The Journal of Immunology
Distinct Cell Death Programs in Monocytes Regulate Innate
Responses Following Challenge with Common Causes of
Invasive Bacterial Disease
Steve J. Webster, Marc Daigneault, Martin A. Bewley, Julie A. Preston,
Helen. M. Marriott, Sarah R. Walmsley, Robert C. Read, Moira K. B. Whyte, and
David H. Dockrell
Peripheral blood monocytes represent the rapid response component of mononuclear phagocyte host defense, generating vigorous
but finite antibacterial responses. We investigated the fate of highly purified primary human monocytes following phagocytosis of
different bacteria. Exposure to high bacterial loads resulted in rapid loss of cell viability and decreased functional competence. Cell
death typically involved classical apoptosis. Exposure to high numbers of Escherichia coli and Klebsiella pneumoniae induced
nonapoptotic death with loss of cell membrane integrity, marked disruption of phagolysosomes, and caspase-1 activation, while
a subset of cells also released caspase-1–regulated extracellular traps. Classical apoptosis increased if extracellular bacterial
replication was reduced and decreased if intracellular ATP levels were reduced during these infections. Both classical
apoptosis and the alternative forms of cell death allowed monocytes, whose functional competence was exhausted, to down-
regulate reactive oxygen species and proinflammatory cytokine responses. In contrast, sustained stimulation of glycolytic metab-
olism and mitochondrial oxidative phosphorylation, with associated hypoxia inducible factor-1a upregulation, maintained
intracellular ATP levels and prolonged monocyte functional longevity, as assessed by maintenance of phagocytosis, reactive
oxygen species production, and proinflammatory cytokine generation. Monocyte innate responses to bacteria are short-lived
and are limited by an intrinsic program of apoptosis, a response that is subverted by overwhelming infection with E. coli and
K. pneumoniae or bacterial stimulation of cell metabolism. In this regard, the fate of monocytes following bacterial challenge more
closely resembles neutrophils than macrophages.The Journal of Immunology, 2010, 185: 2968–2979.
bone marrow precursors,arerecruited to sites ofinflammation during
but, being less numerous than polymorphonuclear phagocytes, their
principal roles are in tissue homeostasis and in maintenance of the
poolof residenttissuemacrophages(3).Forcertain infections,mono-
irculating peripheral blood monocytes represent a pleio-
from differentiated macrophages but their roles in the context of high
During bacterial infection monocytes perform distinct roles in
the innate host response. Monocytes actively phagocytose bacteria
and promptly kill ingested organisms as part of the rapid response
component of the mononuclear phagocyte limb of innate immunity
(5). They demonstrate high-output secretion of proinflammatory
cytokines, such as IL-1b and TNF-a, in response to engagement of
TLR4 or other pattern recognition receptors. Ingestion of particles
intracellular ATP (6, 7). Lysosomal-mediated intracellular degrada-
macrophages, monocytes have a lower concentration of mitochon-
dria, which places them at risk fordepleting intracellular ATPlevels
rapidly during antibacterial innate responses (9). Monocytes thus
have a greater reliance on constitutive glycolytic metabolism for
maintenance of intracellular ATP, in comparison with tissue macro-
on oxidative phosphorylation by mitochondria (10).
During the interaction of myeloid cells with bacterial pathogens
a variety of cell death processes have been described, which fre-
quently benefit host defense. Apoptosis is characterized by nuclear
condensation and fragmentation, DNA cleavage, cell shrinkage,
and preservation of membrane integrity, and it is frequently, al-
though not always, viewed as an anti-inflammatory death process
(11, 12). In contrast, pyroptosis, which also involves nuclear con-
densation and DNA cleavage, is associated with caspase-1 activa-
tion and membrane rupture and may therefore be viewed as a more
inflammatory death process (11). More recently, neutrophils have
been shown to use a death program termed extracellular trap osis
Department of Infection and Immunity, Medical School, University of Sheffield,
Sheffield, United Kingdom
Received for publication March 10, 2010. Accepted for publication June 21, 2010.
This work was supported in part by a Wellcome Trust Senior Clinical Fellowship (no.
076945 to D.H.D.). S.R.W. is supported by a Wellcome Trust Clinician Scientist
Fellowship (no. 078244).
Address correspondence and reprint requests to Dr. David H. Dockrell, Department
of Infection and Immunity, Medical School, University of Sheffield, LU107, Royal
Hallamshire Hospital, Glossop Road, Sheffield S10 2JF, United Kingdom. E-mail
The online version of this article contains supplemental material.
Abbreviations used in this paper: DCF, 29,79-dichloro-dihydrofluorescein diacetate; 2-
DG, 2-deoxyglucose; Eco, E. coli; ETosis, extracellular trap osis; HIF, hypoxia-
inducible factor; Kpn, K. pneumoniae; LDH, lactate dehydrogenase; DCm, inner mi-
tochondrial transmembrane potential; Mi, mock infection; MOI, multiplicity of in-
negative; +ve, positive.
(ETosis) in which nuclear DNA, histones, and serine proteases are
released as an antimicrobial strategy (13). Factors that may in-
fluence the death process include the bacterial strain, bacterial
load, and the activation state of the cell, with apoptotic and
nonapoptotic death processes occurring after the same infection
depending on the prevailing conditions (11, 14).
The paradigm of phagocyte cell death during antibacterial host
defense is provided by neutrophils, which undergo apoptosis after a
period of sustainedphagocytosis and killing (15). In contrast to tissue
macrophages, monocytes have enhanced susceptibility to apoptosis
(16–19) and a relatively short lifespan of 1–2 d in the circulation (20,
21). Proinflammatory stimuli and growth factors, however, are potent
prosurvival stimuli for monocytes and reverse the intrinsic suscepti-
bility to constitutive apoptosis (22, 23). Phagocytosis and killing of
a component of innate host defense (24). Alternatively, early cell
death of macrophages may be a pathogen-driven mechanism by
which the immune system is evaded and can take various forms
ranging between apoptosis, pyroptosis, or necroptosis, with varying
consequences for the overall inflammatory response (11). In some
forms of severe sepsis, for example, meningococcal disease, extre-
mely high counts of bacteria can be present within the bloodstream,
and the highest counts are associated with the most severe manifes-
tations of disease (25). Comparatively little is known concerning the
bacterial challenge, and their altered susceptibility to apoptosis sug-
gests that responses may differ from differentiated macrophages.
In view of their central role in the inflammatory response to
overwhelming bacterial infection, we have investigated the cell fate
of monocytes following exposure to a range of bacteria and demon-
death processes, concomitant with exhaustion of functional compe-
bacterial stimulation of monocyte metabolism prevents apoptosis,
enabling cell survival and prolonging proinflammatory responses.
Materials and Methods
Monocyte isolation and culture
Human PBMCs were isolated by Ficoll Paque (GE Healthcare, Buck-
inghamshire, U.K.) density centrifugation of whole blood, donated by
healthy volunteers. The South Sheffield Research Ethics Committee ap-
proved the studies, and subjects gave written, informed consent. Mono-
cytes were enriched from freshly isolated PBMCs using a MACS
monocyte isolation kit II and MACS LS columns (Miltenyi Biotec, Au-
burn, CA), yielding an average 98% purity. Purified monocytes were
cultured for 1 h in RPMI 1640 culture medium (Lonza, Basel, Switzerland)
with 2 mM L-glutamine (Life Technologies, Rockville, MD) containing
10% human AB serum (First Link, Brierley Hill, U.K.) in 24-well plates
(Costar; Sigma-Aldrich, St. Louis, MO) with or without coverslips at 5 3
105cells/well or 25-cm2flasks (Costar, Sigma-Aldrich) at 2 3 106cells
per flask to allow monocyte adherence. The media was then replaced with
RPMI 1640 with 2 mM L-glutamine containing 10% heat-inactivated FCS
(Bioclear), maintained at 5% CO2at 37˚C and cells used within 12h.
Neisseria meningitidis (MC58, B:15:P1.7.16b serogroup B), Klebsiella
pneumoniae subsp. pneumoniae (ATCC 43816), Escherichia coli (C29,
ocytes were mock infected or exposed to the indicted bacteria at an MOI of
colony counts were estimated at 4 h by gentamicin killing assay, and (C)
monocyte trypan blue exclusion was assessed by brightfield microscopy. The
by ANOVAwith Dunnett’s posttest comparison. Mi, mock infection.
Bacterial challenge results in loss of monocyte viability. Mon-
images of monocytes, stained with toluidine blue, following mock infection (original magnification 311,500) or infection with N. meningitidis (original
magnification 314,000), K. pneumoniae (original magnification 311,500), or S. pneumoniae (original magnification 311,500) at an MOI of 10 for 12 h
obtained using an FEI Tecnai transmission electron microscope.
Nuclearmorphologyby transmissionelectron microscopy in monocytes exposed to bacteria. Representativetransmission electronmicroscopy
The Journal of Immunology2969
2970MONOCYTE DEATH IN BACTERIAL INFECTION
group 2 capsular serotype K54) (26), Neisseria lactamica (Y92-1009, se-
quence type 3493 complex 613), and serotype 2 Streptococcus pneumoniae
(D39 strain, NCTC 7466) were the strains studied. In specific experiments,
the unencapsulatedstrainof MC58, ¢13,was examined to increasebacterial
internalization (27). All bacteria were grown overnight on blood agar with
the exception of N. lactamica, which was grown on chocolate blood agar.
(28). N. lactamica was grown in tryptone broth (Oxoid, Basingstoke, U.K.),
E. coli in brain heart infusion, and K. pneumoniae or N. meningitidis in
Mueller-Hinton broth (Oxoid). S. pneumoniae stocks were prepared and
opsonized as described previously (28). Adherent monocytes were infected
for various time periods with each bacterium at a multiplicity of infection
(MOI) of 10 CFU per monocyte (unless otherwise stated) (28). Intracellular
colony counts were determined using a gentamicin killing assay (29).
All flow cytometric measurements were made using a four-color FACS-
Calibur (BD Biosciences, Mountain View, CA). Forward and side scatter
light was used to identify “viable” monocyte populations based on size
and granularity. Ten thousand events were recorded and all data were
analyzed using FlowJo software, version 8.8.4 (Tree Star, Ashland, OR).
Brightfield and fluorescence microscopy
Brightfield and fluorescent images of whole cell morphology, nuclear mor-
microscope with a Zeiss 363/1.4 oil objective (Zeiss, Oberkochen, Ger-
many).Extracellular DNAstructureswerestained with 5mMHoechst33342
with a 1/200 dilution of rabbit anti-human histone H2A.Z Ab (no. 2718;
Cell Signaling Technology, Beverly, MA) at 4˚C overnight before staining
with a 1/100 dilution of a goat anti-rabbit IgG FITC-conjugated secondary
Ab (Sigma-Aldrich) for 1 h at room temperature. Images were processed by
LSM software and AxioVision 4.7.2 software (Zeiss). Quantification was
provided by analyzing 300 cells per field. In certain experiments, 10 mM of
vehicle control was added 30 min prior to infection. In select experiments,
with a 403/0.7 objective lens (Leica Microsystems, Wetzlar, Germany). Im-
age processing involved use of LAS AF software (Leica Microsystems).
Transmission electron microscopy
Monocytes (2 3 106) were mock infected or challenged with bacteria for
12 h. Cells were centrifuged at 1000 3 g for 5 min, washed three times in
PBS, and fixed in ice-cold 3% glutaraldehyde, 0.1 M phosphate buffer over-
night at 4˚C. The cell pellets were then washed in 0.1 M phosphate buffer
twice. Secondary fixation was carried out in 2% aqueous osmium tetroxide
for 2 h at room temperature and washed twice in 0.1 M phosphate buffer.
Specimens were then dehydrated through a graded series of ethanol: 75%
ethanol for 15 min, 95% ethanol for 15 min, 100% ethanol for 15 min twice
followed by 100% ethanol dried over anhydrous copper sulfate for 15 min.
The specimens were then placed in propylene oxide for 15 min twice. In-
filtration was accomplished by placing the pellets in a 0.5:0.5 mixture of
propylene oxide and Araldite resin overnight at room temperature. The pel-
lets were then left in Araldite resin for 6 h at room temperature and finally
on a Reichert Ultracut E ultramicrotome and stained with 1% toluidine blue
in 1% borax. Sections were examined using an FEI Tecnai transmission
electron microscope at an accelerating voltage of 80 kV and micrographs
were taken using a Gatan digital camera.
Determination of monocyte viability
The cell culture media from mockinfected or bacteria-exposed monocytes
were analyzed for lactate dehydrogenase (LDH) release using a com-
mercially available kit (Cytotox 96; Promega, Madison, WI) following
the manufacturer’s instructions. For trypan blue dye exclusion, mock
and infected monocytes were harvested by cell scraping and stained with
0.2% (w/v) trypan blue dye (Sigma-Aldrich). Cell counts were made using
a hemocytometer by brightfield microscopy.
Morphological analysis of cell death
Monocytes on coverslipswere fixed in 2% (w/v) paraformaldehyde (Sigma-
following the manufacturer’s instructions and counterstained with DAPI
containing mounting medium (Vector Laboratories, Burlingame, CA) (28).
Nuclear morphology was analyzed by fluorescence microscopy, assessing
300 cells per field.
Measurement of loss of inner mitochondrial transmembrane
Monocytes were stained with 10 mM 5,59,6,69-tetrachloro-1,19,3,39-
tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Molecular Probes,
Eugene, OR) for 30 min at 37˚C, 4 h and 12 h postinfection to measure
loss of inner mitochondrial transmembrane potential (Dcm) by flow cytom-
etry (29). Loss of Dcmwas determined by the loss of red fluorescence.
Analysis of loss of lysosomal acidification
Monocyte cultures, in the presence or absence of gentamicin, were washed
three times with PBS at 4 h postinfection, incubated at 37˚C in RPMI 1640
containing 5 mM acridine orange (Sigma-Aldrich) for 15 min, washed, and
resuspended in ice-cold PBS for analysis by flow cytometry with loss of
orange fluorescence recorded as a marker of loss of lysosomal acidification.
Latex bead phagocytosis
Green fluorescent carboxylate-modified latex beads with a mean diameter of
phagocytosis. To account for extracellular binding, F-actin–dependent
phagocytosis was inhibited by adding 2 mM cytochalasin D (Sigma-
Aldrich) to control samples for 30 min (37˚C, 5% CO2) prior to infection
(30). The extent of phagocytosis was determined by flow cytometry with an
increase in fluorescence associated with internalized latex beads.
Determination of intracellular ATP concentration
Infected monocytes were treated with 100 mg/ml gentamicin for 1 h to kill
extracellular bacteria. The media was then replaced with serum-free RPMI
1640 for 5, 10, 15, and 30 min and the cells were lysed in 100 ml of 1% (v/v)
NP-40. Cell lysates were boiled for 10 min to inactivate ATPase activity.
Intracellular ATP was determined using a commercially available biolumi-
nescent kit following the manufacturer’s instructions (ATP bioluminescent
assay kit; Sigma-Aldrich) with results normalized to numbers of viable
cells for each culture, as assessed by the numbers of adherent cells without
altered cell morphology (31). Luminescence was measured using a Fusion
universal microplate analyzer (Packard Instrument, Meriden, CT) and ana-
lyzed by Fusion instrument control application software, version 4.00. To
determine the effect of oxidative phosphorylation or glycolysis on ATP
generation, cultures were treated with either 50 mM oligomycin (Sigma-
Aldrich), an inhibitor of mitochondrial respiration, 10 mM 2-deoxy-D-
glucose (Sigma-Aldrich), aninhibitorofglycolysis, ora combination ofboth
inhibitors 30 min prior to cell lysis (or at the indicated time if different).
Detection of reactive oxygen species
Production of intracellular reactive oxygen species (ROS) was measured
using the cell-permeable molecule 29, 79-dichloro-dihydrofluorescein diac-
etate (DCF; Sigma-Aldrich) (32). Monocytes were preincubated with DCF
nucleus C, Representative brightfield (grayscale image) or fluorescent images of DAPI (blue image)- and TUNEL (green image)-stained monocytes following K.
The totalpercentageof TUNEL-positive monocytes(D),thepercentageofTUNEL-positivemonocyteswithfragmentednuclei(E),andthepercentageofTUNEL-
with Dunnett’s posttest comparison. Bacterial CFUs in these experiments are shown in Supplemental Table 1A. Mi, mock infection; +ve, positive.
Varying forms of cell death areobserved in monocytes exposedto bacteria.A and B, Representative fluorescent images of DAPI (blue images)- and
The Journal of Immunology2971
for 30 min before infection for the indicated time periods. Cells were
washed in PBS and then analyzed by flow cytometry. To determine extra-
cellular ROS production, monocytes preincubated with DCF for 30 min
were infected for 12 h, supernatants centrifuged at 8000 3 g for 10 min,
then transferred to a white-walled, 96-well plate (Costar; Sigma-Aldrich),
and fluorescence was detected using a Fusion universal microplate analyzer
software, version 4.00. As a positive control for ROS, monocytes were
treated with 1 mM PMA (Sigma-Aldrich) for 1 h before analysis and to
measure the production of ROS by S. pneumoniae, bacteriawere cultured in
the absence of monocytes using the same dose of bacteria as used to infect
Cytokine bead array
Monocyte culture supernatants were collected 12 h postinfection and then
Cytokines were analyzed using a BD CBA Flex set (IFN-g, TNF-a, IL-1b,
IL-6, IL-8, IL-10 and IL-12p70) and measured using a BD FACSArray
bioanalyzer (BD Biosciences, Mountain View, CA). The limits of detection
were 1.8 (IFN-g), 1.2 (TNF-a), 2.3 (IL-1b), 1.6 (IL-6), 1.2 (IL-8), 0.13
(IL-10), and 0.6 pg/ml (IL-12p70).
SDS-PAGE and Western blotting
described (29). Protein concentration was determined using a modified
Lowry assay (DC protein assay; Bio-Rad, Hercules, CA) and equal protein
and blotted onto nitrocellulose membrane (Bio-Rad). Blots were incubated
with anti–caspase-1 (Abcam, Cambridge, U.K.), anti–hypoxia-inducible
factor (HIF)-1a (Abcam), or anti-actin (Sigma-Aldrich) Abs for 12 h at
Glostrup, Denmark) and ECL (EZ-ECL; Geneflow, Staffordshire, U.K.).
Cell lysates from MCF-7 cells cultured for 24 h in normoxia (normal in-
cubator 5% CO2) or hypoxia (3 kPa O2), maintained using an Invivo 400
hypoxic work station (Ruskinn, Pencoed, U.K.) with a 5% CO2/balance N2
gas mix, were used as negative and positive controls for HIF-1a.
All data were recorded as mean 6 SEM unless otherwise stated. Statistical
testing was performed using GraphPad Prism 5.02 software (GraphPad
Software, San Diego, CA) with relevant statistical tests described in the
figure legends. Significance was defined as p , 0.05.
Bacterial infection is associated with loss of micromolecule
transport by monocyte cell membranes
We first examined whether the initial interaction with bacteria altered
permeability of monocytes to trypan blue, an assay often regarded as
a marker of loss of cell viability but that strictly measures altered cell
membrane transport of micromolecules (33). The bacteria studied
included pathogens, which are leading causes of invasive disease and
sepsis, as well as a commensal organism, N. lactamica, which is
shielded from the adaptive immune system by a polyclonal IgM re-
comparison with N. meningitidis (34–36). All of these bacteria are
phagocytosed and killed by mononuclear phagocytes without in-
tracellular persistence. We found increasing numbers of extracellular
bacteria with time in each culture, from 4 to 20 h postinfection (Fig.
1A). We demonstrated that monocytes could internalize each bacte-
rium, as shown by intracellular bacterial colony counts 4 h postin-
to stimulate phagocytosis, as has been previously shown (28). In-
ternalization was lowest for N. meningitidis. We found that all
microorganisms tested, with the exception of N. meningitidis, sig-
nificantly reduced monocyte cell membrane transport of micro-
molecules 4–12 h postinfection, as shown by trypan blue staining
(Fig. 1C). The levels and timing of this effect varied between bac-
teria, but these findings suggested altered cellular homeostasis after
sustained bacterial challenge in most infections.
Bacterial infection can induce apoptotic and nonapoptotic cell
We hypothesized that altered levels and kinetics of trypan blue
staining might reflect the existence of distinct monocyte cell death
mechanisms after bacterial challenge and that responses to specific
bacteria might favor particular forms of cell death. To address this,
we focused on morphological and biochemical changes, recog-
nizing that while both apoptosis and pyroptosis induce nuclear
condensation and DNA fragmentation, apoptosis frequently in-
volves nuclear fragmentation, whereas pyroptosis induces promi-
nent disruption of the cell membrane (11, 12). Examination of
nuclear morphology by electron microscopy revealed that some
bacteria (e.g., S. pneumoniae) induce features of apoptosis such as
nuclear fragmentation, but that other infections, such as K. pneu-
moniae, do not (Fig. 2). In keeping with the cell viability data, we
observed that monocytes exposed to N. meningitidis had normal
cell morphology. Further assessment with fluorescence micros-
copy enabled the combined assessment of nuclear morphology
and DNA strand breaks, as detected by TUNEL staining. At 12 h
postinfection with S. pneumoniae, we observed nuclear fragmenta-
tion (Fig. 3A, 3B). In contrast, K. pneumoniae infection produced
TUNEL-positive cells but not nuclear fragmentation (Fig. 3C). The
percentage of monocytes with DNA strand breaks and fragmented
(apoptotic) nuclei and with DNA strand breaks but without nuclear
fragmentation were quantified after each infection (Fig. 3D–F). As
shown, most of the TUNEL-positive monocytes following S. pneu-
moniae or N. lactamica exposure had nuclear fragmentation (Fig.
breaks in these infections was consistent with apoptotic cell death.
In contrast, after exposure to E. coli or K. pneumoniae, most mono-
cytes were TUNEL-positive but lacked fragmented apoptotic nu-
clear morphology (Fig. 3F).
We next determined the integrity of the cell membrane after ex-
posure to each bacterium. As shown in Fig. 3C, some bacteria were
associated with permeabilization of the cell membrane. For exam-
we measured LDH release (Fig. 3G), a marker of significant mem-
brane disruption (37). Only E. coli and K. pneumoniae exposure
that these infections were associated with a nonapoptotic cell death
mechanism, with absence of nuclear fragmentation and membrane
permeabilization being features of pyroptosis (11).
The monocyte death processes following exposure to E. coli
and K. pneumoniae are influenced by bacterial numbers
Different forms of programmed cell death can occur following
identical stimuli (38), and we next investigated whether the non-
was related to bacterial numbers. We repeated experiments in which
bacterial numbers were reduced by early addition of antimicrobials.
As shown for E. coli, this approach reduced both extracellular and
intracellular bacterial burden (Fig. 4A). Addition of antimicrobials
resulted in similar levels of nuclear fragmentation following
S. pneumoniae, E. coli, or K. pneumoniae challenge (Fig. 4B).
Following E. coli and K. pneumoniae exposure in the absence of
apoptotic nuclear morphology suggested a death mechanism distinct
from classical apoptosis (39). Loss of lysosomal acidification can be
detected by a reduction in orange fluorescence of acridine orange,
(12, 40). We observed a greater percentage of cells with loss of
2972MONOCYTE DEATH IN BACTERIAL INFECTION
lysosomal acidification after exposure to E. coli and K. pneumoniae
(Fig. 4C). Antimicrobial treatment also reduced the percentage
ofcellswith lossoflysosomalacidificationat4 hfollowing E.colior
K. pneumoniae challenge.
Monocytes release extracellular traps after exposure to some
a feature of neutrophil ETosis (13). We performed fluorescence
microscopy of monocytes using the DNA-binding agent Hoechst
33342 and anti-histone mAbs to determine whether there were
any features of this death process in our cultures. Column-purified
monocytes, which lacked neutrophil contamination, demonstrated
release of extracellular DNA and histones following exposure to
of extracellular material were noted emanating from cell nuclei and
were associated with extracellular bacteria, and the frequency
of cells showing this feature increased as the infectious challenge
was increased (Fig. 5B). This finding is reminiscent of the extracel-
have not been observed in monocytes (13, 41, 42). Increased extra-
cellular bacterial numbers therefore contributed to the percentage
of monocytes showing features of pyroptosis or ETosis after expo-
sure to E. coli or K. pneumoniae.
Some nonapoptotic death programs, associated with bacterial
infection, involve intracellular caspase-1 activation, and this is
a key feature of pyroptosis but not usually of classical apoptosis
(11). We performed Western blots on cell lysates for the active
(p10) fragment of caspase-1 and were able to demonstrate that
E. coli and K. pneumoniae infection was associated with marked
caspase-1 activation 12 h after challenge, unlike other infections
(Fig. 5C). Inhibition of caspase-1 markedly reduced the appear-
ance of extracellular traps (Fig. 5D, 5E).
Preservation of intracellular ATP prolongs monocyte
Having shown that early changes in the transport of micro-
molecules were associated with the development of a variety of
death processes following bacterial challenge, we next investigated
if changes in cell homeostasis involved cell metabolism, initially
measuring Dcmas a marker of mitochondrial homeostasis (43)
and measuring intracellular ATP. One hour after exposure, some
bacterial infections induced loss of Dcmin some cells, but no
infection was associated with a marked reduction in the concen-
tration of intracellular ATP in the population of cells (Supple-
mental Fig. 1). After 4–12 h, all of the bacteria studied induced
significant loss of Dcm(Fig. 6A). Despite this, N. meningitidis
infection differed from other infections in that intracellular ATP
concentration in the population of viable cells 12 h postinfection
was maintained at levels similar to those observed in mock-
infected cells, after normalization of the number of viable cells to
the level of those detected in the mock infected culture (Fig. 6B).
Viable monocytes after N. lactamica exposure also maintained
intracellular ATP levels, but at lower levels than after N. menin-
gitidis exposure. We addressed whether ATP preservation was
dependent on glycolytic metabolism or oxidative phosphorylation.
Mock infected monocytes required the simultaneous inhibition of
both oxidative phosphorylation and glycolysis to deplete intracel-
lular ATP and to inhibit ATP-dependent functions such as phago-
cytosis (Fig. 6C, 6D). Intracellular ATP was maintained at levels
close to normal following inhibition of either oxidative phosphor-
ylation or glycolytic metabolism in monocytes exposed to
N. meningitidis. Maintenance of intracellular ATP following ex-
posure to N. meningitidis occurred in association with upregula-
tion of HIF-1a (Fig. 6E), an important factor upregulating a range
of metabolic pathways in mononuclear phagocytes (44).
Levels of bacterial internalization were lower for N. meningitidis
than for non-Neisseria species (Fig. 1B). To exclude the possibility
that maintenance of intracellular ATP merely reflected differing rates
of delivery of bacteria to phagolysosomes, and hence reduced ATP
requirements for phagocytosis and killing, we used an unencap-
sulated mutant, since N. meningitidis capsule has been shown to
decrease adherence and phagolysosomal compartmentalization, and
increased the infecting dose (45). The unencapsulated mutant re-
sulted in comparable intracellular ATP concentrations, despite
with features distinct from classical apoptosis. A, The intracellular and
extracellular CFUs per milliliter of monocyte culture lysate or culture su-
pernatant were estimated 6 h after exposure to E. coli in the presence or
absence of gentamicin added 2 h postinfection (n = 4). pp , 0.05; pppp ,
0.001, unpaired t test comparing extracellular versus intracellular;††p ,
without gentamicin. B, Monocytes were mock infected or exposed to N.
meningitidis, K. pneumoniae, E. coli, N. lactamica, or S. pneumoniae at an
MOI of 10, and gentamicin was added at 4 h postinfection. The percentage
of monocytes with fragmented nuclei was recorded at 12 h (n = 3). pp ,
0.05; ppp , 0.01, ANOVA with Dunnett’s posttest versus mock infection.
C, Monocytes were mock infected or exposed to bacteria as in B in the
presence or absence of gentamicin, added 1 h postinfection. The percentage
of monocytes with loss of lysosomal acidification was recorded at 4 h by
flow cytometry (n = 4). pp , 0.05; pppp , 0.001, ANOVAwith Dunnett’s
posttest versus mock infection;†††p , 0.001, two-way ANOVAwith Bon-
ferroni posttest comparing with gentamicin versus without gentamicin.
LLA, loss of lysosomal acidification; Mi, mock infection.
Large numbers of K. pneumoniaeand E. coli causecell death
†††p , 0.001, unpaired t test comparing with gentamicin versus
The Journal of Immunology 2973
increased internalization, and also comparable low levels of apopto-
sis to the parental strain, suggesting capsular inhibition of internal-
ization did not explain the monocyte survival (Supplemental Fig. 2).
To determine whether there was any relationship between ATP levels
and the mechanism of cell death, we studied monocytes exposed to
K. pneumoniae incubated in the presence or absence of gentamicin.
To some of these cultures we added inhibitors of glycolytic metab-
olism and oxidative phosphorylation. As shown in Supplemental
Fig. 3, addition of gentamicin increased the apoptotic morphology
(as in Fig. 4B), in association with a less marked drop in intracellular
ATP than seen in the absence of gentamicin. Inhibitors of ATP
generation increased the percentage of cells with the pyroptotic ap-
pearance, but they reduced the apoptotic appearance of cells with
fragmented nuclei, despite the presence of antibiotics.
Prolonged monocyte survival allows sustained innate immune
In view of the sustained metabolic competence of monocytes after
exposure to N. meningitidis, we next addressed whether these cells
also showed sustained evidence of innate immune function, mea-
coli; Kpn, K. pneumoniae; Mi, mock infection; Nme, N. meningitidis; Spn, S. pneumoniae.
E. coli can trigger extracellular trap release from monocytes. A, Representative fluorescent image of DNA stained with Hoechst 33342 and anti-
2974 MONOCYTE DEATH IN BACTERIAL INFECTION
suring their capacity to phagocytose opsonized latex beads, gen-
erate ROS, as detected by flow cytometry with DCF, and express
cytokines, as determined by cytometric bead array. All infections
were associated with comparable phagocytosis of latex beads to
the mock infected cultures at 1 h (Supplemental Fig. 4A). We
found that only after exposure to N. meningitidis and N. lactamica
were monocytes able to continue to phagocytose particles at levels
comparable to the mock infected cultures 12 h postinfection (Fig.
7A). All infections stimulated generation of ROS from monocyte
cultures early after bacterial challenge (Fig. 7B). In the case of
S. pneumoniae, there was also evidence of extracellular ROS ex-
pression, in keeping with the known capacity of this organism to
generate ROS (46) (Supplemental Figs. 4B, 5). By 12 h, ROS pro-
duction remained noteworthy in the N. meningitidis–exposed cul-
tures (Fig. 7C), in keeping with the ongoing capacity of these
cultures to phagocytose bacteria. Other bacterial infections dif-
fered in their capacity to stimulate intracellular ROS production
at this time. Cultures associated with monocyte apoptosis (e.g.,
S. pneumoniae and N. lactamica) also stimulated ROS production
in the cells retaining viability. E. coli or K. pneumoniae exposure
resulted in no detectable intracellular ROS in cultures 12 h after
challenge even though intracellular ROS was detectable at earlier
time points following exposure to these bacteria.
In keeping with the sustained viability of monocytes and the con-
tinued capacity to phagocytose opsonized particles, we observed that
monocytes were still producing significant quantities of proin-
was unlike the situation with most of the other infections investi-
gated, which demonstrated minimal cytokine production, or, in the
case of E. coli exposure levels below the limit of detection (data not
shown), by 12 h postinfection. N. lactamica–exposed monocytes
retained functional capacity despite significant levels of apoptosis.
These cells produced a different pattern of cytokines to N. meningi-
tidis–challenged monocytes. While TNF-a and IL-8 levels were sim-
ilar, levels of IFN-g, IL-1b, IL-6, and IL-12 were lower and those of
IL-10 were greater. This demonstrated that prolonged survival of
monocytes following bacterial challenge has a significant impact on
the resulting pattern of cytokine release.
Investigation of cell death mechanisms in highly purified primary
human monocytes challenged with bacteria has been a compara-
tively neglected area in comparison with other myeloid cells, in-
cluding macrophages (11). We demonstrate a rapid loss of cell
viability during the first 12 h of exposure to bacteria. Some
infections (E. coli and K. pneumoniae) induce nonapoptotic death.
exposed to N. meningitidis, K. pneumoniae, E. coli, N. lactamica, or S. pneumoniae at an MOI of 10, and at 4 or 12 h the percentage of cells with loss of
Dcmwas estimated by flow cytometry (n = 6). ppp , 0.01; pppp , 0.001, versus mock infection, ANOVA with Dunnett’s posttest. B, Monocytes were
mock infected or exposed to bacteria as in A, and 12 h postinfection intracellular ATP levels were estimated by bioluminescence. K. pneumoniae (5 min,
p , 0.01), S. pneumoniae (5 min, p , 0.001), and E. coli (10 min, p , 0.05) were significantly different from mock infection. At 30 min, there was no
significant difference between N. meningitidis or N. lactamica and mock infection (n = 3). Significance was determined by two-way ANOVA with
Bonferroni posttest. C, Monocytes were mock infected or challenged with N. meningitidis or S. pneumoniae at an MOI of 10 for 12 h. For the last 30
min of incubation, 2-deoxyglucose and/or oligomycin was added to the indicated wells prior to lysis, and intracellular ATP levels were estimated by
bioluminescence (n = 6). pp , 0.05; pppp , 0.001, all comparisons versus samples without inhibitors for that infection, ANOVAwith Dunnett’s posttest. D,
The percentage of monocytes phagocytosing opsonized fluorescent latex beads 1 h following treatment with or without 2-deoxyglucose and oligomycin, as
in C (n = 3). ppp , 0.01, versus samples without inhibitors, ANOVAwith Dunnett’s posttest. E, Representative Western blot probed for HIF-1a and b-actin
from monocytes following 12 h of mock infection or exposure to N. meningitidis or S. pneumoniae at an MOI of 10. The positive control was a lysate from
MCF-7 cells cultured in normoxia, and the negative control was a lysate from MCF-7 cells cultured in hypoxia. The blot is representative of three
independent experiments. Bacterial CFUs in these experiments are shown in Supplemental Table 1B. 2-DG, 2-deoxyglucose; Mi, mock infection; Nme, N.
meningitidis; Spn, S. pneumoniae; +ve, positive control; -ve, negative control.
Monocytes exposed to N. meningitidis preserve intracellular ATP levels and retain phagocytic capacity. A, Monocytes were mock infected or
The Journal of Immunology 2975
Only monocytes exposed to N. meningitidis maintained significant
viability by 12 h postinfection. Loss of cell viability terminated
antibacterial innate responses. The prolonged immune compe-
tence of monocytes exposed to N. meningitidis, however, comes at
the cost of an extended proinflammatory immune response.
constitutive caspase-dependent apoptosis (19, 20, 22). During inter-
action with bacteria, monocytes phagocytose bacteria briskly and
kill these in their phagolysosomes, and thus phagolysosomal escape
is an important bacterial survival strategy (47–49). Monocytes
phagocytose bacteria with slightly less efficiency than neutrophils,
but microbicidal killing appears comparable (5). The lysosomal
system of monocytes is less developed than in tissue macrophages,
lacking the capacity for more prolonged microbial killing (9, 50).
Monocytes are therefore more reliant on acute microbicidal strate-
gies, such as ROS-dependent killing, than are macrophages, and
they are better equipped to control extracellular bacteria that can
be rapidly killed in phagolysosomes than those that rely on pro-
longed intracellular killing inphagolysosomes (7). The bioenergetic
demands of rapid bacterial internalization and intracellular killing
stress phagocytes (6, 51), and monocytes have a lower density of
mitochondria than differentiated macrophages (9). This places
monocytes under considerable bioenergetic stress, and we show
herein that the fate of monocytes is more similar to that of neutro-
phils than the differentiated macrophage.
Our findings emphasize the variety of cell death processes that
the need for careful characterization (52). Trypan blue positivity did
not correlate closely with specific cell death features,suggesting that
subtle disruption of micromolecular transport may be observed
without otherfeatures of loss of viability (33).Byanalysis ofnuclear
morphology and membrane integrity we identified infections pre-
dominantly associated with apoptotic cell death, as evidenced by
specific features of apoptosis, such as cell shrinkage, nuclear frag-
mentation, and preservation of cell surface membrane integrity (24,
39), whereas for E. coli or K. pneumoniae challenge DNA fragmen-
tation was associated with cytolysis but not nuclear fragmentation.
Caspase-1 activation, a feature of pyroptosis but not usually of mac-
rophage apoptosis, was also observed (11), and loss of lysosomal
acidification occurred in a greater percentage of cells in these infec-
of phagosomes with lysosomes, impairment of hydrogen ion pumps,
or lysosomal membrane permeabilization (53–55), although we did
not distinguish which of these mechanisms contributed in this case.
nonapoptotic death processes (56, 57) and caspase-1 activation by
“inflammasomes,” containing nucleotide binding and oligomeriza-
tion domain-like receptor family members, activated by the release
of microbial components into the cytosol (58).
from monocytes and of association of bacteria with these extracel-
lular structures following E. coli and K. pneumoniae infection. The
release of DNA, to form extracellular traps containing histones and
proteases, is observedfrom neutrophils and has also been described
for basophils (13, 41, 42). To our knowledge, a similar process has
not been described for monocytes. The large disruption of
the cellular membranes, we observed with these infections, would
facilitate the release of intracellular contents, including DNA, from
these cells. This process increased in proportion to the infectious
inoculum and was reduced by caspase-1 inhibition. Extracellular
traps were not identified after S. pneumoniae exposure in keeping
which degrades extracellular DNA (59).
Individual microorganisms can mediate more than one death
process (11, 52). Bacterial numbers, cell activation state, and, as we
suggest, energy state may be other interrelated factors. We have not
addressed activation state inthisstudy, butBergsbaken and Cookson
made the important observation that activation of macrophages can
convert apoptosis to caspase-1–dependent pyroptosis (14). Macro-
phage activation by LPS reduces apoptosis (60) and could have re-
duced apoptosis following E. coli and K. pneumoniae infection. The
have previously shown that apoptosis in differentiated macrophages
not specifically examine the influence of intracellular, as opposed to
processes, observed after E. coli and K. pneumoniae exposure, were
associated with higher numbers of extracellular bacteria but not
phagocytosis and production of ROS. A, The percentage of monocytes
phagocytosing opsonized fluorescent latex beads following 12 h mock
infection or exposure to N. meningitidis, K. pneumoniae, E. coli, N. lac-
tamica, or S. pneumoniae at an MOI of 10 (n = 6). ppp , 0.01; pppp ,
0.001, versus mock infection, ANOVA with Dunnett’s posttest. The per-
centage of monocytes with detectable intracellular ROS (B) 1–4 h and (C)
12 h after mock infection or exposure to N. meningitidis, K. pneumoniae,
E. coli, N. lactamica, or S. pneumoniae (MOI of 10). ROS was measured
by flow cytometry following incubation with DCF (n = 3). pp , 0.05,
ANOVA with Dunnett’s posttest. Bacterial CFUs in these experiments are
shown in Supplemental Table 1B. Mi, mock infection.
N. meningitidis–exposed monocytes demonstrate sustained
2976 MONOCYTE DEATH IN BACTERIAL INFECTION
higher numbers of intracellular bacteria 4 h postinfection. Neverthe-
less, reducing extracellular and intracellular bacterial numbers by
antimicrobial treatment increased rates of apoptosis, relative to
pyroptosis, while increasing the infectious dose increased rates of
extracellular trap formation for these infections. Survival of mono-
cytes following N. meningitidis exposure was associated with lower
intracellular bacterial numbers but was not altered by increasing in-
tracellular bacterial numbers. These conclusions emphasize that
unique microbial factors interact with bacterial burden and cellular
factors to determine the fate of cells.
of cell death. Apoptosis requires intracellular ATP, but processes
with a high affinity for ATP, such as many of the executors of ap-
optosis, are only compromised when cellular levels are depleted
100-fold (62). If there is not a source for this low level of ATP, then
necroptosis, a necrosis-like cell death, results in membrane disrup-
tion with the potential release of phagocytosed bacteria and inflam-
matory mediators (12, 39). Little is known of the exact energy
requirements of pyroptosis, but caspase-1 activation also requires
are so low, estimation of these differential energy requirements will
require ultrasensitive assays. Nevertheless, our preliminary analy-
sis suggests that by further depleting cellular ATP the death pro-
gram shifts from apoptosis to pyroptosis. Monocytes must balance
the large energy requirements of their rapid antibacterial response
and their relatively limited capacity to generate ATP with the need
to execute the most appropriate cell death program while there are
still sufficient ATP reserves for its completion.
Execution of a program of apoptosis allows a monocyte, which
has exhausted its functional capacity to kill ingested bacteria, to
enhance intracellular bacterial killing (64, 65) and terminate
proinflammatory responses (66). Increased cell activation, a fall in
intracellular ATP below the levels required for apoptosis, and cy-
tosolic sensing of microbial products would favor pyroptosis (11,
14, 63). Pyroptosis is known to facilitate control of intracellular
bacteria, which escape into the cytoplasm (67). Extracellular ATP
release could activate the same process in bystander cells (68). This
phlogistic death process could be viewed as a compromise in over-
whelming infection in which the monocyte although terminating its
own inflammatory response by cell death does not terminate the
overall tissue inflammatory response, as would happenwith apopto-
sis, but instead ensures its perpetuation with a further round of
inflammatory cell recruitment. In contrast, extracellular traps would
ensure that unphagocytosed extracellular bacteria could be con-
tained (13, 41, 42). Linking extracellular bacterial numbers to in-
levels of cytosolic microbial products or intracellular ATP would
provide the monocyte with an effective mechanism by which the
apoptotic program could be overridden when the antibacterial ca-
pacity of the phagolysosomal compartment is overrun.
Infection with N. meningitidis, the meningococcus, was distinct
since it resulted in sustained monocyte viability in association with
maintenance of intracellular ATP. Preservation of intracellular ATP
levels was the result of activation of both glycolytic metabolism
and oxidative phosphorylation and was associated with upregula-
tion of HIF-1a (44). This response appeared to be intrinsic to N.
meningitidis since intraphagolysosomal loading with an unencap-
sulated mutant (45) failed to reduce ATP or enhance apoptosis.
Exposure to N. meningitidis resulted in sustained innate responses.
This is noteworthy since marked proinflammatory cytokine release
is central to the pathogenesis of meningococcal disease (69). A
subpopulation of viable monocytes after N. lactamica infection
maintained intracellular ATP, continued to phagocytose latex
beads,and showed distinctcytokine expressionwithreducedproin-
flammatory cytokine expression but enhanced IL-10 responses.
When the cytokine expression was normalized to numbers of ad-
herent viable cells, the levels of IL-1b, IL-6, and IL-12 became
comparable between N. lactamica and N. meningitidis and the lev-
els of TNF-a and IL-8 became greater for N. lactamica infection,
but the increase in IL-10 expression for N. lactamica infection was
even more accentuated (data not shown). In addition to showing
increased levels of trypan blue positivity, N. lactamica infection
with evidence that apoptosis was the predominant mechanism
mock infection or exposure to N. meningitidis, K. pneumoniae, N. lactamica, or S. pneumoniae at an MOI of 10 is shown. Cytokine levels in the culture
media were determined by cytokine bead array: IFN-g (A), TNF-a (B), IL-1b (C), IL-6 (D), IL-8 (E), IL-10 (F), and IL-12p70 (G) (n = 8). pp , 0.05; ppp ,
0.01; pppp , 0.001, ANOVA with Tukey’s posttest. Mi, mock infection.
Sustained proinflammatory cytokine production by monocytes exposed to N. meningitidis. Cytokine production by monocytes 12 h after
The Journal of Immunology2977
of cell death. N. lactamica is more readily internalized than N.
meningitidis, but the simultaneous ingestion of apoptotic bodies
would be predicted to deactivate proinflammatory cytokine
responses and enhance IL-10 release (66). In contrast, maintenance
of intracellular ATP and failure to induce apoptosis in N. meningi-
tidis–exposed cultures would be anticipated to have both direct
effects by sustaining cytokine production in the monocytes ingest-
of apoptotic bodies to downregulate proinflammatory cytokine
responses. Additionally, the persistent generation of factors such
as ROS would contribute to tissue injury (70). Failure to engage
bacteria may underpin the proinflammatory features of meningo-
coccal infection, providing an important illustration of the impor-
tance of cell death in limiting persistent high-level innate responses
In summary, we provide evidence that highly purified human
monocytes engage an apoptotic program to downregulate innate
responses to bacteria. The host response to some infections may
prevent this process and substitute an alternative death process,
which aims to contain extracellular bacteria by ensuring re-
cruitment of additional inflammatory cells. Failure to engage either
of these programs is likely to result in persistent innate responses,
which place the host at risk for sepsis and multiorgan failure as
observed in meningococcal sepsis.
Note added in proof. During revision of this paper, we became
aware of the publication of a paper by Bartneck et al. (71) de-
scribing the production of extracellular traps by monocytes in
response to nanoparticles.
We thank Chris Hill for help in generating transmission electron micro-
graphs and Mumtaz Virji for sharing the unencapsulated (¢13) strain of
MC58 N. meningitidis.
The authors have no financial conflicts of interest.
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