Hypoxia and Inflammation

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Mechanisms of Disease
Robert S. Schwartz, M.D., Editor
Review article
n engl j med 364;7 nejm.org february 17, 2011
656
Hypoxia and Inflammation
Holger K. Eltzschig, M.D., Ph.D., and Peter Carmeliet, M.D., Ph.D.
From the Department of Anesthesiology,
University of Colorado Denver, Aurora
(H.K.E.); the Department of Anesthesiol-
ogy and Intensive Care Medicine, Tübin-
gen University Hospital, Tübingen, Ger-
many (H.K.E.); and Vesalius Research
Center VIB, and Vesalius Research Cen-
ter, K.U. Leuven — both in Leuven, Bel-
gium (P.C.). Address reprint requests to
Dr. Eltzschig at the Department of Anes-
thesiology, University of Colorado Denver,
12700 E. 19th Ave., Mailstop B112, Re-
search Complex 2, Rm. 7124, Aurora, CO
80045, or at holger.eltzschig@ucdenver
.edu.
N Engl J Med 2011;364:656-65.
Copyright © 2011 Massachusetts Medical Society.
M
ammals have oxygen-sensing mechanisms that help them
adapt quickly to hypoxia by increasing respiration, blood flow, and survival
responses. If an inadequate supply of oxygen persists, additional mecha-
nisms attempt to restore oxygenation or help the body adapt to hypoxia.
1
These
other mechanisms rely on oxygen-sensing prolyl hydroxylases (PHDs), which hydrox-
ylate prolines in the alpha subunit of the hypoxia-inducible transcription factor (HIF).
This transcription factor is a heterodimer with two subunits: HIF-1α or HIF-2α and
HIF-1β (or aryl hydrocarbon receptor nuclear translocator [ARNT] protein). HIF-1α
is ubiquitous, whereas HIF-2α is restricted to certain tissues.
1
In this review, we show the ways in which the PHD–HIF system affects inflam-
matory processes. We discuss the regulation of immune responses by hypoxia-
induced signaling, outline molecular aspects of the cross-talk between hypoxia and
inflammation, and illustrate the link between hypoxia and inflammation in in-
flammatory bowel disease, certain cancers, and infections.
Hy pox ia -Induced Infl amm at ion
The concept that hypoxia can induce inflammation has gained general acceptance
from studies of the hypoxia signaling pathway. In persons with mountain sickness,
for example, levels of circulating proinflammatory cytokines increase, and leakage
of fluid (“vascular leakage”) causes pulmonary or cerebral edema.
1-3
Increased se-
rum levels of interleukin-6, the interleukin-6 receptor, and C-reactive protein — all
markers of inflammation — were increased in healthy volunteers who spent 3 nights
at an elevation higher than 3400 m.
4
At 8400 m, healthy climbers ascending Mount
Everest had severe hypoxemia (partial pressure of arterial oxygen [PaO
2
], 25 mm Hg).
Alveolar–arterial oxygen differences were elevated in these climbers, a finding that
is consistent with subclinical high-altitude pulmonary edema.
3
Moreover, vascular
leakage, accumulations of inflammatory cells in multiple organs, and elevated serum
levels of cytokines occur in mice after short-term exposure to low oxygen concen-
trations.
5-9
The development of inflammation in response to hypoxia is clinically relevant.
Ischemia in organ grafts increases the risk of inflammation and graft failure or
rejection.
10
In patients undergoing kidney transplantation, the renal expression of
toll-like receptor (TLR) 4 — an extracellular receptor for bacterial lipopolysaccha-
ride — was shown to correlate with the degree of ischemic injury. In this study,
donor kidneys with a loss-of-function TLR4 allele, as compared with donor kidneys
that bore a functional allele of the TLR4 gene, had a higher rate of immediate graft
function.
10
Moreover, increases in pulmonary cytokine levels and TLR expression
was shown to correlate with greater ischemic injury of transplanted lungs and loss
of graft function.
11,12
In the setting of obesity, an imbalance between the supply
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of and demand for oxygen in enlarged adipocytes
causes tissue hypoxia and an increase in inflam-
matory adipokines in fat. The resultant infiltration
by macrophages and chronic low-grade systemic
inflammation promote insulin resistance.
13
Taken
together, these clinical studies indicate that hy-
poxia promotes inflammation (Fig. 1).
Infl amm ation a nd T issu e
Hy poxi a
Just as hypoxia can induce inflammation, inflamed
lesions often become severely hypoxic. As a result
of the steep oxygen gradient between the anaero-
bic intestinal lumen and the metabolically active
lamina propria mucosae, intestinal epithelial cells
are normally hypoxic.
14
In inf lammatory bowel
disease, not only does the entire mucosa becomes
even more hypoxic,
14
but surgical specimens of
the inflamed intestine contain elevated levels of
HIF-1α and HIF-2α.
15
Contributors to tissue hypoxia during inflam-
mation include an increase in the metabolic
demands of cells and a reduction in metabolic
substrates caused by thrombosis, trauma, com-
pression (interstitial hypertension), or atelectasis
(airway plugging). Moreover, multiplication of in-
tracellular pathogens can deprive infected cells of
oxygen.
16
We stress that in the case of inflamed
tissue, hypoxia is not a bystander but instead can
influence the environment of the tissue, particu-
larly by regulating oxygen-dependent gene ex-
pression.
HIF a nd Ox ygen Sensor s
Cellular adaptations to hypoxia rely on the tran-
scription factor HIF, which is inactive when oxy-
gen is abundant but is activated in hypoxic condi-
tions (Fig. 2).
1,17
Oxygen-dependent hydroxylation
of prolyl residues in HIF-1α or HIF-2α in the HIF
heterodimer by PHDs creates a binding site for
the von Hippel–Lindau (VHL) gene product, which
is a component of the E3 ubiquitin ligase com-
plex; the binding of the VHL gene product to
HIF-1α (or HIF-2α) culminates in the destruction
of the α subunit in proteasomes.
18
In addition,
hydroxylation of asparagyl residues in HIF-1α (or
HIF-2α) by factor-inhibiting HIF — an oxygen-
dependent asparagyl hydroxylase — reduces the
transcriptional activity of HIF.
19
The functions of
both hydroxylases (PHDs and factor-inhibiting
HIF) depend on oxygen.
1,17
Germline mutations
in the PHD2 gene have been found in association
with familial erythrocytosis and with a syndrome
of familial erythrocytosis with paraganglioma
20
;
inactivating mutations of both copies of the VHL
gene cause Von Hippel–Lindau disease (which is
characterized by hemangioblastomas, clear-cell re-
nal carcinomas, and pheochromocytomas).
18
HIF can be activated under normoxic condi-
tions, which allows the initiation of an inflam-
matory response before tissues become hypoxic.
Examples of this mechanism are the increase in
HIF-1α transcription by bacterial lipopolysaccha-
ride
21
and the stabilization of HIF-1α when reac-
tive oxygen species and reduced cellular iron in-
hibit prolyl hydroxylase.
22,23
The phenotype of mice with HIF-1α deficiency
differs from that of mice with HIF-2α deficiency,
which implies that these components of the HIF
transcription-factor polypeptides have different
target genes.
24
The HIF2A gene in certain forms
of familial erythrocytosis has a gain-of-function
mutation, which probably causes normoxic stabi-
lization of the HIF-2α protein.
25
Hy pox ia Signa ling a nd NF -κB
Members of the nuclear factor κB (NF-κB) family
of transcription factors regulate inflammation and
orchestrate immune responses and tissue homeo-
stasis.
26-28
Members of this family interact with
members of the PHD–HIF pathway in ways that
link inflammation to hypoxia (Fig. 2).
29
Studies
of a mouse model of inflammatory bowel disease
indicate that PHDs have a regulatory role in the
antiapoptotic effects of NF-κB in intestinal in-
flammation.
30,31
The hypoxia of intestinal ischemia
reperfusion activates NF-κB in intestinal epithe-
lial cells, which in turn increases the production
of tumor necrosis factor α (TNF-α), a proinflam-
matory cytokine, but simultaneously attenuates
intestinal epithelial apoptosis.
32
Additional inter-
actions between hypoxia and inflammation are
seen in the IκB kinase complex, a regulatory com-
ponent of NF-κB (Fig. 2),
30
and in the regulation
of HIF-1α transcription by NF-κB before and
during inflammation.
33,34
Hypoxia amplifies the
NF-κB pathway by increasing the expression and
signaling of TLRs, which enhance the production
of antimicrobial factors and stimulate phagocy-
tosis, leukocyte recruitment, and adaptive im-
munity.
35

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Hy pox ia Signa ling a nd Inna te
Immunit y
The initial defense against pathogens relies on
the activation of neutrophils, macrophages, mast
cells, dendritic cells, and natural killer cells. These
cells of the innate immune system can rapidly
eradicate pathogens and transmit signals that
amplify the adaptive immune response. Myeloid
cells have HIF-dependent ways of functioning in
the oxygen-depleted conditions of hypoxic mi-
croenvironments.
36
HIF-1α–null phagocytes can-
not efficiently eliminate bacterial loads but in-
stead form persistent ulcerative lesions.
36,37
HIF-1α regulates several functions of myeloid
cells (Fig. 3).
38
It allows myeloid cells to gener-
ate ATP in oxygen-deprived inflamed tissues,
thereby stimulating the aggregation, motility,
invasiveness, and bactericidal activity of myeloid
cells.
36,37
HIF-1α also prolongs the lifespan of
neutrophils in hypoxic conditions by inhibiting
apoptosis.
39
In von Hippel–Lindau disease, neu-
trophils are characterized by reduced apoptosis
and enhanced phagocytosis of bacteria under
normoxic conditions, presumably owing to the
failure to degrade HIF-1α.
40
Hy pox ia a nd Ada pti ve I mmun it y
HIF-1α also influences adaptive immunity.
41
Mice
with HIF-1α-deficient lymphocytes have elevated
levels of anti–double-stranded DNA antibodies
and rheumatoid factor in serum, as well as pro-
teinuria and deposits of IgG and IgM in the kid-
Inflammation in Hypoxic Conditions Hypoxia in Inflammatory Conditions
Pulmonary edema Acute lung injury
Colitis
Infections with pathogens
Cancer
Organ transplantation
Adipose tissue
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Figure1.LinksbetweenHypoxiaandInf lammation.
Shown is an overview of clinical conditions characterized primarily by tissue hypoxia that causes inflammatory changes (left) and inflam-
matory diseases leading to tissue hypoxia (right).

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ney.
42
Increased production of HIF-1α in T cells
induces a shift from a type 1 helper T-cell (Th1)
phenotype, which enhances functions of macro-
phages and cytotoxic T cells, to a type 2 helper
T-cell (Th2) phenotype, which inhibits Th1-medi-
ated microbicidal actions of T cells by increasing
production of interleukin-10 and decreasing
interferon-γ levels.
43
HIF also influences regula-
tory T cells,
44
a specialized subgroup of inhibitory
T cells.
45
Hypoxia-induced signaling pathways
stimulate the differentiation and proliferation of
regulatory T cells
44
and increase extracellular lev-
els of adenosine,
46
which protects tissues by re-
straining effector functions of T cells.
47
Epithe li al R es ponses t o H yp ox ic
Infl am mat ion
Activation of the PHD–HIF pathway promotes the
resolution of mucosal inflammation in mice.
48
Hypoxia-induced changes in gene expression by
epithelial cells help to promote mucosal barrier
function (e.g., through activation of intestinal tre-
foil factor)
49
or to increase the production by the
High oxygen
HIF-β
Iκ-Bα
IKKβ
PHD/FIH
Inactive
IKKβ
Iκ-Bα
p50 p65
p50
pp
p65
p50 p65
p50 p65
Angiogenesis
metabolism
NF-κB
TLR
Proteasomal
degradation
HIF-α
OH
Proline
hydroxylase
Asparagine
hydroxylase
OH
HIF NF-κB
HRE
Low oxygen
HIF mRNA
HIF Protein
Nucleus
Cytosol
Inflammation
pp
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Figure2.SchematicOverviewoftheMolecularInteractionbetweentheHIFand(Canonical)NF-κBPathways.
In hypoxic conditions (left), hypoxia-inducible factor (HIF) α and HIF-β subunits translocate to the nucleus, where they bind as heterodi-
mers to a hypoxia response promoter element (HRE), inducing transcription of numerous genes, including those of nuclear factor κB
(NF-κB) and toll-like receptors (TLRs). In normoxia, HIF-α is hydroxylated by prolyl hydroxylases (PHDs) and factor-inhibiting HIF (FIH)
and is thereby targeted for proteasomal degradation (in the case of PHDs) or rendered transcriptionally less active (in the case of FIH;
not shown here). In resting cells (right), NF-κB, a heterodimer consisting of p50 and p65 subunits, is inactive in the cytosol because it is
associated with nuclear factor of kappa light polypeptide gene enhancer in B cells alpha (IκBα), a regulatory component of NF-κB. At the
time of cellular activation, the beta subunit of the IκB kinase complex (IKKβ) phosphorylates the inhibitor IκBα, which thereby becomes
degraded and liberates NF-κB for translocation in the nucleus, where it can activate the transcription of inflammatory genes as well as
of HIF (genes involved in tissue protection and homeostasis are not shown). PHDs and FIH regulate NF-κB activation by controlling the
activity of IKKβ.

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epithelium of antiinflammatory signaling mole-
cules such as adenosine.
50
These adaptive re-
sponses to hypoxia are activated during muco-
sal inflammation and promote the resolution of
inflammatory bowel disease
14,51-53
or acute lung
injury.
5,6,54-58
In mice with targeted deletion of
HIF-1α in intestinal epithelia, as compared with
mice that have intact HIF-1α throughout, more
severe colitis develops after exposure to trinitro-
benzene sulfonic acid. In contrast, in mice with
inflammatory bowel disease and elevated HIF lev-
els due to deficiency of the VHL gene, as com-
pared with control animals, weight loss, disease
activity, and histologic signs of intestinal inflam-
mation are all reduced.
14
In mice with colitis that
is chemically induced by oral administration of
dextran sulfate sodium, treatment with pharma-
cologic compounds that enhance stabilization of
HIF reduces intestinal inflammation.
51,52
Several studies have shown that hypoxia en-
hances the enzymatic conversion of precursor nu-
cleotides such as ATP, adenosine diphosphate, or
AMP to adenosine,
7,59
thereby elevating extracel-
lular levels of adenosine, an antiinflammatory
signaling molecule involved in restraining innate
immune responses.
54,60
A single-nucleotide poly-
morphism in CD39, an enzyme required for ex-
tracellular generation of adenosine, is associated
with low levels of CD39
61
; in a case–control study,
this genetic variant was observed in patients with
High oxygenLow oxygen
Pathogens
and necrosis
Epithelium
Adenosine
Increase in
Increase in
Decrease in
Dendritic cell
Neutrophil
Mast cell
Treg cell
CD4+ helper T cell
CD8+ T cell
Macrophage
Blood
vessel
Adaptive immune system
Innate immune system
Phagocytosis
Bacterial killing
Antimicrobial activity
Antigen presentation
Release of permeability factors and
cytokines
Hypoxic survival
Invasiveness
Endothelial adhesion
M2 macrophage polarization
Release of inflammatory cytokines
T-cell effector activity
Th1 polarization
Differentiation
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Figure3.InfluenceofHypoxiaontheInnateandAdaptiveImmuneSystems.
In the example shown in this schematic overview, the epithelium (left) is breached by invading pathogens, leading to tissue damage; as
a result, innate immune cells mount a host defense response, which is amplified by recruited adaptive immune cells. In general, hypoxia
amplifies the activity of innate immune cells while suppressing the response of the adaptive immune system, in part by promoting dif-
ferentiation of regulatory T cells and negatively regulating the function of CD4+ helper T (Th) cells and CD8+ cytotoxic T cells and the
polarization of type 1 Th (Th1) cells. By negatively regulating adaptive immunity, hypoxia prevents excessive activation of the immune
host defense, which might otherwise lead to collateral tissue damage.

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Crohn’s disease more frequently than it was seen
in healthy subjects.
61
HIF stimulates the production of extracellular
adenosine
62,63
and suppresses both its uptake into
the intracellular compartment and its intracellu-
lar metabolism.
64,65
HIF also enhances adenosine-
receptor signaling by increasing the expression
on the cell surface of adenosine receptors
63,66,67
— an effect that attenuates immune responses,
vascular fluid leakage, and neutrophil accumula-
tion in the presence of myocardial, renal, he-
patic, or intestinal ischemia or acute lung inju-
ry.
50,56,68,69
HIF-dependent induction of the axon
guidance signal netrin-1 in epithelia interferes
with the entry of inflammatory cells into hypoxic
organs by enhancing extracellular adenosine sig-
naling events.
5
Other studies have shown that HIF
also attenuates epithelial inflammation through
induction of epithelial decay-accelerating factor
(which clears epithelia from neutrophils)
70
and
induction of barrier-protective genes in the case
of experimentally induced colitis or hypoxia.
14,49
Ca nce r
Concentrations of oxygen in solid tumors, as com-
pared with those in normal tissues, are frequently
reduced.
71
Solid tumors contain increased levels
of HIF-1α and HIF-2α, and these elevated levels
correlate with cancer-related death.
71
Elevated lev-
els of HIF-1α and HIF-2α in biopsy specimens of
prostate tumors have been associated with an ad-
verse clinical course.
72
Hypoxia in a solid tumor
stabilizes HIF through hypoxia-dependent inhi-
bition of PHDs. Similarly, oncogenes, or the loss
of function of tumor-suppressor genes, result in
the stabilization of HIF, as happens in the case of
the VHL tumor-suppressor gene. In von Hippel–
Lindau disease, inactivating germline mutations of
the VHL tumor-suppressor gene increase the risk
of renal-cell carcinoma and other tumors.
73
Hy-
poxia and inflammation meet at several points in
the setting of cancer (Fig. 4). Activation of HIF in
a hypoxic tumor or in stromal cells within the
tumor augments tumor vascularization.
24,74
This
increase in vascularization changes the morpho-
logic characteristics of tumor vessels and their
endothelial lining in ways that compromise oxy-
gen delivery.
75
Inflammatory cells also contrib-
ute to anomalies of vessels in tumors by releas-
ing vascular endothelial growth factor.
In mice, haplodeletion of PHD2 attenuates tu-
mor-vessel leakiness and vascular distortion while
improving tumor-vessel architecture (“vascular
normalization,” as defined by more sharply demar-
cated boundaries and branching points of tumor
vessels)
76
and tumor oxygenation.
77
This change
is associated with a reduction in tumor invasive-
ness and in the risk of metastasis.
77
This finding
suggests that endothelial cells use PHDs to sense
and correct imbalances in oxygen delivery. Anti-
PHD2 agents may offer a new approach to treat-
ing cancer, since they improve the architecture
and function of tumor vessels.
78
Experimental evidence indicates that inhibi-
tion of HIF within the inflamed tumor core at-
tenuates the growth and vascularization of tu-
mors and enhances the sensitivity of tumors to
radiation.
79
In contrast, inhibition of PHD2 and
stabilization of HIF within the tumor vascula-
ture may play an important role in tumor thera-
py, if the means can be found to selectively di-
rect inhibitors of PHD to the tumor vasculature
and inhibitors of HIF to the hypoxic core.
Infec tions
Stabilization of HIF and induction of HIF-depen-
dent genes occur during infections with pathogens.
For example, infection with Bartonella henselae —
the causative agent of bacillary angiomatosis —
is associated with stabilization of HIF-1α and the
transcription of genes that typically become tran-
scribed in hypoxic conditions.
16
In infected cells,
changes in oxygen consumption, as well as cel-
lular hypoxia and decreased ATP levels, correlate
with HIF stabilization and the release of angio-
genic factors during bacillary angiomatosis.
16
Sta-
bilization of HIF during infections can also be
oxygen-independent.
80
For example, under nor-
moxic conditions, iron uptake by bacteria attenu-
ates PHD activity, stabilizes HIF-1α, and induces
the expression of genes targeted by HIF.
81-83
Sta-
bilization of HIF-1α has been found in liver-biopsy
specimens obtained from patients with chronic
hepatitis C
84
and in skin-biopsy specimens ob-
tained from patients with cutaneous infections
caused by Staphylococcus aureus, varicella–zoster vi-
rus, human herpesvirus 8, or Candida albicans.
85
Pathogens may highjack the host’s HIF path-
way for their own advantage. Pseudomonas aerugi-
nosa rapidly inactivates the adenosine that host
cells produce in an HIF-dependent manner, thus
depriving the host epithelium of the actions of

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extracellular adenosine signaling that promote in-
testinal barrier function during inflammation and
hypoxia.
62,86-88
During infection with group A streptococcus
or P. aeruginosa, HIF-1α in immune cells induces
inflammation that helps to eliminate the patho-
Survival
metabolism
proliferation
EMT
invasion
metastasis
Angiogenic
signals
Chemokines
Cytokines
Recruitment
Activation
Vessel
Chemokines
Cytokines
Polarization
Angiogenic
signals
Tumor cell
PHD
Inflammatory
signals
Oncogenes
Hypoxia
Hypoxia
TLR TLR
Necrotic
cancer cell
NF-κB
HIF
Leukocyte
NF-κB
HIF
PHD2+/+
Decreased HIF-2α
Abnormal vessel,
hypoperfusion Oxygenation
PHD2+/–
Increased HIF-2α
Normal vessel,
better perfusion
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Figure4.SchematicOverviewoftheLinkbetweenHypoxiaandInflammationinCancer.
In tumor cells, oncogenes, inflammatory signals (mediated in part through toll-like receptors [TLRs]), and hypoxia activate nuclear factor κB
(NF-κB) and hypoxia-inducible factor (HIF) 1α (which activate each other). These factors induce a gene program that recruits and acti-
vates leukocytes (through release of chemokines and cytokines), stimulates angiogenesis and the formation of an abnormal vasculature
and endothelium (through release of angiogenic signals), and increases tumor-cell invasion, metastasis, epithelial-to-mesenchymal tran-
sition (EMT), survival, proliferation, and metabolic reprogramming. In leukocytes, hypoxia also activates NF-κB and HIF-1α; endogenous
ligands, released from necrotic cancer cells, activate TLRs upstream of NF-κB and HIF-1α, and HIF-1α up-regulates TLR expression.
A resultant gene-expression profile leads to the production of cytokines and angiogenic signals and skews their polarization phenotype.
Tumor vessels with two prolyl hydroxylase (PHD) domain 2 (PHD2) alleles have an abnormal endothelium, are hypoperfused, and cause
tumor hypoxia, which fuels tumor-cell invasiveness and metastasis. In contrast, tumor vessels lacking one PHD2 allele have increased
HIF-2α levels, which result in an up-regulation of factors that counteract the development of tumor endothelial abnormalities; this, in
turn, results in improved tumor-vessel perfusion and oxygenation and, secondarily, reduced metastasis.

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gen.
37
In mice lacking HIF-1α, bactericidal activ-
ity is decreased in myeloid cells, and the systemic
spread of infection cannot be contained.
37
CONCLUSIONS
Hypoxia and inflammation are intertwined at the
molecular, cellular, and clinical levels. Oxygen-
sensing mechanisms and hypoxia signaling are
potential therapeutic targets for the treatment of
inflammatory diseases. The value of such ap-
proaches could be tested in patients with acute
lung injury, myocardial ischemia, inflammatory
bowel disease, or cancer. Targeting hypoxia-depen-
dent signaling pathways could also help attenuate
organ failure due to ischemia in patients under-
going major surgery or alleviate hypoxia-driven
graft inflammation after solid-organ transplan-
tation.
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