Murine Lung Cancer Increases CD4+ T Cell Apoptosis and Decreases Gut Proliferative Capacity in Sepsis

Abstract

Background
Mortality is significantly higher in septic patients with cancer than in septic patients without a history of cancer. We have previously described a model of pancreatic cancer followed by sepsis from Pseudomonas aeruginosa pneumonia in which cancer septic mice have higher mortality than previously healthy septic mice, associated with increased gut epithelial apoptosis and decreased T cell apoptosis. The purpose of this study was to determine whether this represents a common host response by creating a new model in which both the type of cancer and the model of sepsis are altered.

Methods
C57Bl/6 mice received an injection of 250,000 cells of the lung cancer line LLC-1 into their right thigh and were followed three weeks for development of palpable tumors. Mice with cancer and mice without cancer were then subjected to cecal ligation and puncture and sacrificed 24 hours after the onset of sepsis or followed 7 days for survival.

Results
Cancer septic mice had a higher mortality than previously healthy septic mice (60% vs. 18%, p = 0.003). Cancer septic mice had decreased number and frequency of splenic CD4+ lymphocytes secondary to increased apoptosis without changes in splenic CD8+ numbers. Intestinal proliferation was also decreased in cancer septic mice. Cancer septic mice had a higher bacterial burden in the peritoneal cavity, but this was not associated with alterations in local cytokine, neutrophil or dendritic cell responses. Cancer septic mice had biochemical evidence of worsened renal function, but there was no histologic evidence of renal injury.

Conclusions
Animals with cancer have a significantly higher mortality than previously healthy animals following sepsis. The potential mechanisms associated with this elevated mortality differ significantly based upon the model of cancer and sepsis utilized. While lymphocyte apoptosis and intestinal integrity are both altered by the combination of cancer and sepsis, the patterns of these alterations vary greatly depending on the models used.

Full text

RESEARCH ARTICLE
Murine Lung Cancer Increases CD4+ T Cell
Apoptosis and Decreases Gut Proliferative
Capacity in Sepsis
John D. Lyons
1
, Rohit Mittal
1
, Katherine T. Fay
1
, Ching-Wen Chen
1
, Zhe Liang
1
, Lindsay
M. Margoles
2
, Eileen M. Burd
3
, Alton B. Farris
3
, Mandy L. Ford
4☯
, Craig M. Coopersmith
1☯
*
1Department of Surgery and Emory Critical Care Center, Emory University School of Medicine, Atlanta, GA,
United States of America, 2Department of Internal Medicine and Emory Critical Care Center, Emory
University School of Medicine, Atlanta, GA, United States of America, 3Department of Pathology and
Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, United States of America,
4Department of Surgery and Emory Transplant Center, Emory University School of Medicine, Atlanta, GA,
United States of America
☯These authors contributed equally to this work.
*cmcoop3@emory.edu
Abstract
Background
Mortality is significantly higher in septic patients with cancer than in septic patients without a
history of cancer. We have previously described a model of pancreatic cancer followed by
sepsis from Pseudomonas aeruginosa pneumonia in which cancer septic mice have higher
mortality than previously healthy septic mice, associated with increased gut epithelial apo-
ptosis and decreased T cell apoptosis. The purpose of this study was to determine whether
this represents a common host response by creating a new model in which both the type of
cancer and the model of sepsis are altered.
Methods
C57Bl/6 mice received an injection of 250,000 cells of the lung cancer line LLC-1 into their
right thigh and were followed three weeks for development of palpable tumors. Mice with
cancer and mice without cancer were then subjected to cecal ligation and puncture and sac-
rificed 24 hours after the onset of sepsis or followed 7 days for survival.
Results
Cancer septic mice had a higher mortality than previously healthy septic mice (60% vs.
18%, p = 0.003). Cancer septic mice had decreased number and frequency of splenic
CD4+ lymphocytes secondary to increased apoptosis without changes in splenic CD8+
numbers. Intestinal proliferation was also decreased in cancer septic mice. Cancer septic
mice had a higher bacterial burden in the peritoneal cavity, but this was not associated with
alterations in local cytokine, neutrophil or dendritic cell responses. Cancer septic mice had
PLOS ONE | DOI:10.1371/journal.pone.0149069 March 28, 2016 1/20
OPEN ACCESS
Citation: Lyons JD, Mittal R, Fay KT, Chen C-W,
Liang Z, Margoles LM, et al. (2016) Murine Lung
Cancer Increases CD4+ T Cell Apoptosis and
Decreases Gut Proliferative Capacity in Sepsis. PLoS
ONE 11(3): e0149069. doi:10.1371/journal.
pone.0149069
Editor: Philip Alexander Efron, University of Florida,
UNITED STATES
Received: August 26, 2015
Accepted: January 27, 2016
Published: March 28, 2016
Copyright: © 2016 Lyons et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: This work was supported by funding from
the National Institutes of Health (GM104323,
GM095442, GM072808, GM109779, GM113228).
The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: Dr. Coopersmith is the
president of the Society of Critical Care Medicine.

biochemical evidence of worsened renal function, but there was no histologic evidence of
renal injury.
Conclusions
Animals with cancer have a significantly higher mortality than previously healthy animals fol-
lowing sepsis. The potential mechanisms associated with this elevated mortality differ sig-
nificantly based upon the model of cancer and sepsis utilized. While lymphocyte apoptosis
and intestinal integrity are both altered by the combination of cancer and sepsis, the pat-
terns of these alterations vary greatly depending on the models used.
Introduction
Sepsis is the leading causes of death among critically ill patients in the United States with
between 230,000 and 370,000 people dying of the disease annually [1]. Patients with malig-
nancy are nearly ten times more likely to develop sepsis than the general population [2], and
cancer represents the most common co-morbidity in septic patients[3–5]. Sepsis is also the
leading cause of ICU admission in patients with cancer[6,7]. Importantly, cancer is also the co-
morbidity associated with the highest risk of death in sepsis, with hospital mortality exceeding
50% in patients with cancer and either severe sepsis or septic shock[5,7–9].
The etiology behind the increased mortality seen in cancer patients who develop sepsis com-
pared to previously healthy patients who develop sepsis is multifactorial[2,10]. While some
deaths are related to immunosuppression caused by cancer treatment such as chemotherapy or
radiation, others are likely related to a reduced ability of the host to appropriately respond to
infection in the setting of chronic systemic changes related to underlying malignancy. Animal
models of cancer, in isolation, demonstrate that not only is the tumor microenvironment
altered, but that systemic T cell exhaustion and generalized immune suppression are also
induced by cancer[11]. Further, the host response to a non-lethal infection is markedly altered
following cancer, with phenotypic exhaustion in T cells associated with increasing expression
of co-inhibitory receptors[12].
There are numerous similarities in the host response to both cancer and sepsis[13]. In an
attempt to understand why hosts with cancer have increased mortality following sepsis com-
pared to previously healthy hosts, we have described a model of pancreatic cancer followed by
sepsis from Pseudomonas aeruginosa pneumonia[14]. Mortality was higher in cancer septic
mice than previously healthy mice and this was associated with a decrease in T lymphocyte
apoptosis and an increase in both gut epithelial apoptosis and bacteremia. Interestingly, pre-
venting lymphocyte apoptosis—a strategy associated with uniform success in other pre-clinical
models of sepsis—was associated with increased mortality in cancer septic mice[15].
Despite having a greater understanding of the pathophysiology of sepsis than ever before
[16–18], there has been a remarkable inability to translate preclinical models of sepsis into
effective treatments at the bedside, where management is generally supportive yet non-selec-
tive, with the exception of targeted antimicrobial therapy[19]. One reason (of many) for the
failure of pre-clinical trials to translate into effective therapies for sepsis is that animal studies
are performed in a homogenous previously healthy population, whereas human studies are
performed on heterogeneous patients frequently with multiple co-morbidities. As such, we
sought to determine whether our previous pre-clinical findings in cancer and sepsis would be
generalizable if we altered both the type of cancer and the model of sepsis. To examine this, we
Murine Lung Cancer and Sepsis
PLOS ONE | DOI:10.1371/journal.pone.0149069 March 28, 2016 2/20
This does not alter the authors' adherence to PLOS
ONE policies on sharing data and materials.

developed a new clinically relevant model of lung cancer followed by sepsis induced by cecal
ligation and puncture.
Materials and Methods
Animals
Male and female C57BL/6 mice were used in all experiments, with gender matching between
experimental and control groups. Animals were 6–8 weeks of age prior to initiation of experi-
ments. A subset of animals were then injected with tumor cells (details below) and all mice
were then watched for an additional three weeks before a subset were subjected to cecal ligation
and puncture (CLP, also detailed below) at which time they were watched for 1–7 days depend-
ing on whether they were used for non-survival or survival experiments. Thus animals were a
minimum of 9 weeks old and a maximum of 12 weeks old at time of sacrifice. Experiments
were performed in accordance with the National Institutes of Health Guidelines for the Use of
Laboratory Animals and were approved by the Institutional Animal Care and Use Committee
at Emory University School of Medicine (Protocol DAR-2001875-082815BN). All animals
were housed in an approved university animal facility and were given free access to food and
water throughout. Animals that were injected with tumor cells were monitored to ensure that
tumors did not ulcerate and did not impede animal ambulation according to the Emory
IACUC guidelines for tumor burden. Following CLP, all animals received buprenorphine post-
operatively in an attempt to minimize animal suffering. For non-survival studies, animals were
sacrificed 24 hours post-operatively via asphyxiation by CO2 or exsanguination under deep
ketamine anesthesia. A different subset of animals was followed for survival for 7 days post-
operatively. During this survival experiment, animals were checked twice daily. In addition to
observing the same endpoints outlined above surrounding tumor growth, animals were also
checked to determine if they were moribund related to operation. Animals that either met
tumor endpoints or were moribund were sacrificed using humane endpoints. Moribund ani-
mals were identified as follows: a) surgical complications unresponsive to immediate interven-
tion (wound dehiscence, bleeding, infection), b) medical conditions unresponsive to treatment
such as self-mutilation, severe respiratory distress, icterus, major organ failure or intractable
diarrhea, or c) clinical or behavioral signs unresponsive to appropriate intervention persisting
for 1 day including significant inactivity, labored breathing, sunken eyes, hunched posture,
piloerection/matted fur, one or more unresolving skin ulcers, and abnormal vocalization when
handled. Animals that survived 7 days post-operatively were sacrificed at the conclusion of this
experiment using asphyxiation by CO2.
Cancer model
A murine lung carcinoma cell line (LLC1, American Type Culture Collection, Manassas, VA)
was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% gluta-
mine, 1% penicillin-streptomycin and 1%4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
The rationale for using a lung cancer cell line is that lung cancer has the second highest mortal-
ity for solid tumors in septic patients (2) (pancreatic cancer is the highest and was used for our
prior experiments in sepsis and cancer). After expansion, cancer cells were dissociated from
growth flasks via incubation with 0.25% trypsin, washed, centrifuged for 10 minutes at 1500
RPM and then re-suspended in phosphate buffered solution (PBS) to a final concentration of
250,000 cells per 0.2 ml (live cells selected via trypan blue examination). Mice randomized to
receive cancer had a single subcutaneous injection of 250,000 tumor cells along the right inner
thigh (cancer group) and were followed for 3 weeks prior to CLP to allow for tumor growth.
Control mice were unmanipulated and thus had no intervention prior to CLP (previously
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healthy group) but were watched for an identical time as cancer mice so animals would be age
matched.
Sepsis model and experimental groups
A subset of cancer mice and previously healthy mice were then subjected to CLP, an established
model of polymicrobial peritonitis[20]. Briefly, under isoflurane anesthesia, a small midline
abdominal incision was made, and the cecum was exteriorized and ligated below the ileocecal
valve, avoiding intestinal obstruction. The cecum was punctured twice with a 25 gauge needle
and squeezed gently to extrude a small amount of stool. After placing the cecum back in the
abdomen, the abdominal wall was closed in layers. Immediately following CLP, mice received
subcutaneous injections of a) fluids (1ml of 0.9% saline) to account for insensible losses, b)
antibiotics (50 mg/kg of ceftriaxone, Sigma-Aldrich, St. Louis, MO and 35 mg/kg metronida-
zole, Apotex Corp, Weston, FL) to mimic the clinical scenario where septic patients receive
antimicrobial therapy and c) pain medication (0.1 mg/kg buprenex, McKesson Medical, San
Francisco, CA) to minimize pain and suffering. Animals were either followed 7 days for sur-
vival or sacrificed at 24 hours for sample collection. Antibiotics were re-dosed at 12, 24 and 36
hours after surgery in survival studies.
We have previously published an extensive immunological assessment of lung cancer in
unmanipulated mice [11] so did not repeat those studies. However, we did not previously have
data on many of the outcomes assayed in this study and so performed experiments in both sep-
tic and non-septic animals where appropriate, in order to understand the impact of cancer in
isolation, sepsis in isolation, and the combination of cancer and sepsis. The following is the ter-
minology used for each experimental group: a) unmanipulated (mice that received neither can-
cer nor sepsis), b) cancer (mice that received tumor cell injection alone), c) previously healthy
septic (mice that underwent CLP alone without prior intervention), and d) cancer septic (mice
with tumor cell injection followed three weeks later by CLP).
Leukocyte analysis
Phenotypic flow cytometric analysis of leukocytes was performed on processed cellular suspen-
sions of splenocytes (whole spleens removed at time of sacrifice) or peritoneal fluid (2.5ml PBS
injected into peritoneum and withdrawn after 5 seconds of gentle agitation). The number of
cells per ml of suspension was calculated utilizing a Nexcelom Auto Cellometer, the results of
which were used to determine absolute cell numbers. The following antibodies were used to
stain cells prior to analysis: for peritoneal fluid, anti-GR1.1 FITC (BD Bioscience, San Jose,
CA), anti-Cd11b PerCP (BioLegend, San Diego, CA), anti-Cd11c PeCy7 (eBioscience, San
Diego, CA), and anti-MHC II APC Cy7 (eBioscience); for splenocyte phenotyping, anti-F4/80
PerCP (BioLegend), anti-Cd11c PeCy7 (eBioscience), anti-Cd11b APC (eBioscience), anti-
B220 Alexa700 (BD Bioscience), and anti-MHC II APC Cy7 (eBioscience).
To determine the frequency of apoptotic lymphocytes, splenocytes were collected from sac-
rificed animals and processed to a suspension of 1x10
7
cells/ml, and 1x10
6
splenocytes were
then processed using a commercially available Annexin V and 7-AAD kit (BioLegend) follow-
ing manufacturer’s instructions. Cells were then stained with anti-CD4-PO (Invitrogen, Carls-
bad, CA), anti-CD8-PB (eBioscience) to determine the frequency of pro-apoptotic CD4 and
CD8 T cells. A gating strategy excluded dead cells staining positive for 7-AAD from analysis.
For samples undergoing intracellular cytokine staining, 1x 10
6
splenocytes were plated into
a 96-well plate. Cells were suspended and incubated in RPMI 1640 culture medium and stimu-
lated for four hours utilizing phorbol 12-myristate 13-acetate (30ng/mL) and ionomycin
(400ng/mL) with 10μg/mL of Brefeldin A at 37°C. After stimulation, the cells were stained with
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anti-CD4 PacBlue (BD Bioscience), anti-CD8 PacOrange (Life Technologies Carlsbad, CA),
anti-IL-2 FITC (BD Bioscience), anti-IL-4 PE (eBioscience), anti-CD44 PerCP (BioLegend),
anti-CCR4 PeCy7 (BioLegend), anti-CXCR3 APC (eBioscience), and anti-IFNy Alexa700 (BD
Bioscience). An LSR II flow cytometer (BD Biosciences) was used to run all samples and FlowJo
10.0.8r1 software (Tree Star, San Carlos, CA) was used to analyze all data.
Intestinal permeability
At 19 hours following CLP, mice were gavaged with 0.5 ml of fluorescein isothiocyanate-dex-
tran (FD4) at a concentration of 22mg/ml in PBS (Sigma-Aldrich)[21,22]. Plasma collected at
time of sacrifice 5 hour later was then diluted with an equal volume of PBS, and FD4 concen-
tration was determined by flourospectrometry with excitation/emission wavelengths of 485/
520 nm with a standard curve of serial dilutions (BioTex Synergy HT, Winooski, VT).
Intestinal proliferation
Proliferating intestinal epithelial cells were stained with 5-Bromo-2’deoxyuridine (BrdU). At
90 minutes prior to sacrifice, mice received intraperitoneal injections of BrdU (5mg/ml in 0.9%
saline, Sigma-Aldrich) to label S-phase cells [23,24]. Jejunal tissue was then fixed in 10% forma-
lin for 24 hours before being embedded in paraffin and slide-mounted in 5μm sections. Slides
were then deparaffinized, rehydrated, and incubated in 1% hydrogen peroxide for 15 minutes
before being heated in a pressure cooker in antigen decloaker (Biocare Medical, Concord, CA)
for 45 minutes. Protein block (Dako, Carpinteria, CA) was performed for 30 minutes at room
temperature and slides were incubated overnight at 4°C with rat monoclonal Anti-BrdU
(1:500; Accurate Chemical and Scientific, Westbury, NJ). Samples were then incubated with
goat anti-rat antibody (1:500; Accurate Chemical & Scientific) and streptavidin horseradish
peroxidase (1:500; Dako), each for an hour at room temperature. Diaminobenzidine (DAB)
was used to develop slides for 2–3 minutes, and counterstaining was performed with hematox-
ylin. BrdU-positive cells were quantified in 100 contiguous, well-oriented intestinal crypts.
Intestinal apoptosis
Apoptosis of intestinal epithelial cells was quantified using two complementary techniques:
active caspase-3 staining and morphologic analysis of hematoxylin-eosin stained sections
[25,26]. Sections were treated as above with antigen decloaker and were then blocked with 20%
normal goat serum (Vector Laboratories, Burlingame, CA). Slides were incubated overnight at
4°C with rabbit anti-caspase-3 (1:100; Cell Signaling, Beverly, MA), and then with goat anti-
rabbit biotinylated antibody (1:500; Vector Laboratories) and streptavidin horseradish peroxi-
dase (1:500; Dako) for one hour each at room temperature. Slides were developed with DAB
and then counterstained. Caspase-3 positive cells were counted in 100 contiguous intestinal
crypts.
Apoptotic cells were identified on hematoxylin-eosin-stained sections by identifying charac-
teristic morphological changes including cell shrinkage with condensed and fragmented nuclei
and quantifying them in 100 contiguous crypts.
Villus length
Villus length was measured as the distance in μm from the crypt neck to the villus tip in 12 con-
secutive well-oriented jejunal villi using Nikon Elements imaging software- EIS-Elements BR
3.10 (Nikon Instruments, Melville, NY).
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Bacterial cultures
Quantitative cultures of whole blood and peritoneal fluid were prepared from serial 10-fold
dilutions of samples in sterile 0.9% saline. A 100 μl aliquot of undiluted sample and each dilu-
tion from 10
−1
to 10
−3
was plated on blood agar plates (Remel, Lenexa, KS) and incubated at
35°C in a 5% CO
2
atmosphere for 24 hours. Colony counts were obtained from plates contain-
ing fewer than 300 colonies. The number of colony-forming units (CFUs) per ml of original
sample was determined by multiplying the number of colonies by the reciprocal of the dilution
counted and adjusted for the volume of sample plated.
Cytokines
Whole blood, peritoneal fluid and bronchoalveolar lavage (BAL) fluid were collected and then
centrifuged at 10,000 RPM for 10 minutes. The supernatant from each was then collected and
analyzed for cytokine concentrations using a 6-plex cytokine bead array according to manufac-
turer instructions (Bio-Rad Laboratories, Hercules, CA).
Renal function
Whole blood was centrifuged at 10,000 RPM for 10 minutes. Serum creatinine was then mea-
sured using a creatinine microplate assay (Oxford Biomedical Research, Rochester Hills, MI)
while blood urea nitrogen (BUN) was determined using a urea nitrogen colorimetric detection
kit (Arbor Assays, Ann Arbor, MI). Whole kidneys were removed and fixed in 10% formalin
for 24 hours before paraffin fixation and sectioning. After hematoxylin-eosin staining, sections
were assessed for injury by a pathologist blinded to sample identity (ABF). Animal weights
were measured at time of CLP and immediately prior to sacrifice to assess potential weight loss
due to dehydration.
Liver injury
Liver injury was evaluated by both serum liver enzymes and histology. Alanine aminotransfer-
ase (ALT) was measured on a Beckman AU480 chemistry auto-analyzer (Beckman Diagnos-
tics, LaBrea, CA) following manufacturer instructions. In addition, portions of whole liver
were removed and fixed in 10% formalin for 24 hours prior to paraffin embedding. Sections
were then slide-mounted and stained with hematoxylin-eosin for analysis by a pathologist
(ABF) blinded to sample identity.
Lung injury
For histologic analysis, lungs of animals were flushed with 1ml of 10% formalin, and sections
were then removed and fixed in 10% formalin for 24 hours prior to being embedded in paraf-
fin. Lung sections were then stained with hematoxylin-eosin and examined by a pathologist
(ABF) blinded to sample identity.
Myeloperoxidase (MPO) activity was also assessed in BAL fluid. The trachea was irrigated
with 1 ml of PBS, and fluid was then withdrawn and centrifuged at 10,000 RPM for 10 minutes.
Substrate buffer containing 0.0005% hydrogen peroxide and O-dianisidine was added to the
supernatant, and MPO activity was assayed over 6 minutes at wavelength 460 (BioTek Synergy
HT, Winooski, VT). MPO activity was calculated as optical density/minute per μl of BAL fluid.
Lung fluid protein concentration was measured by analyzing samples treated with protein
assay reagent (Thermo Scientific, Rockford, IL) at 660 nm in conjunction with a standard
curve of bovine serum albumin.
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Complete blood counts
Whole blood collected at time of sacrifice was collected in anti-coagulant-lined blood tubes and
was analyzed for hemoglobin concentration, leukocyte count, and platelet count on a Heska
HemaTrue
1
veterinary hematology analyzer per manufacturer guidelines (Heska, Loveland, CO).
Statistics
All data were analyzed using the statistical software program Prism 6.0 (GraphPad, San Diego,
CA) and are presented as mean ± SEM. Data were tested for Gaussian distribution using the
D'Agostino-Pearson omnibus normality test. Two -way comparisons on data with a Gaussian
distribution were performed using the Student’s t-test. Two-way comparisons on data that did
not have a Gaussian distribution were performed using the Mann-Whitney test. Multi-group
comparisons were analyzed via one-way ANOVA, followed by the Tukey post-test. Survival
was analyzed using the Log-Rank test. A p value of <0.05 was considered to be statistically sig-
nificant throughout.
Results
Mice that received subcutaneous injections of murine lung cancer cells developed well-circum-
scribed solitary tumors at the site of injection three weeks later (average size 1.5 cm in diame-
ter). Post-mortem review of lung histology demonstrated microscopic metastatic disease in
some animals, although no gross tumor spread was seen in any animals at time of sacrifice.
The presence of pre-existing lung cancer worsens mortality following
sepsis
Seven-day mortality was 18% in previously healthy septic mice. In contrast, seven-day mortal-
ity was 60% in cancer septic mice (Fig 1).
The presence of pre-existing lung cancer decreases CD4+ lymphocytes
but not CD8+ lymphocytes following sepsis
Cancer septic mice had a decrease in both the frequency and absolute numbers of splenic
CD4+ lymphocytes compared to previously healthy septic mice (Fig 2A and 2B). In addition,
Fig 1. Effect of cancer on survival from sepsis. Previously healthy mice and those given an injection of
LLC1 cells three weeks earlier (n = 15-17/group) were subjected to CLP. Cancer septic mice had significantly
higher mortality than previously healthy septic mice (p = 0.003).
doi:10.1371/journal.pone.0149069.g001
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cancer septic mice had a higher frequency of annexin-positive staining CD4+ lymphocytes
compared to previously healthy septic mice, suggesting increased apoptosis was responsible for
the loss of CD4+ cells (Fig 2C and 2D). In contrast, while the frequency of splenic CD8+ lym-
phocytes was higher in cancer septic mice (Fig 3A), this was likely due to the decrease in CD4
+ cells since no difference was seen in CD8+ cell numbers (Fig 3B) or annexin staining (Fig 3C
and 3D) between cancer septic mice and previously healthy septic mice. CD4+ cell expression
of the Th1 marker CXCR3 was increased in cancer septic mice (Fig 4A and 4C), while expres-
sion of the Th2 marker CCR4 was not different between previously healthy septic mice and
cancer septic mice (Fig 4B and 4C). Stimulated production of Th1 effector cytokine IFN-γby
CD4+ T cells was not impacted by cancer (Fig 4D, 4F and 4G) while production of the Th2
effector IL-4 was significantly decreased in cancer septic mice (Fig 4E, 4F and 4G).
The presence of pre-existing lung cancer decreases ability to clear local
infection
Peritoneal bacterial burden was higher in cancer septic mice than previously healthy septic mice
(Fig 5A). This was not associated with changes in Interleukin (IL)-1β, IL-6, IL-10, IL-13, MCP-1,
Fig 2. Effect of cancer on splenic CD4+ T cells following sepsis. Cancer septic mice had a significantly lower frequency of CD4+ T cells as a percentage
of total CD3+ T cells (A, p = 0.02, n= 7–9) as well as a decrease in the total number ofCD4+ T cells (B, p = 0.03, n = 8–10) compared to previously healthy
septic mice. This was associated withan increase in annexin-positive CD4+ T cells in cancer septicmice (C, p = 0.04, n = 7–8). A representative flow cytometry
histogram demonstrates increased annexin staining in cancer septic (blue) CD4+ T cells compared to previously healthy septic (red) CD4+ T cells (D).
doi:10.1371/journal.pone.0149069.g002
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TNF-α,orIFN-γin the peritoneal fluid (Fig 5B–5H). Peritoneal fluid also contained similar per-
centages of neutrophils (Fig 6A) and dendritic cells (Fig 6B) between previously healthy septic
mice and cancer septic mice. Dendritic cell activation as determined by MHC II expression on
cells from peritoneal fluid was also similar between the groups (Fig 6C). Splenic dendritic cell fre-
quency and activation were also unaffected by the presence of cancer (Fig 6D and 6E). There
were no differences in absolute numbers of neutrophils or dendritic cells (data not shown).
Serum cytokines were similar between cancer septic mice and previously healthy septic
mice except for an increase in MCP-1 in cancer septic mice (Fig 7). No difference in bacterial
burden was identified in quantitative blood cultures between previously healthy septic mice
and cancer septic mice (data not shown).
The presence of pre-existing lung cancer decreases crypt proliferation
but does not alter other components of intestinal integrity following sepsis
Cancer in isolation decreases crypt proliferation compared to unmanipulated mice. Sepsis in
isolation also decreases crypt proliferation compared to unmanipulated mice. The combination
Fig 3. Effect of cancer on splenic CD8+ T cells following sepsis. Cancer septic mice had a significantly increased frequency of CD8+ T cells as a
percentage of total CD3+ T cells (A, p = 0.007, n = 8–10). However, there was no statistically significant change in total number of CD8+ cells (B, p = 0.10,
n=8–10) or annexin-positive CD8+ T cells (C, p = 0.31, n = 7–10) suggesting the change in percentage of CD8+ cells was related to the decrease in CD4
+ cells. A representative flow cytometry histogram demonstrates no difference in annexin staining in cancer septic (blue) and previously healthy septic (red)
CD8+ T cells (D).
doi:10.1371/journal.pone.0149069.g003
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of cancer and sepsis causes a further disproportionate decrease in crypt proliferation compared
to sepsis alone as cancer septic mice have lower proliferation than previously healthy septic
mice (Fig 8A–8C).
In contrast, villus length is not affected by cancer in isolation, is decreased as has been previ-
ously shown by sepsis (26), but is not different between previously healthy septic mice and can-
cer septic mice (Fig 8D). Cancer and sepsis also did not impact intestinal permeability or crypt
apoptosis compared to sepsis alone (data not shown).
The presence of pre-existing lung cancer worsens biochemical kidney
function without causing liver injury following sepsis
Neither cancer nor sepsis in isolation affected renal function as BUN and Cr levels were similar
in unmanipulated, cancer and previously healthy septic mice. In contrast, both BUN and Cr
were higher in cancer septic mice (Fig 9A and 9B); however kidneys appeared grossly normally
Fig 4. Effect of cancer on Th1 and Th2 markers and cytokine production following sepsis. Cancer septic mice had a modest increase in CD4+ T cell
expression of the Th1 marker CXCR3 (A, p = 0.01, n = 9–10) but did not have a statistically significant change in expression of the Th2 marker CCR4 (B,
p = 0.21, n = 9–10). A representative flow cytometry plot for both markers is included (C). Stimulated production of IFN-γwas not statistically different in CD4
+ T cells from cancer septic mice (D, p = 0.17, n = 9–10); however, stimulated production of IL-4 was lower in CD4+ T cells from cancer septic mice (E,
p = 0.006, n = 9–10). Representative flow cytometry plots for both unstimulated and stimulated IFN-γand IL-4 are shown for previously healthy septic mice
(F) and cancer septic mice (G).
doi:10.1371/journal.pone.0149069.g004
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histologically (data not shown). No differences in body weight were identified between previ-
ously healthy septic mice and cancer septic mice (Fig 9C).
Basal levels of ALT were similar in unmanipulated and cancer mice. While sepsis increased
serum ALT levels, this was not affected by the presence of cancer since ALT was similar
between cancer septic and previously healthy septic mice (Fig 9D). Further, there were no dif-
ferences in liver histology between any of the groups (data not shown).
The presence of pre-existing lung cancer decreases BAL MPO and
increases BAL protein following sepsis
MPO activity was lower in BAL fluid in cancer septic mice compared to previously healthy sep-
tic mice (Fig 10A). In contrast, BAL protein was higher in cancer septic mice compared to
previously healthy septic mice (Fig 10B). BAL cytokines were generally independent of the
presence of cancer although BAL IL-10 was lower in cancer septic mice (Fig 10C–10G). Despite
differences noted in BAL fluid, no differences were identified in inflammation score or percent-
age of inflammatory cells between cancer septic and previously healthy septic mice on histo-
logic examination (data not shown).
Fig 5. Effect of cancer on peritoneal bacteria and local inflammatory response following sepsis. Cancer septic mice had higher levels of bacteria in
their peritoneal cavities than previously healthy septic mice (A, p = 0.005, n = 8–9). This change was not associated with differences in concentration of
peritoneal IL-1β(p = 0.32), IL-6 (p = 0.18), IL-10 (p = 0.52), IL-13 (p = 0.97), MCP-1 (p = 0.67), TNF-α(p = 0.40), or IFN-γ(p = 0.27) (B-H, n = 8 for all groups).
doi:10.1371/journal.pone.0149069.g005
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Fig 6. Effect of cancer on local neutrophil and dendritic cell responses following sepsis. Cancer septic mice and previously healthy septic mice had
similar frequencies of neutrophils (A, p = 0.52, n = 7/group) and dendritic cells (B, p = 0.52, n = 7/group)in their peritoneal fluid, and there was no difference in
the frequency of dendritic cell MHC II expression between the two groups (C, p = 0.52, n = 7/group). Cancer septic mice and previously healthy septic mice
also had similar frequencies of dendritic cells (D, p = 0.73, n = 9–10) and activated dendritic cells (E, p = 0.83, n = 9–10) in splenocytes.
doi:10.1371/journal.pone.0149069.g006
Fig 7. Effect of cancer on systemic cytokines following sepsis. No significant differences were noted in IL-1β(p = 0.10), IL-6 (p = 0.63), IL-10 (p = 0.24),
IL-13 (p = 0.07), or TNF-α(p = 0.99) (B-F, n = 7-8/group). In contrast, increased MCP-1 was detected in the serum of cancer septic mice compared to
previously healthy septic mice (E, p = 0.04, n = 7–8).
doi:10.1371/journal.pone.0149069.g007
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The presence of pre-existing lung cancer does not alter complete blood
counts following sepsis
Cancer, in isolation, causes anemia as hemoglobin levels were significantly lower in cancer
mice than unmanipulated mice (Fig 11A). However, sepsis, in isolation did not alter hemoglo-
bin levels and there was no statistically significant difference in hemoglobin levels between pre-
viously healthy septic mice and cancer septic mice. Cancer, in isolation, did not change total
leukocyte count although sepsis, in isolation, decreased total leukocyte count (Fig 11B). The
combination of sepsis and cancer did not alter total leukocyte count further since it was similar
Fig 8. Effect of cancer on intestinal proliferation following sepsis. Previously healthy septic mice (A) had qualitatively higher levels of proliferation than
cancer septic mice (B, BrdU-positive crypt cells stain brown). Quantitatively, both cancer in isolation and sepsis in isolation decrease crypt proliferation (C,
p = 0.02 and 0.008 respectively compared to unmanipulated mice). The combination of cancer and sepsis further decreased intestinal proliferation, moreso
than was seen with either variable in isolation (p<0.001 previously healthy septic vs. cancer septic, n = 8–10 for all groups in panel C). In contrast, while villus
length was decreased by sepsis (but not cancer) in isolation (p = 0.008), there was no difference in villus length between previously healthy septic and cancer
septic mice (D, p>0.99, n = 8–9 for all groups).
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between previously healthy septic mice and cancer septic mice. Neither cancer nor sepsis in iso-
lation led to a statistically significant change in platelet count compared to unmanipulated ani-
mals, and similarly, there were no statistically significant differences in platelet count between
previously healthy septic mice and cancer septic mice (Fig 11C).
Fig 9. Effect of cancer on renal and liver function following sepsis. Neither cancer nor sepsis in isolation impacted BUN or Cr levels (A, B). However, the
combination of both insults worsens renal function as both BUN and Cr were higher in cancer septic mice than previously healthy septic mice (p = 0.004 and
p<0.0001 respectively, n = 8 for all groups). Body weights were similar in all groups, regardlessof the presence of cancer or a septic insult 24 hours earlier
(C). While cancer had no impact on liver function as assayed by ALT, sepsis, in isolation, increased ALT compared to unmanipulated mice (D, p<0.001).
However, this was not worsened by malignancy as cancer septic mice and previously healthy septic mice had similar ALT levels (p>0.99, n = 8–10 for all
groups).
doi:10.1371/journal.pone.0149069.g009
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Discussion
Similar to the human condition and our previous mouse model of cancer followed by sepsis,
this study demonstrated that the presence of a malignancy significantly worsens survival from
sepsis. However, parameters associated with increased mortality varied greatly between mice
injected with lung cancer cells followed by CLP in this study and our prior findings of mice
injected with pancreatic cancer cells followed by Pseudomonas aeruginosa pneumonia. In this
study, we noted increased apoptosis in splenic CD4+ T lymphocytes, decreased crypt prolifera-
tion, decreased local infection clearance, increased systemic MCP-1, worse renal function,
lower BAL MPO activity, higher BAL protein and lower BAL IL-10 in cancer septic mice com-
pared to previously healthy septic mice. In contrast, in mice with pancreatic cancer cells fol-
lowed by Pseudomonas aeruginosa pneumonia, we found decreased apoptosis in both T and B
lymphocytes, increased gut epithelial apoptosis, increased bacteremia without alterations in
local infection, and higher BAL IL-6 and IL-10 compared to previously healthy septic mice.
Remarkably, except for elevated mortality, there was essentially no overlap in the associated
abnormalities between lung cancer/CLP and pancreatic cancer/pneumonia mice.
Fig 10. Effect of cancer on lung inflammation following sepsis. MPO activity in BAL fluid was decreased in cancer septic mice compared to previously
healthy septic mice (A, p<0.0001, n = 8). In contrast, protein concentration in BAL fluid was higher in cancer septic mice (B, p = 0.04, n = 6–8). BAL cytokines
were similar between mice for IL-1β(p = 0.47), IL-6 (p = 0.59), IFN-γ(p = 0.99) and TNF-α(p = 0.11) but IL-10 levels were lower in cancer septic mice
(p = 0.0005, n = 9–10 for all groups).
doi:10.1371/journal.pone.0149069.g010
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Whether a common host response exists in sepsis is controversial [27–29]. Our results can
be interpreted that the mechanisms responsible for mortality vary significantly, at least in part,
with tumor type and sepsis model. Alternatively, since our results are associative, it is possible
that none of the abnormalities detected in either model of cancer and sepsis is actually respon-
sible for the increased mortality. While we cannot rule out that possibility, it is reasonable to
attempt to put the findings described herein into context of existing literature.
The concept that T lymphocyte derangements play a crucial role in mediating mortality
from sepsis is now over a decade old. Multiple studies demonstrate that preventing T cell apo-
ptosis improves survival following CLP [30–32]. The findings that a) CD4+ lymphocytes are
decreased and b) Annexin V staining is increased are consistent with a more profound immu-
nosuppressive state in mice with lung cancer followed by CLP compared to previously healthy
mice subjected to the same insult. While this would typically be thought of as maladaptive, it
should be noted that prevention of lymphocyte apoptosis worsened survival in mice overex-
pressing Bcl-2 in lymphocytes as well as in BIM knockouts following pneumonia in mice with
pancreatic cancer [15], so the functional significance of increased CD4+ lymphocyte apoptosis
in cancer septic mice needs to be examined further in future experiments.
Altered Th1 and Th2 responses have also been described in sepsis [17,33], and it seemed
plausible that cancer-mediated increases in T cell exhaustion could potentially induce a shift
towards a Th2 response during sepsis [12]. However, our results do not neatly fit this hypothe-
sis. Although we demonstrated a modest increase in expression of CXCR3 on CD4+ T cells in
cancer septic mice (which may suggest a skewing towards a Th1 response), the biological sig-
nificance of this is unclear since we found no associated differences in production of the Th1
effector IFN-γby CD4+ T cells. In addition, we did not detect a difference in expression of the
Th2 marker CCR4 but did observe decreased production of the Th2 effector IL-4 from CD4
+ cells taken from cancer septic mice. These somewhat conflicting data suggest that while there
may be imbalances in the Th1/Th2 response following sepsis in animals with cancer, the
changes are likely not a simple shift directly toward one phenotype or another, and the degree
to which those changes impact mortality is yet to be determined.
Fig 11. Effect of cancer on hemoglobin, leukocyte count and platelet count following sepsis. Cancer, in isolation, decreased serum hemoglobin levels
(A, p<0.0001); however, this was not impacted by sepsis and there was no statistically significant difference between cancer septic and previously healthy
septic mice (p = 0.14, n = 5-9/group). In contrast, while cancer did not impact leukocyte count (B), sepsis, in isolation caused a decrease in in leukocyte count
(p = 0.02). No statistically significant difference in leukocyte count was seen between cancer septic mice and previously healthy mice (p = 0.99, n = 5-9/
group). Platelet count was similar between unmanipulated mice and those with cancer or sepsis in isolation (C). No statistically significant difference in
platelet count was seen between cancer septic mice and previously healthy mice (p = 0.78, n = 5-9/group).
doi:10.1371/journal.pone.0149069.g011
Murine Lung Cancer and Sepsis
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There were significantly higher levels of bacteria present in the peritoneal cavity 24 hours
after CLP in cancer septic mice. Even though local cytokine, neutrophil, and dendritic cell pro-
files were similar between cancer septic and previously healthy septic mice, it is plausible that
the basal immunosuppressive state caused by sepsis predisposes animals to increased local
infection. How this suppression is functionally enacted requires further investigation in future
studies. Notably, polymicrobial sepsis, as would typically be seen following fecal peritonitis, is
associated with higher mortality in patients with cancer and sepsis[9].
Gut integrity is altered by sepsis, with increased apoptosis and permeability as well as
decreased proliferation and villus length. These and other alterations in gut integrity can result
in distant organ injury and propagation of systemic inflammation, resulting in the hypothesis
that the gut is the "motor" driving critical illness [16,34,35]. Although multiple parameters of
gut integrity were altered by sepsis in isolation in this study, the majority of these were similar
between cancer septic mice and previously healthy septic mice. One exception was gut prolifer-
ation, which was decreased by both cancer and sepsis in isolation but disproportionately
decreased by the combination of both insults. In light of the complex function and architecture
of the gut, it is possible that the marked decrease in gut proliferation seen in cancer septic mice
resulted in downstream functional changes within the intestine, resulting in either local or dis-
tant injury. Since the gut is a continuously renewing organ that replaces itself on average every
3–5 days[35], future experiments are required to determine how long the decreased prolifera-
tion induced by the combination of sepsis and cancer persists.
Both BUN and Cr (markers of renal dysfunction) are similar in unmanipulated mice, cancer
mice and previously healthy septic mice, suggesting neither cancer nor sepsis impacts renal
function. However, BUN and Cr are statistically higher in cancer septic mice compared to previ-
ously healthy septic mice. We do not believe that the modest degree of biochemical acute kidney
injury seen at 24 hours is solely responsible for mortality seen in cancer septic mice, and it is
questionable if the small absolute difference in serum BUN/creatinine values is biologically
meaningful, especially given the absence of histologic abnormalities in the kidneys in all groups.
It is possible that renal function worsens throughout the course of sepsis in this model and
therefore contributes to mortality more significantly at later time points. Of note, the etiology of
renal dysfunction that is seen exclusively in cancer septic mice remains to be determined.
Finally, MPO activity, protein and IL-10 were all altered in BAL fluid of cancer septic mice
although this was not accompanied by differences in histologic lung inflammation or other
BAL cytokines between previously healthy septic mice and cancer septic mice. While lowered
MPO activity and decreased levels IL-10 may suggest a dampened pulmonary inflammatory
response, more detailed assays would be needed to determine if this is actually the case. In
addition, since studies have shown that pulmonary disease does not represent a significant
cause of death in mice subjected to CLP[36], the functional significance of these findings is
unclear.
This study has a number of limitations. While a number of abnormalities were identified
that are associated with increased mortality, we cannot conclude that any of them are causative
without performing additional mechanistic studies. Next, all non-survival studies were per-
formed at a single timepoint (24 hours), so it is likely that our experimental design missed tem-
poral trends that would be important towards understanding the relationship between cancer
and sepsis, especially since there appear to be different inflammatory states depending on how
far out a host is from their septic insult [37]. Next, although we have attempted to compare our
results to our previous study on pancreatic cancer/pneumonia, there are three variables that
are different in this manuscript—type of cancer, sepsis model and the fact that micro-meta-
static disease was noted in this study with LLC-1 cells whereas Pan02 pancreatic cancer cells
are not associated with metastatic disease. To determine which of these are most responsible
Murine Lung Cancer and Sepsis
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for differences, changing only a single variable at a time would be required. Next, tumor cells
were injected into the thigh of murine recipients and thus do not replicate the development of
an in-situ lung cancer. Mice with naturally occurring tumors could potentially be exposed to
local tumor-related factors that cause a different inflammatory milieu than is seen in our
model. Further a more gradual tumor onset growth could potentially alter dynamics of tumor-
immune cell interaction, leading to chronic inflammatory changes not seen after only three
weeks of tumor growth. Finally, tumors tend to develop in aged patients, whereas this study
examined mice that were 9–12 weeks old prior to the onset of sepsis. It is unclear how well
young mice function as surrogates for aged patients, and our experimental design precluded us
from assessing the impact of age on the pathophysiology of cancer and sepsis.
Despite these limitations, our data yield new insights into a clinically relevant model of both
a common cancer and a common cause of sepsis, and the interplay between the two insults.
Further research is required to determine if the multiple pathophysiologic abnormalities identi-
fied herein are important in mediating the increased mortality seen when sepsis occurs in the
setting of cancer, and the role of specific tumors or types of sepsis in mediating this complex
interaction.
Author Contributions
Conceived and designed the experiments: JDL MLF CMC. Performed the experiments: JDL
RM ZL LMM EMB ABF KF CWC. Analyzed the data: JDL RM ZL LMM EMB ABF MLF CMC
KF CWC. Contributed reagents/materials/analysis tools: EMB ABF MLF CMC. Wrote the
paper: JDL CMC. Revised the manuscript: RM ZL LMM EMB ABF MLF KF CWC.
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