Cell Host & Microbe
Article
Host Inactivation of Bacterial Lipopolysaccharide
Prevents Prolonged Tolerance Following
Gram-Negative Bacterial Infection
Mingfang Lu,1,* Alan W. Varley,1 Shoichiro Ohta,2 John Hardwick,1 and Robert S. Munford1,*
1Infectious Disease Division, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-9113, USA
2Department of Laboratory Medicine, Saga Medical School, 5-1-1 Nabeshima, Saga, Saga 849-8501, Japan
*Correspondence: mingfang.lu@utsouthwestern.edu (M.L.), robert.munford@utsouthwestern.edu (R.S.M.)
DOI 10.1016/j.chom.2008.06.009
SUMMARY
A transient state of tolerance to microbial molecules
accompanies many infectious diseases. Such toler-
ance is thought to minimize inflammation-induced
injury, but it may also alter host defenses. Here we
report that recovery from the tolerant state induced
by Gram-negative bacteria is greatly delayed in mice
that lack acyloxyacyl hydrolase (AOAH), a lipase that
partially deacylates the bacterial cell-wall lipopoly-
saccharide (LPS). Whereas wild-type mice regained
normal responsiveness within 14 days after they re-
ceived an intraperitoneal injection of LPS or Gram-
negative bacteria, AOAH-deficient mice had greatly
reduced proinflammatory responses to a second
LPS injection for at least 3 weeks. In contrast, LPS-
primed Aoah- knockout mice maintained an anti-in-
flammatory response, evident from their plasma
levels of interleukin-10 (IL-10). LPS-primed Aoah-
knockout mice experiencing prolonged tolerance
were highly susceptible to virulent E. coli challenge.
Inactivating LPS, an immunostimulatory microbial
molecule, is thus important for restoring effective
host defenses following Gram-negative bacterial
infection in animals.
INTRODUCTION
As key sensors and effectors of innate immunity, phagocytes
sense conserved microbial molecules and mount inflammatory
responses that destroy invading microorganisms. For hours to
a few days after such an encounter, however, the cells’ proin-
flammatory responses to the same and other microbial mole-
cules may be greatly reduced. This period of tolerance (also
called reprogramming, since some anti-inflammatory responses
are usually preserved; Henricson et al. [1993]; Shnyra et al.
[1998]), is thought to minimize inflammation-induced damage
during recovery from infectious diseases. Although tolerance in-
duced by Gram-negative bacteria or their LPS (endotoxin) has
been studied most intensively, a similar phenomenon has also
been noted in response to other bacterial agonists (Cross,
2002; Medvedev et al., 2006; Wang et al., 2003) and during
some viral infections (Didierlaurent et al., 2008).
Cell Host & Microbe 4, 293–302, September 11, 2008 ª2008 Elsevier Inc. 293
Tolerance to LPS has been documented in several clinical set-
tings: adults with pyelonephritis (McCabe, 1963), the alveolar
macrophages of individuals who have smoked endotoxin-con-
taining tobacco (Chen et al., 2007), and the blood leukocytes
of children with bacterial meningitis (Helminen and Vesikari,
1990a), adults with alcoholic cirrhosis (Von Baehr et al., 2000),
and adults with sepsis (McCall et al., 1993; Faist et al., 1988;
Munoz et al., 1991). Perhaps most significantly, tolerance in
blood leukocytes has been one of the most consistently ob-
served correlates of infection-induced immunosuppression in
humans (West and Heagy, 2002). Tolerance has also been noted
in the blood cells of uninfected patients following major trauma
(West and Heagy, 2002), with greater tolerance noted in those
who had elevated plasma endotoxin levels (Kelly et al., 1997).
In an investigation of patients with severe sepsis, the blood
monocytes of patients with Gram-negative bacterial infections
showed greater tolerance than did those from patients with
Gram-positive infections (Munoz et al., 1991).
Whereas much is now known about how LPS initiates reprog-
ramming in both monocyte macrophages and neutrophils (Med-
vedev et al., 2006; Wysocka et al., 2001; Cross, 2002; Cavaillon
et al., 2003; McCall and Yoza, 2007; Foster et al., 2007), the
mechanisms that terminate the reprogrammed (tolerant) state
and restore host defenses are poorly understood (McCall and
Yoza, 2007; Wysocka et al., 2005). Indeed, the duration of toler-
ance may vary greatly. In children with Haemophilus influenzae
type b meningitis, leukocyte hyporesponsiveness to LPS re-
solved as the patients recovered over a few days (Helminen
and Vesikari, 1990b). In contrast, adult cancer patients, who re-
ceived a bolus intravenous injection of LPS remained tolerant for
almost 2 weeks (Engelhardt et al., 1991), and the peripheral
blood leukocytes of septic adults have often been reprog-
rammed for many days or weeks after apparently successful
treatment of the inciting infection (Weighardt et al., 2000). In sep-
tic patients, recovery from tolerance has been associated with
survival (Munoz et al., 1991; Weighardt et al., 2000). Understand-
ing how tolerance is terminated in vivo thus might suggest ways
to decrease the duration of postinfection immunosuppression
and improve outcome.
Here we show that recovery from tolerance elicited by LPS or
Gram-negative bacteria in vivo requires acyloxyacyl hydrolase
(AOAH), a macrophage, dendritic cell, and neutrophil lipase
that inactivates LPSs by removing secondary fatty acyl chains
from the lipid A moiety (Munford and Hall, 1986). The duration
and degree of tolerance in vivo correlated with the presence of

fully acylated LPS in peritoneal macrophages. Moreover, mice
experiencing prolonged tolerance were highly susceptible to
E. coli challenge. These results suggest that killing Gram-nega-
tive bacteria does not allow full restoration of effective host
defenses: to prevent prolonged immune suppression, the
dominant bacterial ‘‘signal’’ molecule, LPS, must also be
inactivated.
RESULTS
AOAH Allows Recovery from Tolerance Induced
by LPS or Gram-Negative Bacteria In Vivo
To investigate a possible role for AOAH in recovery from toler-
ance, we gave a small intraperitoneal dose of LPS (10 mg) or
PBS to wild-type (Aoah+/+) and AOAH-deficient (Aoah / ) mice
and measured their responses to a second LPS challenge
(20 mg) given 5, 10, or 22 days later. Aoah / and Aoah+/+ mice
did not differ in their acute cytokine responses to the first LPS in-
jection (see data for PBS-primed mice in Figures 1A–1C), and the
mice in both groups were tolerant on day 5 after the initial LPS
dose. In contrast, whereas wild-type mice regained their ability
to respond normally to a second LPS dose within 5 to 10 days,
LPS-primed Aoah / mice had greatly reduced plasma IL-6,
TNF, and RANTES responses to a second dose for the entire
22 day period after the initial LPS exposure (Figures 1A, 1B,
and S1). In keeping with previous reports that LPS-induced
294 Cell Host & Microbe 4, 293–302, September 11, 2008 ª2008 Elsevier Inc.
Cell Host & Microbe
LPS Deacylation Limits Microbial Tolerance In Vivo
Figure 1. Aoah / Mice Experience Pro-
longed LPS-Induced Tolerance In Vivo
(A–C) Aoah / and Aoah+/+ mice received intra-
peritoneal injections of PBS or LPS (10 mg) in
PBS on day 0. On days 5, 10, or 22, separate
groups of mice (n = 4–11 mice/group) were chal-
lenged intraperitoneally with 20 mg LPS. Cytokine
or chemokine levels were measured in plasma
samples collected 4 hr later. Combined data
from 4 experiments.
(D and E) Intact Gram-negative bacteria also in-
duce prolonged tolerance in Aoah / mice. Mice
were primed intraperitoneally with 1.5 3 107 col-
ony-forming units of an avirulent strain of E. coli
K12. Fourteen days later they were challenged
intraperitoneally with LPS, and plasma samples
were collected 1 hr (TNF) and 4 hr (IL-6) later for
cytokine analysis (n = 8–10, data combined from
two experiments).
(F) Aoah / mice recover from tolerance induced
by an agonistic antibody to MD-2—TLR4. Mice
were injected intraperitoneally with 1 mg UT12
IgG or heat inactivated (HIA) UT12 on day 0. On
days 5 and 21, separate groups of mice were chal-
lenged intraperitoneally with 20 mg LPS. RANTES
levels were measured in plasma 4 hr after LPS
challenge (n = 4 to 5). Closed symbols, Aoah+/+;
open symbols, Aoah / ; squares, UT12 priming;
triangles, HIA UT12 priming. Similar results were
observed using TNF and IL-6 as response read-
outs (data not shown) and in a second experiment
(n = 4 to 5). Differences from PBS control group
(or HIA UT12 group) of same genotype: * p < 0.05;
**p < 0.01; *** p < 0.001. Error bars represent ± 1
SEM. Note log scales.
macrophage reprogramming alters responses along both the
MyD88-dependent and -independent arms of the intracellular-
signaling pathway downstream of TLR4 (Bagchi et al., 2007;
Sato et al., 2002), in vivo responses to LPS challenge that were
MyD88-dependent (TNF [Figure S1], IL-6) or MyD88-indepen-
dent (RANTES from Björkbacka et al., 2004), MCP-1 (Mata-
Haro et al., 2007) [Figure S1]) were impaired. AOAH is thus re-
quired for mice to recover their ability to mount several normal
proinflammatory responses to LPS. In contrast, LPS-primed
Aoah / mice produced slightly higher plasma concentrations
of IL-10, a major anti-inflammatory cytokine, than did the other
groups (Figure 1C). Retention of a normal IL-10 response to
LPS despite greatly reduced proinflammatory responses sug-
gests that the tolerant animal’s reactions to LPS are reprog-
rammed, not globally depressed (Henricson et al., 1993; Shnyra
et al., 1998; Medvedev et al., 2006).
Since Gram-negative bacteria also contain nonLPS activators
of innate immune responses (peptidoglycan, lipoproteins, and
others), it was important to know whether AOAH is also required
to terminate tolerance induced by infection with intact Gram-
negative bacteria. We gave a sublethal infectious dose (1.5 3
107 colony-forming units [cfus]) of an avirulent E. coli strain to
Aoah+/+ and Aoah / mice and challenged them 14 days later
with an intraperitoneal injection of either a virulent E. coli strain
(O18K1, 3 3 107 cfus) or 20 mg LPS. As shown in Figures 1D
and 1E, Aoah / mice previously exposed to E. coli had
Cell Host & Microbe
LPS Deacylation Limits Microbial Tolerance In Vivo

significantly lower plasma IL-6 and TNF responses to the LPS

challenge than did Aoah+/+ mice; similar results were seen fol-
lowing virulent E. coli challenge (Figure S2). These findings con-
firm the ability of AOAH to terminate tolerance induced by whole
Gram-negative bacteria and support the conclusion that LPS is
the dominant Gram-negative ‘‘molecular pattern’’ recognized
by animal cells (Somerville et al., 1996; Munford, 2008).

MD-2—TLR4 Activation by an Agonistic Monoclonal

Antibody Does Not Induce Prolonged Tolerance
in AOAH-Deficient Animals
It is possible that AOAH prevents prolonged tolerance through
a mechanism that does not involve its ability to deacylate LPS.
To test this hypothesis, we gave Aoah+/+ and Aoah / mice an
intraperitoneal injection of a monoclonal antibody that stimulates
cells via MD-2—TLR4 in the absence of LPS (Ohta et al., 2006)
and tested their responses to an intraperitoneal dose of LPS
given 5 days or 21 days later. We found identical responses for
the two genotypes: in both Aoah+/+ and Aoah / mice, tolerance
on day 5 was followed by recovery by day 21 (Figure 1F, com-
pare with the responses to a priming dose of LPS in Figure 1B).
AOAH is thus important for recovery from tolerance induced by
its substrate, LPS, but not from tolerance induced by a nonLPS
agonist that activates the same signaling pathway.

LPS-Induced Antibodies Are Not Required to Prolong

Tolerance in AOAH-Deficient Mice
Since Aoah / mice have greatly exaggerated polyclonal IgM
and IgG3 antibody responses to LPS (Lu et al., 2005), we tested
the hypothesis that antibody-mediated LPS neutralization pre-
vents responses to the second LPS challenge in primed animals
(Greisman et al., 1969). Mice that lack both B cells (Kitamura
et al., 1991) and AOAH (mMT, Aoah double knockouts) exhibited
prolonged LPS tolerance in the absence of circulating IgM and
IgG (Figure 2). B cells or antibody responses to the priming
LPS thus do not account for persistent LPS tolerance in
Aoah / mice.
Peritoneal Macrophages Also Exhibit Reprogrammed
Responses to Microbial Ligands
Macrophages harvested from the peritoneal cavities of LPS-
primed Aoah / mice were also reprogrammed. Re-exposure
to LPS ex vivo was associated with greatly diminished LPS-in-
duced MyD88-dependent (TNF, IL-6, IL-1b [data not shown])
and MyD88-independent (RANTES, nitric oxide production)
responses (Figures 3A and 3B). In addition, the production
of TNF, IL-6 (data not shown), and RANTES was reduced in
response to Micrococcus luteus, a TLR2- dependent stimulus,
and there was significantly impaired RANTES production after
stimulation with a MyD88-independent TLR3 agonist, poly I:C
(Alexopoulou et al., 2001) (Figure 3A). LPS thus induced pro-
longed cross-tolerance (or ‘‘heterotolerance,’’ Dobrovolskaia
et al. [2003]) to other microbial agonists in Aoah / animals. In
contrast, whereas LPS-primed Aoah / macrophages produced
wild-type levels of IL-10 when incubated with Micrococcus
luteus, their IL-10 response to LPS was approximately one-third
that of macrophages from primed Aoah+/+ mice (Figure 3C). A
more extensive survey of peritoneal macrophage responses to
LPS is presented in Table S1.
Figure 2. Prolonged Tolerance Is Not Antibody Mediated
(A and B) Aoah / , mMT, and Aoah / mMT mice were primed with LPS intra-
peritoneally on day 0 and challenged with LPS on day 14. Plasma samples
were collected 4 hr later, and levels of IL-6 (A) and IL-10 (B) were measured
by ELISA. Combined data from three experiments, n = 5–15 mice/group, are
shown. Error bars represent ± 1 SEM. The differences from PBS control group
of the same genotype are *** p < 0.001.
In Aoah / Animals, Tolerance Can Be Induced by Very
Low LPS Doses and Last for Two Months
LPS-induced tolerance was dose-dependent (Figure 3D); as little
as 0.1 mg intraperitoneal LPS induced tolerance for 2 weeks in
Aoah / peritoneal macrophages, whereas 10 mg or more was
required in Aoah+/+ animals. Moreover, tolerance lasted for at
least 2 months in the absence of AOAH (Figure 3E). Aoah /
mice of another genetic background (C3H/HeN) also were toler-
ant for 14 days or more following exposure to 10 mg LPS intraper-
itoneally, excluding a strain-specific effect of the Aoah / muta-
tion (data not shown).
Recombinant AOAH Prevents Prolonged Tolerance
in Aoah / Animals
To confirm that AOAH deficiency allows prolonged LPS-induced
tolerance, we expressed recombinant human AOAH in AOAH-
deficient mice using a gutted adenoviral vector, Ad.CMV-
hAOAH. Fourteen days after viral infection, the mice were given

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LPS Deacylation Limits Microbial Tolerance In Vivo

/
Figure 3. Reprogramming Persists in Explanted Peritoneal Macrophages from LPS-Primed Aoah Mice
(A–C) Aoah / and Aoah+/+ mice were given LPS i.p. (primed mice) or PBS (control mice) on day 0 and on day 10. Ten days after the second injection, peritoneal
macrophages were isolated and treated ex vivo with LPS, Micrococcus luteus (ML), or poly I:C for 6 hr (A and C) or 24 hr (B). RANTES (A), nitrite (B), and IL-10 (C)
were measured in culture supernatants.
(D) LPS induction of tolerance in vivo is dose dependent. Mice (n = 3–6) received various doses of E. coli O14 LPS or PBS intraperitoneally on day 0. On day 14,
isolated peritoneal macrophages were exposed to 1 mg/ml LPS for 6 hr, and medium IL-6 was measured. Error bars represent ± 1 SEM.
(E) Responses to LPS of macrophages explanted from mice primed with 10 mg LPS 8 weeks previously. In (A–E), significant differences from PBS-primed mice of
same genotype are indicated by * p < 0.05; **p < 0.01; *** p < 0.001.
(F) Recombinant AOAH prevents prolonged tolerance in Aoah / mice. Mice that had received an intravenous injection of 1 3 108 transgene equivalents of
Ad.CMV-hAOAH or Ad.CMV-eGFP 14 days previously were primed intraperitoneally with PBS or 1 mg LPS. Peritoneal macrophages were isolated 14 days later
and stimulated with LPS ex vivo. The *** denote p < 0.001 versus other groups. Data were combined from two independent experiments. The symbol designations
in (A) are used in the other panels. Here, the open circles represent LPS-primed Aoah / mice that had received Ad.CMV-hAOAH.

either 1 mg LPS or PBS intraperitoneally. When they were har-
vested 2 weeks later and stimulated with LPS ex vivo, macro-
phages from LPS-primed Aoah / mice that had received
Ad.CMV-hAOAH produced 20-fold more IL-6 and TNF than did
macrophages from Aoah / mice that had received a control
virus, Ad.CMV-eGFP (Figure 3F), and their ability to produce
several other cytokines and chemokines in response to LPS
was also restored (Table S1). Providing recombinant AOAH
thus prevented prolonged tolerance in Aoah / animals.
Retention of Acylated LPS by Macrophages
Is Associated with Prolonged Tolerance In Vivo
Ten days after intraperitoneal priming there were more macro-
phages, neutrophils, and B cells in the peritoneal washes of
Aoah / mice that had been primed with LPS than in the other
groups (Table S2). Flow cytometric examination of peritoneal
cells 10 days after intraperitoneal injection of a fluorescent LPS
preparation (FITC-LPS) revealed that the FITC-LPS was associ-
ated almost exclusively with F4/80+ cells (macrophages)
(Figure 4A). Approximately 60% to 70% of the recovered macro-
phages was FITC+ in both Aoah / and Aoah+/+ mice. The mean
fluorescence intensity was similar for macrophages of both ge-
notypes. To measure the uptake and deacylation of LPS by peri-
toneal cells in vivo, we used [3H/14C]LPS. Ten days after intraper-
itoneal injection of 10 mg radiolabeled LPS, more of the LPS
remained associated with peritoneal cells in Aoah / mice,
which had more macrophages (Figure 4B, left); very little of
this LPS had been deacylated (Figure 4B, right). In contrast,
the [3H/14C]LPS found in Aoah+/+ cells lacked one-third of its
3
H-acyl chains, consistent with loss of the two secondary acyl
chains that are released by AOAH (Shao et al., 2007). Collec-
tively, these findings suggest that the macrophages from LPS-
primed Aoah / and Aoah+/+ mice contained similar amounts
of LPS per cell when they were studied 10 days later; in Aoah+/+
macrophages, the LPS had lost one-third of its acyl chains,
whereas in Aoah / macrophages it remained fully acylated. Im-
portantly, production of TNF following LPS exposure ex vivo was
inversely related to the amount of cell-associated 14C-LPS
(Figure 4C). Prolonged tolerance thus correlated directly with
the retention of fully acylated LPS by peritoneal macrophages.

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LPS Deacylation Limits Microbial Tolerance In Vivo
Figure 4. Fully Acylated LPS Remains Macrophage-Associated in
Primed Aoah / Mice and Induces Prolonged Reprogramming
(A) Flow cytometric analysis of peritoneal monocyte macrophages harvested
10 days after intraperitoneally injection of FITC-LPS, LPS, or FITC glycine.
(B) Peritoneal cells were harvested from Aoah+/+ and Aoah / mice that had
received intraperitoneal injections of 10 mg [3H/14C]LPS 10 days earlier. Recov-
ery of LPS (left) and its deacylation (percentage loss of 3H dpm, right) are
shown. AOAH removes two of the six acyl chains. Error bar represents 1
SEM and n = seven to eight mice/group.
(C) Ten days after LPS intraperitoneal injection, there was an inverse correla-
tion between the amount of TNF produced following LPS stimulation of ex-
planted macrophages ex vivo and the amount of radiolabeled LPS associated
with the explanted cells.
Cell Host & Microbe 4, 293–302, September 11, 2008 ª2008 Elsevier Inc. 297
Prolonged Tolerance Is Immunosuppressive
To find out how prolonged in vivo tolerance influences host de-
fense, we compared the ability of Aoah / and Aoah+/+ mice to
resist a virulent E. coli O18 strain (Cross et al., 1995) two weeks
after a priming dose of LPS. Whereas no PBS-primed Aoah+/+
mice succumbed to the bacterial challenge, 90% of the LPS-
primed Aoah / mice did not survive (Figure 5A). When we
repeated the bacterial challenge and measured cfus in various
organs 24 hr later, we recovered 102- to 103-fold more cfus
from the blood of LPS-primed Aoah / mice than from mice in
the other experimental groups (Figure 5B). A similar pattern
was observed in the liver, spleen, lung, kidney and peritoneal
fluid (data not shown). In the absence of AOAH, LPS priming
thus severely impairs the ability to control E. coli infection.
The impact of prolonged tolerance was evident shortly follow-
ing the infectious challenge. One hour after challenge with 3 3
107 E. coli O18K1, LPS-primed Aoah / mice had lower plasma
TNF (Figure 5C) and IL-6 (data not shown) than did animals in
the other groups; the IL-10 response was more variable
(Figure 5D). Similar results were found when mice had been
primed with live E. coli and then challenged with the virulent
O18K1 strain (Figure S2). Animals that retained fully acylated
LPS in peritoneal macrophages thus mounted sluggish pro-in-
flammatory cytokine responses when challenged with live bac-
teria. Twenty-four hours after infection, the plasma and perito-
neal levels of TNF and IL-6 (data not shown) were directly
related to the numbers of bacteria found in these fluids (Figures
5E and 5F). Uncontrolled infection in LPS-primed Aoah / mice
thus can induce inflammatory responses that likely contribute to
lethality.
DISCUSSION
We studied the duration of endotoxin tolerance in vivo and found
strong evidence that terminating tolerance to Gram-negative
bacteria requires enzymatic inactivation of their major TLR
ligand, LPS. The inactivating enzyme, AOAH, is produced by
monocyte macrophages, neutrophils, dendritic cells (Lu et al.,
2003), and renal cortical epithelial cells (Feulner et al., 2004). It
removes the secondary acyl chains that are required for LPS
recognition by MD-2—TLR4, the LPS-signaling receptor (Mun-
ford and Varley, 2006). LPS deacylation occurs over many hours
in vivo and does not appreciably diminish the acute inflammatory
response to LPS or Gram-negative bacteria. On the other hand,
persistently acylated LPS can induce prolonged, exaggerated
antibody responses (Lu et al., 2005), hepatomegaly (Shao
et al., 2007), and low-grade elevations in circulating levels of cer-
tain cytokines (IL-10) (B. Shao, unpublished data). Here we found
that AOAH deficiency has a profound impact on the intensity and
duration of endotoxin tolerance. The LPS dose required to in-
duce prolonged reprogramming in animals that could not deacy-
late LPS was at least 20-fold lower than that needed in wild-type
mice (Figure 3D), and the tolerant state lasted for at least 2
months (Figure 3E). The intensity of the tolerant state, as as-
sessed by the production of TNF by explanted macrophages,
was directly related to the amount of macrophage-associated
acylated LPS (Figure 4C). Prolonged tolerance did not occur
in Aoah / animals when MD-2—TLR4 was activated by an
agonistic monoclonal antibody (Figure 1F), providing strong

evidence that the enzyme’s ability to inactivate LPS is essential
to its ability to limit tolerance.
In the categorization developed by Greisman (Greisman et al.,
1969), early endotoxin tolerance is mediated by changes in
cellular responsiveness to LPS (this phase was later attributed
to macrophages (Freudenberg and Galanos, 1988), whereas
(late) tolerance that lasts more than a few days is caused by neu-
tralization of the challenge LPS dose by antibodies raised in re-
sponse to the initial LPS exposure. The latter phenomenon
seemed a likely explanation for prolonged tolerance in Aoah /
mice, which react to low doses of LPS by mounting robust
polyclonal IgM and IgG3 responses that include antibodies to
many different LPSs (Lu et al., 2005). Indeed, we also found in-
creased numbers of B cells in the peritoneal washes of LPS-
primed Aoah / mice (Table S1). We were thus surprised to
find that prolonged LPS reprogramming occurred in Aoah /
mice that lack B cells (Figure 2) and was also present in macro-
phages that had been removed from the antibody-rich in vivo
environment (Figure 3). In animals that cannot deacylate LPS,
prolonged endotoxin tolerance is not mediated by antibodies
or B cells.
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LPS Deacylation Limits Microbial Tolerance In Vivo
Figure 5. LPS-Primed Aoah / Mice Are
More Susceptible to E. coli Challenge
(A) Groups of mice received a priming intraperito-
neal dose of 10 mg E. coli O14 LPS or PBS. Ten to
14 days later, they were challenged intraperitone-
ally with 800 cfus E. coli O18K1. Data combined
from three independent experiments (n = 15–19)
are shown. Each survival curve is different from
the others at p % 0.01 (Mantel-Cox).
(B) In another experiment, cultures were per-
formed 24 hr after intraperitoneal inoculation of
800 cfus E. coli. The figure shows the cfus recov-
ered from cultures of blood.
(C and D) In a third experimental design, 5 3 107
cfus E. coli were injected intraperitoneally, and
plasma cytokine levels were measured 1 hr later.
At this time point, plasma TNF levels (C) were sig-
nificantly lower in primed Aoah / mice than in the
other groups, whereas plasma IL-10 was more
variable (D). Data combined from two experi-
ments, each with n = 4. Error bars = 1 SEM.
(E and F) Twenty-four hours postchallenge (800
cfus), in contrast, the concentrations of TNF in
the blood (E) correlated (F) with the number of
recovered bacteria in (B). Similar correlations
were observed between IL-6 and cfus and for
both peritoneal fluid and blood (data not shown).
* p < 0.05; **p < 0.01; *** p < 0.001.
Many previous investigators have
found that early endotoxin tolerance
(lasting hours or a few days) can boost
host defenses (Lehner et al., 2001; Freu-
denberg et al., 2001; Cross, 2002; Cavail-
lon et al., 2003; Echtenacher and Mannel,
2002). Enhanced resistance to infectious
challenge during early tolerance might
have several mechanisms: the ability of
TNF (produced in response to the priming
dose of LPS) to boost host defenses
(Echtenacher and Mannel, 2002; Cavaillon et al., 2003), a greater
influx of neutrophils into the site of bacterial inoculation (Lehner
et al., 2001), diminished neutrophil apoptosis (Feterowski et al.,
2001), interferon-mediated hypersensitivity to LPS (Freudenberg
et al., 2001), or increased production of antimicrobial proteins
(Foster et al., 2007). In contrast, other groups have found that
early tolerance can diminish host defenses. In particular, toler-
ance induced by intraperitoneal infection or intraportal injection
of LPS can diminish antibacterial resistance in the lungs of
mice (Mason et al., 1997; Deng et al., 2006), pointing to complex
interactions between the systemic and local compartments (Fit-
ting et al., 2004). In all of these studies, microbial challenge was
administered within a few hours or days of the priming exposure.
The interval between primary LPS exposure and bacterial
challenge was much longer in our studies than in those previ-
ously reported. Remarkably, for at least two weeks after LPS
priming, AOAH-deficient animals had increased numbers of in-
traperitoneal B cells and macrophages (Table S2), suggesting
that the fully acylated LPS remains stimulatory in vivo. Nonethe-
less, they mounted sluggish proinflammatory (TNF, IL-6) re-
sponses when challenged with live bacteria (Figures 4C and
Cell Host & Microbe
LPS Deacylation Limits Microbial Tolerance In Vivo
S2), and were more likely to die following intraperitoneal bacterial
challenge (Figure 4A). Moreover, macrophages explanted from
LPS-primed Aoah / mice produced significantly lower levels
of many chemokines and cytokines when they were stimulated
ex vivo with LPS (Figure 3 and Table S1). The immunodeficiency
observed here seems quite similar to that described by Cross
et al., who found that TLR4-dependent inflammatory responses
were indispensable for host defense versus virulent E. coli chal-
lenge (Cross et al., 1995). In the present case, it seems likely that
a dampened, sluggish initial response allowed bacteria to prolif-
erate to levels that then induced sustained, harmful levels of
proinflammatory mediators.
Several previous observations have suggested that persis-
tence of MD-2 —TLR4 agonists can prolong tolerance in vivo.
Whereas a single intravenous dose of LPS could induce toler-
ance in humans for less than 14 days (Engelhardt et al., 1991),
daily administration of LPS maintained tolerance for as long as
30 days (Greisman et al., 1963) and weekly injections maintained
tolerance for as long as 42 days (Engelhardt et al., 1991). In mice,
which generally recover from LPS-induced tolerance within 5 to
7 days (Wysocka et al., 2001), the agonistic UT12 monoclonal
antibody to MD-2/TLR4 induced tolerance that lasted at least 9
days (Ohta et al., 2006). These reports and the inverse correlation
found here between ex vivo TNF production and macrophage-
associated LPS (Figure 4C) suggest that persistent TLR4 activa-
tion is sufficient to perpetuate, at least in part, mechanisms that
reprogram cellular responses to LPS and several other microbial
agonists. Remarkably, LPS-primed Aoah / mice may not re-
gain normal responsiveness to LPS. For convenience we termi-
nated almost all experiments 10 to 14 days after priming, a time
at which significant differences between Aoah / and Aoah+/+
mice were consistently found. LPS-primed Aoah / mice re-
mained tolerant in vivo for at least 22 days (Figure 1), however,
and explanted macrophages were reprogrammed when they
were studied 8 weeks after the priming LPS dose (Figure 3E).
In other studies we have found that LPS-exposed Aoah /
mice develop lymph node enlargement (Lu et al., 2005) and he-
patomegaly (Shao et al., 2007) that lasted at least 6 and 4 weeks,
respectively. Enzymatic deacylation may thus be required to
terminate many different responses to LPS in vivo.
We found that providing AOAH in an adenoviral vector could
prevent prolonged LPS-induced tolerance in Aoah / animals
(Figure 2F). This result raises the possibility that increasing
AOAH activity in vivo might hasten the recovery of host defenses
following tissue-invasive Gram-negative bacterial diseases and
help prevent secondary infections. Enhancing AOAH activity
might also improve pulmonary defenses in individuals whose al-
veolar macrophages have been tolerized by the endotoxin in to-
bacco smoke (Chen et al., 2007), enhance responses to infection
in patients with hepatic cirrhosis, whose circulating monocytes
may be tolerized by gut-derived endotoxin (Von Baehr et al.,
2000), or diminish systemic immune activation in patients with
human immunodeficiency virus disease (Brenchley et al.,
2006). It should also be worthwhile to look for enhanced infection
susceptibility related to differences in AOAH expression among
such individuals (Gao et al., 2007).
Although exogenously-administered alkaline phosphatase
can evidently inactivate LPS in vivo in rodents (Beumer et al.,
2003; Koyama et al., 2002; Poelstra et al., 1997; van Veen
Cell Host & Microbe 4, 293–302, September 11, 2008 ª2008 Elsevier Inc. 299
et al., 2005), until recently there was only indirect evidence that
endogenous phosphatases play this role. Inactivation of LPS
by an intestinal alkaline phosphatase has now been reported to
prevent gut-mucosal inflammation in zebrafish (Bates et al.,
2007), which do not produce AOAH, suggesting that LPS de-
phosphorylation may be an important mechanism for inactivating
the large amounts of LPS found in the intestine. Our findings in-
dicate, in contrast, that enzymatic deacylation plays a distinctive
role in inactivating LPS in extraintestinal tissues, even in animals
that produce endogenous phosphatases (Peterson and Mun-
ford, 1987; Lu et al., 2005; Shao et al., 2007; Vaishnava and
Hooper, 2007). Most importantly, we have provided evidence
that disabling this potent microbial agonist is required to restore
effective antibacterial host defenses following Gram-negative
bacterial infection in vivo. It is possible that host mechanisms
for inactivating other microbial molecules, such as bacterial lipo-
peptides and peptidoglycan fragments (Hedl et al., 2007) or viral
TLR agonists (Didierlaurent et al., 2008), may also play a role in
terminating infection-induced immunosuppression in animals.
EXPERIMENTAL PROCEDURES
Mice
Targeted disruption of mouse Aoah was accomplished as described (Lu et al.,
2003). The mutation was backcrossed eight generations into the C57Bl/6 and
C3H/HeN backgrounds. All mice were kept in a specific pathogen-free barrier
facility. mMT Aoah / double knockouts, obtained by crossing mMT with
Aoah / mice, lacked serum IgM and IgG. Mouse protocols were approved
by the Institutional Animal Care and Utilization Committee of The University
of Texas Southwestern Medical Center (Dallas, TX).
Reagents
E. coli O14 LPS was prepared by the phenol-chloroform-petroleum ether
method (Galanos et al., 1969). Rc S. typhimurium LPS (3H-labeled fatty acyl
chains and 14C-labeled glucosamine backbone) was prepared as described
previously (Munford and Erwin, 1992); 1 mg had approximately 150,000 dpm
3
H and 10,000 dpm 14C. We measured the ratio of 3H dpm to 14C dpm in
cell lysates to estimate percent deacylation (Shao et al., 2007). FITC-LPS
was prepared as described by Tobias et al. (1995). LPS from E. coli O14
was resuspended (2 mg/ml) in 0.1 M borate, pH 10.5. Five micrograms radio-
labeled LPS were added so that the concentration of the final product could be
calculated. Ten milligrams of solid FITC was then added to 2.5 ml suspension
and incubated for 3 hr at 37 C. A 10-fold excess of glycine was added to stop
the reaction. The suspension was dialyzed (1000 MW cut off) against PBS until
the dialysis buffer was no longer yellow. The FITC-LPS was then precipitated
by adding a 2-fold excess of ethanol. The pellet was washed three times with
70% ethanol and resuspended in PBS. The degree of labeling was 0.17 FITC/
LPS (mol/mol). Glycine-FITC was made by mixing glycine with FITC in PBS.
The solution was diluted so that its OD494 was the same as that of the FITC-
LPS. The FITC mean fluorescence intensity (MFI) of peritoneal cells did not
change after adding trypan blue, which quenches extracellular FITC. To en-
hance intracellular FITC, cells were permeabilized with 0.5% saponin (Sigma)
and then stained with Qdot 565 Goat Anti-Fluorescein Conjugate (Invitrogen).
Murine monoclonal anti-MD-2/TLR4 antibody, UT12 (Ohta et al., 2006), was
prepared from mouse ascites using a T-GEL MacroPAC Kit from SCIPAC (Sit-
tingbourne, Kent, UK). Purity was ascertained by SDS-PAGE with Coomassie
blue staining. The purified mAb was approximately 500-fold more potent
than heat-denatured mAb (95 C, 5 min) at eliciting TNF release from C56Bl/
6 (Tlr4+/+) murine macrophages, and it did not stimulate Tlr4 / macrophages
to release TNF (data not shown).
Gutted Adenoviral Vectors
A vector that expresses human AOAH (gAd.CMV-hAOAH) was prepared by
cloning the 1.7 kb AOAH cDNA into a gutted-Ad shuttle plasmid, pACPar-
entCmF.CMVp-hGHTerm, as an EcoRI fragment. The product was linearized

with PmeI and cotransformed with undigested gutted-Ad packaging plasmid
(pAd4) into E. coli strain BJ5183 (a gift from B. Vogelstein, Johns Hopkins
School of Medicine) to allow homologous recombination (He et al., 1998),
which results in the transfer of the expression cassette and Kan marker from
the shuttle plasmid into the packaging plasmid. Kanamycin-resistant clones
were screened by digestion with PacI, and clones showing the expected pat-
tern were verified by transformation into 293 cells followed by assaying for
AOAH activity. The finished packaging plasmid was then digested with PacI
to liberate the complete gutted-Ad genome, which was transformed into
116cre cells (Parks et al., 1996). High titer, purified gAd.CMV-hAOAH was
then prepared by the cre-lox system of Graham and coworkers (Parks et al.,
1996) (6) as modified by Palmer and Ng (Palmer and Ng, 2003). A control virus
that expresses eGFP (cDNA from Clontech, Palo Alto, CA) was produced in the
same way. The viral preps were assayed by quantitative PCR to determine the
number of transgene equivalents per milliliter. In preliminary experiments, we
found that sustained production of AOAH in vivo occurred from 2 to 4 weeks
after intravenous injection of Ad.CMV-hAOAH.
In Vivo and Ex Vivo Experiments
Mice were injected intraperitoneally with 10 mg E. coli 014 LPS in 300 ml PBS,
1 mg UT12 mAb in PBS, 1.5 3 107 E. coli LCD25 (Munford et al., 1992) or
PBS only. This E. coli inoculum contains approximately 150 ng LPS (Ingraham
et al., 1983). They were challenged on later dates with 20 mg E. coli O111 LPS in
300 ml PBS intraperitoneally. Tail-vein blood was obtained 1 and 4 hr after chal-
lenge. EDTA was used as anticoagulant. Live bacterial challenge was per-
formed using E. coli O18K1, a virulent strain provided by A. Cross (University
of Maryland School of Medicine). For ex vivo experiments, mice were primed
with E. coli 014 LPS or PBS intraperitoneally; on some occasions, two priming
doses were given. Ten or 14 days after the last priming dose, mice were eutha-
nized with CO2, and their peritoneal cells were flushed with 5 ml PBS containing
3 mM EDTA. After adherence for 16 hr and washing with complete RPMI
(cRPMI) medium (Lu et al., 2005), the cells were treated with 1 mg/ml of E. coli
O111 LPS (Sigma), 40 mg/ml of killed Micrococcus luteus cells (Sigma), or
10 mg/ml of poly I:C (Sigma) for 6 hr. The media were harvested for cytokine as-
says, and the cells were washed prior to lysis in PBS 0.1% Triton X-100 to mea-
sure cell protein (Biorad). In results not shown, the TNF response to polyI:C was
very low in all of the study groups, indicating that the preparation used here was
endotoxin free, and Micrococcus luteus cells induced similar production of TNF
by Tlr4+/+ and Tlr4 / murine macrophages, as expected for a TLR2 agonist.
Assays
The RANTES, TGFb, and MIP-1a ELISA Kits were from R&D Systems. The IL-1b
ELISA Kit from eBioscience and the Griess Reagent Kit were from Invitrogen.
Other cytokines and chemokines were measured using ELISA Kits from Bec-
ton-Dickenson or a Searchlight Array (Thermo Scientific Life Science Research
Products, Rockford, IL). Flow cytometry and data analysis were performed as
described (Lu et al., 2005). Fc binding was blocked with 0.5 mg/ml
of normal mouse IgG (Caltag). Cells were then incubated with antibodies for
1 hr on ice, washed, and analyzed using FACScan (Becton-Dickenson) with
Flow Jo Software (TreeStar, Inc.). Anti-mouse F4/80-FITC (Clone CI:A3-1)
was from Serotec; Anti-mouse F4/80 (Biotin and PE) (Clone BM8) was from
Caltag. CD11b Clone M1/70, Gr-1 Clone RB6-8C5, B220 Clone RA3-6B2,
CD5 Clone 53-7.3, CD3 Clone 145-2C11, NK1.1 Clone PK136, and CD11c
Clone HL3 were all from Becton-Dickenson.
Statistics
Unpaired Student’s t test (two-tailed) was used for comparisons between
groups. Log-rank (Mantel-Cox) test was used for survival-curve analysis.
SUPPLEMENTAL DATA
Supplemental Data include two tables and two figures and can be found online
at http://www.cellhostandmicrobe.com/cgi/content/full/4/3/293/DC1/.
ACKNOWLEDGMENTS
This work was supported by grant AI18188 from the National Institute of Al-
lergy and Infectious Diseases and the Jan and Henri Bromberg Chair in Internal
300 Cell Host & Microbe 4, 293–302, September 11, 2008 ª2008 Elsevier Inc.
Cell Host & Microbe
LPS Deacylation Limits Microbial Tolerance In Vivo
Medicine, UT-Southwestern Medical Center. We thank F. L. Graham and
B. Vogelstein for essential reagents; David Farrar, Beth Levine and Lora
Hooper for helpful advice; and Baomei Shao for titrating the adenovirus preps.
Received: March 6, 2008
Revised: June 2, 2008
Accepted: June 30, 2008
Published: September 10, 2008
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