for myeloid cell-mediated
inflammation. Cell 112, 645–657 (2003). doi: 10.1016/S0092-
8674(03)00154-5; pmid: 12628185
31. R. Ostuni et al., Latent enhancers activated by stimulation in
differentiated cells. Cell 152, 157–171 (2013). doi: 10.1016/
j.cell.2012.12.018; pmid: 23332752
32. O. Warburg, Metabolism of tumours. Biochem. Z. 142, 317
33. C. H. Chang et al., Posttranscriptional control of T cell effector
function by aerobic glycolysis. Cell 153, 1239–1251 (2013).
doi: 10.1016/j.cell.2013.05.016; pmid: 23746840
34. J. C. Rodríguez-Prados et al., Substrate fate in activated
macrophages: A comparison between innate, classic, and
alternative activation. J. Immunol. 185, 605–614
(2010). doi: 10.4049/jimmunol.0901698; pmid: 20498354
35. A. S. Zinkernagel, C. Peyssonnaux, R. S. Johnson, V. Nizet,
Pharmacologic augmentation of hypoxia-inducible factor-1a
with mimosine boosts the bactericidal capacity of phagocytes.
J. Infect. Dis. 197, 214–217 (2008). doi: 10.1086/524843;
36. S. Paust et al., Critical role for the chemokine receptor CXCR6
in NK cell-mediated antigen-specific memory of haptens and
viruses. Nat. Immunol. 11, 1127–1135 (2010). doi: 10.1038/
ni.1953; pmid: 20972432
37. J. W. van der Meer, M. Barza, S. M. Wolff, C. A. Dinarello, A low
dose of recombinant interleukin 1 protects granulocytopenic
mice from lethal Gram-negative infection. Proc. Natl. Acad.
Sci. U.S.A. 85, 1620–1623 (1988). doi: 10.1073/pnas.85.5.1620;
38. P. Aaby et al., Randomized trial of BCG vaccination at birth
to low-birth-weight children: Beneficial nonspecific effects
in the neonatal period? J. Infect. Dis. 204, 245–252 (2011).
doi: 10.1093/infdis/jir240; pmid: 21673035
39. A. Yáñez et al., Detection of a TLR2 agonist by hematopoietic
stem and progenitor cells impacts the function of the
macrophages they produce. Eur. J. Immunol. 43, 2114–2125
(2013). doi: 10.1002/eji.201343403; pmid: 23661549
40. J. A. Hamilton et al., Hypoxia enhances the proliferative
response of macrophages to CSF-1 and their pro-survival
response to TNF. PLOS ONE 7, e45853 (2012). doi: 10.1371/
journal.pone.0045853; pmid: 23029275
41. B. Everts et al., Commitment to glycolysis sustains survival
of NO-producing inflammatory dendritic cells. Blood 120,
1422–1431 (2012). doi: 10.1182/blood-2012-03-419747;
42. Y. Masuda, T. Togo, S. Mizuno, M. Konishi, H. Nanba, Soluble
b-glucan from Grifola frondosa induces proliferation and
Dectin-1/Syk signaling in resident macrophages via the
GM-CSF autocrine pathway. J. Leukoc. Biol. 91, 547–556
(2012). doi: 10.1189/jlb.0711386; pmid: 22028332
43. H. Li, R. Durbin, Fast and accurate short read alignment
with Burrows-Wheeler transform. Bioinformatics 25,
1754–1760 (2009). doi: 10.1093/bioinformatics/btp324;
44. N. A. Rao et al., Coactivation of GR and NFKB alters the
repertoire of their binding sites and target genes.
Genome Res. 21, 1404–1416 (2011). doi: 10.1101/
gr.118042.110; pmid: 21750107
45. Y. Zhang et al., Model-based analysis of ChIP-Seq (MACS).
Genome Biol. 9, R137 (2008). doi: 10.1186/gb-2008-9-9-r137;
46. Guide for the Care and Use of Laboratory Animals (National
Academies Press, Washington, DC, ed. 8, 2011).
S.-C.C., J.Q., and M.G.N. were supported by a Vici grant of
the Netherlands Organization of Scientific Research and ERC
Consolidator grant 310372 (both to M.G.N). C. W. is supported
by funding from the European Research Council under
the European Union’s Seventh Framework Programme
(FP/2007-2013)/ERC grant agreement 2012-322698). Y.L. is
supported by Veni grant 863.13.011 of the Netherlands
Organization for Scientific Research. R.A.C. and K.M.S.
were supported by National Institute of General Medical
Sciences grant 5P30GM103415-03 (William Green, PI) and
1P30GM106394-01 (Bruce Stanton, PI), and National Institute of
Allergy and Infectious Diseases grant R01AI81838 (R.A.C., PI).
R.A.C./K.M.S. thank B. Berwin for the S. aureus. R.J.X funded by
DK43351, DK097485, Helmsley Trust, and JDRF. The detailed
data have been deposited in the GEO database with accession
Figs. S1 to S15
Tables S1 and S2
10 January 2014; accepted 28 August 2014
1250684-8 26 SEPTEMBER 2014 • VOL 345 ISSUE 6204
RESEARCH | RESEARCH ARTICLE