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A Central Role for Free Heme in the Pathogenesis of Severe Sepsis

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  • Chronic Diseases Research Center (CEDOC) / Faculty of Medical Sciences (FCM), NOVA University of Lisbon

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Low-grade polymicrobial infection induced by cecal ligation and puncture is lethal in heme oxygenase-1-deficient mice (Hmox1(-/-)), but not in wild-type (Hmox1(+/+)) mice. Here we demonstrate that the protective effect of this heme-catabolizing enzyme relies on its ability to prevent tissue damage caused by the circulating free heme released from hemoglobin during infection. Heme administration after low-grade infection in mice promoted tissue damage and severe sepsis. Free heme contributed to the pathogenesis of severe sepsis irrespective of pathogen load, revealing that it compromised host tolerance to infection. Development of lethal forms of severe sepsis after high-grade infection was associated with reduced serum concentrations of the heme sequestering protein hemopexin (HPX), whereas HPX administration after high-grade infection prevented tissue damage and lethality. Finally, the lethal outcome of septic shock in patients was also associated with reduced HPX serum concentrations. We propose that targeting free heme by HPX might be used therapeutically to treat severe sepsis.
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DOI: 10.1126/scitranslmed.3001118
, 51ra71 (2010);2 Sci Transl Med
, et al.Rasmus Larsen
A Central Role for Free Heme in the Pathogenesis of Severe Sepsis
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SEPSIS
A Central Role for Free Heme in the Pathogenesis
of Severe Sepsis
Rasmus Larsen,
1
Raffaella Gozzelino,
1
Viktória Jeney,
1
László Tokaji,
1
Fernando A. Bozza,
2,3
André M. Japiassú,
2,3
Dolores Bonaparte,
1
Moisés Marinho Cavalcante,
1
* Ângelo Chora,
1
Ana Ferreira,
1
Ivo Marguti,
1
Sílvia Cardoso,
1
Nuno Sepúlveda,
1,4
Ann Smith,
5
Miguel P. Soares
1
(Published 29 September 2010; Volume 2 Issue 51 51ra71)
Low-grade polymicrobial infection induced by cecal ligation and puncture is lethal in heme oxygenase-1deficient
mice (Hmox1
/
), but not in wild-type (Hmox1
+/+
) mice. Here we demonstrate that the protective effect of this heme-
catabolizing enzyme relies on its ability to prevent tissue damag e caused by the circulating free heme released from
hemoglobin during infection. Heme administration after low-grade infectioninmicepromotedtissuedamageand
severe sepsis. Free heme contributed to the pathogenesis of severe sepsis irrespective of pathogen load, revealing
that it compromised host tolerance to infection. Development of lethal forms of severe sepsis after high-grade infec-
tion was associated with reduced serum concentrations of the heme sequestering protein hemopexin (HPX), whereas
HPX administration after high-grade infection prevented tissue damage and lethality. Finally, the lethal outcome of
septic shock in patients was also associated with reduced HPX serum concentrations. We propose that targeting free
heme by HPX might be used therapeutically to treat severe sepsis.
INTRODUCTION
Severe sepsis is a disease with limited treatment options that kills more
than half a million individuals peryearintheUnitedStatesalone(1).
Severe sepsis can develop from an unfettered immune response to mi-
crobial infection that leads to a systemic refractory drop in blood pres-
sure, disseminated intravascular coagulation, multiple end-stage organ
failure, and eventually, death (2). The physiological and molecular mech-
anisms that underlie the pathogenesis of severe sepsis remain poorly
understood (2).
In most cases of microbial infection, the innate and adaptive im-
mune systems allow for pathogen clearance and a return to homeosta-
sis (3). In some cases, however, this host defense strategy, referred to
as resistance to infection (46), can lead to irreversible tissue damage
and compromise host viability (7). An alternative host defense
strategy, referred to as tolerance to infection (5, 6), can limit disease
severity irrespective of pathogen load (46). Host genes conferring to l -
erance to infecti on include the stress-responsive enzyme heme oxygenase-
1(Hmox1), as previously demonstrated for malaria, the disease caused
by Plasmodium infection (8).
Heme oxygenase-1 (HO-1) acts as the rate-limiting enzyme in the
breakdown of heme (Fe protoporphyrin IX; FePPIX) into equimolar
amounts of biliverdin, iron (Fe), and carbon monoxide (9). Induction
of HO-1 expression in response to stress caused by microbial infection
suppresses the development of severe sepsis in mice (10). This safe-
guarding action can act irrespective of pathogen load, relying instead
on the cytoprotective effect of HO-1 against the excess free heme
produced via hemolysis during infection.
Free heme induces programmed cell death in response to proin-
flammatory agonists, as demonstrated for tumor necrosis factor (TNF)
(8, 11). We refer to this phenomenon as heme sensitization to
programmed cell death, because the cyto toxic effect of free heme is
revealed only in the presence of other cytotoxic agonists (8, 11).
The molecular mechanism underlying the cytotoxic effect of free heme
relies on its pro-oxidant activity (8, 11), driven by the divalent Fe atom
contained within its protoporphyrin IX ring, which can promote the
production of free radicals via Fenton chemistry (12).
Giventhatfreehemecancausetissuedamageandhencecompro-
mise host tolerance to infection, we investigated whether limiting this
deleterious effect can be used for therapeutic purposes to enhance host
tolerance against microbial infec tion s and prevent the developm ent of
severe sepsis.
RESULTS
HO-1 affords host tolerance against
polymicrobial infection
Severe sepsis was produced in BALB/c mice by low-grade poly-
microbial infection induced by cecal ligation and puncture (CLP).
Using quantitative reverse transcription polymerase chain reaction
(RT-PCR), we measured the expression of the Hmox1 gene and found
that it was induced in peritoneal infiltrating leukocytes, liver, lung, and
kidney at various time points after CLP (Fig. 1A). Mortality increased
from 13% in wild-type (Hmox1
+/+
) mice to 80% in Hmox1-deficient
(Hmox1
/
) mice when both were subjected to low-grade CLP (Fig. 1B).
Similar results were ob tained in BALB/c severe combined immuno-
deficient (SCID) mice lacking B and T lymphoc ytes (Fig. 1B), demon-
strating that the protective effect of HO-1 is not dependent on adaptive
immunity. The mortality of heterozygous Hmox1
+/
mice was similar
1
Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal.
2
Intensive Care Unit, Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo
Cruz, 21040-900 Rio de Janeiro, Brazil.
3
DOr Institute for Research and Education,
22281-100 Rio de Janeiro, Brazil.
4
Center of Statistics and Applications of the University of
Lisbon, Campo Grande, 1749-016 Lisbon, Portugal.
5
Division of Molecular Biology and
Biochemistry, University of Missouri, 5007 Rockhill Road, Kansas City, MO 64110, USA.
*Present address: Universidade Federal do Rio de Janeiro Campus MacaéInstituto
Macaé de Metrologia e Tecnologia, Macaé, Rio de Janeiro 27930-560, Brazil.
To whom correspondence should be addressed. E-mail: mpsoares@igc.gulbenkian.pt
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to that of Hmox1
+/+
mice(Fig.1B).ThehighermortalityofHmox1
/
versus Hmox1
+/+
mice was not attributable to the surgical procedure
per se, as Hmox1
/
mice did not succumb to sham laparotomy,
which mimicked CLP without polymicrobial infectio n.
The mortality of Hmox1
/
mice after CLP was associated with the
development of multiple end-stage organ failure, a hallmark of severe
sepsis (2). Plasma concentrations of aspartate aminotransferase (AST),
blood urea nitrogen (BUN), and creatinine phosphokinase (CPK),
markers of liver, kidney, and muscle dysfunction, respectively, were sig-
nificantly increased in infected Hmox1
/
versus Hmox1
+/+
mice (Fig.
1C). Liver, kidney, and cardiac damage were confirmed by histolog-
ical detection of centrolobular necrosis, tubular epithelial necrosis, and
myocardial necrosis, respectively (Fig. 1D). This demonstrates that in-
duction of HO-1 expression in response to polymicrobial infection
limits tissue damage and the development of severe sepsis.
Exacerbated mortality of Hmox1
/
versus Hmox1
+/+
mice did not
result from higher pathogen (bacterial) load, as assessed by comparing
the number of colony-forming units (CFUs) in the peritoneum and blood
(Fig.1E),aswellasintheliver,spleen,kidneys,andlungs(fig.S1).
Hmox1
/
mice also succumbed when challenged with heat-killed
bacteria (60% mortality), whereas Hmox1
+/
and Hmox1
+/+
mice did
not (0% mortality) (Fig. 1F). This demonstrates that HO-1 affords tol-
erance against polymicrobial infection (5, 6) independently of its pre-
viously reported antimicrobial activity (10).
Production of several cytokines involved in the pathogenesis of se-
vere sepsis [for example, TNF, interleukin-6 (IL-6), and IL-10] was
similar in Hmox1
/
versus Hmox1
+/
or Hmox1
+/+
mice subjected to
low-grade CLP (fig. S2, A, D, and G). Likewise, peritoneal or bone marrow
derived monocytes/macrophages (Mø) from Hmox1
/
versus Hmox1
+/+
mice produced similar amounts of IL-6 when exposed in vitro to bac-
terial lipopolysaccharide (LPS) or to live bacteria (fig. S2, E and F),
while producing slightly but significantly higher amounts of TNF
when exposed to LPS (fig. S2B) but not to live bacteria (fig. S2C). Higher
production of IL-10 also occurred in Hmox1
/
versus Hmox1
+/+
exposed to LPS or to live bacteria (fig. S2, H and I). Because HO-1 reg-
ulates the expression of a subset of cytokines, including IL-10 (fig. S2,
H and I) in response to bacterial agonists such as LPS (fig. S2H) or live
bacteria (fig. S2I), we cannot exclude that this effect might contribute
to the protect ive mechanism by which HO-1 suppresses the patho-
genesis of severe sepsis.
When exposed to LPS and interferon-g (IFN-g), peritoneal
from naïve Hmox1
/
mice produced slightly but significantly high-
er amounts of nitric oxide (NO) than did Hmox1
+/+
peritoneal
(fig. S3A). Whether reduced NO production contributes to the protec-
tive action of HO-1 remains to be established.
HO-1 prevents free heme from eliciting severe sepsis
Free heme, the substrate of HO-1 activity, is cytotoxic to red blood
cells and causes hemolysis (13). This produces cell-free hemoglobin
andeventuallymorefreeheme(14) (that is, heme not contained with-
in the heme pockets of hemoglobin). This definition of free heme does
not preclude the association of heme with other proteins or lipids in a
manner that does not control its ability to induce oxidative stress (11).
We asked whether increased mortality of Hmox1
/
mice subjected to
polymicrobial infection was associated with increased hemolysis, as
well as with the accumulation of cell-free hemoglobin and/or free
heme in plasma (11). When subjected to low-grade CLP, Hmox1
/
mice, but not Hmox1
+/+
mice, accumulated extracellular hemoglobin
(Fig.2A)andfreehemeinplasma(Fig. 2B), whereas plasma concentra-
tions of the hemoglobin-binding protein haptoglobin (15)(Fig.2A)and
the heme-binding protein hemopexin (HPX) (16) were decreased (Fig. 2B).
Fig. 1. HO-1 affords tolerance against polymicrobi al infection in mice. (A)
Hmox1 messenger RNA (mRNA) expression in peritoneal leukocytes (Perit.
leu.), lung, liver, and kidney after low-grade CLP in BALB/c mice, as deter-
mined by quantitative RT-PCR. Data are shown as mean ± SD (n =3per
group). (B) Survival of Hmox1
+/+
(n =15),Hmox1
+/
(n =12),Hmox1
/
(n =
10), SCID.Hmox1
+/+
(n =5),andSCID.Hmox1
/
(n =5)BALB/cmiceafter
low-grade CLP. (C) Serological markers of organ injury in Hmox1
+/+
(n =15
to 17), Hmox1
+/
(n =10to13),andHmox1
/
(n = 6) BALB/c mice 24 hours
after low-grade CLP. Data are shown as mean ± SD. Dashed lines indicate
basal plasma concentrations in naïve wild-type BALB/c mice. IU, international
units. (D) Representative examples of hematoxylin and eosin (H&E)stained
liver, kidney, and heart tissues from Hmox1
+/+
and Hmox1
/
mice after low-
grade CLP. Magnification, ×400. Arrows indicate red blood cells (RBCs) asso-
ciated with vascular congestion and/or thrombosis. CV, coronary vessel; M,
myocardium; PV, portal vein; G, glomerulus. (E) Bacterial load (CFU) in the
peritoneum and blood of mice subjected to low-grade CLP (12 hours after
CLP). Circles represent individual mice. Bars represent median values. ns, not
significant. (F) Survival of Hmox1
+/+
(n =7),Hmox1
+/
(n =6),andHmox1
/
(n = 5) BALB/c mice after intraperitoneal administration of heat-killed bacte-
ria (Bact.). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
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We then asked whether accumulation of free heme in plasma
contributes to the pathogenesis of severe sepsis. Heme administration
to wild-type (Hmox1
+/+
) mice subjected to low-grade CLP led to se-
vere sepsis (77% mortality) (Fig. 2C), characterized by multiple end-
stage organ failure, as revealed serologically by increased AST, BUN,
and CPK plasma concentrations (Fig. 2D). Organ damage was con-
firmed histologically (Fig. 2E). Heme administration to naïve wild-type
(Hmox1
+/+
) mice, although not lethal per se (0% mortality), elicited
kidney, but not liver or cardiac, damage (Fig. 2D). Heme administra-
Fig. 2. H O-1 prevents heme-driven severe sepsis. (A)Hemoglobinand
haptoglobin plasma concentrations in Hmox1
+/+
(n =10),Hmox1
+/
(n =
11), and Hmox1
/
(n = 6) BALB/c mice 12 hours after low-grade CLP. (B)
Free heme and HPX plasma concentrations in Hmox1
+/+
(n =10),Hmox1
+/
(n =11),andHmox1
/
(n = 6) BALB/c mice, 12 hours after low-grade CLP.
(C)SurvivalofHmox1
+/+
BALB/c mice after low-grade CLP. When indicated,
mice received vehicle (n = 6), protoporphyrin IX (NaPPIX; n =8),orheme
(FePPIX; n = 13). Protoporphyrins were administered (15 mg/kg ip) at 2, 12,
and 24 hours after CLP. Dotted line shows statistical comparison of vehicle-
and FePPIX-treated animals. (D) Measurement of serum AST, BUN, and CPK.
Mice were treated as in (C), and serum biochemistry was analyzed 24 hours
after low-grade CLP. Data are shown as mean ± SD (eight to nine mice per
group). IU, international units. (E) Representative examples of H&E staining
(magnification, ×400) of tissue samples taken 24 hours after low-grade CLP
from mice that received heme described as in (C). Arrows indicate red
blood cells (RBC). PV, portal vein; G, glomerulus. Samples are representative
results of three mice in each group. (F) Bacterial load in the peritoneum and
blood of mice (n = 12 per group) treated as described in (C), 12 to 24 hours
after CLP. Veh, vehicle; Heme, FePPIX. (G) Survival of BALB/c wild-type
(Hmox1
+/+
) mice after intraperitoneal administration of a sublethal bolus
of heat-killed bacteria (E. coli ) followed by heme administration as in (C)
(eight mice per group). Circles represent individual mice. Bars represent
median values. *P <0.05;**P < 0.01; ***P < 0.001; ns, not significant.
Fig. 3. Free heme promotes the pathogenesis of severe sepsis. (A) Survival
of wild-type (Hmox1
+/+
) BALB/c mice subjected to sham laparotomy (n =3),
low-grade (LG) (n = 15) CLP, or high-grade (HG) (n =11)CLP.(B to E)He-
moglobin (B), haptoglobin (C), free heme (D), or HPX (E) plasma concentra-
tions in naïve (n = 6 to 8) mice or 12 hours after sham laparotomy (n =10),
LG CLP (n =6),orHGCLP(n = 11 to 12). Circles represent individual mice.
Bars represent median values. (F) Survival of wild-type (Hmox1
+/+
)BALB/c
mice subjected to high-grade CLP. Mice received purified rabbit HPX (50
mg/kg ip; n = 9), purified rabbit polyclon al IgG (50 mg/kg ip; n = 16), or
vehicle (intraperitoneally, PBS; n = 7) at 2, 12, 24, and 36 hours after CLP. (G)
Serological markers of organ injury in mice treated as in (F). Measurements
were made in serum from IgG-treated mice at the time of death (36 hours)
and in HPX-treated mice at the end of the experiment (day 11). Results
shown are the mean ± SD (n = 5 to 6 mice per group). (H)Representative
H&E staining (magnification, ×400) in mice treated as in (F). Samples are
representative of three mice. CV, coronary vessel; G, glomerulus. M, myo-
cardium. Samples are representative results of three mice in each group.
HPX-treated mice in (G) and (H) were analyzed 11 days after CLP. Control
IgG-treated mice in (G) and (H) were analyzed 24 to 36 hours after CLP
(time of death). *P <0.05;**P <0.01;***P < 0.001; ns, not significant.
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tion was also not lethal in mice subjected to sham laparotomy (0% mor-
tality). Moreover, iron-free protoporphyrin IX failed to cause organ
damage or to precipitate severe sepsis when administered to mice
subjected to low-grade CLP (0% mortality) (Fig. 2C). These observa-
tions demonstrate that free heme can precipitate the onset of severe
sepsis in mice subjected to an otherwise benign (nonlethal) poly-
microbial infection. They also reveal that the kidney is particularly vul-
nerable to the damaging effects of free heme.
The number of CFUs in the peritoneum and blood was similar in
mice subjected to low-grade CLP whether or not they received heme
thereafter (Fig. 2F). This demonstrated that the ability of free heme to
precipitate severe sepsis in mice (Fig. 2C) was not associated with
increasedpathogenload(Fig.2F),thusrevealingthatfreehemecom-
promised host tolerance against polymicrobial infection. This notion
was strongly supported by the observation that administration of free
heme to wild-type (Hmox1
+/+
) mice subjected to a sublethal dose of
heat-killed bacteria led to 100% mortality, as compared to 12.5% mor-
tality in control mice receiving vehicle (Fig. 2G).
We then asked whether the deleterious effect of free heme could be at-
tributed to its previously described action on polymorphonuclear (PMN)
cells (17). The numbers of peritoneal-infiltrating CD45
+
CD11b
+
GR1
+
PMN cells in Hmox1
/
mice subjected to low-grade CLP were two to
threefold higher than those in Hmox1
+/
and Hmox1
+/+
mice (figs. S5 and
S6,AandB).Thiswasnotthecaseforperitonealnaturalkiller(NK),T,or
B cells (fig. S6, C to E). Expression of the phagocytic NADPH (reduced
form of nicotinami de adenine dinucleotide phosphate) oxidase gp 91
phox
in peritoneal infiltrating leukocytes was also higher in Hmox1
/
versus
Hmox1
+/+
mice (fig. S5B). This effect was attributed to the increased num-
bers of PMN cells in Hmox1
/
versus Hmox1
+/+
mice and was associated
w i t h en hance d oxidative activity in peritoneal leukocytes from Hmox1
/
mice relative to Hmox1
+/+
mice (fig. S5, C and D). Whereas heme admin-
istration to naïve Hmox1
+/+
mice can elicit peritoneal PMN cell infiltra-
tion (fig. S5E) (17), this effect was negligible when heme was administered
to mice subjected to low-grade CLP (fig. S5E). Although these data sug-
gest that heme-driven PMN cell activation does not play a major role in
the pathogenesis of severe sepsis, we cannot exclude that other put at i v e
effects of free heme on PMN cells, such as
degranulation, might act in a detrimental
manner to promote the pathogenesis of se-
vere sepsis.
Free heme is a critical
component in the pathogenesis
of severe sepsis
When subjected to high-grade CLP
(>90% mortality) (Fig. 3A), wild-type
(Hmox1
+/+
) mice displayed abnormal
red blood cell morphology (poikilocytosis)
(fig . S4). This was associated with the
accumulation of cell-free hemoglobin
in plasma (Fig. 3B), compared to mice
subjected to low-grade CLP (<20% mor-
tality) (Fig. 3, A and B, and fig. S4). More-
over, there was a decrease in haptoglobin
plasma concentrations in Hmox1
+/+
mice
subjected to high-grade CLP compared
to mice subjected to low-grade CLP (Fig.
3C), confirming that hemolysis occurs in
response to high-grade but not low-grade
infection. Similarly, the concentratio n of
free heme in plasma increased (Fig. 3D),
whereas HPX plasma concentration de-
creased (Fig. 3E), in mice subjected to
high- grade relative to l ow-grade CLP.
Given that mortality in response to poly-
microbial infection is associated with
high concentrations of free heme and
low concentrations of HPX in plasma,
we hypothesized that one might be able
to prevent the onset of severe sepsis by
restoring HPX plasma concentration,
so that HPX is available to neutralize
the rising amounts of free heme. Admin-
istration of purified HPX to wild-type
( Hmox1
+/+
) mice subjecte d to high-
grade CLP reduced the mortality level
Fig. 4. The oxidative effect of free heme sensitizes hepatocytes to programmed cell death. (A) Primary
BALB/c hepatocytes were either untreated (NT) or exposed to heme (5 µM, 1 hour) plus mouse recom-
binant TNF (5 ng/ml, for 16 hours), antibody against Fas (0.5 mg/ml, for 4 hours), H
2
O
2
(125 mM, for 8 hours),
or the ONOO
donor SIN-1 (100 mM, for 24 hours). Production of free radicals was determined by flow
cytometry with CM-H
2
DCFDA. 2nd signal refers to TNF, antibody against Fas, H
2
O
2
, or ONOO
, as spe-
cified for each panel. (B) Percentage of cell death in primary hepatocytes treated as in (A). When in-
dicated (+), hepatocytes were pretreated with the antioxidant NAC (10 mM, for 4 hours). Cell viability
was determined by crystal violet staining. (C) Percentage of cell death in primary hepatocytes treated
as in (A). When indicated (+), hepatocytes were transduced with a LacZ- or Hmox1-encoding Rec.Ad.
Cell viability was determined as in (B). Data are representative of three independent experiments with
hepatocytes isolated from different mice. Bars indicate mean ± SD of n = 5 to 6 independent samples
per group. *P < 0.05; **P < 0.01; ***P < 0.001.
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to 22%, compared to 86 and 69% in control mice that received phosphate-
buffered saline (PBS) (the HPX vehicle) or immunoglobulin G (IgG),
respectively (Fig. 3F). The protective effect of HPX was associated with
the return of liver, kidney, and cardiac function to homeostatic levels, as
assessed by AST, BUN, and CPK plasma concentrations, respectively (Fig.
3G), and as confirmed histologically (Fig.3H).Incontrast,controlmice
that received a nonheme-binding protein (IgG) after high-grade CLP
succumbed to liver, cardiac, and kidney failure, as assessed by AST,
BUN, and CPK plasma concent rations, respectively (Fig. 3G).
Free heme elicits programmed cell death
We have previously shown that free heme can promote programmed
cell death in response to TNF (8). We asked whether this effect is
extended to other agonists involved in the pathogenesis of severe sep-
sis. Because hepatic failure is a central component of severe sepsis, we
tested whether free heme induces oxidative stress and TNF-mediated
programmed cell death in primary mouse hepatocytes in vitro. When
exposed to free heme, hepatocytes did not produce significant amounts
of intracellular free radicals, as assessed by flow cytometry with a
broad free radical probe (Fig. 4A). However, when exposed to free heme
and TNF (Fig. 4A) (8) or free heme plus Fas cross-linking (which acti-
vates the Fas signaling transduction pathway), hepatocytes produced
large amounts of intracellular free radicals (Fig. 4A). This effect was
not observed when hepatocytes were exposed to free heme and oxidiz-
ing agents such as hydrogen peroxide (H
2
O
2
)orperoxynitrite(ONOO
),
which are sufficient to cause free radical accumulation in hepatocytes
(Fig. 4A). Primary hepatocytes did not undergo programmed cell death
when exposed to TNF, Fas cross-linking, H
2
O
2
, or ONOO
(Fig. 4B)
(8), whereas programmed cell death was readily induced in cells treated
with free heme first and then TNF, Fas cross-linking, H
2
O
2
,orONOO
(Fig. 4B). These observations suggest that the redox activity of the heme
Featomunderliesitscytotoxicity,presumablybycatalyzingthepro-
duction of free radicals through Fenton c h e m is try (12). Consistent with
this hypothesis, the antioxidant N-ac e t yl - cysteine (NAC) protected hepa-
tocytesfromprogrammedcelldeathinthepresenceoffreehemeand
TNF, Fas ligand, H
2
O
2
,orONOO
(Fig. 4B). These observations reveal
that the pathological effects of free heme, namely, its ability to synergize
with other cytotoxic agonists to cause tissue damage, can be extended to
a variety of agonists other than TNF (11), including some previously
implicated in the pathogenesis of severe sepsis.
Transduction of hepatocytes with a recombinant adenovirus (Rec.Ad.)
that expresses HO-1 (fig. S7) was protective against programmed cell
death in the presence of free heme and TNF, Fas cross-linking, H
2
O
2
,
or ONOO
, when compared with control hepatocytes transduced
with a Rec.Ad. that expresses LacZ (Fig. 4C). We have previously
shown that the cytoprotective effect of HO-1 is associated with inhi-
bition of free radical production (8), suggesting that HO-1 acts as an
antioxidant to suppress the cytotoxic effects of free heme (8).
Heme triggers HMGB1 release in vitro and in vivo
We reasoned that the cytotoxic effect of free heme might precipitate
severe sepsis by eliciting tissue damage per se, as well as by promoting
the release of high mob ility group box 1 (HMGB1), an endogenous
proinflammatory ligand (18) involved in the pathogenesis of severe
sepsis (19) and previously linked to HO-1 (20). In untreated primary
hepatocytes, HMGB1 expression was mainly restricted to the nucleus
(Fig. 5A). However, when hepato cytes were exposed in vitro to free
heme plus TNF, HMGB1 was translocated from the nucleus to the
Fig. 5. Free heme triggers the release of HMGB1 from hepatocytes. (A)
HMGB1 (red) and DNA (blue) in mouse Hepa1-6 hepatocytes exposed to ve-
hicle (Nontreated), free heme (40 µM, for 1 hour), TNF (50 ng/ml, for 3 hours),
or heme (40 µM, for 1 hour) plus TNF (50 ng/ml, for 3 hours). Magnification,
×400. Images are representative of three independent experiments. One nu-
cleus per field is outlined (dotted line). (B) HMGB1 was measured by Western
immunoblotting of proteins in the supernatants of primary mouse (BALB/c)
hepatocytes that were exposed to heme (5 µM, for 1 hour) and TNF (5 ng/ml,
for 16 hours) in culture. A representative result from two independent
experiments is shown. NS, nonspecific band. (C)HMGB1wasmeasuredby
Western immunoblotting of proteins in the supernatants of mouse Hepa1-6
hepatocytes exposed to heme and TNF as described in (A). When indicated,
cells were pretreated with the antioxidant NAC (10 mM; for 4 hours). A rep-
resentative result from two independent experiments is shown. (D)HMGB1
was measured by Western immunoblotting of proteins in the supernatants
of mouse Hepa1-6 hepatocytes treated with heme and TNF as in (A) and
either transduced or not transduced with LacZ or Hmox1 Rec.Ad. Blots are
representative of two independent experiments. (E)HMGB1staininginthe
liver and kidney from Hmox1
+/+
and Hmox1
/
mice 24 hours after CLP. One
of three representative samples are shown. Samples were counterstained
with hematoxylin. Magnification, ×40 0. Arrows ind icate representative nuclei
from which HMGB1 underwent full translocation from the nucleus to the
cytoplasm and extracellular space. (F and G) HMGB1 and Ig heavy chain
(IgG) were detected by Western blot in the peritoneal cavity (F) or plasma
(G) of Hmox1
+/+
, Hmox1
+/
,orHmox1
/
mice 12 hours after low-grade CLP.
Numbers indicate individual animals (n = 3 per genotype).
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cytoplasm (Fig. 5A) and released extracellularly (Fig. 5B). This was not
the case when hepatocytes were exposed to either free heme or TNF
alone (Fig. 5, A and B). Extracellular HMGB1 release was suppressed
by the antioxidant NAC (Fig. 5C), as well as by HO-1 overexpression
(Fig. 5D). These observations reveal that the oxidative effect of free
heme promotes HMGB1 release from hepatocytes, an effect sup-
pressed by HO-1.
Next, we asked whether HO-1 would prevent HMGB1 release fr o m
damaged tissue in vivo (18), as suggested in previous studies (20). In
Hmox1
/
mice subjected to low-grade CLP there was tran slocation
of HMGB1 into the cytoplasm, as assessed in the liver and kidney
(Fig. 5E), whereas translocation was less pronounced in wild-type
(Hmox1
+/+
) mice (Fig. 5E). This effect was associated with the systemic
releaseofHMGB1intotheperitoneumandplasmaofHmox1
/
rel-
ative to Hmox1
+/+
mice (Fig. 5, F and G). The relative amount of per-
itonealorplasmaIgGwasunchangedinHmox1
/
versus Hmox1
+/+
mice (Fig. 5, F and G).
HPX neutralizes the cytotoxic effect of free heme
We next sought to determine whether HPX, which binds tightly to
heme (K
d
<1 pM) in a manner that dampens its pro-oxidant activity
(21 ), can also prevent heme-induced tissue damage (Fig. 3, F to H)
(16). The ability of free heme to sensitize hepatocytes in vitro such that
they produced high amounts of free radicals in response to TNF was
inhibited when heme was bound to HPX (Fig. 6A). Moreover, the
ability of free heme to sensitize hepatocytes to undergo programmed
cell death in response to TNF was also inhibited once heme was
bound to HPX (Fig. 6B). Similar results were obtained with primary
human hepatocytes in that HPX prevented heme sensitization to
programmed cell death in response to TNF (Fig. 6C). Accordingly,
HPX-bound heme also failed to promote HMGB1 release from pri-
mary mouse hepatocytes in vitro (Fig. 6D). Similarly, HPX-bound
heme also failed to promote HMGB1 release from primary human he-
patocytes in response to TNF (Fig. 6E). The increased molecular weight
of the cell-free HMGB1 released from primary human hepatocytes is
most likely attributed to posttranscriptional HMGB1 modifications,
such as phosphorylation. Together, these observations suggest that
HPX suppresses the cytoto xic effects of free heme in both mouse and hu-
man hepatocytes.
Low HPX serum concentration is associated with organ
dysfunction and fatal outcome in septic shock patients
Given that HPX plasma concentration is reduced in mice that suc-
cumb to severe sepsis (Fig. 3E), we asked whether this would also
be the case in patients that succumb to septic shock (22). In a cohort
of 52 patients (table S1), HPX serum concentration within 48 hours of
presentation with septic shock was positively associated with patient
survival time (Fig. 6F). That is, patients with lower HPX serum con-
centrations succumbed at earlier time points compared to patients
with higher HPX serum concentrations (Fig. 6F). This observation
allowed us to extrapolate the probability of survival/mortality as a
function of HPX serum concentration (Fig. 6G), in keeping with the
ob se r v at i on th a t HPX serum concentration within 48 hours of septic
shock diagnosis was higher in patients that survived septic shock com-
pa r ed to no n s u r vi v o r s (Fig . 6H) . Fi n a l ly , there was an inverse correlation
between HPX serum concentration and severity of organ dysfunction,
Fig. 6. HPX suppresses the cytotoxic effect
of free heme. (A) Primary BALB/c hepato-
cytes were untreated (NT) or exposed to
heme (5 µM) or HPX-heme complexes (5 µM,
for 1 hour) and TNF (5 ng/ml, for 16 hours).
Production of free radicals was determined
by flow cytometry with the broad free rad-
ical probe CM-H
2
DCFDA. (B)PrimaryBALB/c
hepatocytes were treated as in (A), exposed
to heme (5 µM), HPX (5 µM), or heme-HPX
complexes (5 µM, for 1 hour) and, when in-
dicated, to TNF (5 ng/ml; for 16 hours). Cell
viability was determined by crystal violet
staining. Results shown are the mean ± SD
from six samples in one of two independent
experiments with hepatocytes pooled from
three mice. (C)Primaryhumanhepatocytes
were exposed to heme (10 µM; 1 hour) or
heme-HPX complexes (10 µM, 1 hour), and
then to TNF (10 ng/ml, 8 hours). Cell viability
was determined by crystal violet staining. Re-
sults shown are the mean ± SD from six sam-
ples in one experiment representative of four independent experiments. (D)
HMGB1wasmeasuredbyWesternblottingofproteinsinthesupernatantsof
primary mouse (BALB/c) hepatocytes treated as in (A). Blots are representative
of two independent experiments. (E) HMGB1 measured by Western blotting in
the supernatants of primary human hepatocytes treated as in (C). (F) Survival
time (see Materials and Methods) of patients developing septic shock versus
prediction of best-fitted model. The solid line refers to the expected median
survival time as a function of HPX serum concentration at the time of septic
shock diagnosis, predicted by the best model for survival time (based on
lognormal distribution). Gray circles represent individuals that succumbed dur-
ing hospitalization (nonsurvivors). White circles represent individuals that sur-
vived septic shock and left the hospital at the times indicated. P <0.05forthe
respective effect. (G) Expected mortality probability at day 28, plotted as a
function of HPX serum concentration at the time of septic shock diagnosis, pre-
dicted by the best model for survival time (based on logn ormal dis tribution). (H)
Box plot representation of HPX serum concentration at the time of septic shock
diagnosis in a cohort of 52 patients, including survivors (n =34)andnonsurvi-
vors (n = 18). Data represent mean [interquartile range (IQR), 25 to 75%]. *P< 0.05.
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as defined by the sequential organ failure assessment (SOFA) score (23)
(table S2). Overall, these observations support the notion that HPX
serum concentration defines the extent of tissue damage (organ dys-
function) and hence the outcome of septic shock in humans.
DISCUSSION
An infected host has two distinct evolutionarily conserved defense
strategies that limit disease severity. The best-characterized of these
two strategies relies on the capacity of the host immune system to con-
tain and reduce its pathogen load. This defense strategy is referred to
as host resistance to infection (6). The other defense strategy acts ir-
respective of pathogen load and relies instead on limiting tissue dam-
agecauseddirectlyorindirectlybythepathogenand/orbytheimmune
response elicited by that pathogen. This defense strategy, referred to as
host tolerance to infection (6), has long been recognized in plants (5),
but only more recently in flies (6)andmice(24).
Blood-borne pathogens can cause hemolysis (25) and hence lead to
accumulation of extracellular hemoglobin in the circulation. Oxidation
of cell-free hemoglobin can be highly deleterious to the host in at least
three ways. First, it can exacerbate inflammation (26). Second, it can
release heme (Figs. 2, A and B, and 3D) (14),aputativesourceofiron
that can promote microbial growth (27). Third, as shown herein, free
heme can be highly cytotoxic in the presence of proinflammatory ago-
nists (Fig. 4B) (11), causing irreversible tissue damage and organ fail-
ure (Fig. 2, D and E), the hallmarks of severe sepsis (2)(Fig.7).
That heme-driven tissue damage can contribute to the patho-
genesis of severe sepsis is suggested by four independent lines of ev-
idence. First, exacerbated mortality of Hmox1
/
mice subjected to
microbial infection (Fig. 1B) correlated with the accumulation of free
heme in the plasma (Fig. 2B). Second, administration of free heme to
wild-type (Hmox1
+/+
) mice subjected to low-grade (nonlethal) microbi-
al infection was sufficient to elicit a lethal form of severe sepsis (Fig. 2C).
Third, free heme accumulated in the plasma of wild-type (Hmox1
+/+
)
mice subjected to high-grade (lethal), but not low-grade, microbial in-
fection (Fig. 3, A and D). Fourth, sequestration of free heme by HPX
suppressed the development of severe sepsis in wild-type (Hmox1
+/+
)
mice subjected to high-grade microbial infection (Fig. 3, F to H).
There are a number of cyto protect ive mechanisms against the del-
eterious effects of free heme (8, 11, 16, 28). These include the plasma
protein HPX, which binds free heme and neutralizes its oxidative (Fig. 6A)
and hence cytotoxic (Fig. 6, B and C) effects (16)(Fig.7).Asshown,
the lethal outcome of severe sepsis in mice (Fig. 3E) and septic shock
in humans is associated with decreased concentration of circulating
HPX (Fig. 6F). Therefore, we reasoned that administration of exoge-
nous HPX might be used therapeutically to increase tolerance to in-
fection and hence prevent the development of severe sepsis in mice,
which we found to be the case (Fig. 3, F to H). It is likely that a similar
approach will translate into the treatment of severe sepsis in humans,
given the observation that low concentrations of circulating HPX are
associated with septic shock lethality in humans as well (Fig. 6, F to H).
One cannot exclude at this point that in addition to preventing the cyto-
toxic effects of free heme, HPX (29) might exert anti-inflammatory
effects that contribute to its protective effect.
The salutary effect of HPX most probably requires the expres-
sion of HO-1 to catabolize HPX-bound heme (Fig. 7). This would
explain why, despite the presence of HPX, HO-1deficient (Hmox1
/
)
mice are highly susceptible to immune-mediated inflammatory dis-
eases (30), including endotoxic shock (31) and polymicrobial infection
(10 ) (Fig. 1B), in which lack of adequate HPX-bound heme catabolism
leads to irreversible tissue damage, end-stage organ dysfunction, and
eventually to death (32, 33).
The protective effect of HO-1 against polymicrobial infection has
previously been attributed to the antimicrobial activity of carbon mon-
oxide (10), one of the end products of heme catabolism carried out by
HO-1 (9). This would suggest that HO-1 affords some level of re-
sistance against polymicrobial infection. Two independent lines of ev-
idence support the notion that HO-1 also affords tolerance against
polymicrobial infection: First, mice that can induce the expression
of HO-1 (Hmox1
+/+
) in response to polymicrobial infection (Fig. 1A)
survive(Fig.1B)whensubjectedtothesamepathogenload(Fig.1E)
that kills Hmox1
/
mice (Fig. 1B). Second, Hmox1
/
but not Hmox1
+/+
mice succumb to death even when challenged with heat-killed bacteria
(Fig. 1F) (31, 33). These observations provide mechanistic evidence for
the proposed protective effect of HO-1 expression in the outcome of
severe sepsis and septic shock (34).
The mechanism by which HO-1 affords tolerance against microbi-
al infections relies to a large extent on its ability to suppress the del-
Fig. 7. Role of free heme in the pathogenesis of severe sepsis. The patho-
genesis of severe sepsis is associated with hemolysis, which involves the re-
lease of hemoglobin (Hb) from red blood cells (RBC). Oxidation of cell-free Hb
leads to the release of its prosthetic heme groups. This pathological event
can be prevented by the acute-phase protein haptoglobin (HPT), whereas
free heme can be captured by the acute-phase protein HPX. Once the con-
centration of HPT and/or HPX in serum decreases below a certain threshold
level, free heme accumulates in plasma and can sensitize cells in parenchy-
mal tissues to undergo programmed cell death in response to a variety of
proinflammatory agonists. This leads to the release of endogenous proin-
flammatory ligands from damaged tissues, for example, HMGB1. Expression
of the stress-respon sive enzyme HO-1 in parenchymal cells affords cytopro-
tection against free heme, thus suppressing tissue damage and ultimately
multiple-organ dysfunction/failure.
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eterious effect of free heme (Fig. 4, A and B) produced during the
course of infection (Figs. 3D and 7). This notion is strongly supported
by the observation that HO-1 protected mouse cells from the cytotoxic
effectsoffreeheme(Fig.4C),andshould prevent irreversible tissue
damage, multiple organ dysfunction, and host death (Fig. 1, C and D).
Because this protective effect of HO-1 does not interfere with patho-
gen load (Fig. 1E), we conclude that HO-1 promotes, whereas extra-
cellular heme compromises, host tolerance to polymicrobial infection.
The findings from the studies reported here potentially can be
translated into several clinical applications for monitoring and treat-
ment of sepsis. In the clinical setting , monitori ng the patients levels of
circulatinghemeand/orHPXmightbeusedtopredictthelikelihood
of a fatal outcome in each case of severe sepsis (Fig. 6G). In addition,
the development of strategi es that mitigate the deleterious effects of
free heme might also be used therapeutically to prevent the all-too-
common lethal outcome of severe sepsis.
MATERIALS AND METHODS
Mice and genotyping
BALB/c, BALB/c.SCID, BALB/c.Hmox1
+/
,andBALB/c.SCID.Hmox1
+/
mice were maintained under specific pathogen-free conditions ac-
cording to the Animal Care Committee of the Instituto Gulbenkian
de Ciência. All animal protocol s were approved by the Direcção Geral
de Veterinaria of the Portuguese government. BALB/c.Hmox1
+/
mice were generated originally by S. F. Yet (Pulmonary and Critical
Care Division, Brigham and Womens Hospital, Boston, MA) by dis-
ruptionofexon3intheHmox1 locus (35). Mice were backcross ed 10
times into the BALB/c background. Heterozygous (Hmox1
+/
)breed-
ing pairs yield ~8% viable and otherwise healthy homozygous HO-1
deficient mice (35). Littermate Hmox1
-/+
and Hmox1
+/+
mice w e r e
used as controls. Mice were genotyped by PCR. Briefly, a 400base-pair
(bp) PCR product spanning the 5 flanking region of the neomycin
complementary DNA (cDNA) in the Hmox1 locus was amplified
from genomic DNA with the following primers: 5-TCTTGAC-
GAGTTCTTCTGAG-3 and 5-ACGAAGTGACGCCATCTGT-3
(35). For the endogenous Hmox1 locus, 5-GGTGACAGAAGAGGC-
TAAG-3 and 5-CTGTAACTCCACCTCCAAC-3 primers were
used to amplify a 456-bp product. PCRs were repeated at least two
times before experiments were performed and were carried out after
experiments to confirm genotypes.
Cell culture
Primary mouse peritoneal leukocytes were obtained by peritoneal la-
vage with ice-cold apyrogen PBS (Sigma). Briefly, leukocytes were
washed in PBS and resuspended in RPMI 1640 Glutamax I (Gibco)
supplemented with 5% fetal bovine serum, penicillin (50 U/ml), and
streptomycin (50 mg/ml) (Life Technologies). For cytokine measure-
ments, cells (2.5 × 10
4
) were plated in flat-bottom 96-well microtiter
plates (Techno Plastic Products AG) (100 µl, 2 hours, 37°C); nonad-
herent cells were removed, and adherent cells, that is, Mø, were acti-
vated with bacterial LPS (Sigma, Escherichia coli serotype 0127:B8) for
6 or 24 hours. Bone marrow cells were incubated for 6 days in RPMI
1640 Glutamax I (Gibco), 10% fetal calf serum, 30% L929 supernatant
[as macrophage colony-stimulating factor (M-CSF) source]. The bone
marrow-derived macrophages (BMDMs) were seeded (16 hours) in
six-well plates (3 × 10
5
cells per well) in RPMI, 3.3% FCS, and 5%
L929 supernatant. BMDMs were incubated with live Gram-positive
(En terococc us subsp. isolated from mouse intestine) or Gram-negative
(E. coli DH5a) bacteria (8 hours), after which cell culture supernatant
was collected and centrifuged (5 min, 1200 rpm, 4°C) to remove cells
and bacteria (5 min, 10,000 rpm, C). Cell-free supernatants were
stored at 80°C until used. Hepa1-6 cells (C57L mouse hepatoma;
American Type Culture Collection) were seeded in DMEM (Invitrogen),
10% FCS, penicillin, and streptomycin (20 U/ml, Invitrogen). All cells
were incubated at 37°C, 95% humidity, and 5% CO
2
.
Protoporphyrins
Heme (iron protoporphyrin; FePPIX; Frontier Scientific ) and proto-
porphyrin IX (protoporphyrin IX disodium salt; NaPPIX; Frontier
Scientific) were dissolved in 0.2 M HCl, and pH was adjusted to 7.4
with sterile 0.2 M NaOH.
Primary hepatocytes
Primary mouse hepatocytes were isolated as described (36). Briefly,
liversfromnaïveBALB/cmicewereperfusedthroughtheportalvein
(5 ml/min, 10 min, 37°C) with liver perfusion medium (Invitrogen),
and the tissue was disrupted. Cells were filtered (100 mm), washed
(Williams E medium; 4% FCS) (Invitrogen), pelleted (100g,30s,20°C),
and resuspended (Williams E medium, 4% FCS). Hepatocytes were
isolated in a Percoll gradient (1.06/1.08/1.12 g/ml; 750g,20min,20°C)
(GE Healthcare), resuspended (Williams E medium; 4% FCS), centri-
fuged (2 × 200g, 10 min, 4°C), resuspended (WilliamsEmedium;
4% FCS), and seeded onto gelatin (0.2%)coated plates. The medium
was replaced after 4 hours, and experiments were performed 24 to 48
hours thereafter. Primary human hepatocytes were cultured in hepa-
tocyte culture medium as detailed by the supplier (Lonza).
Heme sensitization assays
Hepatocytes were seeded and exposed to heme (5 mM, 1 hour) in
Hanks Balanced Salt Solution (Invitrogen) without serum to avoid
potential heme scavenging by serum proteins, as described (8). Sub se-
quently, hepatocytes were washed (PBS) and challenged in Dulbeccos
modified Eagles medium, 10% FCS (Hepa1-6), or 4% FCS (primary
hepatocytes), with human recombinant TNF (5 to 40 ng/ml, 3 to 16
hours; R&D Systems), Fas ligand (Jo2 antibody against CD 95, 0.5 mg/ml,
4 hours; BD Biosciences), H
2
O
2
(125 mM, 8 hours; Sig ma), or 3-
morpholinosydnonimine (SIN-1; 100 mM, 24 hours; Sigma). Cell via-
bility was assessed by crystal violet assay, as described (37). Heme
(FePPIX; Frontier Scientific) was dissolved in sterile 0.2 M NaOH at
alkaline pH and adjusted to pH 7.4 with sterile 0.2 M HCl. Iron-free
protoporphyrin (NaPPIX, Frontier Scientific) was dissolved in sterile
0.2 M HCl at acidic pH, and pH was adjusted to 7.4 with sterile 0.2 M
NaOH. Aliquots were stored at 80°C until use.
Cytokines and NO measurements
TNF, IL-6 and IL-10 were quantified by enzyme-linked immunosorbent
assay (ELISA) according to the manufacturersinstructions(Becton
Dickinson). NO was measured with a Griess colorimetric assay (38).
CLP
CLP was performed as described elsewhere (39, 40). Briefly, mice were
anesthetized [ketamine (120 mg/kg)/xylazine (16 mg/kg) intra-
peritoneally (ip)]. Under sterile conditions, a 1-cm incision was made
parallel to the midline, and the cecum was exteriorized and ligated
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(sterile 3-0 Mersilk sutures; Ethicon) immediately distal to the ileoce-
cal valve (reducing the lumen by 50 to 60% for low-grade CLP and 80
to 90% for high-grade CLP). The cecum was punctured once with a
23-gauge needle (low-grade CLP) or twice with a 21-gauge needle
(high-grade CLP) and its content was extruded by applying pressure
andreinsertedintotheabdominalcavity. The peritoneal wall was su-
tured with sterile 3-0 Dafilon sutures (Braun) and the skin was closed
with a surgical staple (Autoclip 9 mm; Becton Dickinson). A single
dose of saline was injected subcutaneo usly (1 ml per animal) for fluid
resuscitation. After the surgical procedure, animals were maintained at
37°C (30 min) and received antibiotics intraperitoneally (imipenem-
cilastatin, Tienam, MSD; 0.5 mg per animal) 2 hours after the surgical
procedure and every 12 hours during 72 hours.
Colony-forming units
Peritoneal fluid was obtained by peritoneal lavage with 5 ml of sterile
PBS (Sigma). Organs were weighed and homogenized under sterile
conditions in 0.5 ml of PBS with Dounce tissue grinders (Sigma).
Serial dilutions of blood, peritoneal lavage, and homogenized organs
were immediately plated on Trypticase Soy Agar II plates supplemen-
ted with 5% Sheep Blood (Becton Dickinson). CFUs were counted af-
ter 24 hours of incubation at 37°C.
cDNA synthesis and LightCycler analysis
Total RNA was extracted with RNeasy Protect Mini Kit (Qiagen) and
reverse-transcribed (SuperScriptII RNase H
reverse transcriptase;
Invitrogen) with random hexamer primers (Invitrogen) as follows: 70°C
for 10 min, 37°C for 50 min, and 95°C for 5 min (RoboCycler Stratagene).
HO-1 primers are 5-TCTCAGGGGGTCAGGTC-3 (forward) and 5-
GGAGCGGTGTCTG GGATG-3 (reverse). The reaction was carried out
with 1 ml of cDNA and 3 pmol of each primer, 2 mM MgCl
2
,and1×
FastStart DNA SYBR Green I mix (Roche). The thermal cycler program
was composed of 1 cycle at 95°C for 10 min, 45 cycles at 95°C for 15 s, 58°C
for 5 s, and 72°C for 16 s, with transition rates of 20°C/s. PCR products
were quantified by LightCycler Real-Time quantitative PCR software
(Roche). Cycle numbers in the log-linear phase were plotted against the
logarithm of template DNA. External standardization was performed
with full-length HO-1 cDNA. Hypoxanthine-guanine phosphoribosyl-
transferase (HPRT) was used to normalize cDNA levels (41).
Flow cytometry
Leukocytes were washed and blocked in calcium- and magnesium-free
PBS containing 2% FCS (v/v). After incubation (30 min, C) with
fluorochrome-conjugated monoclonal antibodies directed against
CD11b (clone M1/70), IAd (clone AMS-32.1), GR1 (clone 1A8), CD49b
(clone DX5), a/bTCR (clone H57-597), or CD19 (clone 1D3) (BD
Biosciences, Pharmingen), cells were washed twice with PBS and 2%
FCS (v/v) and acquired in a FACScan or FACSCalibur with CellQuest
software (BD Biosciences). Dead cells were excluded from the analysis
with propidium iodide. Analysis was done with FlowJo software (Tree
Star Inc.). Cellular free radical generation was determined by incubat-
ing cells (10 mM, 15 min, 37°C, 95% humidity, 5% CO
2
) with the broad
free radical probe 5-(and 6)-chloromethyl-27-dichlorodihydrofluo scein
diacetate acetyl ester (CM-H
2
DCFDA; Molecular Probes).
Immunofluorescence
Hepa1-6 cells, seeded and treated (as described above) on glass cover
slips (Paul Marienfeld GmbH & Co.), were fixed (4% paraformal-
dehyde, 30 min), permeabilized (0.1% Triton X-100, 20 min), blocked
(PBS, 10% goat serum, 20 min), and incubated overnight at 4°C with
rabbit antibody against human HMGB1 (Abcam, ab18256; 0.5 mg/ml)
or control rabbit IgG (Sigma) in PBS and 10% goat serum. Alexa 568
conjugated goat antibody against rabbit IgG (5 mg/ml; Invitrogen) was
used as secondary antibody. Nuclear DNA was stained with Hoechst
33342 (10 m g/ml,PBS,20min;Invitrogen),andcellsweremountedin
Vectashield (Vector Laboratories). Images were captured with a fluo-
rescence microscope (Leica, DMRA2) equipped with UV light and
Evolution MP 5.0 Color Camera (Media Cybernetics). Images were
analyzed with ImageJ software (National Institutes of Health).
Histology and immunohistology
Tissue samples were processed and stained essentially as described
(42). HMGB1 was detected in paraffin-embedded, formalin-fixed
sections (5 µm) after microwave antigen retrieval [0.01 M citrate
buffer (pH 6.0) 20 min] with rabbit antibody against human HMGB1
(Becton Dickinson, 556528) (0.5 mg/ml,4°C,overnight).RabbitIgGwas
detected with biotin-conjugated donkey antibody against rabbit sec-
ondary antiserum (1:1000; Jackson Immunoresearch) and streptavidin-
conjugated horseradish peroxidase amplification kit (Vectastain Elite
ABCKit,VectorLabs).Signal was revealed with 3,3-diaminobenzidine
(DAB). Sections were counterstained with Harris hematoxylin. Nega-
tive controls were performed by omitting the primary antibody or
with a nonspecific rabbit polyclonal antibody. Images were obtained
and analyzed as described above.
Serum biochemistry
Blood was collected in tubes with heparin after cardiac puncture, cen-
trifuged (2 × 5 min, 1600g). AST, BUN, and CPK were measured
according to the protocols of the International Federation of Clinical
Chemistry, as described (4345), by spectrophotometric analysis
(modular DP; Roche-Hitachi, Echev arne Laboratories). Plasma HPX
and haptoglobin were determined by ELISA (Life Diagnostics). Plas-
ma hemoglobin was determined by spectroscopy at a wavelength of
577 nm (l
577
). Total plasma heme was measured with the 3,3,5,5
tetramethylbenzidine (TMB) peroxidase assay (BD Biosciences) at
l
655
. Purified hemoglobin was used as standard for plasma hemoglo-
bin and heme measurements. Blood smears were fixe d in methanol
and stained with Giemsa stain, and images were obtained and ana-
lyzed as described above.
HPX
Intact apo-HPX was isolated from rabbit serum as described (46). Pur-
ified HPX binds heme as assessed by absorbance and circular dichro-
ism spectroscopy of the apoprotein or the oxidized and reduced
heme-HPX complexes; the concentration of the protein and equi-
molar heme binding were quantified with published procedures and
extinction coefficients (47). Neither the apo-HPX nor the heme-HPX
complex is toxic for cells in vitro even at high concentrations (48). Mice
received purified HPX (50 mg/kg ip) at 2, 12, 24, and 36 hours after CLP.
Western blotting
Proteins were prepared and subjected to electrophoresis essentially
as described before (49). For HMGB1 detection in peritoneal fluid
and in serum, samples were ultrafiltered with Centrico n 100 columns
(Millipore) and precipitated with trichloroacetic acid (TCA), washed
twice in acetone, dried, dissolved in urea (8 M), and added to SDS
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polyacrylamide gel ele ctrophoresis ( SDS-PAGE) loading buffer.
Primary hepatocyte and Hepa1-6 culture supernatants were concen-
trated on Vivaspin 500 columns (10-kD molecular mass cuto ff;
Vivascience AG) resulting in up to ~10-fold concentration. HMGB1
was detected with polyclonal antibody (Abcam, ab18256; 0.1 mg/ml).
HO-1 was detected with a rabbit polyclonal antibody against human
HO-1 (1:2.500; SPA-895, StressGen). Monoclonal antibodies were used
to detect a-tubulin (T9026, 1:5.000 dilution; Sigma) and inducible NO
synthase (Becton Dickinson). Primary antibodies were detected with
horseradish peroxidaseconjugated donkey antibody against rabbit,
goat antibody against mouse, or rabbit antibody against mouse IgG
secondary antibodies (Pierce, Rockford). Peroxidase activity was visu-
alized with the SuperSignal chemiluminescent detection kit (Pierce)
according to the manufacturersinstructionsandstoredintheformof
photoradiographs (BiomaxTMMS, Eastman Kodak) or with the
Image Station 440CF (Kodak). Digital images were obtained with an
image scanner equipped with Adobe Photoshop software.
Septic shock patients
We analyzed the plasma concentration of HPX in 52 patients under-
going septic shock, as defined by the American College of Chest Phy-
sicians (ACCP)Society of Critical Care Medicine (SCCM) consensus
criteria (22). Patients were treated according to standard recommen-
dations (50), including aggressive fluid resuscitation, broad-spectrum
antibiotic therapy over the first 24 hours, vasoactive agents, and at
least one intravenous dose of hydrocortisone. Blood samples were
collected on the first, second, third, fifth, and seventh day after septic
shock diagnosis. Blood was collected on ice between 1000 and 1200
hours with an arterial line or a peripheral vein, and plasma was col-
lected by centrifugation (800g, 15 min, 4°C), aliquoted, and stored
(70°C) until analysis. Organ dysfunction was defined by the SOFA
score on the basis of daily measurements (23). The outcome analyzed
was 28th-day hospital mortality. The study protocol was approved by
the institutional review board of each participating center (University
Hospital of Federal University of Rio de Janeiro, Hospital Quinta
DOr, Casa de Saúde São José, Rio de Janeiro, Brazil). All patients,
or their legal surrogates, gave written informed consent before any
study-related procedures.
Statistical analysis
The comparison of two independent samples was assessed by the
Students t test and the Mann-Whitney test for Gaussian and non-
Gaussian distributed samples, respectively. To compare more than
two samples, we performed analysis of variance (ANOVA) or Kruskal-
Wallis tests for Gaussian and non-Gaussian distributed samples, re-
spectively. Comparison of different survival curves for the variously
treated animals was done by the nonparametric log-rank test. For
pairwise comparisons, the Bonferroni correction was used to ensure
the overall significance level. Regression models were applied to de-
scribe genotype-based data, and statistical signif icance prese nted
throughout the paper refers to additive effects. Kolmogorov-Smirnov
and Shapiro-Wilk tests were performed to assess the normality of the
samples under analysis. Regression models were applied to describe
genotype-based data. In all data sets, the following model equation
was applied: Y = a + b ×genotype+c × heterozygote, where Y denotes
the variable under analysis, with logarithmic transformation when ap-
propriate; a isthebaselinereferringtotheHmox1
/
mean; b is the
mean effect of adding an Hmox1
+/+
allele in the genotype (additive
effect); c is the deviation of heterozygote mean from a single additive
effect; genotype is an explanatory variable denoting the genotype
coded as 0, 1, and 2 (0, 1, 2 Hmox1
+/+
alleles, respectively); and
Hmox1
+/
is the binary variable indicating the heterozygote genotype.
Model validation was done by a thorough residual analysis, which in-
cluded testing normality of the residuals and visual inspection of any
trend in the residuals across genotypes. Statistical significance refers to
additive effects in the regression analysis. Kolmogorov-Smirnov and
Shapiro-Wilk tests were performed to infer whether data could come
from normal distributions. All statistical tests were done at 5% signif-
icance level with InStat and R software (51).
For human data, a survival analysis was performed with the pack-
age available in the R software (51). For each patient, survival time was
computedbythedifferencebetweenthetimeofpatientinclusionin
the intensive care unit and the respective closing date of the hospital
record. Patients that left hospital after treatment were considered as
right-censored observations for the respective survival time. Because
the survival time could be approximated by a lognormal distribution,
several survival regression models based on such probability distribu-
tions were fitted to the data. HPX was included in the models as an
explanatory variable with either the firstorthelasttimepointmeasure
available for a patient. The statistical significance of this explanatory
variable in the models was assessed by the traditional z-score tests. A
correlation analysis between SOFA score and HPX at different time
points was also performed with Spearmanscoefficient.
SUPPLEMENTARY MATERIAL
www.sciencetranslationalmedicine.org/cgi/content/full/2/51/51ra71/DC1
Fig. S1. Effects of HO-1 on bacterial load.
Fig. S2. Modulation of cytokine production by HO-1.
Fig. S3. HO-1 regulates the production of NO in peritoneal macrophages.
Fig. S4. Red blood cell morphology in mice subjected to CLP.
Fig. S5. HO-1 modulates PMN cell activation in response to CLP.
Fig. S6. Infiltrating leukocytes after CLP.
Fig. S7. Adenoviral overexpression of HO-1 in hepatocytes.
Table S1. Characteristics of septic shock patients.
Table S2. Correlations between HPX serum concentration and SOFA.
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52. Acknowledgments: We thank S. F. Yet (Pulmonary and Critical Care Division, Brigham and
Womens Hospital) for providing Hmox1 breeding pairs from which Hmox1
/
mice were
derived; E. Tolosano (University of Torino, Torino, Italy) for critical review of the manuscript;
S. Rebelo (Instituto Gulbenkian de Ciência) for invaluable help in breeding the Hmox1
/
mice;
K. Rish, R. Lovelace, and R. Helston (University of Missouri) for technical expertise in the HPX
isolation and characterization; and T. Davis (University of Missouri) for heme-HPX preparation.
Funding: This work was supported by Fundação para a Ciência e Tecnologia (Portugal) grants
SFRH/BPD/25436/2005 and PTDC/BIO/70815/2006 (to R.L.); SFRH/BPD/44256/2008 (to R.G.);
SFRH/BD/11816/2003 (to L.T.); SFRH/BD/3106/2000 (to A.C.); SF RH/BPD/21707/2005 and
PTDC/SAU MII/71140/ 2006 (to A.F.); SFHR/BD/33218/2007 (to I.M.); POCTI/SAU-MNO/56066/
2004, POCTI/BIA-BCM/56829/2004, PTDC/BIA-BCM/101311/2008, and PTDC/SAU-FCF/100762/
2008 (to M.P.S.); as well as GEMI Fund Linde Healthcare (to M.P.S.), the European Community,
Sixth Framework grant LSH-2005-1.2.5-1 (to M.P.S.), and Marie Curie FP7-PEOPLE-2007-2-1-IEF,
GASMALARIA (to V.J.). A.S. is supported by research incentive funds from the University of
Missouri at Kansas City, MO, USA. F.A.B. is a research scholar supported by Conselho Nacional
de Desenvolvimento Científico e Tecnológico and FAPERJ, Brazil. Author contributions: R.L.
performed most of the experimental work with help from A.F. and S.C. R.G. performed the
RESEARCH ARTICLE
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on October 25, 2010stm.sciencemag.orgDownloaded from
experiments defining the cytotoxic effect of free heme. V.J. performed the experiments estab-
lishing heme and hemoglobin concentrations in plasma. A.C. and I.M. performed flow cytom-
etry analysis. F.A.B. and A.M.J. conduced the clinical study with septic shock patients. M.M.C.
and D.B. set up the CLP model in the laboratory.A.S. suppliedthe purified HPX and heme-HPX,
gave critical advise on its use, and contributed to writing of the manuscript. M.P.S. formulated
the hypothesis that free heme might play pivotal roles in the pathogenesis of severe sepsis,
designed the experimental approach, and wrote the manuscript with help from R.L. R.L. and R.
G. contributed to the study design. All author s read and appro ved the manus cript.
Competing interests: The authors declare that they have no competing interests.
Submitted 29 March 2010
Accepted 3 September 2010
Published 29 September 2010
10.1126/scitranslmed.3001118
Citation: R. Larsen, R. Gozzelino, V. Jeney, L. Tokaji, F. A. Bozza, A. M. Japiassú, D. Bonaparte,
M. M. Cavalcante, Â. Chora, A. Ferreira, I. Marguti, S. Cardoso, N. Sepúlveda, A. Smith, M. P. Soares,
A central role for free heme in the pathogenesis of severe sepsis. Sci. Transl. Med. 2, 51ra71
(2010).
RESEARCH ARTICLE
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... Since severe cases of HUS frequently require dialysis and pose the risk of progression to chronic and end-stage kidney disease [4,7], it is important to further elucidate the underlying mechanisms involved in HUS pathogenesis and to identify molecular targets for new potential therapeutic approaches. There is evidence that free heme propagates disease progression in hemolytic disorders and life-threatening infections [8][9][10], including sepsis [8], malaria [10] and sickle cell disease [11][12][13][14]. The degradation of heme into equimolar amounts of ferrous iron, carbon monoxide (CO) and biliverdin is catalyzed by heme oxygenases (HO) which are intracellular enzymes found in humans and mice [15]. ...
... Since severe cases of HUS frequently require dialysis and pose the risk of progression to chronic and end-stage kidney disease [4,7], it is important to further elucidate the underlying mechanisms involved in HUS pathogenesis and to identify molecular targets for new potential therapeutic approaches. There is evidence that free heme propagates disease progression in hemolytic disorders and life-threatening infections [8][9][10], including sepsis [8], malaria [10] and sickle cell disease [11][12][13][14]. The degradation of heme into equimolar amounts of ferrous iron, carbon monoxide (CO) and biliverdin is catalyzed by heme oxygenases (HO) which are intracellular enzymes found in humans and mice [15]. ...
... While the ubiquitously expressed HO-1 isoform is induced during inflammatory or oxidative conditions, HO-2 is constitutively expressed in various tissues [16]. Nevertheless, it has been demonstrated that HO-2 is less effective in exerting a cytoprotective function against substantial quantities of free heme during hemolysis [17,18], whereas HO-1 allows the efficient recycling of heme-bound iron under hemolytic conditions which would otherwise cause oxidative stress [19] and inflammation [8,20]. The induction of HO-1, encoded by the gene heme oxygenase-1 (Hmox1), is associated with various anti-inflammatory, anti-proliferative as well as anti-apoptotic responses in answer to diverse stress conditions [16,17]. ...
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Hemolytic-uremic syndrome (HUS) is a systemic complication of an infection with Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli, primarily leading to acute kidney injury (AKI) and microangiopathic hemolytic anemia. Although free heme has been found to aggravate renal damage in hemolytic diseases, the relevance of the heme-degrading enzyme heme oxygenase-1 (HO-1, encoded by Hmox1) in HUS has not yet been investigated. We hypothesized that HO-1, also important in acute phase responses in damage and inflammation, contributes to renal pathogenesis in HUS. The effect of tamoxifen-induced Hmox1 gene deletion on renal HO-1 expression, disease progression and AKI was investigated in mice 7 days after HUS induction. Renal HO-1 levels were increased in Stx-challenged mice with tamoxifen-induced Hmox1 gene deletion (Hmox1R26Δ/Δ) and control mice (Hmox1lox/lox). This HO-1 induction was significantly lower (−43%) in Hmox1R26Δ/Δ mice compared to Hmox1lox/lox mice with HUS. Notably, the reduced renal HO-1 expression was associated with an exacerbation of kidney injury in mice with HUS as indicated by a 1.7-fold increase (p = 0.02) in plasma neutrophil gelatinase-associated lipocalin (NGAL) and a 1.3-fold increase (p = 0.06) in plasma urea, while other surrogate parameters for AKI (e.g., periodic acid Schiff staining, kidney injury molecule-1, fibrin deposition) and general disease progression (HUS score, weight loss) remained unchanged. These results indicate a potentially protective role of HO-1 in the pathogenesis of Stx-mediated AKI in HUS.
... Whether this reflects a normal situation in children or reflects underlying disease is not clear. Either way, this level of CFH has been shown to elicit cell permeability and injury [10,11,18]. Notably, hemopexin levels were higher in all patients immediately post-CPB to the extent that the ratio with CFH was above one, a condition expected to prevent CFH toxicity. ...
... This understanding stems largely from hemolytic disease (e.g., sickle cell disease) and experiences with CFH-based blood substitutes [19][20][21]. More recent evidence suggests that hemolysis plays a role in non-hemolytic acuate inflammatory diseases, especially in critical care settings (e.g., sepsis, infection, stored RBC transfusion, and associated end-organ injury), leading to a renewed focus on understanding various species that are released during hemolysis and the mechanisms linking these to increased inflammation and oxidative tissue injury [18,[22][23][24][25][26][27][28]. It is now clear that in addition to CFH, free heme and iron are all independent mediators of toxic effects associated with hemolysis. ...
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Background/Objectives: Hemolysis has been associated with acute kidney injury (AKI) in infants and neonates after surgery involving cardiopulmonary bypass (CPB). Erythrocyte hemolysis and subsequent end-organ injury have been shown to be a complex process involving the liberation of multiple molecules that mediate the loss of nitric oxide and oxidative damage. This study assesses the association of multiple products of erythrocyte hemolysis with the evolution of AKI in neonates and infants undergoing CPB surgery. Methods: Blood and urine samples were collected at multiple time points before and after CPB and stored within an institutional biorepository. Twenty-one patients with AKI were matched with twenty-one non-Aki patients based on demographic and case complexity data. Results: Samples were analyzed for cell-free hemoglobin, heme, non-transferrin-bound iron, haptoglobin, hemopexin, and nitrite/nitrate. NGAL and KIM-1 were measured to index AKI. Cell-free hemoglobin was higher, haptoglobin was lower, and haptoglobin:hemoglobin ratio was lower in AKI compared to non-AKI patients. Conclusions: AKI in neonates and infants after CPB is associated with a pre and postoperative decrease in serum haptoglobin. These results confirm the need for future studies to prevent injury from hemolysis during CPB and potentially identify at-risk patients with decreased haptoglobin levels before surgery if delay is an option.
... [66][67][68] Increased circulating free heme levels have been linked to a higher risk of hemolytic disorders, such as sickle cell anemia, and regulated EC death in patients with severe sepsis. 66,69 Moreover, the excessive release of free heme under pathological conditions is associated with EC barrier dysfunction. In macrophages, heme or the oxidized form of hemoglobin stimulates NLRP3, resulting in the production of the inflammatory factor IL-1β. 70,71 In ECs, heme, rather than hemoglobin, activates the NLRP3 inflammasome, resulting in the release of IL-1β ( Figure 2). ...
... 79 Heme, an important iron-containing compound in the human body, can induce the release of HMGB1 and produce a synergistic effect with it, both participating in the pathogenic mechanism of sepsis. 69 Notably, HMGB1 can affect enzymes involved in heme metabolism, such as HO-1, promoting ferroptosis in ECs. 80 These DAMPs, as part of the DAMP family, influence each other through various pathways and play a pro-inflammatory synergistic role in sepsis. ...
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Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Endothelial cells (ECs) are an important cell type typically affected in sepsis, resulting in compromised barrier function and various forms of regulated cell death (RCD). However, the precise mechanisms underlying sepsis-induced EC damage remain unclear. This review summarizes the recent research progress on factors and mechanisms that may affect the permeability and RCD of ECs under septic conditions, including glycocalyx, damage-associated molecular patterns, and various forms of RCD in ECs, such as apoptosis, pyroptosis, ferroptosis, and autophagy. This review offers important insights into the underlying mechanisms of endothelial dysfunction in sepsis, aiming to contribute to developing small-molecule targeted clinical therapies.
... We further demonstrate that IL6 and DEX-mediated induction of the APR is partially protective in CLP. This was somewhat expected because several acute phase proteins (APPs) have protective activities in sepsis and sepsis-like models (Libert et al, 1994a;Hochepied et al, 2000;Van Molle et al, 1997;Dalli et al, 2014;Libert et al, 1996;Janz et al, 2013;Larsen et al, 2010). IL6, via STAT3 activation, also affects the degradation and synthesis of fatty acids in the liver, depending on the context (Gavito et al, 2016). ...
... Besides their recognized anti-inflammatory and/or antimicrobial functions, APPs serve diverse physiological roles. Notably, haptoglobin and hemopexin are pivotal in the clearance of hemoglobin and heme released during hemolysis (Smith and McCulloh, 2015;Larsen et al, 2010), while IL6 and APR stimulate hepatocellular regeneration (Moshage, 1997;Streetz et al, 2000;Cressman et al, 1996;Greenbaum et al, 1998). To conclude, our data suggest that HNF4α dysfunction in sepsis prevents an adequate APR towards IL6, potentially compromising liver regeneration and the removal of hemoglobin and heme during hemolysis. ...
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In sepsis, limited food intake and increased energy expenditure induce a starvation response, which is compromised by a quick decline in the expression of hepatic PPARα, a transcription factor essential in intracellular catabolism of free fatty acids. The mechanism upstream of this PPARα downregulation is unknown. We found that sepsis causes a progressive hepatic loss-of-function of HNF4α, which has a strong impact on the expression of several important nuclear receptors, including PPARα. HNF4α depletion in hepatocytes dramatically increases sepsis lethality, steatosis, and organ damage and prevents an adequate response to IL6, which is critical for liver regeneration and survival. An HNF4α agonist protects against sepsis at all levels, irrespectively of bacterial loads, suggesting HNF4α is crucial in tolerance to sepsis. In conclusion, hepatic HNF4α activity is decreased during sepsis, causing PPARα downregulation, metabolic problems, and a disturbed IL6-mediated acute phase response. The findings provide new insights and therapeutic options in sepsis.
... During sepsis-induced anemia, cell-free hemoglobin (CFH) is released by erythrocytes through a series of mechanisms [4]. Traditionally, free heme and CFH-damaged tissue have been observed to worsen sepsis in lipopolysaccharide-induced systemic inflammatory animal models [5,6]. Clinical studies have shown that higher levels of CFH in patients with sepsis are associated with lower survival rates [7,8]. ...
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Background: Sepsis is a leading cause of mortality in intensive care units (ICUs). Cell-free hemoglobin (CFH) released during sepsis interacts with lysosomal enzymes from neutrophils and macrophages. This study aims to examine the association of LVV-hemorphin-7 (LVV-H7), cathepsin D, and cathepsin G with sepsis and shock in ICU patients. Methods: A prospective observational cohort study was conducted in the medical ICU of a tertiary referral hospital in Taiwan. The patients with an acute increasing sequential organ failure assessment (SOFA) score ≥ 2 between 2022 and 2023. Blood samples from 40 healthy controls were obtained from the hospital biobank. CFH metabolites, including LVV-H7 and lysosomal enzyme cathepsin D and cathepsin G, were compared between the sepsis (definite and probable) and non-sepsis (possible sepsis) groups. Multivariate logistic regression analyzed factors associated with sepsis and shock. Results: Among 120 patients, 75 were classified as septic and 45 as non-septic. Significant differences were observed in CFH, cathepsin D, cathepsin G, and LVV-H7 levels between sepsis and non-sepsis groups. LVV-H7 was a significant predictor for sepsis (adjusted OR [aOR] 1.009, 95% CI 1.005–1.013; p < 0.001) and shock (aOR 1.005, 95% CI 1.002–1.008; p < 0.05). Cathepsin G predicted non-shock (aOR 0.917, 95% CI 0.848–0.991; p < 0.05), while cathepsin D predicted septic shock (aOR 1.001, 95% CI 1.000–1.002; p < 0.05). Conclusions: LVV-H7, cathepsin D, and cathepsin G are associated with the classification of sepsis and shock episodes in critically ill patients with elevated SOFA scores.
... Previous studies have suggested that sepsis patients often have elevated CFH plasma levels, which are independently associated with a higher risk of death [6,19]. Animal studies have also supported these findings [20]. Shaver et al. evaluated the effect of CFH on renal function in an experimental sepsis model, suggesting that CFH exacerbates AKI [21]. ...
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Background Sepsis-associated acute kidney injury (SA-AKI) is common and associated with poor outcomes in critically ill patients. Acetaminophen is often used as an antipyretic and analgesic drug, but the association of acetaminophen use with mortality and recovery of renal function in SA-AKI patients remain unclear. We aimed to investigate the association between acetaminophen use and outcomes in SA-AKI patients. Methods This is a retrospective cohort study based on the MIMIC-IV database. Adult patients with SA-AKI were included in the analysis. The exposure was acetaminophen use within 7 days after the onset of SA-AKI. The primary outcome was 28-day mortality. Secondary outcomes included ICU mortality, in-hospital mortality, 90-day mortality, 1-year mortality, and renal recovery. Cox proportional hazards regression models were used to estimate the hazard ratio (HR) with 95% confidence interval (CI) for mortality. Logistic regression models were used to estimate the odd ratio (OR) with 95% CI for renal recovery. Results 6752 patients with SA-AKI were included, and 3892 (57.6%) patients received acetaminophen. Acetaminophen use was associated with decreased 28-day mortality (HR 0.69, 95% CI 0.63–0.75), ICU mortality (HR 0.56, 95% CI 0.50–0.63), in-hospital mortality (HR 0.62, 95% CI 0.57–0.69), 90-day mortality (HR 0.73, 95% CI 0.68–0.79), and 1-year mortality (HR 0.62, 95% CI 0.57–0.69). Acetaminophen use also was associated with improved renal recovery (OR 1.15, 95% CI 1.04–1.28). Conclusions Acetaminophen use is associated with decreased mortality and improved renal recovery in SA-AKI patients.
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The mechanisms of bacterial killing by neutrophil extracellular traps (NETs) are unclear. DNA, the largest component of NETs was believed to merely be a scaffold with antimicrobial activity only through the charge of the backbone. Here, we demonstrate for the first time that NETs DNA is beyond a mere scaffold to trap bacteria and it produces hydroxyl free radicals through the spatially concentrated G-quadruplex/hemin DNAzyme complexes, driving bactericidal effects. Immunofluorescence staining showed potential colocalization of G-quadruplex and hemin in extruded NETs DNA, and Amplex UltraRed assay portrayed its peroxidase activity. Proximity labeling of bacteria revealed localized concentration of radicals resulting from NETs bacterial trapping. Ex vivo bactericidal assays revealed that G-quadruplex/hemin DNAzyme is the primary driver of bactericidal activity in NETs. NETs are DNAzymes that may have important biological consequences.
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Red blood cells (RBCs), traditionally recognized for their role in transporting oxygen, play a pivotal role in the body's immune response by expressing TLR9 and scavenging excess host cell-free DNA. DNA capture by RBCs leads to accelerated RBC clearance and triggers inflammation. Whether RBCs can also acquire microbial DNA during infections is unknown. Murine RBCs acquire microbial DNA in vitro and bacterial-DNA-induced macrophage activation was augmented by WT but not Tlr9-deleted RBCs. In a mouse model of polymicrobial sepsis, RBC-bound bacterial DNA was elevated in WT but not in erythroid Tlr9-deleted mice. Plasma cytokine analysis in these mice revealed distinct sepsis clusters characterized by persistent hypothermia and hyperinflammation in the most severely affected subjects. RBC-Tlr9 deletion attenuated plasma and tissue IL-6 production in the most severe group. Parallel findings in human subjects confirmed that RBCs from septic patients harbored more bacterial DNA compared to healthy individuals. Further analysis through 16S sequencing of RBC-bound DNA illustrated distinct microbial communities, with RBC-bound DNA composition correlating with plasma IL-6 in patients with sepsis. Collectively, these findings unveil RBCs as overlooked reservoirs and couriers of microbial DNA, capable of influencing host inflammatory responses in sepsis.
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Stressed mammalian cells up-regulate heme oxygenase 1 (Hmox1; EC 1.14.99.3), which catabolizes heme to biliverdin, carbon monoxide, and free iron. To assess the potential role of Hmox1 in cellular antioxidant defense, we analyzed the responses of cells from mice lacking functional Hmox1 to oxidative challenges. Cultured Hmox1(-/-) embryonic fibroblasts demonstrated high oxygen free radical production when exposed to hemin, hydrogen peroxide, paraquat, or cadmium chloride, and they were hypersensitive to cytotoxicity caused by hemin and hydrogen peroxide. Furthermore, young adult Hmox1(-/-) mice were vulnerable to mortality and hepatic necrosis when challenged with endotoxin. Our in vitro and in vivo results provide genetic evidence that up-regulation of Hmox1 serves as an adaptive mechanism to protect cells from oxidative damage during stress.
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Objective: In 2003, critical care and infectious disease experts representing 11 international organizations developed management guidelines for severe sepsis and septic shock that would be of practical use for the bed-side clinician, under the auspices of the Surviving Sepsis Campaign, an international effort to increase awareness and improve outcome in severe sepsis. Design: The process included a modified Delphi method, a consensus conference, several subsequent smaller meetings of subgroups and key individuals, teleconferences, and electronic-based discussion among subgroups and among the entire committee. Methods: We used a modified Delphi methodology for grading recommendations, built on a 2001 publication sponsored by the International Sepsis Forum. We undertook a systematic review of the literature graded along five levels to create recommendation grades from A to E, with A being the highest grade. Pediatric, considerations were provided to contrast adult and pediatric management Results: Key recommendations, listed by category and not by hierarchy, include early goal-directed resuscitation of the septic patient during the first 6 hrs after recognition; appropriate diagnostic studies to ascertain causative organisms before starting antibiotics; early administration of broad-spectrum antibiotic therapy; reassessment of antibiotic therapy with microbiology and clinical data to narrow coverage, when appropriate; a usual 7-10 days of antibiotic therapy guided by clinical response; source control with attention to the method that balances risks and benefits; equivalence of crystalloid and colloid resuscitation; aggressive fluid challenge to restore mean circulating filling pressure; vasopressor preference for norepinephrine and dopamine; cautious use of vasopressin pending further studies; avoiding low-dose dopamine administration for renal protection; consideration of dobutamine inotropic therapy in some clinical situations; avoidance of supranormal oxygen delivery as a goal of therapy; stress-dose steroid therapy for septic shock; use of recombinant activated protein C in patients with severe sepsis and high risk for death; with resolution of tissue hypoperfusion and in the absence of coronary artery disease or acute hemorrhage, targeting a hemoglobin of 7-9 g/dL; appropriate use of fresh frozen plasma and platelets; a low tidal volume and limitation of inspiratory plateau pressure strategy for acute lung injury and acute respiratory distress syndrome; application of a minimal amount of positive end-expiratory pressure in acute lung injury/acute respiratory distress syndrome; a semirecumbent bed position unless contraindicated; protocols for weaning and sedation/analgesia, using either intermittent bolus sedation or continuous infusion sedation with daily interruptions/lightening; avoidance of neuromuscular blockers, if at all possible; maintenance of blood glucose <150 mg/dL after initial stabilization; equivalence of continuous veno-veno hemofiltration and intermittent hemodialysis; lack of utility of bicarbonate use for pH >7.15; u