Cutting Edge: Essential Role of Hypoxia Inducible
Factor-1? in Development of
Carole Peyssonnaux,*†Pilar Cejudo-Martin,* Andrew Doedens,* Annelies S. Zinkernagel,†
Randall S. Johnson,2* and Victor Nizet2†‡
Sepsis, the leading cause of death in intensive care units,
reflects a detrimental host response to infection in which
bacteria or LPS act as potent activators of immune cells,
including monocytes and macrophages. In this report, we
show that LPS raises the level of the transcriptional regu-
lator hypoxia-inducible factor-1? (HIF-1?) in macro-
phages, increasing HIF-1? and decreasing prolyl hydrox-
ylase mRNA production in a TLR4-dependent fashion.
myeloid lineage, we demonstrate that HIF-1? is a critical
production of inflammatory cytokines, including TNF-?,
IL-1, IL-4, IL-6, and IL-12, that reach harmful levels in
the host during early sepsis. HIF-1? deletion in macro-
phages is protective against LPS-induced mortality and
blocks the development of clinical markers including hy-
potension and hypothermia. Inhibition of HIF-1? activ-
ity may thus represent a novel therapeutic target for LPS-
induced sepsis. The Journal of Immunology, 2007, 178:
2). Important biological mediators of sepsis are inflammatory
cytokines including TNF-?, IL-1, and IL-6, which are released
macrophages, because the transfer of LPS-sensitive macro-
phages renders previously LPS-resistant mice sensitive to sepsis
(3). No single agent or treatment strategy has shown sufficient
cytokine activation patterns of sepsis. We recently identified
novel and essential roles of the transcriptional regulator hypox-
epsis is a major and increasing cause of mortality and
morbidity throughout the world with an annual inci-
dence of 2.4–3.0 cases per 1,000 in the population (1,
ia-inducible factor (HIF)31? (HIF-1?) in controlling macro-
phage inflammatory responses in the skin and joints (4) and
promoting their phagocytic function (5). In this study we hy-
pothesized that HIF-1? could play an important role in medi-
ating the inflammatory responses during LPS-induced sepsis.
Using mice with a targeted deletion of HIF-1? in macrophages
(4, 5), we examined this hypothesis in vitro and in vivo, dem-
onstrating the potential relevance of HIF-1? as a target for sep-
Materials and Methods
Transgenic HIF flox, VEGF flox, and LysM-cre alleles were individually back-
crossed to ?99% C57BL/6 before breeding to obtain the indicated genotypes.
These mice and inbred mouse strains HIFflox/flox/LysMcre and VEGFflox/flox/
LysMcre (backcrossed in a pure C57BL/6 genetic background) and inbred
mouse strains CH3/HeOuJ and CH3/HeJ (obtained from The Jackson Labo-
ratory) were handled by approved protocols of the University of California San
Diego Institutional Animal Care and Use Committee (La Jolla, CA). For com-
parisons of targeted myeloid cell gene deletions driven by the LysM promoter,
knockout mice were compared with the corresponding flox/flox littermates
(designated wild type (WT)). Eight- to 10-wk-old male HIFflox/flox/LysMcre or
VEGFflox/flox/LysMcre males were injected i.p. with 15 mg/kg Escherichia coli
LPS serotype 0111:B4 (Sigma-Aldrich) or saline solution. Mice were moni-
pressure and heart beat rate, tail cuff measurements were made by using the
Kent Scientific XBP1000 system. Mean blood pressure was determined by the
following method: diastolic pressure ? [(systolic pressure ? diastolic pres-
sure) ? 3]. Surface temperatures were measured with a ThermoVision A20
thermal infrared camera (FLIR Systems).
Measurement of cytokine levels
Blood samples were drawn 90 min or 4 h after LPS injection by retro-orbital
bleeding. Mouse TNF-? and IL-6 were assayed by using the ELISA kit from
eBioscience and vascular endothelial growth factor (VEGF) was assayed with
measured by the ProteoPlex murine cytokine array kit (EMD Biosciences).
Western blot studies
Bone marrow-derived macrophages of CH3/HeOuJ and CH3/HeJ mice were
isolated as described (5). LPS (100 ng/ml) or log-phase Pseudomonas aeruginosa
*Division of Biological Sciences and†Department of Pediatrics, School of Medicine and
‡Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San
Diego, La Jolla, CA 92093
Received for publication October 10, 2006. Accepted for publication April 16, 2007.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grants CA82515 (to R.S.J.)
(to V.N.). A.Z. is supported by Swiss National Foundation Grant PPBZHB-108365 and
P.C.-M. is supported by a fellowship from the Ministerio de Educacion y Ciencia (Spain).
2Address correspondence and reprint requests to Dr. Randall S. Johnson, Molecular Bi-
ology Section, Division of Biological Sciences, University of California San Diego, 9500
Victor Nizet, School of Medicine, Skaggs School of Pharmacy and Pharmaceutical Sci-
ences, University of California San Diego, 9500 Gilman Drive, MC 0687, La Jolla, CA
92093-0687; E-mail address: email@example.com
3Abbreviations used in this paper: HIF, hypoxia-inducible factor; PHD, prolyl hydroxy-
lase; VEGF, vascular endothelial growth factor; WT, wild type.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
or Salmonella typhimurium were added (at a multiplicity of infection of five
bacteria per cell) to bone marrow-derived macrophages for 4 h. Cells harvested
and extracted with radioimmune precipitation assay buffer were probed using
rabbit anti-mouse HIF (Novus Biological) at 1/1000.
Real-time quantitative RT-PCR
The RNA of macrophages exposed to 100 ng/ml LPS (Sigma-Aldrich) for 4 h
was isolated using TRI Reagent (Molecular Research Center). First-strand syn-
thesis and real-time PCR were performed as described (5) in a TaqMan Uni-
versal SYBR Green Master Mix (Applied Biosystems) using specific primers
with the following sequences (rates normalized to the expression level of ribo-
somal RNA): HIF-1, 5?-GAAACGACCACTGCTAAGGCA-3? (forward)
and 5?-GGCAGACAGCTTAAGGCTCCT-3? (reverse); prolyl hydroxylase
(PHD) 1, 5?-GGCCAGTGGTAGCCAACATC-3? (forward) and 5?-GTG
GCATAGGCTGGCTTCAC-3? (reverse); PHD2, 5?- TGACCACACCTC
TCCAGCAA-3? (forward) and 5?- CTGCCAACAATGCCAAACAG-3? (re-
verse); and PHD3, 5?-GGTGGCTTGCTATCCAGGAA-3? (forward) and
The significance of experimental differences was evaluated by two-way
by the construction of Kaplan-Meier plots and use of the log-rank test.
All animal experiments were approved by the University of California San Di-
ego Institutional Animal Care and Use Committee and performed using ac-
cepted veterinary standards.
Results and Discussion
HIF-1? is induced by LPS in a TLR4-dependent fashion
Cellular HIF-1? levels are controlled in part through PHD-
mediated proteolytic degradation in which oxygen and iron
serve as cofactors (6). Recently, we and others have demon-
strated that bacteria or LPS induce HIF-1? stabilization even
under normoxic conditions (5, 7–9). LPS is a microbial activa-
tor of TLR4, a pattern recognition molecule critical for initiat-
ing innate immune signaling cascades and proinflammatory re-
sponses (10). To determine whether the HIF-1? response to
bone marrow of WT mice or those possessing a spontaneous
mutation in TLR4 (Tlr4Lps-d) (11). By Western blot analysis
we found that the loss of TLR4 clearly reduced the level of
macrophage HIF-1? induced following exposure to LPS and
two Gram-negative bacterial species, S. typhimurium and P.
aeruginosa (Fig. 1A). Real-time RT-PCR analysis strongly
suggested that part of the TLR4-dependent increase in
HIF-1? levels following LPS exposure occurs at the tran-
scriptional level (Fig. 1B). Although differences in mRNA
stability cannot be definitively excluded, a significant in-
crease in the promoter activity of HIF-1? after LPS treat-
ment has recently been described (7).
To further understand why LPS can raise HIF-1? levels
even under normoxia, we explored a second mechanism for
LPS-induced HIF-1? stabilization—expression of the three
HIF-1? PHDs (PHD1–3) that catalyze degradation of the
transcriptional regulator. LPS stimulation significantly de-
creased levels of PHD2 and PHD3 mRNA in WT macro-
phages but not in the TLR4-macrophages (Fig. 1C); PHD1
expression was not affected. Because these PHDs are non-
equilibrium enzymes (i.e., they do not catalyze the reverse
reaction), enzyme abundance is an important determinant of
the rate of substrate hydroxylation and, thus, cellular
HIF-1? levels (12). We conclude that the TLR4-dependent
increase in HIF-1? after LPS stimulation reflects a combi-
nation of increased HIF-1? transcription and decreased
HIF-1? degradation mediated by PHD2 and PHD3. The
PHD2 isoform is known to play a key nonredundant role in
HIF-1? regulation (13), and a similar HIF-1? stabilization
mechanism that works through selective inhibition
of PHD2 expression in response to TGF-?1 has recently
been described (14).
Given our observation that macrophage HIF-1? levels are
increased in response to LPS, we predicted that HIF-1?
transcriptional regulation could influence the characteristic
pattern of inflammatory gene expression attributed to LPS
activation and TLR4 signaling. As a preliminary validation
of this principle, we exposed isolated macrophages from
WT and HIF1? myeloid null mice to LPS and measured the
transcript levels for the signature cytokine IL-6 by real-time
RT-PCR. HIF1? null macrophages showed a significant
reduction in IL-6 mRNA compared with WT (Fig. 1D),
suggesting that the induction of HIF1? could represent a
functional intermediary in the LPS/TLR4 activation
extracts from WT and Tlr4Lps-dbone marrow-derived
macrophages stimulated for 4 h with LPS, S. typhimurium
(ST) or P. aeruginosa (PA). B and C, Real-time PCR for
HIF-1? expression (B) or PHD-1, PHD-2, and PHD-3
marrow-derived macrophages; samples were normalized
individually to each PHD isoform control. D, Real-time
PCR for IL-6 expression on LPS-stimulated WT and
HIF-1? myeloid null bone marrow-derived macrophages.
Studies were performed in triplicate and repeated twice
with similar results; a representative experiment shown.
LPS induces HIF-1? expression through a
7517The Journal of Immunology
LPS-induced cytokine release in vivo is influenced by HIF-1?
To confirm the significance of our observations in vivo, we in-
jected WT and HIF-1? macrophage null mice with 15 mg/kg
determination of several cytokine levels by ELISA (Fig. 2). Cy-
upon LPS challenge. The loss of macrophage expression of
HIF-1? led to significant decreases in the production of
TNF-?, IL-1?, IL-1?, and IL-12 but did not affect the pro-
duction of IFN-? or the anti-inflammatory cytokines IL-4 and
IL-10. TNF-? and IL-1? are “proximal” cytokines well known
to trigger many of the clinical manifestations of LPS-induced
sepsis (reviewed in Ref. 15), whereas IL-6 levels correlate to
(16, 17). Neutralizing the Abs to IL-12 can reduce LPS-in-
duced mortality in mice, indicating this less well studied cyto-
kine also plays a significant role in the systemic LPS response
(18, 19). We conclude that HIF-1? in macrophages contrib-
utes to the expression of several key cytokines implicated in the
pathogenesis of LPS-induced sepsis.
Deletion of HIF-1? is protective against LPS-induced sepsis
symptomatology and lethality
We hypothesized that the diminished responsiveness of
HIF-1? null macrophages to LPS-induced inflammatory cyto-
kine activation would be reflected in decreased LPS-induced
sepsis symptomatology in the corresponding knockout mice.
Two hallmark clinical manifestations of the LPS-induced sys-
temic activation of cytokines, including TNF-?, in the mouse
induced hypothermia, with mutant mice possessing an average
surface temperature 2°C greater than that of WT mice (Fig.
3A). Significant (p ? 0.0005) protection against LPS-induced
hypotension was also observed in the HIF-1? myeloid null
pared with 41 ? 2 for the WT controls (Fig. 3B). With an im-
provement of this hemodynamic parameter, the shock index,
defined as the ratio of the heart rate to systolic blood pressure,
was significantly decreased in HIF-1? myeloid null mice com-
pared with the WT controls (p ? 0.0004) (Fig. 3C).
duction of the inflammatory cytokines TNF-?, IL-6, IL-
1?, IL-1?, IL-4, and IL-12. A, TNF-? and IL-6 levels
measured by ELISA 90 min after LPS injection. B, Cyto-
ray 1 h 30 min and 4 h after LPS injection; n ? 5 animals
per group. Studies were repeated twice with similar results;
a representative experiment is shown.
HIF-1? contributes to macrophage pro-
mean blood pressure (B), and shock index (C) of WT
mean blood pressure ? diastolic pressure ? [(systolic
pressure ? diastolic pressure) ?3]. HR, Heart rate; BP,
blood pressure. D, Survival following i.p. LPS injection
in WT and HIF-1? myeloid null or VEGF myeloid
null-mice (n ? 12 per group). E, VEGF levels in serum
of HIF-1? myeloid null mice 4 h after LPS challenge;
n ? 5 animals per group. The study was performed in
triplicate and repeated twice with similar results; a rep-
resentative experiment is shown.
HIF-1? contributes to LPS-induced
7518 CUTTING EDGE: ESSENTIAL ROLE OF HIF-1? IN SEPSIS
The correlation of LPS-induced symptomatology with le- Download full-text
thality was also established. Whereas LPS challenge of the WT
mice produced 82% mortality by 48 h, HIF-1? myeloid null
mice experienced only 14% mortality by day 5 (p ? 0.002)
bidity and mortality (24). However, the deletion of VEGF in
the myeloid lineage does not rescue LPS-induced mortality
(Fig. 3E). Moreover, the loss of macrophage expression of
HIF-1? did not by itself produce a significant decrease in the
total production of VEGF by the mouse 4 h after LPS stimula-
tion (Fig. 3E), with the understanding that VEGF production
in other cell types (e.g., vascular smooth muscle, hepatocytes)
remained unaffected in our targeted knockout mice. We con-
flammatory cytokines, and not VEGF, plays a more important
function in influencing the sepsis phenotype.
An interplay of HIF-1? and NF-?B regulatory pathways in
macrophage activation is revealed by our findings and other re-
cent data. Frede et al. (7) found that LPS induced HIF-1? in
human macrophages in vitro at normoxia through enhanced
transcription of the HIF-1? gene. In this study, LPS induced
upstream of the HIF-1? transcriptional start site, and the inhi-
bition of NF-?B abolished LPS-induced HIF-1? target gene
expression. Although NF-?B is an important downstream ef-
fector of the HIF-1?-dependent response in neutrophils to an-
oxia (25), we did not observe differences in NF-?B, IKK?, or
I?B? expression in HIFlox/lox/LysMcre macrophages vs WT af-
ter LPS stimulation (data not shown), indicating that the
NF-?B pathway per se is not affected by HIF-1? deletion in
macrophages. Thus, anoxia and infection/inflammation may
induce HIF-dependent and HIF-independent NF-?B path-
ways, respectively. Lastly, it is possible that some proinflamma-
tory cytokines could be directly activated by HIF; for example,
we observe a hypoxia response element site GCGTG 5? of the
IL-6 gene and two hypoxia response element sequences in tan-
dem 3? of the TNF-? gene in the mouse genome (hypoxia re-
sponse elements in the classical HIF target gene erythropoietin
are also located in the 3? flanking region).
In conclusion, we have identified an additional global regu-
latory function of HIF-1? in the inflammatory function of
macrophages. HIF-1? is activated by LPS in a TLR4-depen-
dent fashion and contributes to the cytokine activation, symp-
knowledge, this represents the first linkage of the HIF-1? tran-
scriptional regulator with TLR pattern recognition and should
tant pathways in the regulation of macrophage activation. Pre-
vious work has prompted discussion of HIF-1? modulation as
a pharmacologic approach for the treatment of chronic inflam-
matory disorders or the augmentation of innate immune func-
tion (26–28). Our studies suggest that HIF-1? may addition-
ally represent a novel therapeutic target for ameliorating the
aberrant cytokine activation pattern and poor prognosis of pa-
tients with LPS-induced sepsis.
We acknowledge Christian Stockmann for helpful suggestions and Adam Bou-
tin for assistance in temperature and blood pressure measurements.
The authors have no financial conflict of interest.
2. Martin, G. S., D. M. Mannino, S. Eaton, and M. Moss. 2003. The epidemiology of
sepsis in the United States from 1979 through 2000. N. Engl. J. Med. 348:
Infect. Immun. 51: 891–895.
4. Cramer, T., Y. Yamanishi, B. E. Clausen, R. Pawlinski, N. Mackman, V. Haase,
2003. HIF-1? is essential for myeloid cell-mediated inflammation. Cell 112: 645–657.
5. Peyssonnaux, C., V. Datta, T. Cramer, A. Doedens, E. A. Theodorakis, R. L. Gallo,
N. Hurtado-Ziola, V. Nizet, and R. S. Johnson. 2005. HIF-1? expression regulates
the bactericidal capacity of phagocytes. J. Clin. Invest. 115: 1806–1815.
lases. Novartis Found. Symp. 272: 15–36.
7. Frede, S., C. Stockmann, P. Freitag, and J. Fandrey. 2006. Bacterial lipopolysaccha-
ride induces HIF-1 activation in human monocytes via p44/42 MAPK and NF-?B.
Biochem. J. 396: 517–527.
8. Blouin, C. C., E. L. Page, G. M. Soucy, and D. E. Richard. 2004. Hypoxic gene
activation by lipopolysaccharide in macrophages: implication of hypoxia-inducible
factor 1?. Blood 103: 1124–1130.
9. Kempf, V. A., M. Lebiedziejewski, K. Alitalo, J. H. Walzlein, U. Ehehalt, J. Fiebig,
S. Huber, B. Schutt, C. A. Sander, S. Muller, G. Grassl, et al. 2005. Activation of
hypoxia-inducible factor-1 in bacillary angiomatosis: evidence for a role of hypoxia-
inducible factor-1 in bacterial infections. Circulation 111: 1054–1062.
10. Palsson-McDermott, E. M., and L. A. O’Neill. 2004. Signal transduction by the li-
popolysaccharide receptor, Toll-like receptor-4. Immunology 113: 153–162.
identification of the Toll-4 receptor as a candidate gene in the critical region. Blood
Cells Mol. Dis. 24: 340–355.
12. Appelhoff, R. J., Y. M. Tian, R. R. Raval, H. Turley, A. L. Harris, C. W. Pugh,
PHD1, PHD2, and PHD3 in the regulation of hypoxia-inducible factor. J. Biol.
Chem. 279: 38458–38465.
prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1?
in normoxia. EMBO J. 22: 4082–4090.
14. McMahon, S., M. Charbonneau, S. Grandmont, D. E. Richard, and C. M. Dubois.
2006. Transforming growth factor ?1 induces hypoxia-inducible factor-1 stabiliza-
tion through selective inhibition of PHD2 expression. J. Biol. Chem. 281:
15. Blackwell, T. S., and J. W. Christman. 1996. Sepsis and cytokines: current status.
Br. J. Anaesth. 77: 110–117.
16. Remick, D. G., G. R. Bolgos, J. Siddiqui, J. Shin, and J. A. Nemzek. 2002. Six at six:
interleukin-6 measured 6 h after the initiation of sepsis predicts mortality over 3 days.
Shock 17: 463–467.
17. Oberholzer, A., S. M. Souza, S. K. Tschoeke, C. Oberholzer, A. Abouhamze,
J. P. Pribble, and L. L. Moldawer. 2005. Plasma cytokine measurements augment
prognostic scores as indicators of outcome in patients with severe sepsis. Shock 23:
18. Wysocka, M., M. Kubin, L. Q. Vieira, L. Ozmen, G. Garotta, P. Scott, and
G. Trinchieri. 1995. Interleukin-12 is required for interferon-? production and le-
thality in lipopolysaccharide-induced shock in mice. Eur. J. Immunol. 25: 672–676.
of the Stat6 pathway contributes to the suppression of cell-mediated immunity and
death in sepsis. Surgery 128: 133–138.
20. Kozak, W., C. A. Conn, J. J. Klir, G. H. Wong, and M. J. Kluger. 1995. TNF soluble
receptor and antiserum against TNF enhance lipopolysaccharide fever in mice.
Am. J. Physiol. 269: R23–R29.
21. Leon, L. R., A. A. White, and M. J. Kluger. 1998. Role of IL-6 and TNF in thermo-
regulation and survival during sepsis in mice. Am. J. Physiol. 275: R269–277.
22. Silva, A. T., K. F. Bayston, and J. Cohen. 1990. Prophylactic and therapeutic effects
of a monoclonal antibody to tumor necrosis factor-? in experimental Gram-negative
shock. J. Infect. Dis. 162: 421–427.
23. Forsythe, J. A., B. H. Jiang, N. V. Iyer, F. Agani, S. W. Leung, R. D. Koos, and
G. L. Semenza. 1996. Activation of vascular endothelial growth factor gene transcrip-
tion by hypoxia-inducible factor 1. Mol. Cell. Biol. 16: 4604–4613.
24. Yano, K., P. C. Liaw, J. M. Mullington, S. C. Shih, H. Okada, N. Bodyak,
P. M. Kang, L. Toltl, B. Belikoff, J. Buras, et al. 2006. Vascular endothelial growth
25. Walmsley, S. R., C. Print, N. Farahi, C. Peyssonnaux, R. S. Johnson, T. Cramer,
A. Sobolewski, A. M. Condliffe, A. S. Cowburn, N. Johnson, and E. R. Chilvers.
2005. Hypoxia-induced neutrophil survival is mediated by HIF-1?-dependent
NF-?B activity. J. Exp. Med. 201: 105–115.
26. Zarember, K. A., and H. L. Malech. 2005. HIF-1?: a master regulator of innate host
defenses. J. Clin. Invest. 115: 1702–1704.
27. Strieter, R. M. 2003. Mastering innate immunity. Nat. Med. 9: 512–513.
28. Nathan, C. 2003. Immunology: oxygen and the inflammatory cell. Nature 422:
7519The Journal of Immunology