Cell Host & Microbe

Article

Salmonella Require the Fatty Acid RegulatorPPARd for the Establishment of a MetabolicEnvironment Essential for Long-Term PersistenceNicholas A. Eisele,1,4 Thomas Ruby,1,4 Amanda Jacobson,1 Paolo S. Manzanillo,2 Jeffery S. Cox,2 Lilian Lam,1

Lata Mukundan,3 Ajay Chawla,3 and Denise M. Monack1,*1Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA2Department of Microbiology and Immunology, Program in Microbial Pathogenesis and Host Defense3Cardiovascular Research Institute

University of California, San Francisco, San Francisco, CA 94143, USA4These authors contributed equally to this work

*Correspondence: dmonack@stanford.eduhttp://dx.doi.org/10.1016/j.chom.2013.07.010

SUMMARY

Host-adapted Salmonella strains are responsible fora number of disease manifestations in mammals,including an asymptomatic chronic infection in whichbacteria survive within macrophages located in sys-temic sites. However, the host cell physiology andmetabolic requirements supporting bacterial persis-tence are poorly understood. In a mouse model oflong-term infection, we found that S. typhimuriumpreferentially associates with anti-inflammatory/M2macrophages at later stages of infection. Further,PPARd, a eukaryotic transcription factor involvedin sustaining fatty acid metabolism, is upregulatedin Salmonella-infected macrophages. PPARd defi-ciency dramatically inhibits Salmonella replication,which is linked to the metabolic state of macro-phages and the level of intracellular glucose availableto bacteria. Pharmacological activation of PPARdincreases glucose availability and enhances bac-terial replication in macrophages and mice, whileSalmonella fail to persist in Ppard null mice. Thesedata suggest that M2 macrophages represent aunique niche for long-term intracellular bacterialsurvival and link the PPARd-regulated metabolicstate of the host cell to persistent bacterial infection.

INTRODUCTION

Host-adapted strains of Salmonella enterica cause systemic

infections and have the ability to persist within host tissues for

long periods of time despite the induction of a robust immune

response (Monack et al., 2004b). For example, Salmonella

enterica serovar Typhi (S. typhi) causes a systemic infection

(typhoid fever), which involves colonization of the reticuloendo-

thelial system (House et al., 2001; Monack et al., 2004a). Typhoid

fever affects more than 20 million people per year, resulting in

greater than 220,000 deaths annually (Crump et al., 2004;

Cell Hos

Woc-Colburn and Bobak, 2009). Upon the resolution of acute

disease, a significant percentage (1%–6%) of patients become

asymptomatic chronic carriers of S. typhi. Chronically infected

hosts are critical reservoirs of infection and transmit disease to

naive individuals, thus posing a significant threat to public health

(Lawley et al., 2008; Parry et al., 2002). From the bacterial

perspective, persistent infection is essential for microbial sur-

vival in nature. To date, very little is known about the molecular

mechanisms driving chronic Salmonella infections of mamma-

lian hosts. Thus, a better understanding of long-term systemic

Salmonella colonization should provide insight into bacterial

survival strategies, as well as the host factors organisms are

exploiting in order to persist.

Salmonella enter the host through the gastrointestinal tract,

where bacteria translocate across the epithelial barrier either by

invading microfold (M) cells overlying Peyer’s patches, or by

being taken up by CD-18-expressing immune cells (Haraga

et al., 2008). In order for the infection to extend beyond the intes-

tinal mucosa, bacteria survive and replicate within macrophages

by creating a privileged intracellular niche that allows organisms

toevadeadaptive immune responses.Wehavepreviously shown

in a murine model of long-term S. typhimurium infection that

bacteria persist within macrophages residing in systemic tissues

(Monack et al., 2004a). However, very little is known about the

physiological state of the macrophages that harbor Salmonella.

Macrophages can change their physiology in response to envi-

ronmental cues, giving rise to different populations of cells with

distinct functions depending on the local concentration of cyto-

kines. For example, highly microbicidal macrophages (referred

to as classically activated or M1 macrophages) polarize in

response to the Th1 cytokine IFN-g, and the tissue healing,

anti-inflammatory macrophages (also known as alternatively

activated or M2 macrophages) are elicited by the Th2 cytokines

IL-4 and IL-13 (Gordon and Martinez, 2010). Following infection,

there is frequently a shift from Th1 to Th2 type immunity as the

adaptive immune response transitions from acute to chronic

phases. This shift is associated with a change in the ratio of

pro- to anti-inflammatory macrophages at sites of inflammation

and may be a common feature following infection or tissue

damage that is important for the resolution of inflammation and

wound healing (Fairweather and Cihakova, 2009).

t & Microbe 14, 171–182, August 14, 2013 ª2013 Elsevier Inc. 171

Cell Host & Microbe

Persistent Salmonella Infection Requires PPARd

Over the last decade, the unique transcriptional signature of

alternatively activated or M2 macrophages has been defined

(Gordon, 2003;Mosser and Edwards, 2008). Genes induced dur-

ing alternative activation include arginase-1, various phagocytic

receptors and lectins, chemokines, cytokines, and transcrip-

tional regulators. Acquisition and maintenance of this phenotype

require the metabolic regulators peroxisome proliferator-acti-

vated receptors (PPARs) PPARg, PPARd, and the coactivator

protein PGC-1b (PPARg coactivator-1b) (Bouhlel et al., 2007;

Kang et al., 2008; Odegaard et al., 2007, 2008). PPARs bind fatty

acids and fatty acid metabolites in the cytosol, then translocate

to the nucleus to facilitate a transcriptional program that

responds to fuel availability (Brunmair et al., 2006; Xu et al.,

1999). Recent studies indicate that the ability of immune cells

to perform their effector functions is tightly linked to, and

controlled by, their metabolic state (Biswas and Mantovani,

2012). For example, classically activated macrophages require

anaerobic glycolysis to fuel the microbicidal program (Biswas

and Mantovani, 2010). In contrast, IL-4 induces uptake and

oxidation of fatty acids and biogenesis of mitochondria, pro-

grams that are transcriptionally controlled by PPARs in macro-

phages (Huang et al., 1999; Odegaard et al., 2007).

There have been several studies characterizing the role of

PPARs during microbial infection. For example, PPARg- and

PPARd-dependent arginase-1 induction has been shown to

enhance intracellular growth of Leishmaniamajor (Gallardo-Soler

et al., 2008). Similarly, macrophage-specific PPARg-deficient

mice are more resistant to L. major and Listeria monocytogenes

(Abdullah et al., 2012; Odegaard et al., 2007). Although these

results suggest that M2 macrophages and PPARs contribute to

the ability of intracellular pathogens to cause persistent infec-

tions, the molecular mechanisms are unknown.

To better understand the molecular mechanisms involved in

facilitating the persistence of facultative intracellular bacterial

pathogens, we characterized the host response during chronic

S. typhimurium infection in our mouse model of long-term infec-

tion (Monack et al., 2004a). We found that the initial response

toward infection encompasses a proinflammatory/Th1 cytokine

response, followed by an anti-inflammatory/Th2 cytokine

response. We further show that S. typhimurium preferentially

associates with anti-inflammatory/M2 macrophages at later

stages of infection, indicating that these cells might represent

a unique niche for the long-term intracellular survival of bacteria.

In addition, we demonstrate that PPARd is upregulated in

Salmonella-infected macrophages. In the absence of PPARd,

bacteria are unable to replicate in macrophages due to a

decreased availability of glucose, and PPARd-deficient mice

are unable to sustain persistent S. typhimurium infection. Taken

together, these data describe a molecular mechanism by which

a host metabolic program drives persistent colonization by an

intracellular bacterial pathogen, and suggest unique therapeutic

targets for the disruption of chronic Salmonella carriage.

RESULTS

Salmonella Associates with Anti-Inflammatory/M2-likeMacrophages In VivoTo gain functional insights into the host pathways exploited by

S. typhimurium during the establishment and maintenance of

172 Cell Host & Microbe 14, 171–182, August 14, 2013 ª2013 Elsevi

persistent systemic colonization, we orally infected 129/SvJ

Nramp1+/+mice andmonitored immune responses and bacterial

loads at various time points after infection. To characterize the

immune response during the course of infection, a large panel

of cytokines in the mesenteric lymph nodes (MLNs) were

measured at 0, 5, 15, and 42 days postinfection (Figure 1A). In

addition, the relative composition of macrophage subclasses

was measured during the first 60 days postinfection (Figure 1B).

Concomitant with increasing concentrations of Th2 cytokines in

persistently infected MLNs (Figure 1A), there was an increase in

the percentage of CD301+ macrophages (a transmembrane

C type lectin expressed on anti-inflammatory/M2 macrophages)

in the spleen and MLNs over time, with maximum levels

observed at 30 days after infection (Figure 1B). An increase in

the percentage of the CD301+ macrophage population corre-

lated with an increase in the levels of S. typhimurium colonizing

the spleen and MLNs (Figure 1B). We also observed that

S. typhimurium colocalized with cells expressing two addi-

tional anti-inflammatory/M2 macrophage markers, Ym-1 (see

Figure S1A online) and Arginase I (data not shown), in frozen

spleen sections 30 days postinfection. Furthermore, a higher

percentage of CD301+ macrophages were associated with

S. typhimurium compared to other (CD301�) macrophages

(19.1% ± 5.2% versus 1.46% ± 0.4%, respectively) (Figure 1C).

The level of Salmonella was higher in the CD301+ macrophage

population compared to the CD301� population as determined

by an increase in themean fluorescent intensity (MFI) (Figure 1D).

We define these infected cells as anti-inflammatory/M2-like

macrophages because they express some of the markers

commonly used to distinguish M2 cells from M1 cells (CD301,

Ym-1, and Arginase I). In accordance with these data, a previous

study suggests that Salmonella survives in hemophagocytic

macrophages (Nix et al., 2007), phagocytes that have taken up

leukocytes and erythrocytes (Fame et al., 1986). Indeed, we

and others have found that these macrophages also express

markers of anti-inflammatory/M2 macrophages (Figures S1B

and S1C) (McCoy et al., 2012). To test whether S. typhimurium

exploits IL-4-dependent signaling in macrophages, we infected

wild-type (WT) and conditional knockout mice defective in IL-4/

IL-13 signaling in macrophages (Il-4raLoxP/LoxP-LysmCre) and

found significantly higher levels of Salmonella in the MLNs of

WT-infected mice 28 days postinfection (Figure 1E). Thus, these

data demonstrate that Th2 cytokine signaling in macrophages

during chronic infection contributes to persistent bacterial

colonization.

Salmonella Replication Is Enhanced withinIL-4-Stimulated Primary MacrophagesSince IL-4 has a major role in establishing an anti-inflammatory

phenotype in macrophages, we analyzed Salmonella replication

in IL-4-stimulated macrophages ex vivo to determine whether

the increase in Salmonella-infected anti-inflammatory/M2-like

cells in host tissues correlated with increased survival and/or

replication in these cells. The numbers of intracellular

S. typhimurium strain 12023 increased over time in both unsti-

mulated and IL-4-stimulated bone marrow-derived macro-

phages (BMDMs) and RAW 264.7 macrophage-like cells

(Figure 2A and Figure S2A). Similar results were seen for the

SL1344 strain we used for in vivo experiments (data not shown).

er Inc.

Figure 1. S. typhimurium Resides in Anti-Inflammatory/M2-like Macrophages during Chronic Infection

(A) Biphasic immune response in infected MLNs. Concentrations of cytokines were determined by multiplex analysis at the indicated time points after infection.

Each block corresponds to one cytokine value in one mouse. Results from one representative experiment are shown. ‘‘1’’ and ‘‘2’’ indicate the two clusters of

cytokines identified based on similar expression profiles along the time course of infection. Cluster 1 contains proinflammatory cytokines, and cluster 2 shows

anti-inflammatory factors.

(B) Macrophages from the spleen and MLNs were identified based on CD301 cell-surface expression in CD11b+ F4/80+ macrophages. Percentage of CD301+

cells relative to the total number of cells from the corresponding tissue (gray bars, left y axis), and Salmonella colonization level from the same organs (solid black

line, right y axis) are shown. Data presented are representative of two independent experiments.

(C) Splenocytes were isolated at 30 dpi, and CD301+ macrophages were identified based on CD301 expression of CD11b+ F4/80+ Ly6G cells. Salmonella-

positive gates were drawn based on plots from samples stained with an appropriate isotype control antibody. Numbers indicate the percentage of positive cells in

each gate. The data presented are representative of three independent experiments.

(D) Salmonella-associated mean fluorescence intensity (MFI) associated with CD301+ or CD301� macrophages from the spleen at 30 dpi. Graph shows the

means ± SD of five mice and are representative of at least three independent experiments.

(E) WT or Il-4raLoxP/LoxP-LysmCre BALB/c mice were infected orogastrically with 109 CFU S. typhimurium SL1344DaroA, and bacterial loads in the MLNs were

quantified at 28 dpi. Shown is the geometric mean determined from two independent experiments. All other data are presented as mean ± SD. *p < 0.05, **p <

0.01. Figure is related to Figure S1.

Cell Host & Microbe

Persistent Salmonella Infection Requires PPARd

However, there were significantly higher levels of bacteria at

24 hr postinfection in IL-4-stimulated cells compared to the un-

stimulated cells. As expected, S. typhimurium did not replicate

in IFN-g-stimulated macrophages (Figure 2A and Figure S2A).

To validate that cytokine treatment skewed the activation state

of the macrophage during infection, we monitored macrophage

activation bymeasuring extracellular nitric oxide (NO) production

and cytoplasmic arginase activity (Figures S2B and S2C). In

addition, there were no significant differences in the amount of

LDH released from cytokine-treated BMDMs infected with

S. typhimurium for 26 hr, indicating that the differences in the

levels of S. typhimurium recovered from infected BMDMs were

not due to differences in host cell death (Figure 2B). Notably,

Cell Hos

IL-4 stimulation of the human-derived monocytic cell line

THP-1 also supported higher levels of intracellular replication

of the human-specific pathogen S. typhi (Figure S2D).

We also observed that the level of CD301 expressed on the

surface of BMDMs varies, suggesting that macrophage activa-

tion states are heterogeneous within an infected cell culture (Fig-

ure S2E). Thus, we first separated CD301+ and CD301� cells

from infected IL-4-stimulated BMDMs and compared the level

of S. typhimurium replication over time in these two populations.

Although the percentage of macrophages containing bacteria

was similar between the two populations after 2 hr of infection

(CD301+ = 3.4 ± 0.8, CD301� = 2.8 ± 0.6 CFU/macrophage, Fig-

ure 2C), CD301+ macrophages harbored significantly higher

t & Microbe 14, 171–182, August 14, 2013 ª2013 Elsevier Inc. 173

Figure 2. Enhanced S. typhimurium Repli-

cation within IL-4-Stimulated Cells

(A) Salmonella replication in BMDMs treated as

shown. At the indicated time points, cells were

lysed and bacterial loads were enumerated.

(B) Cell death was determined by measuring LDH

release at 26 hpi and is represented as the per-

centage of total cell death in lysed control cells.

(C) Relative proportion of Salmonella-associated

cells in the CD301+ and CD301� populations from

IL-4-stimulated BMDMs at 2 hpi.

(D) Salmonella MFI of CD301+ or CD301� macro-

phages at 26 hpi.

(E) Fold replication of Salmonella in CD301+ and

CD301� populations. Data are presented as

mean ± SD. *p < 0.05, **p < 0.01. Graphs show the

means of triplicate samples and are representative

of at least three independent experiments. Fig-

ure is related to Figure S2.

Cell Host & Microbe

Persistent Salmonella Infection Requires PPARd

levels of S. typhimurium compared to CD301� cells after 26 hr

(Figure 2D). This result was also observed for CD301+ and

CD301� cells present in an untreated, infected BMDM cell

culture (Figure 2E, Figure S2F), albeit to lower levels than in

cells treated with IL-4, indicating that cytokine stimulation is

needed for establishment of the full M2 phenotype. Taken

together, these results suggest that the intracellular environment

of CD301+ macrophages is well suited to accommodate

Salmonella replication and that this macrophage subset repre-

sents a unique niche that is permissive for bacterial colonization.

PPARd Is Critical for Salmonella Replication withinIL-4-Stimulated MacrophagesTo investigate a host molecular pathway that might be control-

ling Salmonella replication within IL-4-stimulated macrophages,

we focused on a superfamily of nuclear receptors, PPARs, which

have been implicated in sustaining the anti-inflammatory/M2

phenotype in macrophages (Odegaard et al., 2007, 2008). We

first measured the levels of Ppara, Pparg, and Ppard gene

expression in untreated BMDMs infected with S. typhimurium

174 Cell Host & Microbe 14, 171–182, August 14, 2013 ª2013 Elsevier Inc.

and found that Ppard transcript levels

increased significantly, whereas Ppara

and Pparg mRNA levels decreased (Fig-

ure 3A). We also observed an increase in

PPARd protein expression (Figure S3A).

The mRNA level of Adrp (a gene specif-

ically regulated by PPARd [Mukundan

et al., 2009]) was also significantly higher

in IL-4-stimulated BMDMs infected with

Salmonella, providing further evidence

that activation of PPARd transcriptional

activity is associated with Salmonella

colonization (Figure S3B). In addition,

the level of Salmonella associated

with IL-4-stimiulated macrophages at

26 hr postinfection correlated with an

increased level of PPARd protein expres-

sion (Figure S3C). To confirm that a loss

of PPARd expression inhibits polariza-

tion toward the M2 phenotype during Salmonella infection, we

measured arginase activity and the level of CD301 surface

expression in infected WT and Ppard�/� BMDMs stimulated

with IL-4 (Figures S3D and S3E).

Consequently, we investigated whether PPARd is required for

creating an intracellular environment that supports Salmonella

colonization by comparing the ability of bacteria to replicate

within WT and Ppard�/� BMDMs. Unexpectedly, Salmonella

did not replicate in Ppard�/� BMDMs treated with IL-4, and there

was no increase in macrophage cell death (Figure 3B, Fig-

ure S3F). Although the number of infected cells 24 hr after infec-

tion was similar between IL-4-stimulated WT and Ppard�/�

BMDMs (Figure 3C), the level of Salmonella colonization 24 hr

after infection was higher in WT macrophages (Figure 3D).

Importantly, the level of bacterial invasion at the time of infection

was similar (Figure 3E). Using a complementary approach, we

also determined that the synthetic PPARd agonist GW0742

added to WT BMDMs resulted in increased intracellular

Salmonella replication to an extent similar to IL-4 stimulation

(Figure 3F). These results suggest that Salmonella may take

Figure 3. PPARd Is Required for Salmonella Replication

(A) Upregulation of Ppard mRNA in S. typhimurium-infected BMDMs treated with IL-4. Relative transcript levels for genes encoding Ppar family members are

shown. Data were normalized to uninfected samples for each gene.

(B) Salmonella 12023 replication in IL-4-stimulated WT and Ppard�/� BMDMs.

(C) Percentage of infected cells determined by flow cytometry among WT and Ppard�/� BMDMs at 24 hr post-IL-4 stimulation.

(D) Salmonella-associated MFI in WT and Ppard�/� BMDMs 24 hr following activation with IL-4.

(E) Salmonella-associated MFI in WT and Ppard�/� BMDMs 2 hr after infection, prior to IL-4 stimulation.

(F) Salmonella fold replication in BMDMs unactivated (unact) or stimulated with IL-4 (20 ng/ml), or GW0742 (100 mM), 26 hpi. The data presented are repre-

sentative of at least three independent experiments. *p < 0.05.

Data are presented as mean ± SD. Figure is related to Figure S3.

Cell Host & Microbe

Persistent Salmonella Infection Requires PPARd

advantage of a PPARd-regulated pathway(s) in order to replicate

within IL-4-stimulated macrophages.

Requirement of PPARd for Replication in MacrophagesIs Specific for Salmonella

PPARs regulate broad transcriptional programs that control not

only the expression of important metabolic systems but also

inflammatory responses. Previous studies indicate that macro-

phages deficient in Pparg or Ppard secrete elevated levels of

proinflammatory cytokines, such as TNFa, IL-6, and IL-12,

upon bacterial infection or LPS stimulation, respectively (Abdul-

lah et al., 2012; Odegaard et al., 2008; Rajaram et al., 2010).

Notably, this hyperactive inflammatory phenotype has been

linked to the inhibition of Mycobacterium tuberculosis replica-

tion in human-derived macrophages in which Pparg has

been knocked down (Rajaram et al., 2010). Similarly, Listeria

monocytogenes does not replicate in Pparg�/� BMDMs (Abdul-

lah et al., 2012; Rajaram et al., 2010). To test whether a similar

mechanism may be controlling bacterial replication in Ppard�/�

BMDMs, we first examined the ability of several pathogens to

replicate in IL-4-stimulated macrophages. M. tuberculosis repli-

cated in PPARd-deficient BMDMs to the same extent as in WT

Cell Hos

cells (Figure 4A), suggesting that PPARd and PPARg play

different roles during macrophage infections with this pathogen.

Consistent with these results, Ppard gene expression in BMDMs

did not increase duringM. tuberculosis infection, whereas Pparg

gene expression increased �10-fold (Figure 4B). Similar to

M. tuberculosis, L. monocytogenes and Francisella novicida

replicated in PPARd-deficient BMDMs to the same extent as

in WT BMDMs (Figures 4C and 4D). Although the intracellular

replication of each of these pathogens is known to be limited

by proinflammatory cytokine signaling (Kirimanjeswara et al.,

2008; Shaughnessy and Swanson, 2007; Zuniga et al., 2012),

their replication was not hindered in the absence of PPARd.

These results suggest that, in the context of bacterial infections,

PPARd deficiency does not result in increased levels of proin-

flammatory cytokine production or signaling.

To directly test whether PPARd deficiency leads to increased

cytokine secretion during S. typhimurium infection, we mea-

sured the concentrations of a number of cytokines in the super-

natants of IL-4-stimulated WT and Ppard�/� BMDMs during a

time course of infection (Figure 4E and Figure S4A). There

were no significant differences in the levels of most of the cyto-

kines tested between WT and Ppard�/� BMDMs that were not

t & Microbe 14, 171–182, August 14, 2013 ª2013 Elsevier Inc. 175

Figure 4. Ppard Deficiency Does Not Lead to Elevated Secretion of Proinflammatory Cytokines in S. typhimurium-Infected Macrophages

(A) M. tuberculosis replication in WT and Ppard�/� BMDMs over time.

(B) Ppard and Pparg transcript levels in WT C57BL/6 BMDMs infected with M. tuberculosis. Data were normalized to uninfected cells.

(C) L. monocytogenes replication in WT and Ppard�/� BMDMs over time.

(D) F. tularensis replication in WT and Ppard�/� BMDMs over time.

(E) Cytokine concentrations from supernatants of IL-4-stimulated WT and Ppard�/� BMDMs were determined by multiplex analysis at the indicated time points

following infection. Results from one representative experiment are shown. Asterisk indicates the cluster of proinflammatory cytokines identified based on similar

expression profiles along time course of infection. All other data are presented as themean of triplicate samples ± SD and are representative of three independent

experiments. Figure is related to Figure S4.

Cell Host & Microbe

Persistent Salmonella Infection Requires PPARd

infected, or at early time points after infection (2 hpi, Figure S4A).

Unexpectedly, concentrations of many of the proinflammatory

cytokines were significantly higher in the supernatants taken

from infected IL-4-stimulated WT BMDMs relative to Ppard�/�

cells after 12, 18, or 24 hr (Figure 4E and Figure S4A). These

results further support the conclusion that the inability of

S. typhimurium to replicate in Ppard�/� BMDMs is not due to

increased proinflammatory cytokine-induced bacterial killing

mechanisms. Consistent with these results, the rate of E. coli

killing was the same in WT and Ppard�/� BMDMs (Figure S4B).

Moreover, NO levels were higher in the supernatants of

WT BMDMs compared to Ppard�/� BMDMs infected with

Salmonella (Figure S4C), indicating that the inability of

Salmonella to replicate in Ppard�/� macrophages cannot be

attributed to NO-dependent antimicrobial mechanisms. Taken

together, these results suggest that PPARd regulation of the

cytokine and receptor program expressed by IL-4-stimulated

cells does not restrict the ability of Salmonella to successfully

replicate within these cells.

The Metabolic Program Controlled by PPARd RegulatesSalmonella ReplicationPPARd functions to coordinately regulate the expression of

metabolic programs, most notably fatty acid b-oxidation (Barak

et al., 2002) which becomes dysregulated in Ppard�/� BMDMs

(Figure S5A). We found that the inhibition of fatty acid oxidation

by etomoxir, a carnitine palmitoyltransferase I inhibitor, abro-

gated the enhancing effects of IL-4 on Salmonella replication

(Figure 5A). Etomoxir had no effect on BMDM viability, activation

176 Cell Host & Microbe 14, 171–182, August 14, 2013 ª2013 Elsevi

state, or bacterial growth in broth (Figures S5B–S5E). Taken

together, these results indicate that S. typhimurium replication

in IL-4-stimulated cells is dependent on PPARd-regulated fatty

acid metabolism.

Since PPARd regulates the shift from a glucose-based meta-

bolism in host cells to a fatty acid-based metabolism (Brunmair

et al., 2006), we reasoned that a major carbon source might be

more readily available for Salmonella when PPARd is active. To

test this, we first determined that glucose is a major carbon

source for Salmonella in macrophages. A mutant Salmonella

strain that cannot transport glucose (DptsG, Dglk, and

DmanXYZ) is defective for intracellular replication in BMDMs

(Figure 5B), similar to results obtained when macrophage or

epithelial cell lines were used (Bowden et al., 2009). Furthermore,

we found that intracellular Salmonella acquires glucose from the

macrophage as determined by the ability of bacteria to appro-

priate a fluorescent glucose analog that was administered to

BMDMs during infection (Figures 6B and 6C). We also measured

the expression level of Salmonella genes involved in glucose

transport, since it has previously been shown that bacteria regu-

late the expression of genes involved in glucose metabolism in

relation to the levels of available glucose in the local environment

(Meadow et al., 1990). The expression of Salmonella genes

involved in glucose transport and metabolism was lower when

infecting Ppard�/� macrophages (Figure S6A), suggesting that

Salmonella encounters a lower concentration of glucose in

Ppard�/� BMDMs compared to WT cells. To test this further,

we measured the concentration of glucose in infected WT and

Ppard�/�BMDMs and found that the level of intracellular glucose

er Inc.

Figure 5. Salmonella Is Able to Acquire Glucose from the Host

(A) Salmonella fold replication 24 hr after administration of the indicated

treatment is shown.

(B) Salmonella strain 12023 (plain) or a DptsGDglkDmanXYZ triple mutant

(hatched) fold replication in WT and Ppard�/� BMDMs prestimulated with IL-4

for 24 hr prior to infection. IL-4 wasmaintained in the medium along the course

of infection. Graph shows the fold replication at 24 hr. The data presented are

mean ± SD of triplicate samples and representative of three independent

experiments. *p < 0.05. Figure is related to Figure S5.

Cell Host & Microbe

Persistent Salmonella Infection Requires PPARd

in PPARd-deficient BMDMs was significantly lower compared to

the levels in WT BMDMs (Figures 6A and 6D). These results indi-

cate that PPARd activity influences intracellular glucose levels. In

addition, we stimulated WT BMDMs with a PPARd agonist,

GW0742, and found that specific activation of PPARd increases

the intracellular concentration of glucose (Figure S6B). Conse-

quently, Salmonella acquires significantly less glucose in the

absence of PPARd (Figures 6B and 6C). We also examined the

ability of bacteria to replicate in BMDMs treated with the AMP-

activated protein kinase (AMPK) activator AICAR. AMPK activa-

tion increases glucose transport into cells, resulting in elevated

intracellular concentrations of glucose in both WT and Ppard�/�

BMDMs (Figure 6D) (Merrill et al., 1997). AICAR administered to

infected cells rescued bacterial replication in IL-4-stimulated

Ppard�/� BMDMs in a dose-dependent manner (Figure 6E).

Taken together, these data indicate that Salmonella replication

within IL-4-stimulated BMDMs is regulated by the PPARd-

dependent availability of glucose in macrophages.

PPARd Is Required for Salmonella Persistence In VivoTo determine whether PPARd is essential for the long-term

persistence of Salmonella in systemic host tissues, we

infected WT and Ppard�/� mice orogastrically with 108 CFU

S. typhimurium. Bacteria colonized the spleen and MLNs of

WT and Ppard�/�mice to the same extent at 5 and 10 days post-

infection. However, viable S. typhimurium was not detected in

Ppard�/� tissues 6 weeks following infection, indicating that

S. typhimurium did not persist in Ppard�/� mice (Figure 7A).

These results correlated with the number of CD301+ macro-

phages in the spleen and MLNs, which were significantly lower

inPpard�/� compared toWTmice (Figure 7B).We also evaluated

the ability of S. typhimurium to persistently colonize Ppard�/�

chimeric mice (Figure 7C). While very few bacteria were recov-

ered from the MLNs of lethally irradiated Ppard�/� mice that

were reconstituted with Ppard�/� bone marrow, significantly

higher levels were found in mice transplanted with WT bone

marrow. Thus, Salmonella requires PPARd activity in cells that

Cell Hos

differentiate from the hematopoietic compartment in order to

maintain a persistent infection.

To confirm the role of PPARd in S. typhimurium persistence

and to ensure that the effect of PPARd observed in vivo was

not due to possible differences in mouse genetic backgrounds,

WT mice were injected with the PPARd agonist GW0742 or the

vehicle control every day for 6 days prior to infection with

S. typhimurium. Mice treated with the PPARd agonist had

a significantly higher percentage of CD301+ cells that were

expressing PPARd compared to control mice (Figures 7D

and 7E). Thus, as expected, the levels of Salmonella were sig-

nificantly higher at 20 days postinfection in the spleen and at

15 and 20 days postinfection in the MLNs of GW0742-injected

mice compared to control mice (Figure 7F). These results further

support the role of PPARd in creating an environment conducive

for bacterial colonization, leading to long-term Salmonella

persistence.

DISCUSSION

In this report, we have demonstrated that S. typhimurium prefer-

entially associates with anti-inflammatory/M2 macrophages at

later stages of a systemic murine infection and that PPARd, a

host regulator of fatty acid metabolism in M2 macrophages, is

required for bacterial persistence. A previous study using an

acute model of infection showed a similar association between

the number of M2 macrophages and level of S. typhimurium

colonization in mice but did not demonstrate a significant differ-

ence in intracellular bacterial replication in IL-4-stimulated RAW

264.7macrophages (Bishop et al., 2008). Indeed, the vast major-

ity of Salmonella studies in macrophages have been conducted

usingmacrophage-like cell lines which have been shown to have

markedly different metabolic requirements than primary cells

(Biswas and Mantovani, 2010, 2012; Gordon and Martinez,

2010). Thus, our results suggest that primary anti-inflamma-

tory/M2-like macrophages may represent a more biologically

relevant model to study Salmonella pathogenesis.

We found at least one mechanism underlying enhanced

Salmonella replication in IL-4-stimulated macrophages

that requires PPARd-regulated availability of glucose within

macrophages. Importantly, the replication of M. tuberculosis,

F. tularensis, and L. monocytogenes in IL-4-stimulated Ppard�/�

BMDMs was unimpaired. In addition, we show that

M. tuberculosis infection induced the expression of Pparg, but

not Ppard, suggesting that a requirement for PPARdmay be spe-

cific to Salmonella. We speculate that such specificity could be

due to unique metabolic needs required during infection. In sup-

port of this notion, previous studies indicate thatM. tuberculosis

primarily utilizes fatty acids as a major source of energy within

macrophages (Eisenreich et al., 2010; McKinney et al., 2000;

Pandey and Sassetti, 2008). In contrast, L. monocytogenes

and F. tularensis utilize amino acids as energy sources during

replication withinmacrophages (Alkhuder et al., 2009; Eisenreich

et al., 2010; Eylert et al., 2008; Raghunathan et al., 2010). More-

over, we show here that the inability of Salmonella to replicate in

Ppard�/� BMDMs is due to a decreased concentration of intra-

cellular glucose, a crucial carbon source that is essential for

the replication of S. typhimurium (Bowden et al., 2009). The

reduced availability of glucose for intracellular Salmonella is

t & Microbe 14, 171–182, August 14, 2013 ª2013 Elsevier Inc. 177

Figure 6. Salmonella Acquires Less

Glucose from Macrophages in the Absence

of Ppard

(A) BMDMs were stimulated for 48 hr with IL-4

(20 ng/ml) prior to infection with Salmonella 12023.

Glucose levels were measured at indicated time

points after infection. IL-4 was maintained in

medium throughout the experiment.

(B) 2-NBDG fluorescence intensity measured

within each bacterium, normalized over the volume

of the bacterium. Results are shown for the 12023

Salmonella strain inWT andPpard�/�BMDMs, and

for the 12023DptsGDglkDmanXYZ mutant in WT

BMDMs, 26 hr after infection. Cells were presti-

mulated with IL-4 for 48 hr before infection. Over

100 bacteria were analyzed for each macrophage

genotype. The data presented are mean ± SD and

representative of three independent experiments.

(C) BMDMs were infected with the indicated

Salmonella strains (red) before 2-NBDG (green)

was added to the medium. Images are a projection

of a 10 mm z stack collected through the 633

objective of a confocal microscope.

(D) IL-4-stimulatedWT and Ppard�/�BMDMswere

treated with ddH20 (Vehicle) or 200 mMAICAR, and

intracellular glucose was measured after 24 hr.

The data presented are representative of three

independent experiments ± the SD of triplicate

samples.

(E) IL-4-stimulated WT and Ppard�/� BMDMswere

infected then treated with either ddH20 (Vehicle)

or the indicated concentration of AICAR. Fold

replication was determined at 24 hpi. Data are

combined from three independent experiments,

and the SEM is shown. *p < 0.05, **p < 0.01, ***p <

0.001. Figure is related to Figure S6.

Cell Host & Microbe

Persistent Salmonella Infection Requires PPARd

likely a consequence of the inability of Ppard�/�macrophages to

upregulate the fatty acid metabolic program, resulting in an

increased dependence on glucose utilization by the host cell

for energy. Few studies have focused on the link between

PPAR family members and the ability of intracellular pathogens

to cause infection, but all of them to date have suggested an

indirect role via the regulation of the immune response (Gal-

lardo-Soler et al., 2008; Odegaard et al., 2007). Contrary to this

observation, our data demonstrate that proinflammatory cyto-

kine production by Ppard�/� macrophages is decreased

compared to WT cells during Salmonella infection. Therefore,

our data link the PPARd-regulated metabolic state of the host

cell to bacterial infection.

Since M2 macrophages do not express many of the defense

mechanisms needed to eliminate invading microbes, some

pathogens have evolved strategies to actively polarize cells

toward the M2 phenotype as a virulence mechanism. For

example, Toxoplasma gondii bypasses IL-4 signaling by directly

phosphorylating STAT6 using the rhoptry kinase ROP16

178 Cell Host & Microbe 14, 171–182, August 14, 2013 ª2013 Elsevier Inc.

expressed by type I and III strains, result-

ing in increased parasite replication in

macrophages (Jensen et al., 2011).

In addition, a previous study suggests

that F. tularensis induces alternatively

activated/M2 macrophages and that

increased bacterial survival is dependent on IL-4 (Shirey et al.,

2008). However, several conflicting studies report that IL-4, in

addition to other products secreted by mast cells, inhibits

F. tularensis intracellular replication (Ketavarapu et al., 2008;

Rodriguez et al., 2011; Shirey et al., 2008). Given the importance

of M2 macrophages and PPARd for Salmonella persistence, we

hypothesize that bacteria have evolved mechanisms to actively

modulate the M2 phenotype. Indeed, we show that bacteria

dramatically induce the upregulation of Ppard gene and protein

expression during infection in the absence of IL-4 stimulation.

Thus, future studies aim to identify the bacterial factor(s) required

for the induction of Ppard, and to determine whether Salmonella

directly augments PPARd activity and/or the M2 phenotype to

promote persistence within the mammalian host.

Importantly, we also showed that S. typhimurium does not

persist in PPARd-deficient mice and correlate this to decreased

levels of CD301+ macrophages. Notably, similar levels of bacte-

ria were recovered in the spleens and MLNs of infected WT and

Ppard�/�mice during the acute phase of the infection, when high

Figure 7. PPARd Is Required for Long-Term Carriage of S. typhimurium in Systemic Organs

(A) Salmonella colonization of the spleens and MLNs from WT and Ppard�/� mice was quantified at the indicated time points following oral infection.

(B) Proportion of CD301+ macrophages in spleens and MLNs of infected WT and Ppard�/� mice was assessed by FACS at 42 dpi.

(C) Bacterial loads in the MLNs of chimeric Ppard�/� mice were measured 42 dpi.

(D and E) Proportion of CD301+ (D) and PPARd+ (E)macrophages in the spleens ofmice injectedwith GW0742 or the vehicle control. Graph shows cell percentage

before mice were infected.

(F) PPARd activation increases Salmonella colonization levels of spleen (left) and MLN (right). Data are presented as the geometric mean ± SD. *p < 0.05, **p <

0.01. Each experiment was performed twice with three to five mice per time point for each genotype or treatment.

Cell Host & Microbe

Persistent Salmonella Infection Requires PPARd

levels of proinflammatory cytokines are produced, indicating

that there is no initial defect in the ability of Salmonella to system-

ically colonize Ppard�/�mice. In contrast, lower levels of bacteria

were recovered from Ppard�/� mice during the chronic phase of

the infection, when relatively higher levels of anti-inflammatory

cytokines are produced, supporting the notion that the combina-

tion of PPARd activity and the cytokine environment are required

for the establishment and/or maintenance of the chronic

phase of infection. Although we were able to restore disease in

lethally irradiated Ppard�/� mice reconstituted with WT bone

marrow, we cannot completely exclude the possibility that a

cell type other than macrophages is responsible for Salmonella

persistence in vivo. In the future, it will be prudent to study

Cell Hos

the role of PPARd in different cell types during persistent

Salmonella infection in the appropriate mouse genetic back-

ground (Nramp1+/+).

We have previously shown that IFN-g plays an important role

in controlling the levels of Salmonella in mice (Monack et al.,

2004a). In this study, we showed that chronically infected mice

contained S. typhimurium in systemic tissues within microgranu-

lomas/typhoidal nodules in which the central areas contained

tightly opposed macrophages that contain bacteria. However,

when asymptomatic, chronically infected mice were treated

with an IFN-g-neutralizing antibody, high levels of bacteria

were detected in systemic tissues. Neutralization of IFN-g led

to the disintegration of the typhoidal nodules, and there was

t & Microbe 14, 171–182, August 14, 2013 ª2013 Elsevier Inc. 179

Cell Host & Microbe

Persistent Salmonella Infection Requires PPARd

uncontrolled bacterial replication. These findings indicate that

the balance between the Th1 and Th2 responses had been dis-

rupted when we neutralized IFN-g. This is very similar to what is

seen during persistent M. tuberculosis infection where the bac-

teria are located within macrophages in granulomas and T lym-

phocytes (producing many different cytokines, including IFN-g)

and surround the macrophages infected with M. tuberculosis

to ‘‘wall off’’ the infection (Monack et al., 2004b).

Taken together, our data strongly support the hypothesis that

Salmonella has evolved to survive long-term in M2-like macro-

phages by exploiting the unique metabolism of these cells.

PPARd has emerged as a powerful metabolic regulator in diverse

tissues, highlighting the broad potential of PPARd augmentation

in the treatment of metabolic disease. Its combined activities

make PPARd a key regulator with the potential to therapeutically

target multiple aspects of metabolic syndromes, including

obesity and insulin resistance (Barish et al., 2006). Future exper-

iments investigating the role of PPARd, as well as other PPARs,

during human infection with S. typhi will be important, as there

might be differences in humans compared to mice (Bouhlel

et al., 2009). It is tempting to speculate that Salmonella evolved

to take advantage of host factors critical for the homeostasis

of various host cell processes. Such factors that influence

asymptomatic carriage of S. typhi in humans are unknown.

Thus, future studies seek to determine if there are genetic differ-

ences in S. typhi carriers that influence the recruitment and

activation of macrophages where Salmonella persists within

mammalian hosts.

EXPERIMENTAL PROCEDURES

Mouse Strains and Cell Culture

Female 129x1/SvJ mice (6–8 weeks old, Jackson Laboratories) were used for

in vivo infections and to analyze the interactions between Salmonella andmac-

rophages (Figures 1 and 2). Ppard�/� 129/SvImJmice (Barak et al., 2002) were

utilized to study the role of PPARd in Salmonella persistence. WT littermates

were used as controls. Mice were obtained from Jackson Laboratories to

generate the chimeric mice. 129/SvImJ WT mice were used for the PPARd

agonist injection in mice. WT BALB/c and Il-4raLoxP/LoxP-LysmCre mice were

obtained from Dr. Ajay Chawla (Nguyen et al., 2011).

BMDMswere prepared fromWT and Ppard�/�mice as previously described

(Warren and Vogel, 1985). Briefly, marrows were aseptically removed and

flushed with DMEM. BMDMs were differentiated in DMEM (Invitrogen) with

10% (v/v) heat-inactivated FBS (Hyclone), 10% 3t3-MCSF cell supernatant,

10 mM HEPES (GIBCO), and nonessential amino acids (GIBCO). RAW264.7

cells were cultured in DMEM with 10% (v/v) heat-inactivated FBS. The human

monocyte cell line THP-1 (ATCC TIB-202) was maintained in RPMI 1640

(GIBCO) containing 10% (v/v) heat-inactivated FBS (Invitrogen), 25 mM

HEPES (GIBCO), 2 mM L-glutamine, 1 mM sodium pyruvate, and 55 mM

2-mercaptoethanol. All cell cultures were incubated in a humidified chamber

at 37�C in 5% CO2.

Mouse Infections

Mice were kept under specific pathogen-free conditions in filter-top cages and

were provided sterile water and food ad libitum. Mice were deprived of food for

14 hr before orogastric inoculation. Mice were inoculated orogastrically with

108 CFU/mouse S. typhimurium SL1344. In experiments utilizing BALB/c

mice, the S. typhimurium SL1344DaroA strain was used, and mice were not

deprived of food prior to infection with 109 CFU/mouse (Fang et al., 2005).

Mice were euthanized by CO2 asphyxiation and cervical dislocation, followed

by dissection and harvesting of the indicated organs. Tissues were weighed

and homogenized in PBS, and appropriate dilutions were plated on LB agar

plates to obtain CFUs per gram of tissue.

180 Cell Host & Microbe 14, 171–182, August 14, 2013 ª2013 Elsevi

Macrophage Infections

BMDMswere seeded at a density of 2.53 105 cells per well of a 24-well culture

dish. For infection of WT and Ppard�/� BMDMs, cells were incubated in low-

glucose medium (1 g/L) supplemented with 5% FBS and 5% 3t3-MCSF cell

supernatant 24 hr prior to infection. Low-glucose medium was then used

throughout the time course of infection. Where indicated, cells were treated

with recombinant mouse IL-4 (20 ng/ml, Peprotech) for 24–48 hr prior to infec-

tion (this length had no effect on bacterial survival or replication). AICAR-

treated samples were incubated in medium containing 4.5 g/L glucose.

Salmonella strains from a culture grown overnight were used to infect the mac-

rophages at an moi of 5, and bacteria were centrifuged for 5 min at 50 3 g to

synchronize bacterial uptake. Following a 30 min incubation, cells were

washed and incubated in medium containing 100 mg/ml of gentamicin for

90 min. Cells were then incubated in medium containing 10 mg/ml of genta-

micin until the end of the experiment. Cells were stimulated with recombinant

mIL-4, mIFNg (100 units/ml) (R&D Systems), etomoxir (50 mM, Sigma), or

AICAR (Sigma) 2 hr after infection when switching to gentamicin 10 mg/ml.

Gentamicin-protected bacteria were harvested from macrophages at various

time points by lysis of cells with 1% Triton X-100 and plated for enumeration.

For experiments involving AICAR,Salmonella expressingmCherry was used at

an moi of 20, and intracellular bacteria were counted in macrophages that

were stained as described above. Z stacks taken from 15 random fields in

three replicates at each time point were examined. Fold replication was calcu-

lated by normalizing the CFU concentration from a given time point to the CFU

concentration from the 2 hpi time point. For experiments utilizing

M. tuberculosis, F. novicida, and L. monocytogenes, an moi of 1, 10, and 1

was used, respectively.

To assess bacterial levels as a function of luxCDABE light production, RAW

cells were seeded at a density of 53 104 cells/well in DMEM lacking phenol red

in Optilux 96-well, black-wall, clear-bottommicrotest plates (BD Falcon). Light

production from infected cells was assayed using a Veritas Microplate Lumin-

ometer. The relative light units obtained from luminometer readings were log10

transformed and normalized to levels at 2 hr postinfection to account for var-

iations in bacterial uptake by the macrophages. The data were additionally

normalized to the average value for unactivated cells. A stock culture of

THP-1 cells was maintained as monocyte-like, nonadherent cells. For infec-

tion, cells were seeded at 53 105 cells per well in 24-well tissue-culture dishes

and were differentiated by the addition of 10�7 M phorbol 12-myristate 13-

acetate for 48 hr. Infection with S. typhi was performed as described above

at an moi of 10.

See the Supplemental Experimental Procedures.

SUPPLEMENTAL INFORMATION

Supplemental Information includes six figures, Supplemental Experimental

Procedures, and Supplemental References and can be found with this article

online at http://dx.doi.org/10.1016/j.chom.2013.07.010.

ACKNOWLEDGMENTS

We thank all members of the Monack lab for valuable comments. We also

thank Justin Odegaard for helpful discussions. N.A.E. is supported by Molec-

ular and Cellular Immunobiology training grant NIH T32 AI07290-28. T.R. is

supported by grants from the Philippe Foundation and NIH T32 AI007328.

This work was supported by AI08972 from the National Institute of Allergy

and Infectious Diseases and a Burroughs Wellcome Fund Investigator in the

Pathogenesis of Infectious Diseases award to D.M.M. and by NIH grants

DK081405 and HL076746 and an NIH Director’s Pioneer Award

(DP1AR064158) to A.C. All animal care was in accordance with Stanford Uni-

versity’s A-PLAC committee guidelines. T.R., N.A.E., and D.M.M. designed

research; T.R., N.A.E., A.J., P.S.M., and L.L. performed research; L.M. and

A.C. contributed analytical tools and mice; T.R., N.A.E., A.J., L.L., and

D.M.M. analyzed data; T.R., N.A.E., L.M., A.C., and D.M.M. wrote the paper.

Received: February 27, 2013

Revised: May 10, 2013

Accepted: June 20, 2013

Published: August 14, 2013

er Inc.

Cell Host & Microbe

Persistent Salmonella Infection Requires PPARd

REFERENCES

Abdullah, Z., Geiger, S., Nino-Castro, A., Bottcher, J.P., Muraliv, E., Gaidt, M.,

Schildberg, F.A., Riethausen, K., Flossdorf, J., Krebs, W., et al. (2012). Lack of

PPARg in myeloid cells confers resistance to Listeria monocytogenes infec-

tion. PLoS ONE 7, e37349, http://dx.doi.org/10.1371/journal.pone.0037349.

Alkhuder, K., Meibom, K.L., Dubail, I., Dupuis, M., and Charbit, A. (2009).

Glutathione provides a source of cysteine essential for intracellular multiplica-

tion of Francisella tularensis. PLoS Pathog. 5, e1000284, http://dx.doi.org/10.

1371/journal.ppat.1000284.

Barak, Y., Liao, D., He, W., Ong, E.S., Nelson, M.C., Olefsky, J.M., Boland, R.,

and Evans, R.M. (2002). Effects of peroxisome proliferator-activated receptor

delta on placentation, adiposity, and colorectal cancer. Proc. Natl. Acad. Sci.

USA 99, 303–308.

Barish, G.D., Narkar, V.A., and Evans, R.M. (2006). PPAR delta: a dagger in the

heart of the metabolic syndrome. J. Clin. Invest. 116, 590–597.

Bishop, J.L., Sly, L.M., Krystal, G., and Finlay, B.B. (2008). The inositol phos-

phatase SHIP controls Salmonella enterica serovar Typhimurium infection

in vivo. Infect. Immun. 76, 2913–2922.

Biswas, S.K., and Mantovani, A. (2010). Macrophage plasticity and interaction

with lymphocyte subsets: cancer as a paradigm. Nat. Immunol. 11, 889–896.

Biswas, S.K., and Mantovani, A. (2012). Orchestration of metabolism by mac-

rophages. Cell Metab. 15, 432–437.

Bouhlel, M.A., Derudas, B., Rigamonti, E., Dievart, R., Brozek, J., Haulon, S.,

Zawadzki, C., Jude, B., Torpier, G., Marx, N., et al. (2007). PPARgamma acti-

vation primes human monocytes into alternative M2 macrophages with anti-

inflammatory properties. Cell Metab. 6, 137–143.

Bouhlel, M.A., Brozek, J., Derudas, B., Zawadzki, C., Jude, B., Staels, B., and

Chinetti-Gbaguidi, G. (2009). Unlike PPARgamma, PPARalpha or PPARbeta/

delta activation does not promote human monocyte differentiation toward

alternative macrophages. Biochem. Biophys. Res. Commun. 386, 459–462.

Bowden, S.D., Rowley, G., Hinton, J.C., and Thompson, A. (2009). Glucose

and glycolysis are required for the successful infection of macrophages and

mice by Salmonella enterica serovar typhimurium. Infect. Immun. 77, 3117–

3126.

Brunmair, B., Staniek, K., Dorig, J., Szocs, Z., Stadlbauer, K., Marian, V., Gras,

F., Anderwald, C., Nohl, H., Waldhausl, W., and Furnsinn, C. (2006). Activation

of PPAR-delta in isolated rat skeletal muscle switches fuel preference from

glucose to fatty acids. Diabetologia 49, 2713–2722.

Crump, J.A., Luby, S.P., and Mintz, E.D. (2004). The global burden of typhoid

fever. Bull. World Health Organ. 82, 346–353.

Eisenreich, W., Dandekar, T., Heesemann, J., and Goebel, W. (2010). Carbon

metabolism of intracellular bacterial pathogens and possible links to virulence.

Nat. Rev. Microbiol. 8, 401–412.

Eylert, E., Schar, J., Mertins, S., Stoll, R., Bacher, A., Goebel, W., and

Eisenreich, W. (2008). Carbon metabolism of Listeria monocytogenes growing

inside macrophages. Mol. Microbiol. 69, 1008–1017.

Fairweather, D., and Cihakova, D. (2009). Alternatively activated macrophages

in infection and autoimmunity. J. Autoimmun. 33, 222–230.

Fame, T.M., Engelhard, D., and Riley, H.D., Jr. (1986). Hemophagocytosis

accompanying typhoid fever. Pediatr. Infect. Dis. 5, 367–369.

Fang, F.C., Libby, S.J., Castor, M.E., and Fung, A.M. (2005). Isocitrate lyase

(AceA) is required for Salmonella persistence but not for acute lethal infection

in mice. Infect. Immun. 73, 2547–2549.

Gallardo-Soler, A., Gomez-Nieto, C., Campo, M.L., Marathe, C., Tontonoz, P.,

Castrillo, A., and Corraliza, I. (2008). Arginase I induction by modified lipopro-

teins in macrophages: a peroxisome proliferator-activated receptor-gamma/

delta-mediated effect that links lipid metabolism and immunity. Mol.

Endocrinol. 22, 1394–1402.

Gordon, S. (2003). Alternative activation of macrophages. Nat. Rev. Immunol.

3, 23–35.

Gordon, S., and Martinez, F.O. (2010). Alternative activation of macrophages:

mechanism and functions. Immunity 32, 593–604.

Cell Hos

Haraga, A., Ohlson, M.B., and Miller, S.I. (2008). Salmonellae interplay with

host cells. Nat. Rev. Microbiol. 6, 53–66.

House, D., Bishop, A., Parry, C., Dougan, G., and Wain, J. (2001). Typhoid

fever: pathogenesis and disease. Curr. Opin. Infect. Dis. 14, 573–578.

Huang, J.T., Welch, J.S., Ricote, M., Binder, C.J., Willson, T.M., Kelly, C.,

Witztum, J.L., Funk, C.D., Conrad, D., and Glass, C.K. (1999). Interleukin-4-

dependent production of PPAR-gamma ligands in macrophages by 12/15-

lipoxygenase. Nature 400, 378–382.

Jensen, K.D., Wang, Y., Wojno, E.D., Shastri, A.J., Hu, K., Cornel, L., Boedec,

E., Ong, Y.C., Chien, Y.H., Hunter, C.A., et al. (2011). Toxoplasma polymorphic

effectors determine macrophage polarization and intestinal inflammation. Cell

Host Microbe 9, 472–483.

Kang, K., Reilly, S.M., Karabacak, V., Gangl, M.R., Fitzgerald, K., Hatano, B.,

and Lee, C.H. (2008). Adipocyte-derived Th2 cytokines and myeloid

PPARdelta regulate macrophage polarization and insulin sensitivity. Cell

Metab. 7, 485–495.

Ketavarapu, J.M., Rodriguez, A.R., Yu, J.J., Cong, Y., Murthy, A.K.,

Forsthuber, T.G., Guentzel, M.N., Klose, K.E., Berton, M.T., and

Arulanandam, B.P. (2008). Mast cells inhibit intramacrophage Francisella

tularensis replication via contact and secreted products including IL-4. Proc.

Natl. Acad. Sci. USA 105, 9313–9318.

Kirimanjeswara, G.S., Olmos, S., Bakshi, C.S., and Metzger, D.W. (2008).

Humoral and cell-mediated immunity to the intracellular pathogen

Francisella tularensis. Immunol. Rev. 225, 244–255.

Lawley, T.D., Bouley, D.M., Hoy, Y.E., Gerke, C., Relman, D.A., and Monack,

D.M. (2008). Host transmission of Salmonella enterica serovar Typhimurium is

controlled by virulence factors and indigenous intestinal microbiota. Infect.

Immun. 76, 403–416.

McCoy, M.W., Moreland, S.M., and Detweiler, C.S. (2012). Hemophagocytic

macrophages in murine typhoid fever have an anti-inflammatory phenotype.

Infect. Immun. 80, 3642–3649.

McKinney, J.D., Honer zu Bentrup, K., Munoz-Elıas, E.J., Miczak, A., Chen, B.,

Chan, W.T., Swenson, D., Sacchettini, J.C., Jacobs, W.R., Jr., and Russell,

D.G. (2000). Persistence of Mycobacterium tuberculosis in macrophages

and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406,

735–738.

Meadow, N.D., Fox, D.K., and Roseman, S. (1990). The bacterial phospho-

enolpyruvate: glycose phosphotransferase system. Annu. Rev. Biochem. 59,

497–542.

Merrill, G.F., Kurth, E.J., Hardie, D.G., and Winder, W.W. (1997). AICA riboside

increases AMP-activated protein kinase, fatty acid oxidation, and glucose

uptake in rat muscle. Am. J. Physiol. 273, E1107–E1112.

Monack, D.M., Bouley, D.M., and Falkow, S. (2004a). Salmonella typhimurium

persists within macrophages in the mesenteric lymph nodes of chronically

infected Nramp1+/+mice and can be reactivated by IFNgamma neutralization.

J. Exp. Med. 199, 231–241.

Monack, D.M., Mueller, A., and Falkow, S. (2004b). Persistent bacterial

infections: the interface of the pathogen and the host immune system. Nat.

Rev. Microbiol. 2, 747–765.

Mosser, D.M., and Edwards, J.P. (2008). Exploring the full spectrum of

macrophage activation. Nat. Rev. Immunol. 8, 958–969.

Mukundan, L., Odegaard, J.I., Morel, C.R., Heredia, J.E., Mwangi, J.W.,

Ricardo-Gonzalez, R.R., Goh, Y.P., Eagle, A.R., Dunn, S.E., Awakuni, J.U.,

et al. (2009). PPAR-delta senses and orchestrates clearance of apoptotic

cells to promote tolerance. Nat. Med. 15, 1266–1272.

Nguyen, K.D., Qiu, Y., Cui, X., Goh, Y.P., Mwangi, J., David, T., Mukundan, L.,

Brombacher, F., Locksley, R.M., and Chawla, A. (2011). Alternatively activated

macrophages produce catecholamines to sustain adaptive thermogenesis.

Nature 480, 104–108.

Nix, R.N., Altschuler, S.E., Henson, P.M., and Detweiler, C.S. (2007).

Hemophagocytic macrophages harbor Salmonella enterica during persistent

infection. PLoS Pathog. 3, e193, http://dx.doi.org/10.1371/journal.ppat.

0030193.

t & Microbe 14, 171–182, August 14, 2013 ª2013 Elsevier Inc. 181

Cell Host & Microbe

Persistent Salmonella Infection Requires PPARd

Odegaard, J.I., Ricardo-Gonzalez, R.R., Goforth, M.H., Morel, C.R.,

Subramanian, V., Mukundan, L., Red Eagle, A., Vats, D., Brombacher, F.,

Ferrante, A.W., and Chawla, A. (2007). Macrophage-specific PPARgamma

controls alternative activation and improves insulin resistance. Nature 447,

1116–1120.

Odegaard, J.I., Ricardo-Gonzalez, R.R., Red Eagle, A., Vats, D., Morel, C.R.,

Goforth, M.H., Subramanian, V., Mukundan, L., Ferrante, A.W., and Chawla,

A. (2008). Alternative M2 activation of Kupffer cells by PPARdelta ameliorates

obesity-induced insulin resistance. Cell Metab. 7, 496–507.

Pandey, A.K., and Sassetti, C.M. (2008). Mycobacterial persistence requires

the utilization of host cholesterol. Proc. Natl. Acad. Sci. USA 105, 4376–4380.

Parry, S.M., Palmer, S.R., Slader, J., and Humphrey, T.; South East Wales

Infectious Disease Liaison Group. (2002). Risk factors for salmonella food

poisoning in the domestic kitchen—a case control study. Epidemiol. Infect.

129, 277–285.

Raghunathan, A., Shin, S., and Daefler, S. (2010). Systems approach to inves-

tigating host-pathogen interactions in infections with the biothreat agent

Francisella. Constraints-based model of Francisella tularensis. BMC Syst.

Biol. 4, 118.

Rajaram, M.V., Brooks, M.N., Morris, J.D., Torrelles, J.B., Azad, A.K., and

Schlesinger, L.S. (2010). Mycobacterium tuberculosis activates human

macrophage peroxisome proliferator-activated receptor gamma linking

mannose receptor recognition to regulation of immune responses.

J. Immunol. 185, 929–942.

182 Cell Host & Microbe 14, 171–182, August 14, 2013 ª2013 Elsevi

Rodriguez, A.R., Yu, J.J., Murthy, A.K., Guentzel, M.N., Klose, K.E.,

Forsthuber, T.G., Chambers, J.P., Berton, M.T., and Arulanandam, B.P.

(2011). Mast cell/IL-4 control of Francisella tularensis replication and host

cell death is associated with increased ATP production and phagosomal acid-

ification. Mucosal Immunol. 4, 217–226.

Shaughnessy, L.M., and Swanson, J.A. (2007). The role of the activated

macrophage in clearing Listeria monocytogenes infection. Front. Biosci. 12,

2683–2692.

Shirey, K.A., Cole, L.E., Keegan, A.D., and Vogel, S.N. (2008). Francisella tular-

ensis live vaccine strain induces macrophage alternative activation as a

survival mechanism. J. Immunol. 181, 4159–4167.

Warren, M.K., and Vogel, S.N. (1985). Opposing effects of glucocorticoids on

interferon-gamma-induced murine macrophage Fc receptor and Ia antigen

expression. J. Immunol. 134, 2462–2469.

Woc-Colburn, L., and Bobak, D.A. (2009). The expanding spectrum of disease

due to salmonella: an international perspective. Curr. Infect. Dis. Rep. 11,

120–124.

Xu, H.E., Lambert, M.H., Montana, V.G., Parks, D.J., Blanchard, S.G., Brown,

P.J., Sternbach, D.D., Lehmann, J.M., Wisely, G.B., Willson, T.M., et al. (1999).

Molecular recognition of fatty acids by peroxisome proliferator-activated

receptors. Mol. Cell 3, 397–403.

Zuniga, J., Torres-Garcıa, D., Santos-Mendoza, T., Rodriguez-Reyna, T.S.,

Granados, J., and Yunis, E.J. (2012). Cellular and humoral mechanisms

involved in the control of tuberculosis. Clin. Dev. Immunol. 2012, 193923.

er Inc.