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Monocyte Recruitment, Activation, and

Function in the Gut-Associated Lymphoid


Tissue during Oral Salmonella Infection
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J Immunol 2007; 178:5789-5801; ;


doi: 10.4049/jimmunol.178.9.5789
http://www.jimmunol.org/content/178/9/5789

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References

Anna Rydstrm and Mary Jo Wick

The Journal of Immunology

Monocyte Recruitment, Activation, and Function in the


Gut-Associated Lymphoid Tissue during Oral Salmonella
Infection1
Anna Rydstrom and Mary Jo Wick2

ecruitment of bone marrow-derived phagocytic monocytes is a hallmark of inflammation induced by infection


or other means (1, 2). In response to inflammatory stimuli, monocytes are released from the bone marrow into the blood
and home to tissues (1 8). The recruitment of monocytes and neutrophils from the blood to infected tissues is a requirement for
ensuring host survival to infection (9 13).
Monocytes circulating in the blood are composed of a heterogeneous population of cells that can be divided into distinct phenotypic and functional subsets in humans, rodents, and other species (3, 8, 14, 15). In murine blood, Gr-1lowCCR2lowCX3CR1high
and Gr-1intCCR2highCX3CR1low (int,3 intermediate) monocyte
subpopulations have been described where the latter is more prone
to migrate to inflammatory sites while the former migrates to tissues under steady-state conditions (3, 8, 14, 16). It is suggested
Department of Microbiology and Immunology, Goteborg University, Goteborg,
Sweden
Received for publication June 12, 2006. Accepted for publication February 19, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Swedish Research Council (621-20041378) and the Swedish Foundation for Strategic Research Microbes and Man Program
(A3 01:93/01/01) and was performed at the Mucosal Immunobiology and Vaccine
Center funded by the Swedish Foundation for Strategic Research.
2
Address correspondence and reprint requests to Dr. Mary Jo Wick, Department
of Microbiology and Immunology, Box 435, Goteborg, Sweden. E-mail address:
mary-jo.wick@immuno.gu.se
3
Abbreviations used in this paper: int, intermediate; 7-AAD, 7-aminoactinomycin D;
CCD, cytochalasin D; DC, dendritic cells; eGFP, enhanced GFP; iNOS, inducible NO
synthase; LB, Luria-Bertani; MAdCAM-1, mucosal addressin cell adhesion molecule-1; mono/mac, monocyte/macrophage; MLN, mesenteric lymph node; PP, Peyers patch.

Copyright 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00


www.jimmunol.org

that Gr-1intCCR2highCX3CR1low monocytes are more immature


monocytes newly released from bone marrow that undergo a maturation process and become Gr-1lowCCR2lowCX3CR1high monocytes (8, 14). In addition to maturation, monocytes, under the influence of local stimuli, have the potential to differentiate into
dendritic cells (DC), macrophages, and Langerhans cells (3, 16
25). Thus, recent in vivo studies of monocytes are beginning to
elucidate the complex nature of this pivotal mononuclear phagocyte population in steady-state and inflammatory conditions. However, relatively little is known about the role of the recently identified monocyte subsets in bacterial infections, and no information
is yet available on the function of these populations in the GALT
after oral bacterial challenge.
Salmonella enterica is an intracellular bacterial species that contains several serovars of food and waterborne pathogens, such as
the human pathogen serovar Typhi and its cousin serovar Typhimurium (Salmonella enterica serovar Typhimurium). Although S.
enterica serovar Typhimurium causes a localized infection of the
gastrointestinal tract in humans, it causes a systemic infection resembling typhoid fever in mice. After the oral infection of mice, S.
enterica serovar Typhimurium (or Salmonella for brevity) penetrates the intestinal epithelium using M cell-dependent and independent pathways (26). The first organs targeted by orally acquired
Salmonella are the Peyers patches (PP) and the mesenteric lymph
nodes (MLN) (27, 28), the lymphoid organs that drain the intestine
and initiate immune responses to gastrointestinal Ags. Although
the PP are directly seeded by bacteria that cross the intestinal epithelium, bacteria arrive in the MLN via intestinal lymph, likely
transported by DC (26, 29). The bacteria also spread to the spleen
and liver via the blood (29). The control of Salmonella early during
infection is dependent on IL-12, IFN-, and TNF-, molecules that
enhance the microbicidal capacity of phagocytes, and mice lacking
any of these cytokines are more susceptible to infection (30). The

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Neutrophils, monocytes, and dendritic cells (DC) are phenotypically and functionally related phagocytes whose presence in infected tissues is critical to host survival. Their overlapping expression pattern of surface molecules, the differentiation capacity of
monocytes, and the presence of monocyte subsets underscores the complexity of understanding the role of these cells during
infection. In this study we use five- to seven-color flow cytometry to assess the phenotype and function of monocytes recruited to
Peyers patches (PP) and mesenteric lymph nodes (MLN) after oral Salmonella infection of mice. The data show that CD68highGr1int (intermediate) monocytes, along with CD68intGr-1high neutrophils, rapidly accumulate in PP and MLN. The monocytes have
increased MHC-II and costimulatory molecule expression and, in contrast to neutrophils and DC, produce inducible NO synthase.
Although neutrophils and monocytes from infected mice produce TNF- and IL-1 upon ex vivo culture, DC do not. In addition,
although recruited monocytes internalize Salmonella in vitro and in vivo they did not induce the proliferation of OT-II CD4 T
cells after coincubation with Salmonella expressing OVA despite their ability to activate OT-II cells when pulsed with the OVA323339
peptide. We also show that recruited monocytes enter the PP of infected mice independently of the mucosal addressin cell adhesion
molecule-1 (MAdCAM-1). Finally, recruited but not resident monocytes increase in the blood of orally infected mice, and MHC-II
up-regulation, but not TNF- or iNOS production, occur already in the blood. These studies are the first to describe the accumulation and function of monocyte subsets in the blood and GALT during oral Salmonella infection. The Journal of Immunology,
2007, 178: 5789 5801.

5790

MONOCYTE RECRUITMENT DURING ORAL Salmonella INFECTION

Materials and Methods


Mice
C57BL/6 mice were purchased from Charles River Laboratories. IFN/, IL-12p40/, RAG/ mice and OT-II mice, all backcrossed more
than nine generations on the C57BL/6 background, were bred at the Experimental Biomedicine Animal Facility of Goteborg University, Goteborg, Sweden. Mice were used at 8 to 12 wk of age and provided with food
and water ad libitum. All experiments were performed using protocols
approved by the Swedish governments animal ethical committee and followed institutional animal use and care guidelines.

Bacterial strains
The S. enterica serovar Typhimurium 3181, SR11 derivative 4666 (40)
was used for the studies shown in Fig. 1, A and B, and Figs. 3, 5, and 8. S.
enterica serovar Typhimurium SL1344 and its enhanced GFP (eGFP)-expressing derivative SMO22 were used where bacterial uptake in vivo was
examined (see Fig. 4) (41). Strain 14028r (42) expressing OVA (43) was
used for cytokine production and bacterial uptake experiments (Fig. 2,
CE, and Fig. 6B). Strain 4550 expressing OVA-GFP or GFP (44) was
used for in vitro infection in the Ag presentation assays (see Fig. 7). Strain
8554 (SR11 asdA16 rpsL hisG) was used for in vivo infection of mice
in Fig. 2, C and D, and Figs. 6, 7, and 9. Bacteria were grown in Millers

Luria-Bertani (LB) broth or Lennox (8554) broth overnight at 37C. For


SL1344 and SMO22 the medium was supplemented with antibiotics
(SL1344, 100 g/ml streptomycin; SMO22, 50 g/ml kanamycin and 100
g/ml streptomycin). 14028r/OVA was grown on agar supplemented with
50 g/ml carbenicillin. The bacterial suspension was diluted and the OD
was measured at 600 nm. After centrifugation, the bacteria were resuspended at the appropriate concentration in sterile PBS. The actual bacterial
dose administered was determined by the serial plating of bacteria on LB
agar plates.

Infection of mice
Mice were given 0.1 ml of 1% NaHCO3 intragastrically 10 min before
infection. C57BL/6 or RAG/ mice were then infected intragastrically
with 2 8 107 bacteria in 200 l of PBS. IFN-/ and IL-12/ mice
are more susceptible to Salmonella and were given 5 8 106 bacteria in
200 l PBS intragastrically. Only IFN-/ and IL-12/ mice with a
bacterial load similar to that of wild-type mice were included. In experiments where cell interactions with eGFP-expressing bacteria in vivo were
studied (Fig. 4), mice were infected intragastrically with 109 SMO22 or
SL1344 bacteria in 200 l of PBS. In experiments using 8554 (Fig. 2,
CE, and Figs. 6, 7, and 9), mice were infected intragastrically with 8
108 bacteria in 200 l of PBS. Mice were sacrificed after 25 days and the
bacterial load in PP, MLN, spleen, and blood was determined by plating
serial dilutions of single cell suspensions on LB or Lennox (8554) agar
plates.

Cell preparation
Mice were sacrificed 25 days postinfection and the spleen, MLN, and PP
were removed. Blood was collected from the heart by vascular perfusion
with 5 ml of PBS containing 4 mM EDTA. The organs were digested with
0.45 mg/ml Liberase CI (Roche) in HBSS for 30 min at 37C and pipetted
into a single cell suspension or pressed through nylon mesh (TP filter).
Splenocytes and blood were treated with 0.14 M NH4Cl for 5 min at room
temperature once or twice, respectively, to lyse RBC, and debris was removed by filtration. Cells were then washed three times with HBSS and
resuspended in RPMI 1640 (Invitrogen Life Technologies) with heatinactivated 10% FBS (PAA Laboratories). The total number of viable
cells per organ was determined by trypan blue exclusion. For intracellular cytokine staining, 2 106 cells/ml were resuspended in 5 ml of
complete RPMI 1640 supplemented with 10% FBS and incubated at
37C in the presence of 5 g/ml brefeldin A (Sigma-Aldrich) for 4 h.
For bacterial uptake studies in vivo, CD11b-expressing cells were enriched from PP and MLN pooled from 25 mice by magnetic separation
using autoMACS (Milteny Biotech) after incubation with anti-CD11b
beads (clone M1/70), (Milteny Biotech) according to manufacturers
protocol.

Flow cytometry
Single cell suspensions were washed in FACS buffer (HBSS containing 3%
FBS, 1 mM EDTA, and 10 mM HEPES) that was used throughout the
surface staining procedure. Fc receptors on cells were blocked by incubating with the anti-FcRII/III mAb 2.4G2 for 15 min at 4C. Cells were then
stained for 20 min at 4C with the following mAbs that were biotinylated
or conjugated to FITC, PE, allophycocyanin, allophycocyanin-Cy7, PECy7, Pacific Blue, or Qdot605: anti-CD11c (clone HL3), anti-Gr-1 (clone
RB6-8C5), anti-CD11b (clone M1/70), anti-F4/80 (clone CI:A3-1), antiMHC-II (clone M5/114.15.2), anti-CD80 (clone 16-10A1), anti-CD86
(clone GL1), anti-CD62L (clone MEL-14), anti-NK1.1 (clone PK136), antiCD19 (clone 1D3), anti-B220 (clone RA3-6B2), anti-TCR -chain (H57597), anti-CD4 (clone GK1.5), anti-Ly6C (clone AL-21), and anti-Ly6G
(clone 1A8). The following anti-chemokine receptors were used: antiCCR2 (clone MC21), anti-CCR6 (clone 1C12), anti-CXCR2 (clone
242216), and anti-CXCR3 (clone 220803). The Abs rat IgG1 (clone 8R334), IgG2a (clone R35-95), and IgG2b (clone A95-1) were used as isotype
controls and biotinylated mouse anti-ratIgG2a as the secondary Ab. All
mAb except anti-F4/80 (Caltag Laboratories), anti-CXCR2 and antiCXCR3 (R&D Systems), and anti-CCR2 (generously provided by M.
Mack, University of Regensburg, Regensburg, Germany), were purchased
from BD Pharmingen. Anti-CD4 (clone GK1.5), purified in house, was
conjugated to Qdot605 (Quantum Dot Corporation), and anti-Ly6G was
conjugated to Pacific Blue (Molecular Probes) according to the manufacturers protocol. 7-Aminoactinomycin D (7-AAD; Sigma-Aldrich) was included in all stainings to define viable cells.
For staining with anti-CCR2 and anti-CCR6, cells were first blocked
with 10% normal mouse serum (Sigma-Aldrich) for 15 min and stained
with the primary Ab for 45 min. After washing, cells were incubated with
a secondary biotin-conjugated Ab for 20 min, washed, and blocked with

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production of reactive nitrogen intermediates via the inducible NO


synthase (iNOS) is also important to controlling Salmonella infection (30).
Studies in mice made neutropenic with a depletion strategy using mAb RB6-8C5, which primarily recognizes Ly6G but also
cross-reacts with Ly6C (31), have concluded that neutrophils are
important for host survival to Salmonella (32, 33). Although this
general conclusion is not debated, these studies may also have
depleted monocytes, particularly inflammatory monocytes that express Ly6C and are recognized by RB6-8C5 mAb (3, 7, 8, 34).
Similarly, studies addressing the role of macrophages during Salmonella infection obtained in studies using chemical depletion
methods (35) should be considered with the caveat of monocyte
depletion (8) and the possible effects on bystander phagocytes.
Although the importance of mononuclear phagocytes in host
survival to Salmonella infection is not disputed, the related phenotypic and functional nature of neutrophils, monocytes, and tissue
macrophages (5, 34, 36 39), combined with the recent data revealing monocyte subsets with distinct roles (3, 7, 8), underscores
the need for precise analysis of these populations during infection
with this intracellular pathogen. This is particularly true for the
organs that are the immediate targets of Salmonella after penetration of the intestinal epithelial barrier, the PP, and the MLN.
These issues are addressed here by performing direct ex vivo
five- to seven-color flow cytometry analysis of cells accumulating
in the PP and MLN during the first few days of oral Salmonella
infection of mice. We characterize the influx of monocytes and
neutrophils into the gut-draining lymphoid tissues and show that
recruited monocytes are the major producers of iNOS and TNF-,
most of which is made by uninfected bystander monocytes. We
also examine the capacity of the different myeloid lineage populations recruited to infected PP and MLN to internalize and kill
Salmonella in vitro and in vivo, produce TNF-, IL-1, and IL-12
upon ex vivo culture, and induce the proliferation of OT-II CD4
T cells after coincubation with Salmonella expressing OVA or
pulsed with OVA peptide. In addition, chemokine receptor expression
on the recruited myeloid lineage populations is assessed and the role
of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in recruiting monocytes to infected PP is analyzed. Finally, infection-induced recruitment and activation of monocyte subsets in the blood of
orally infected mice is examined. These studies are the first to characterize recruited monocytes in PP and MLN after oral Salmonella
infection and assess the number and function of their counterparts in
the blood of orally infected mice.

The Journal of Immunology

5791

Bacterial survival assay and Ag processing and presentation


assays
Cell suspensions were made from the MLN, spleen, and blood pooled from
10 mice at day 4 postinfection as described above. Cells from the spleen
and blood were subsequently pooled (to get enough cells to work with) and
depleted of B, T, and NK cells by magnetic separation using autoMACS
(Milteny Biotech) after incubation with a mixture of anti-CD19 beads,
anti-CD90 (Thy1.2) beads, and anti-NK (DX5) beads according to the
manufacturers protocol (Milteny Biotech). MLN cells were likewise depleted of T, B, and NK cells. After staining for flow cytometry, cells were
sorted into CD11chigh DC, Ly6Chigh monocytes, Ly6Clow monocytes, and
Ly6Ghigh neutrophils using a FACSAria flow cytometer (BD Biosciences)
and DIVA software (BD Biosciences). The different cell populations were
seeded at 1.5 105 cells/well in round-bottom 96-well plates in complete
medium (RPMI 1640 Glutamax-1 containing 10% FBS (PAA Laboratories), 2 mM MEM sodium pyruvate, 20 mM HEPES, 0.05 mM 2-ME. and
2.5 g/ml fungizone (all from Invitrogen Life Technologies)). For bacterial
survival assays, sorted Ly6Ghigh neutrophils and Ly6Chigh monocytes were
infected with strain 14028r, centrifuged at 270 g for 4 min, and incubated for 2 h at 37C. The bacteria-to-cell ratio was between 5:1 and 18:1.
After washing three times in complete medium containing 80 g/ml gentamicin, the cells were resuspended in the medium and incubated at 37C
for an additional 1 or 18 h. Cells were lysed with 0.2% Triton X-100 with
vigorous pipetting and incubated at room temperature for 10 min. The
supernatant was then serially diluted and plated onto LB agar plates. Bacterial colonies were counted after overnight incubation at 37 C and the
total number of bacteria recovered was calculated. As controls for determining the number of bacteria not internalized by the cells but viable in the
supernatant, cells were preincubated with 20 g/ml CCD (cytochalasin D)
1 h before the addition of bacteria and present for the initial 2 h of bacterial
infection.
For Ag-presenting assays, titrated numbers of 4550-OVA-GFP or
4550-GFP (no OVA) were added to the cells. Alternatively, the OVA323339
peptide was added at a concentration of 0.1, 1, or 10 g/ml. Cells were
then centrifuged for 270 g for 4 min and incubated at 37 C. After 2 h,
FIGURE 1. Populations differentially expressing Gr-1 and CD68 rapidly accumulate in the MLN and PP of orally infected mice. Cells from
MLN (AC and E) and PP (D) of naive mice and mice infected 5 days
earlier were stained with 7-AAD, anti-CD68, anti-Gr-1, anti-CD11c,
and one of the molecules shown in the histograms (E). Viable (7AAD) cells were then analyzed by five-color flow cytometry. A, Based
on their expression of Gr-1 and CD68, cells were gated into CD68intGr1high (R1) and CD68highGr-1int (R2) cells. B, Dendritic cells were defined as CD11chighGr-1low (R3) cells. C and D, cells from MLN (C) or
PP (D) were gated as viable (7-AAD) R1 and R2 cells as in A and the
absolute number (MLN) and the percentage (PP) of the gated cells were
examined on days (d) 3 and 5 postinfection. Bacterial recovery from the
indicated organ on days 2, 3, and 5 postinfection is shown on the right.
Each time point represents the mean value for a total of 6 7 mice

examined in two independent experiments. Error bars indicate SD. E,


Histograms show surface expression of the indicated molecules on gated
R1, R2, and R3 cells from naive mice and from mice 5 days postinfection.
The upper and lower numbers in the histograms are the mean fluorescence
intensity of gated cells from infected and naive mice, respectively. The
paucity of cells within the R1 gate in naive mice (A) made it impossible to
accurately assess the level of expression of the molecules, and therefore
naive levels were not determined in the R1 histograms. Dashed lines represent appropriate isotype control mAb. In A, B, and E similar results were
obtained from PP (not shown). The data are representative of two or three
independent experiments that examined a total of at least six mice per
group.

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the FcRII/III mAb 2.4G2 for 15 min. Finally, the cells were incubated
with the additional mAbs to stain surface molecules and with streptavidinallophycocyanin for 20 min.
When intracellular staining was performed, the samples were washed
after surface staining and fixed in 2% formaldehyde in PBS for 20 min at
room temperature. After the cells were washed in permeabilization buffer
(HBSS containing 0.5% saponin and 0.5% BSA (Sigma-Aldrich)), which
was subsequently used throughout the staining procedure, they were incubated for 30 min at room temperature with one or more of the following
mAbs: anti-TNF- (clone MP6-XT22), anti-IL12p40 (clone C15.6), antiIFN- (clone 37895.11) (all purchased from BD PharMingen), anti-CD68
(clone FA/11; Serotec), S. enterica serovar Typhimurium O-4 (clone 1E6;
Abcam), and the appropriate isotype control or iNOS Ab (M-19) (Santa
Cruz Biotechnology). Staining with rabbit anti-iNOS was followed by incubation with allophycocyanin-conjugated anti-rabbit IgG (Santa Cruz
Biotechnology) for 30 min at room temperature. An anti-Salmonella O-4
mAb was conjugated to Pacific Blue according to the manufacturers
protocol.
Cells or bacteria (strains SMO22 and SL1344) were acquired on an
LSR-II flow cytometer (BD Biosciences) using DIVA software (BD
Biosciences) and analyzed using Flow Jo software (Tree Star). Whereas
the absolute number of the cell populations is reported for the MLN
and the spleen, the number and size of PP differs in individual mice and
thus the percentage rather than the absolute number of cells in a given
population was calculated.

5792

MONOCYTE RECRUITMENT DURING ORAL Salmonella INFECTION


the cells were washed three times in complete medium containing 80
g/ml gentamicin and resuspended in 100 l of medium with gentamycin.
Finally, 2 105 MACS-purified, CFSE-labeled OVA323339-specific
CD4 T cells from OT-II mice were added. The OT-II CD4 T cells were
negatively purified from pooled spleens plus MLN from naive mice using
a CD4 isolation kit (Milteny Biotech) according to the manufacturers protocol and were labeled with CFSE. For CFSE labeling, cells were incubated with 5 M CFSE (Molecular Probes) in PBS for 8 min at room
temperature. After 3.5 days of coculture at 37C, cells were harvested,
stained with anti-CD4 and anti-TCR, and the percentage of divided CD4
OT-II T cells was determined by flow cytometry.

Measurement of cytokines by ELISA

Adoptive transfer and blocking experiments


Donor monocytes and neutrophils were negatively enriched using
autoMACS and anti-CD49b (clone DX5) beads (Milteny Biotech) from
pooled splenocytes and the blood of 10 15 RAG/ mice infected 3
days earlier with Salmonella 4666. Donor T cells were negatively
enriched from the pooled spleen and MLN of naive C57BL/6 mice
using autoMACS and a CD4 isolation kit (Milteny Biotech). The donor
monocytes, neutrophils, and T cells were then pooled and CFSE labeled
as described above. Cells (10 15 106) containing 35% T cells and
39% CD11bNK1.1 cells were injected i.v. into C57BL/6 mice that
were infected 3 days earlier with 4666. Some of the recipient mice
were given 150 g of the anti-MAdCAM-1 mAb MECA-367 (45) (BD
Pharmingen) i.v. 6 8 h before cell transfer. Control animals received
either 150 g of the isotype control mAb 9B5 (anti-human CD44;
kindly provided by A. Hanninen (University of Turku, Turku, Finland)
or nothing. Twelve to 16 h after adoptive transfer, PP, MLN, spleen,
and blood were removed from recipient mice and stained for flow cytometry as described above.
FIGURE 2. Mono/mac are the main producers of TNF- and iNOS
during early infection with S. enterica serovar Typhimurium. A and B,
cells from the MLN of naive mice or mice orally infected (Inf) 4 days
earlier were stained with 7-AAD and anti-CD68, anti-Gr-1, anti-CD11c,
anti-TNF-, and anti-iNOS and analyzed by six-color flow cytometry.
Intracellular TNF- and iNOS staining of mono/mac (R2 in A), neutrophils (neutr) (R1 in B), and DC (R3 in B), defined as in Fig. 1 were
stained directly ex vivo at day 4 postinfection. The percentages of
TNF-, iNOS, and double positive cells are indicated in the quadrants.
The right dot plot in A shows staining of mono/mac (R2) from naive
mice. The staining of R1, R2, and R3 cells with isotype-matched control
mAb for TNF- and Ab for iNOS resulted in 1% and 0.1% positive
cells, respectively, for all three populations (not shown). The number of
neutrophils (R1) in naive mice was too low to quantify TNF- and
iNOS production. The results shown are representative of five independent experiments with a total of 10 mice. Complete data and statistical
analysis are shown in Table I. CE, TNF- (C) and IL-1 (D and E)
production by monocytes, neutrophils, and DC sorted from infected
mice and cultured ex vivo. Mice were orally infected with Salmonella
and 4 days later cells from the spleen and blood (C and D) or MLN (E)
of 10 mice were pooled and depleted of NK1.1, CD19, and TCR
cells by MACS. Cells were stained for the surface expression of CD11b,
CD11c, Ly6C, and Ly6G and sorted into Ly6ChighLy6Glow monocytes
(purity 93%) and Ly6CintLy6Ghigh neutrophils (purity 91%) as
shown in Fig. 6 and CD11chigh DC (purity 80%) as shown in Fig. 7A
(right dot plot). The sorted cells were incubated in medium alone (unstim, unstimulated; open bars) or pulsed with Salmonella for 2 h (restim,
restimulated; black bars), washed extensively, and incubated in medium
containing gentamicin. Supernatant was collected at 20 h for TNF-

Results
Phagocytic cells accumulate in the GALT after oral infection
The gut-draining lymphoid tissues PP and MLN are the first organs
seeded by Salmonella after oral infection (27). However, little is
known about the changes in the number, phenotype, and function
of phagocytic cell populations, particularly the recently described
monocyte subsets, responding to this oral pathogen in these organs. To address these issues, mice were orally infected with Salmonella and cells from PP and MLN were analyzed by five- to
seven-color flow cytometry early during infection. Populations
with differential expression of CD68 and Gr-1 responding to oral
infection were apparent (Fig. 1). In particular, two populations that
were relatively rare in naive mice, CD68intGr-1high (where int stands
for intermediate) and CD68highGr-1int cells (gate R1 and R2, respectively, in Fig. 1), increased dramatically in the MLN and PP of infected mice starting at day 3 postinfection (Fig. 1, A and C and D).
Although the increase in both populations was large, CD68highGr-1int
(R2) cells remained more abundant than gated R1 cells and comprised
13% of total cells per organ at day 5 postinfection.

and at 36 h for IL- and was measured by ELISA. Results are expressed
as mean SEM (pg/ml) of 13 independent experiments with duplicate
samples. MACS-purified CD11b cells from naive mice are shown as a
naive control. ND, not detected.

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Sorted Ly6Ghigh neutrophils, Ly6Chigh monocytes, and CD11chigh DC were


resuspended in medium or infected for 2 h with 4550-OVA or 14028r,
washed, and treated as described for bacterial survival assays. After 20 36
h, as indicated in the appropriate figure legends, the plates were centrifuged
and the supernatants were frozen at 20C until assayed for cytokine content. The presence of TNF-, IL-1, and IL-12p70 in supernatants was
determined by ELISA following the manufacturers protocol (BD Biosciences). To determine naive cytokine levels, CD11b-expressing cells
were enriched from the spleen of naive mice by autoMACS after incubation with anti-CD11b beads (clone M1/70). The CD11b cells were seeded
at 2 105 cells/well in 96-well plates and incubated for 48 h before
supernatants were frozen at 20C.

The Journal of Immunology


Table I.

5793

Production of iNOS and TNF- in phagocytic cells of naive or Salmonella-infected mice at day 4 postinfection
PP

Cell population

iNOS

TNF-

MLN
a

DP

iNOS

TNF-

Spleen
a

DP

iNOS

TNF-a

DPb

Neutrophils (R1)
Nave
0.4 0.9
10.2 7.6
#
0.2 0.4
5.6 2.9
#
0.5 0.4
1.5 0.8
#
Infected
2.6 3.4 10.8 7.7
#
4.0 4.0 10.4 4.0
#
2.1 2.7 8.4 9.8
#
Mono/mac (R2)
Nave
1.3 1.6
7.9 5.3
#
0.7 0.8
7.1 4.5
#
0.3 0.2
4.6 3.5
#
Infected
31.6 13.4 28.9 10.6 8.9 2.9 19.7 10.0
26.2 5.0
4.8 3.0 19.2 8.9
20.4 10.2 5.2 2.3
DC (R3)
Nave
0.8 1.3
0.8 0.4
#
0.4 0.4
1.9 0.9
#
0.0 0.0
(40)
NDc
Infected
10.6 6.2 1.4 0.5
#
2.9 2.5 3.6 2.5
#
2.2 2.3
(40)
NDc
a
The mean percentage of positive cells for either iNOS or TNF- within the respective cell population SD of groups of 217 mice from 2 4 independent experiments
is shown.
b
DP, Cells double positive for both TNF- and iNOS.
c
ND, Not done.
#; Too few cells (50) to accurately determine; and , p 0.05 and p 0.005, respectively, when compared with infected mono/mac. and , p 0.05 and p 0.005,
respectively, when compared with the same population of cells from naive mice. All p values were calculated using the Mann-Whitney test.

NK1.1 (data not shown). B cells (CD19) and T cells (TCR


increased slightly in MLN and PP early during oral infection. In
MLN, the absolute number of B and T cells increased 1.5-fold and
1.2-fold, respectively, at day 5 postinfection while the number of
NK cells (NK1.1TCR) remained constant (data not shown).
Together, the data in Fig. 1 show that the predominant cellular
change that occurs in the GALT after oral Salmonella infection is
the rapid accumulation of cells with a phenotype distinct from
neutrophils and DC. Moreover, the phenotype of R2 cells suggests
they may be monocytes recruited from the blood, as has been
shown in peritoneal or i.v. inflammation models and in lymph
nodes draining inflamed skin (1 4, 6 8, 37). Because monocyte
refers to a population circulating in the blood and macrophage
indicates a tissue resident differentiated cell, we refer to the cells
that accumulate in infected PP and MLN as monocyte/macrophage
(mono/mac).
The mono/mac that accumulate in PP and MLN are the main
producers of TNF- and iNOS during infection
To assess the function of the myeloid cells recruited to PP and
MLN during oral Salmonella infection, their capacity to produce
TNF- and iNOS, effector molecules important in controlling Salmonella (30), were assessed by direct ex vivo double intracellular
staining. Mono/mac producing TNF- alone, iNOS alone, or both
simultaneously were induced by Salmonella infection (Fig. 2A and
Table I). Mono/mac were the predominant producers of these
anti-bacterial molecules, accounting for 50 70% of all TNF-
and 80 90% of all iNOS cells in MLN. Moreover, 20 25% of
the mono/mac that stain positive for iNOS were also positive
for TNF-.
In contrast to mono/mac, very few neutrophils or DC stained
positive for iNOS (Fig. 2B and Table I). Similar results were also
found in the PP and spleen of orally infected mice (Table I). Some
increase in cells staining positive for intracellular TNF- was apparent among neutrophils and DC in infected mice (Table I).
We also determined the ability of sorted monocytes, neutrophils,
and DC from Salmonella-infected mice to secrete TNF-, IL-1,
and IL-12p70 after 20 h (TNF-) or 36 h of ex vivo culture.
TNF- and IL-1 were detected in the supernatant of cultured
monocytes and neutrophils, whereas DC production of these cytokines was not apparent (Fig. 2, CE). Little if any increase in the
amount of TNF- was detected upon the restimulation of sorted
monocytes with bacteria, and no increase was detected upon DC
restimulation (data not shown). In contrast, the restimulation of
monocytes or neutrophils with bacteria during the ex vivo culture

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To further characterize the two populations responding to oral


Salmonella infection, the expression of a number of surface and
costimulatory molecules was examined (Fig. 1E). The gated
CD68intGr-1high (R1) cells in infected mice lacked F4/80 and
CD11c expression but had a very high level of CD11b. Few cells
within this gate were positive for MHC-II, CD80, or CD86 at day
5 postinfection and the positive cells likely represent some spillover of R2 cells, as the junction between R1 and R2 gates in
infected tissues becomes less absolute than in naive animals (Fig.
1A). A fraction of R1 cells were positive for CD62L. Together,
these data suggest that cells in the R1 gate are neutrophils (34, 38).
Although most cells within the R2 gate of infected MLN were
CD11bhigh, they had several features that distinguished them from
R1 cells. This included the expression of F4/80 and a significant
level of MHC-II by the majority of the gated cells. R2 cells from
infected mice had increased CD80 and CD86 expression compared
with naive mice, a fraction of the cells were CD62L, and some
expressed a low level of CD11c (Fig. 1E). Thus, infection-induced
R2 cells are phenotypically distinct from R1 cells.
Although the very low level of CD11c on gated R2 cells suggested that they were not conventional DC, which in the mouse are
characterized by high surface expression of CD11c, MHC-II, and
costimulatory molecules (46), the phenotype of gated R2 cells was
compared with that of the CD11chigh population in the same infected individual (Fig. 1, B and E). Consistent with previous reports, the CD11chigh cells expressed high levels of MHC-II, CD80,
and CD86. Both myeloid and lymphoid DC are included in this
gate as evidenced by the biphasic expression pattern for CD11b
(Fig. 1E), which corresponds to the CD8 and CD8 subsets
(Ref. 46 and references therein). Moreover, additional studies
showed that 20% of the CD11chighCD11blow DC express CD8,
which is consistent with previous data (46 48). Although most of
the DC expressed CD68 (data not shown), their low Gr-1 expression and the nonoverlapping gating for Gr-1 on R2 vs R3 cells
exclude DC from the R2 gate (Fig. 1, A and B). Instead, most
CD68highGr-1low cells in the PP and MLN of both naive and infected mice (Fig. 1A, cells below the R2 gate) were DC expressing
high levels of CD11c and MHC-II (data not shown). Furthermore,
very few plasmacytoid DC identified as TCRCD19NK1.1
CD11bCD11cintB220Ly6C (48, 49) were detected in naive
MLN (0.06%), and they decreased to 0.01% of the total population in infected MLN. In addition, the CD11bCD11cint phenotype of plasmacytoid DC (48, 49) excludes them from the R1R3
gates. Less than 10% of R1, R2, and R3 cells at day 5 postinfection
were positive for the lymphoid markers CD19, TCR, and

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MONOCYTE RECRUITMENT DURING ORAL Salmonella INFECTION


resulted in enhanced IL-1 production (Fig. 2, D and E). IL-12p70
was not detected in the supernatant of any of the sorted cell types
cultured ex vivo with or without Salmonella (data not shown).
Likewise, an increase in monocytes from the MLN or spleen of
mice infected 3 or 5 days earlier with Salmonella that stained positive for intracellular IL-12p40 above the level detected in naive
mice was not detected. Despite the fact that IL-12p70/IL-12p40
has a role during Salmonella infection (30), the cellular source(s)
of this cytokine remain elusive.
Thus, mono/mac, defined as CD68highGr-1int cells, express
MHC-II and costimulatory molecules (Fig. 1E) and are the main
sources of TNF- and iNOS early during Salmonella infection.
Moreover, the majority of mono/mac in infected tissues produce
TNF- or iNOS rather than both simultaneously.
Impaired expression of iNOS and MHC-II but not TNF- in
infected IFN-/ and IL12p40/ mice

Uptake of eGFP-expressing Salmonella by phagocytic cells


To examine the relative uptake of bacteria by mono/mac and neutrophils in the same individual, mice were orally infected with
Salmonella expressing eGFP and bacteria-associated cells were
identified by flow cytometry. To get enough events to accurately
assess the phenotype of eGFP cells, CD11b cells were first
enriched by positive selection using MACS. In addition, an analysis of eGFP fluorescence negated using FITC-conjugated antiCD68 (Fig. 1A), so an alternate strategy using CD11b, Gr-1, and
F4/80 was used to identify the phagocytic cells (Fig. 4A) (7). To
conclude that this strategy allowed identification of the neutrophil

cells (NK and NKT cells) and T cells (TCRNK1.1) gated as in the
upper dot plot in PP and MLN at day 4 postinfection (upper panel) is
shown. Staining for IFN- in IFN-/ mice is used as a control (lower
row). AE Data were obtained in at least three independent experiments
and each group contains 4 16 mice.

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FIGURE 3. Defective iNOS and MHC-II but not TNF- production


in mono/mac from infected IFN-/ and IL12p40/ mice. Wild-type
(WT), IFN-/, and IL12p40/ mice were orally infected with Salmonella and 4 days (PP and MLN) or 5 days (spleen) later cells were
stained with 7-AAD, anti-CD68, anti-Gr-1, anti-CD11c, and either antiTNF- and anti-iNOS (A and B) or anti-MHC-II (D) and analyzed by
six- (A and B) or five-color (D) flow cytometry. Mono/mac and DC were
gated as in Fig. 1A. A, The dot plots show intracellular staining for
TNF- and iNOS on cells from the MLN of wild-type, IFN-/, and
IL12p40/ mice with a similar bacterial burden (shown in C). The
numbers represent the percent positive cells in the quadrant. B, TNF-
(left) and iNOS (right) production by mono/mac in the PP, MLN, and
spleen of infected wild-type, IFN-/, and IL12p40/ mice with a
similar bacterial burden (shown in C). TNF- and iNOS production in
naive mice is shown in Table I. Error bars indicate SD. The p values
(, p 0.01; , p 0.001) are from the Mann-Whitney U test for
infected C57BL/6 mice compared with infected IFN-/ or
IL12p40/ mice. C, Bacterial recovery from PP, MLN, and spleen 4
days postinfection. D, MHC-II expression by mono/mac in PP and
MLN (two left histograms) and DC in MLN (right histogram) in wildtype, IFN-/, and IL12p40/ mice with a similar bacterial burden
(shown in C) at day 4 postinfection. E, IFN- production by NK1.1

We next asked whether iNOS and TNF- produced by recruited


mono/mac early during infection depended on IFN- and IL-12.
First, the accumulation of mono/mac and neutrophils in the PP,
MLN, and spleen of infected IFN-/ and IL-12p40/ mice
with a bacterial burden similar to that of infected wild-type mice 4
days postinfection was not compromised (data not shown). Likewise, the frequency of mono/mac producing TNF- in PP, MLN,
and spleen was the same in wild-type, IFN-/, and IL-12p40/
mice with a similar bacterial burden (Fig. 3, AC). In contrast,
iNOS production by mono/mac in all three organs in infected IFN/ and IL-12p40/ mice was nearly absent (Fig. 3, A and B).
We also wished to determine whether the infection-induced increase in MHC-II on the mono/mac of infected wild-type mice was
IFN--dependent. Indeed, Salmonella-induced MHC-II up-regulation on mono/mac was severely compromised in IFN-/ and
IL-12p40/ mice (Fig. 3D). This is in contrast to MHC-II expression on DC, which was similar and high on CD11chigh (R3)
cells in infected wild-type and knockout mice. Similar results were
seen in the spleen (data not shown). We next examined the cellular
source(s) of IFN- in the PP and MLN responsible for infectioninduced mono/mac activation. These data revealed that NK and
NKT cells (NK1.1TCR) and some T cells (TCRNK1.1)
are the main producers of IFN- 4 days postinfection (Fig. 3E).
Thus, the induction of iNOS and MHC-II expression but not
TNF- production by mono/mac during oral Salmonella infection
is dependent on IL-12 and IFN-, and NK/NKT cells and T cells
are the predominant sources of IFN- in PP and MLN early during
infection.

The Journal of Immunology

and mono/mac cells studied above, CD11bGr-1highF4/80 and


CD11bGr-1intF4/80 were backgated into a CD68 vs Gr-1 plot
(Fig. 4B). This showed that the F1 and F2 populations defined by
CD11b, Gr-1, and F4/80 identified the majority (6395% in three
independent experiments) of mono/mac (R1) and neutrophils (R2),
respectively, with little cross-contamination between the two populations (Fig. 4, A and B) (7).
The results in Fig. 4C show a slightly higher fraction of eGFP
neutrophils (F1 cells) compared with mono/mac (F2) (1.6-fold
difference) in the PP and MLN of infected wild-type mice. In infected IFN-/ mice, very similar fractions of neutrophils and
mono/mac were eGFP in both PP and MLN (1.2-fold difference).
The somewhat higher fraction of eGFP cells in the MLN of infected IFN-/ mice compared with wild-type mice reflects the
higher number of bacteria recovered from IFN-/ mice in these
experiments (data not shown) due to the compromised capacity to
kill Salmonella in the absence of IFN- (30).
To investigate the accuracy of detecting cell-associated bacteria
in wild-type and IFN-/ mice using eGFP fluorescence as the
readout, two types of experiments were performed. First, the stability of eGFP in CD11b cells from infected wild-type and IFN/ mice was assessed. These data showed similar eGFP stability, as 94 6.3% (n 7) and 94 2.2% (n 6) of bacterial
colonies recovered from CD11b cells isolated from infected
wild-type and IFN-/ mice, respectively, were eGFP. Second,
the detection bacteria by eGFP fluorescence was compared with
the use of an anti-Salmonella O-4 LPS Ab. These studies revealed
that 1.3 4.9% of CD11b cells from PP and MLN pooled from
infected wild-type mice stained positive for O-4 reactivity, while
eGFP fluorescence was detected in 0.4 2% of the cells in the same
infected individuals. Identical percentages were obtained for infected IFN-/ mice. Although the down-regulation of eGFP fluorescence in vivo cannot be eliminated as a contributor to this
difference, the greater reactivity of an anti-LPS Ab is not unexpected because it will recognize LPS on intact bacteria as well as
bacterial degradation products and shed LPS. In contrast, the detection of bacteria using eGFP fluorescence relies on intact protein
emitting fluorescence, a method that will not detect bacteria degraded to a point that negates eGFP fluorescence.
Very few eGFP events were found among TCR or B220
cells in infected tissues of the same animals, demonstrating that
80% of all bacteria-associated cells were either neutrophils or
mono/mac (data not shown). The relationship between bacterial
uptake and effector molecule production was also assessed directly
ex vivo (Fig. 4D). Approximately 30 40% of eGFP CD11bGr1int F2 cells were also iNOS (data not shown), demonstrating the
tendency of bacteria-associated cells to produce this effector molecule. However, while 30 40% of all CD11bGr-1int F2 cells
expressed iNOS (Fig. 3B), only a minor fraction of iNOS or
TNF- CD11bGr-1 cells were eGFP (Fig. 4D). This suggests that most iNOS and TNF--producing cells are bystander
cells, as has been observed with other pathogens (5, 7, 12). In
were gated as in the left dot plot in A. eGFP expression on gated iNOS
(left) or TNF- (right) cells is shown and the percentage of cells is indicated in each plot. E, The histograms show the expression of MHC-II on
eGFP (open histogram) and eGFP (shaded histogram) CD11bGr-1int
cells (gated as in the dot plot) in PP of wild-type (WT) or IFN-/ mice
infected 4 days earlier with eGFP Salmonella. The dotted line represents
staining using the isotype-matched control mAb on eGFP cells (median
value 97162). The numbers show the mean fluorescence intensity for
eGFP (top) and eGFP (bottom) CD11bGr-1int cells in each plot. Cells
were pooled from 2 4 mice. Data are representative of three independent
experiments.

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FIGURE 4. Bacterial uptake in vivo by phagocytic cells in MLN and PP


at day 4 postinfection. Mice were orally infected with Salmonella expressing eGFP or the parental strain not expressing eGFP and after 4 days
CD11b cells were purified by MACS from MLN and PP. MACS-enriched cells were then stained with anti-CD11b, anti-Gr-1, and 7-AAD and
anti-F4/80, anti-MHC-II, anti-TNF-, anti-iNOS, or the appropriate control isotype and analyzed by five-color flow cytometry. A, CD11b-enriched
cells were gated into CD11bGr-1 cells and further gated into F4/80high
(F2) or F4/80low (F1) populations. B, These two populations were then
backgated into a CD68 vs Gr-1 plot to confirm that CD11bGr-1F4/80low
cells correspond to CD68intGr-1high (R1 cells) and that CD11bGr-1F4/
80high cells correspond to CD68highGr-1int (R2 cells). C, The percentage of
eGFP cells within gated neutrophils (F1) and monocytes (F2) in PP (left)
and MLN (right) of a Salmonella-infected mouse 4 days postinfection. The
upper row is from a wild-type mouse infected with Salmonella expressing
eGFP (Sal eGFP) while the middle row is a wild-type mouse infected with
the parental Salmonella strain not expressing eGFP (Sal). The lower row of
dot plots is from an IFN-/ mouse infected with Salmonella expressing
eGFP. Data are representative of five independent experiments. The MannWhitney U test comparing C57BL/6 or IFN-/ mice infected with Salmonella expressing eGFP to mice infected with Salmonella not expressing
eGFP was used for statistical analysis. The value of p 0.05 was obtained
for all groups. Samples contained 34 2550 eGFP events. D,
CD11bGr-1 cells from the MLN and PP of mice infected 4 days earlier

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MONOCYTE RECRUITMENT DURING ORAL Salmonella INFECTION

addition, bacterial uptake did not appear to influence the infectioninduced up-regulation of MHC-II on mono/mac either in wild-type
or IFN-/ hosts (see Fig. 4E for PP; MLN data not shown).
Similarly, bacterial uptake did not restore the ability of mono/mac
from infected IFN-/ mice to produce iNOS (Fig. 3A and data
not shown).
Accumulation of cells in the blood of orally infected mice
Having established that neutrophils and cells with a phenotype
suggesting a relationship to monocytes (3, 9, 13, 34, 50) (our socalled mono/mac population) accumulated and exerted effector
functions in the gut-associated lymphoid tissues of orally infected
mice, we asked when these cells appeared in the blood during
infection and whether they were activated in the circulation. To
this end, the populations defined in PP and MLN based on CD68
and Gr-1 expression were characterized in the blood of naive and
Salmonella-infected mice. CD68 vs Gr-1 staining on blood cells
revealed abundant R1 and R2 populations similar to those found in
the tissues (Figs. 5A and 1A). In naive mice, R1 and R2 were 8 and
4%, respectively, of all leukocytes in blood. Both R1 and R2 populations accumulated in the blood as the infection progressed, increasing 2-fold and 4-fold, respectively, at day 3 postinfection and
2.5-fold and 4.5-fold, respectively, at day 5 (Fig. 5, A and C).
To further characterize the cell populations in blood during infection and compare them with the ones found in infected tissues,
the expression of myeloid markers and costimulatory molecules on
the blood populations was analyzed (Fig. 5D). The phenotype of
gated Gr-1highCD68int (R1) cells in the blood was very homogenous and similar in both infected and naive mice (Fig. 5D). Essentially, all blood R1 cells expressed CD11b and CD62L but
lacked F4/80, CD11c, MHC-II, CCR2, and the costimulatory mol-

ecules CD80 and CD86. In addition, gated R1 cells (as well as R2


and R4 cells) were negative for CD19, TCR, and NK1.1 (data not
shown).
Like their tissue counterparts, blood CD68highGr-1int (R2) cells
from infected mice were positive for CD11b, F4/80, and CCR2
(Fig. 5D and data not shown). The adhesion molecule CD62L was
expressed on some blood R2 cells in naive mice, and the fraction
of CD62L cells increased in the blood of Salmonella-infected
mice. Similar to R2 cells in the MLN of infected mice, R2 cells in
the blood expressed much higher MHC-II in infected compared
with naive animals. In contrast, however, the up-regulation of
CD86 and particularly CD80 on R2 cells in the blood was modest
compared with that seen in infected MLN (Figs. 1E and 5D).
Cells consistent with the phenotype of murine DC present in the
tissues of naive and infected mice (CD11chighMHC-II cells) were
not found in the blood (Fig. 5B and data not shown) (51). However, two populations of monocytes, Gr-1CD62LCCR2 inflammatory and Gr-1CD62LCCR2 resident monocytes, have
been described in mouse blood (3). The Gr-1CD62LCCR2
phenotype of gated R2 cells is consistent with the phenotype of
inflammatory monocytes, while Gr-1CD62L cells in R4 share
features with resident monocytes (Fig. 5, A and D) (3). R2 cells
increased in the blood of infected mice while R4 cells did not (Fig.
5C). Moreover, most of the cells within the R4 gate, putative resident monocytes, express an intermediate level of CD11c and upregulate MHC-II during infection. This suggests a possible relationship to, or capacity to become, DC. Cells within gate R4
appear to be the best candidate for resident monocytes, as the
CD11cint cells in gate R5 in Fig. 5B were mostly (80%)
NK1.1CD11bint NK cells (data not shown), consistent with other

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FIGURE 5. Characterization of myeloid cells in the blood during Salmonella infection. Blood leukocytes from naive mice or from mice infected 4 days
earlier were stained with 7-AAD, anti-CD68, anti-Gr-1, anti-CD11c, and one of the molecules shown in the histograms and analyzed by five-color flow
cytometry. A, Using a gating strategy similar to that used for MLN (Fig. 1A), neutrophils were identified as CD68intGr-1high cells (R1) and monocytes as
CD68highGr-1int cells (R2). The gated R4 cells that are discussed in the text are also indicated. B, Dot plot of Gr-1 vs CD11c on cells from the blood of
a naive and infected mouse showing the absence of CD11chigh cells (R3) and the presence of CD11cint cells (R5). Data are representative of six naive and
10 infected mice examined in three independent experiments. C, Kinetics of R1, R2, and R4 cell accumulation in blood at days (d) 2, 3, and 5 postinfection.
Data are the mean of 4 15 mice per time point from at least two independent experiments. Error bars are the SD. At these time points 100 to a maximum
of 4000 bacteria were found in the blood collected from a mouse. D, Histograms show the expression of surface markers for the indicated cell populations
in infected (open histogram) and naive (filled histogram) mice. Dashed histograms indicate staining with a isotype-matched control mAb. The numbers
show the mean fluorescence intensity for infected (top) and naive (bottom) mice in each plot. All surface molecules were examined in at least two
independent experiments on a total of 2 4 naive and 4 8 infected mice. E, TNF- and iNOS production by R2 cells in the blood. The percentage of positive
cells for either TNF- or iNOS or for both is indicated. The data are representative of two independent experiments with a total of four naive and five
infected mice. The isotype control for TNF- showed 1% positive monocytes (not shown).

The Journal of Immunology

5797

reports (51, 52). The remaining NK1.1 cells in R5 were


CD68highGr-1lowCD11bint and fall within gate R4 in Fig. 5A.
Given the efficient activation of mono/mac to produce large
amounts of iNOS and TNF- and to up-regulate MHC-II in the PP,
MLN, and spleen during infection, we asked whether this activation already occurs in the blood of infected mice. As seen in Fig.
5D, MHC-II was readily up-regulated on R2 cells, putative inflammatory monocytes in blood. However, no Salmonella-induced
iNOS and little TNF- above the level seen in naive mice were
detected in this population (Fig. 5E). Together, the data in Fig. 5
show that two subsets of monocytes resembling resident (R4) and
inflammatory monocytes (R2) can be identified in the blood of
Salmonella-infected mice. R2 but not R4 monocytes increase in
the blood during Salmonella infection and both have increased
MHC-II expression but do not produce anti-bacterial effector molecules. Moreover, neutrophils increase in the blood of Salmonellainfected mice, while cells with the features of tissue DC are not
readily apparent in the blood of naive or infected animals.

To examine the phagocytic activity and killing capacity of


monocytes and neutrophils, these cells were isolated from blood
and spleen at day 4 postinfection, pooled, and depleted of T, B,
and NK cells by MACS. The remaining cells were sorted into
Ly6Chigh monocytes (CD11bLy6GlowLy6Chigh) and neutrophils
(CD11bLy6GhighLy6Cint) (Fig. 6A). Because CD68 is expressed
intracellularly and requires a staining procedure using fixation and
permeabilization that kills the cells, we changed the gating strategy
and used CD11b, Ly6C, and Ly6G to identify mono/mac and neutrophils (7, 8, 31, 34, 37, 53). Anti- TCR, CD19, NK1.1, and
CD11c were also included in the staining to assure that no T cells,
B cells, NK cells, or DC were included in the gates. To conclude
that this strategy allowed identification of the mono/mac and neutrophils previously defined by CD68 and Gr-1, CD11bLy6Glow
Ly6Chigh monocytes and CD11bLy6GhighLy6Cint neutrophils
were backgated into a CD68 vs Ly6G plot (Fig. 6A). This showed
that 79% of the CD11bLy6GlowLy6Chigh cells fell within the
mono/mac (R2) gate and 97% of the CD11bLy6GhighLy6Cint
cells fell into the neutrophil (R1) gate, with little cross-contamination between the two populations (Fig. 6A).
The sorted monocytes and neutrophils were pulsed with Salmonella for 2 h, washed extensively, cultured for an additional 1 or
18 h in medium containing gentamicin, and the intracellular survival of bacteria was examined. After 3 h, both monocytes and
neutrophils had phagocytosed bacteria but the neutrophils were
more effective (Fig. 6B), which is consistent with the in vivo data
(Fig. 4C). After 20 h, few viable bacteria were recovered from the
two populations, showing that most of the phagocytosed bacteria
had been killed. The use of CCD in parallel wells showed that the
majority of bacteria were actively internalized rather than being
attached to the cell surface (Fig. 6B).
Having established that Ly6Chigh monocytes were able to
phagocytose bacteria in vitro, we wanted to examine whether they
or Ly6Clow monocytes from infected mice were able to induce the
proliferation of OT-II CD4 T cells. To examine this, Ly6Chigh
monocytes, Ly6Clow monocytes, and DC from infected mice were
sorted based on expression of CD11b, Ly6C, Ly6G, and CD11c
(Fig. 7A), pulsed ex vivo with Salmonella expressing OVA, and
subsequently cocultured with CFSE-labeled OT-II CD4 T cells.
DC but not Ly6Chigh or Ly6Clow monocytes induced proliferation
of OT-II cells (Fig. 7B). Bacterial titration showed that the T cell
response peaked when DC were stimulated using a 5 to 1 bacteria
to cell ratio, and that Ly6Chigh monocytes were unable to induce a
T cell response at all bacteria to cell ratios tested (Fig. 7C). Fur-

FIGURE 6. Neutrophils and Ly6Chigh monocytes phagocytose and kill


Salmonella in vitro. Mice were infected with 8554 and 4 days later cells
from the spleen and blood of 10 mice were pooled and depleted of NK1.1,
CD19, and TCR cells by MACS. Cells were stained for surface expression of CD11b, CD11c, Ly6C, and Ly6G and sorted into Ly6ChighLy6Glow
monocytes and Ly6CintLy6Ghigh neutrophils as shown in (A, left dot plot).
Purity of the sorted cells was 95% for monocytes and 91% for neutrophils.
A. The indicated Ly6ChighLy6Glow monocytes and Ly6CintLy6Ghigh neutrophils (neut) were back-gated into a Ly6G vs CD68 plot to confirm that
the monocyte and neutrophil populations identified by the Ly6C/G strategy
correspond to those identified using Gr-1 (Ly6G) and CD68 (see Fig. 1A).
B, Sorted monocytes and neutrophils (Neu) were infected ex vivo with
14028r for 2 h in the absence or presence of CCD. The cells were washed
and incubated in medium containing gentamicin for an additional 1 or 18 h.
Cells were then lysed and the number of viable bacteria was determined by
plating. Data are the mean of duplicate samples 1 SD. Similar results
were obtained in two independent experiments.

thermore, DC titration with a fixed bacteria to cell ratio (5 to 1)


showed that proliferation of OT-II cells decreased as DC number
declined (Fig. 7D). The observed proliferation of DC was epitopespecific, because a lack of proliferation was observed when the
cells were pulsed with Salmonella not expressing OVA (Fig. 7D).
Finally, pulsing with the OVA323339 peptide showed that
Ly6Chigh monocytes induced a modest proliferation of OT-II cells,
while both Ly6Clow monocytes and DC efficiently induced proliferation (Fig. 7B).
Together, these results show that Ly6Chigh monocytes and neutrophils phagocytose and kill Salmonella in vitro. Moreover, and in
contrast to DC, neither Ly6Chigh or Ly6Clow monocytes induce
OT-II T cell proliferation after pulsing with Salmonella expressing
OVA although Ly6Clow monocytes induce OT-II proliferation after pulsing with OVA peptide.
Monocytes and neutrophils are recruited to PP despite blocking
of MAdCAM-1
Monocytes and neutrophils in blood express the selectin CD62L
(Fig. 5D) and the 4 integrin (37, 54, 55). CD62L and 4 partnered
with 7, in particular, are ligands for MAdCAM-1. MAdCAM-1 is
expressed on the high endothelial venules of PP and is critical for
lymphocyte homing to this organ (45, 56, 57). To investigate
whether MAdCAM-1 is also involved in monocyte and neutrophil
recruitment to PP during Salmonella infection, blocking studies
were performed. Although T cell recruitment to the PP of Salmonella-infected mice was abrogated in animals treated with the

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Bacterial uptake and Ag presentation capacity

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MONOCYTE RECRUITMENT DURING ORAL Salmonella INFECTION

anti-MAdCAM-1 mAb MECA-367, monocytes and neutrophils


accumulated in PP equally well in MECA-367-treated and untreated mice (Fig. 8). MECA-367 blocked T cell recruitment to PP
but not MLN (Fig. 8. B and C) or spleen (data not shown) as
predicted (45). Thus, unlike T cells, monocytes and neutrophils do
not require the addressin MAdCAM to enter into PP in mice orally
infected with Salmonella.
Chemokine receptor expression on Ly6Chigh monocytes, Ly6Clow
monocytes, neutrophils and DC in blood, PP, and MLN of
Salmonella-infected mice
To gain further insight into entry of myeloid cells into the gut
lymphoid tissues during oral Salmonella infection, we next studied
chemokine receptor expression because these receptor/ligand interactions provide important information that direct cell entry into
lymphoid tissues (58). Thus, CXCR2, CXCR3, and CCR6 expres-

FIGURE 8. Recruitment of adoptively transferred Ly6Chigh monocytes


and neutrophils to PP during oral Salmonella infection occurs despite
blocking MAdCAM-1. A total of 10 15 106 CFSE-labeled monocytes,
neutrophils, and T cells were injected i.v. into C57BL/6 mice orally infected 3 days earlier with 4666. Half of the recipient Salmonella-infected
mice were given anti-MAdCAM-1 mAb (MECA367) i.v. 6 8 h before cell
transfer. Twelve to 16 h later, PP, MLN, spleen, and blood were removed
and stained with anti-CD11b, anti-TCR, anti-CD19, anti-NK1.1, antiLy6C, and anti-Ly6G and analyzed by flow cytometry. A, TCRCD11b
cells were identified as T cells (left) and CD11bTCRCD19NK1.1
cells were further gated into monocytes (Ly6ChighLy6Glow) or neutrophils
(Ly6CintLy6Ghigh) (right) in PP as indicated. B and C, Recruitment of
adoptively transferred T cells, monocytes, and neutrophils to PP on day 4
postinfection after blocking i.v with anti-MAdCAM-1 (MECA 367) (upper
row) or not blocking (lower row) is shown. The percentage of CFSE cells
within the population is indicated in the plots. Mice given an isotype control Ab (9B5; anti-human CD44) gave similar results as nontreated mice
(not shown). Results are from five independent experiments with a total of
8 10 mice per group. Error bars are the SEM. The p value; (, p 0.001)
are from the Mann-Whitney U test for anti-MAdCAM-1-treated mice compared with unblocked or isotype-blocked mice.

sion on myeloid cells from blood, PP and MLN were analyzed by


flow cytometry at day 4 postinfection. Almost all blood neutrophils
express CXCR2, while 20% of neutrophils in PP and MLN express this receptor (Fig. 9). In contrast, few if any Ly6Chigh monocytes in any of the immune compartments examined were
CXCR2. The fraction of cells positive for CXCR3 was relatively
low for all cell types in all tissues examined, except for neutrophils
in MLN where 13% were positive. Finally, CCR6 expression was
only found on DC in PP and MLN. Thus, the high expression of
CCR2 on Ly6Chigh monocytes in blood (R2 cells; Fig. 5D) is consistent with the role of this receptor and its ligand MCP-1 (CCL2)
in the release of these inflammatory monocytes into the blood for
subsequent entry into infected tissues, as described previously (3,
7, 12, 59). Moreover, the data suggest a potential role for CXCR2
in neutrophil migration and CCR6 in the migration of DC.

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FIGURE 7. DC but not Ly6Chigh monocytes (Mo hi) or Ly6Clow monocytes (Mo lo) process and present a Salmonella Ag on MHC class II. Cells
from the blood and spleen of mice infected 4 days earlier with 8554 were
pooled, depleted of B, T, and NK cells, and stained with anti-CD11b,
anti-CD11c, anti-Ly6C, and anti-Ly6G and a mixture of CD19, NK1.1, and
TCR. A, Cells were sorted into Ly6Chigh monocytes, Ly6Clow monocytes,
and CD11chigh DC as shown. B, A total of 1.5 105 cells/well were pulsed
for 2 h with 4550 expressing or not expressing OVA at a 5:1 bacteria to
cell ratio. Alternatively, the cells were pulsed with 1 g/ml OVA323339
peptide for 2 h. The cells were washed, resuspended in medium containing
gentamicin, and 2 105 MACS-purified, CFSE-labeled OT-II CD4 T
cells were added to the wells. After 3.5 days, the division of the CFSElabeled T cells was assessed by flow cytometry. C, DC and Ly6Chigh monocytes were pulsed at different bacteria to cell ratios with 4550 expressing
OVA for 2 h and processed as in B. D, DC infected at a 5:1 bacteria to cell
ratio with 4550 expressing OVA (circles) or not expressing OVA (square)
for 2 h were washed and serially diluted 2-fold. OT-II cells were added and
were processed as in B. Infection of the highest concentration of DC with
4550 not expressing OVA induced 1.6% proliferation of the OT-II cells
(square). Data are representative of 2 4 independent experiments. The purity of the sorted cells was 86% for DC, 93% for Ly6Chigh monocytes, and
82% for Ly6Clow monocytes. Similar results were obtained with monocytes and DC purified from MLN (data not shown).

The Journal of Immunology

5799

Discussion
The origin of monocytes, neutrophils, and DC from a common
myeloid progenitor (36, 39), their overlapping expression pattern
of surface molecules (5, 34, 37, 38), and the differentiation capacity of monocytes (3, 1725) make characterizing tissue-infiltrating
myeloid populations complex. In this study we characterized the
rapid recruitment of CD68highGr-1int and CD68intGr-1high cells to
PP and MLN in the first few days after oral Salmonella infection.
The phenotype of the recruited cells was consistent with their identification as neutrophils (CD68intGr-1high) and inflammatory
monocytes (CD68highGr-1int) (3, 5, 8, 34, 37, 38). Recruited monocytes were numerically dominant over neutrophils in infected tissues and were major producers of the effector molecules important
in controlling Salmonella infection, iNOS and TNF-.
CD68highGr-1int monocytes recruited to PP and MLN during
infection had increased MHC-II, CD80, and CD86 but yet had
phenotypic and functional features distinct from DC in the same
tissue. For example, low CD11c expression was detected on a
small fraction of recruited monocytes compared with high CD11c
expression on DC (40, 46, 47). Furthermore, MHC-II expression
on Gr-1int monocytes was greatly increased during infection in an
IL-12- and IFN--dependent fashion. In contrast, MHC-II expression on CD11chigh DC was very high in both steady-state and
infected organs and was unaffected in the absence of IL-12 or
IFN-. Functionally, recruited monocytes produced iNOS, TNF-,
and IL-1 while DC produced little if any of these molecules.
Double intracellular staining revealed that the majority of monocytes produced either TNF- or iNOS, with relatively few cells
staining positive for both. The production of TNF- and/or iNOS
by monocytes recruited to the PP and MLN of orally infected mice
is consistent with studies of monocytes recruited to the spleen after
the i.v. administration of Listeria monocytogenes (7, 12, 48). The
capacity of neutrophils from Salmonella-infected tissues to release
some cytokines is also consistent with the complex activities of
these cells observed with other pathogens (60, 61).
Our studies using eGFP-expressing Salmonella in vivo revealed
that a relatively small fraction of monocytes producing iNOS or
TNF- were eGFP. The findings that relatively few cells associate with Salmonella early during infection (1%; Fig. 4 and Ref.

47) and a large fraction of all mono/mac produce iNOS or TNF-


(30%) make it unlikely that bacterial uptake and destruction
(which negates eGFP detection) accounts for the predominance of
eGFP cells making iNOS or TNF-. The data rather suggest the
production of TNF- and iNOS by bystander cells, an observation
consistent with other infection models (5, 12). Cells containing
bacteria can respond differently than bystander cells exposed
only to factors in the environment (47). Bacterial association
did not, however, overcome the IFN-/ requirement for recruited monocytes to produce iNOS or up-regulate MHC-II.
Thus, the IFN-/-mediated pathways inducing iNOS and
MHC-II up-regulation during infection cannot be overcome by
alternate pathways induced in bacteria-containing cells, as can
occur for the up-regulation of costimulatory molecules on DC
that cannot respond to TNF- (47).
The Ag presentation capacity of the myeloid lineage cells differed. For example, while recruited (Ly6Chigh) monocytes induced
only marginal proliferation of OT-II CD4 T cells after stimulation with OVA peptide, Ly6Clow monocytes induced robust proliferation. The reason for this differential capacity of Ly6Clow and
Ly6Chigh monocytes is not clear, as both subsets express MHC-II
and costimulatory molecules, albeit at much lower levels than DC.
Effectively inducing T cell proliferation may be a property acquired during monocyte differentiation and is performed only by
the mature Ly6Clow population. Alternatively, iNOS produced by
monocytes could inhibit T cell proliferation (62).
In addition and in contrast to DC, neither Ly6Clow nor Ly6Chigh
monocytes caused OT-II proliferation after coincubation with Salmonella expressing OVA. This was somewhat surprising given the
capacity of the recruited Ly6Chigh monocytes to phagocytose Salmonella and up-regulate MHC-II and costimulatory molecules
during infection. It could be that the Ag-processing machinery in
monocytes is not capable of generating the OVA peptide from
internalized Salmonella expressing OVA, at least within the time
frame examined here. It has also recently been shown that a subset
of DC in PP, those expressing CCR6, are required for CD4 T cell
priming during oral Salmonella infection (28). The activation of
OT-II cells by Salmonella-pulsed MLN DC but not monocytes is
consistent with the role of CCR6 DC in priming naive T cells to

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FIGURE 9. Chemokine receptor expression by neutrophils, Ly6Chigh monocytes, Ly6Clow monocytes, and DC after oral Salmonella infection. C57BL/6
mice were orally infected with 8554 and at 4 days postinfection CD19 cells were MACS-depleted and the remaining cells were stained with 7-AAD
and anti-CD11b, anti-Ly6G, anti-Ly6C, anti-CD11c, a pool of anti-NK1.1, anti-TCR, and anti-CD19, and an Ab against a chemokine receptor (CXCR2,
CXCR3, or CCR6). The expression of chemokine receptors on viable (7-AAD) cells was assessed in the blood, PP, and MLN by seven-color flow
cytometry. Histograms show surface expression of the indicated chemokine receptor on gated Ly6CintLy6Ghigh neutrophils and Ly6ChighLy6Glow monocytes in all organs, on gated Ly6Clow monocytes in blood, and on DC in PP and MLN. Cells were gated as in Figs. 7A and 8A. Filled histograms represent
staining with isotype-matched control Ab. Numbers are the mean value of chemokine receptor-positive cells with the isotype control subtracted (SEM) from
23 independent experiments using 10 infected mice/experiment that were divided into 13 groups.

5800

MONOCYTE RECRUITMENT DURING ORAL Salmonella INFECTION

Acknowledgments
The technical assistance of Kristina Lindgren is acknowledged. We are also
grateful to Roy Curtiss III (Arizona State University, Phoenix, AZ) for
Salmonella 4550, 4666, and 8554, Stephen Schoenberger (La Jolla

Institute for Allergy and Immunology, La Jolla, CA) for OT-II mice, Matthias Mack (University of Regensburg, Regensburg, Germany) for antiCCR2 mAb MC21, Arno Hanninen (University of Turku, Turku, Finland)
for the MECA-367 mAb and the supernatant containing the 9B5 mAb, and
Mats Bemark for purifying the 9B5 mAb.

Disclosures
The authors have no financial conflict of interest.

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