Microbes and Infection, 3, 2001, 11911200

2001 ditions scientifiques et mdicales Elsevier SAS. All rights reserved

S1286457901014794/REV

Cytokines in host defense against Salmonella

Lars Eckmann*, Martin F. Kagnoff
Department of Medicine 0623D, Laboratory of Mucosal Immunology, University of California
San Diego, 9500 Gilman Drive, La Jolla, California 92093-0623, USA

ABSTRACT Cytokines are key communication molecules between host cells in the defense
against the enteric pathogen, Salmonella. Infection with Salmonella induces expression of multiple
chemokines and proinflammatory cytokines in cultured intestinal epithelial cells and macrophages. In
animal models, protective roles have been shown for IL-1, TNF, IFN-, IL-12, IL-18 and IL-15,
whereas IL-4 and IL-10 inhibit host defenses against Salmonella. 2001 ditions scientifiques et
mdicales Elsevier SAS
microbial pathogenesis / innate immunity / chemokines / macrophages / intestinal epithelial cells

1. Introduction
Infections with Salmonella are an important cause of
diarrhea and mucosal inflammation and can lead to severe
systemic disease. Infection is usually initiated by the ingestion of contaminated food. Ingested Salmonella spread
rapidly along the axis of the intestinal tract and invade the
mucosa throughout the intestine. After invading and passing through the intestinal epithelium, Salmonella colonize
the lamina propria and Peyers patches. Salmonella rapidly invade host cells within these sites, especially macrophages, but other cell types may also be invaded. If local
host defenses are insufficient to limit infection to the
intestinal tract, infection can spread systemically to mesenteric lymph nodes, spleen and liver. The outcome of
infection is determined both by bacterial and host factors,
including the virulence of the infecting Salmonella, and
the ability of the host to mount an adequate inflammatory
and immune response, and ultimately the ability of the
host to destroy the pathogen. The host immune and inflammatory response involves multiple cell types that are resident at the site of infection or infiltrate from the circulation, and a complex communication network between
these cells in the form of soluble and cell-bound communication molecules.
Cytokines are a diverse group of secreted proteins that
act as key communication molecules between virtually all
cells in the body. They play a central role in regulating
immune and inflammatory responses, as well as many
other host functions, during infection with pathogens. Studies on the role of specific cytokines in the pathogenesis of
Salmonella infection generally have used two different

*Correspondence and reprints.

E-mail address: leckmann@ucsd.edu (L. Eckmann).
Microbes and Infection
2001, 1191-0

approaches. One group of studies has focused on the

interactions of Salmonella with defined cell types in cell
culture. This approach has the advantage that it allows one
to define the cellular mechanisms by which Salmonella
enters specific cells, and the accompanying cellular
responses, including the altered expression of cytokines.
However, the cell culture approach cannot define the
physiological relevance of such cellular responses. A second group of studies has characterized the role of specific
cytokines in the pathogenesis of Salmonella infections in
vivo by determining the expression of specific cytokines
during the course of infection, and the consequence of
altering cytokine levels for infection in vivo. These studies
have commonly used mouse models of Salmonella infection. This approach allows one to determine the physiological role of a specific cytokine, but is often limited in its
ability to define the cell types and cellular mechanisms
responsible for the cytokine functions. Given these considerations, we have divided this chapter into two major parts,
one dealing with results from cell culture studies and the
other with findings obtained in mouse models of infection.

2. Cell culture studies

Salmonella can enter a wide range of, and possibly all,
nucleated mammalian cells in cell culture. It is likely that
not all of these interactions are pathogenetically important, and it is possible, if not likely, that some cell types that
can be infected in cell culture do not become infected
during the course of Salmonella infection in vivo. Nonetheless, characterization of bacteriahost cell interactions
in cell culture in different cell types is useful as it provides
a baseline from which specific hypotheses can be generated for subsequent in vivo studies in regard to the involvement of specific cytokines and cell types in host defense
against Salmonella.
1191

Forum in Immunology

The most studied and best understood bacteriacell

interactions are those between Salmonella and intestinal
epithelial cells, and Salmonella and macrophages, and the
consequences of these interactions for cytokine expression are described in the following two sections. Interactions of Salmonella with other cell types, whose roles in
Salmonella pathogenesis are less well understood, are
discussed in the last section.
2.1. Intestinal epithelial cells

Ingested Salmonella must cross the intestinal epithelial

barrier to initiate infection of the intestinal lamina propria
and, ultimately, systemic infection. Of the five lineages of
intestinal epithelial cells (i.e. enterocytes, M cells, goblet
cells, enterochromaffin, Paneth cells), M cells and enterocytes are generally thought to be the most important
epithelial cells for bacterial invasion and transcytosis. The
relative importance of these two epithelial lineages as an
entry portal for Salmonella is a matter of debate. After
experimental infection with high doses of Salmonella,
most bacteria are observed within M cells in the small
intestine, indicating that these cells have a higher capacity
to internalize Salmonella compared with surrounding
enterocytes. On the other hand, a lower uptake efficiency
of enterocytes for Salmonella may be compensated for by
their greater numbers, as they greatly outnumber M cells
in the small intestine (by > 100:1). Furthermore, the colon
is markedly devoid of M cells, yet the colonic mucosa can
be efficiently colonized by Salmonella. Cell culture studies on the interactions between Salmonella and intestinal
epithelial cells have generally been performed with model
systems that mimic enterocytes rather than M cells.
Whether or not the findings in enterocytes are applicable
to M cells remains to be established.
Infection of monolayers of human intestinal epithelial
cells with live S. dublin, S. typhimurium or S. typhi
increases the expression of many cytokine genes, as summarized in table I. Most of these are chemokines with
chemoattractant properties for a range of leukocytes, particularly neutrophils (e.g., IL-8, GRO//, ENA-78) and
monocytes/macrophages (e.g., MCP-1). Several of the
chemokines upregulated in intestinal epithelial cells after
Salmonella infection also attract specialized subsets of T
cells (IP-10, Mig, I-TAC) [1] or T cells and immature
dendritic cells (MIP-3) [2]. All of these cell types are an
important part of the inflammatory response after infection
with enteric bacterial pathogens, can be found in close
vicinity to the epithelium acutely after infection, and play
a role in innate and acquired host defense against Salmonella. This indicates that the epithelial chemokine response
can provide a mechanistic explanation for the trafficking
of the various leukocyte subsets to the subepithelial regions
after Salmonella infection. Consistent with this concept,
most of the chemokines that have been studied in this
regard are released from the basolateral side of the epithelium, suggesting that the cellular targets are located in the
lamina propria. A notable exception is the putative
chemokine, PEEC (pathogen-elicited epithelial chemoattractant), which is released in the apical direction from
polarized epithelial cells and may have a role in neutrophil transmigration across the epithelium into the luminal
1192

Eckmann and Kagnoff

space [3]. In addition to chemokines, the intestinal epithelium releases other cytokines that can enhance the effectiveness of local host defense by activating phagocytic
cells (G-CSF, GM-CSF) and mediating other proinflammatory functions (IL-6, TNF). Taken together, the cytokines
released by intestinal epithelial cells after Salmonella infection are well suited to initiate and orchestrate the early
inflammatory events that occur after acute bacterial infection. It will be important, in future studies, to apply this
concept to relevant in vivo models to define the physiological role of epithelial cytokine responses in Salmonella pathogenesis.
In addition to releasing cytokines, intestinal epithelial
cells express multiple cytokine receptors and are targets
for cytokine signaling. It is beyond the scope of this review
to discuss the effects of various cytokines on epithelial cell
functions, but infection with enteroinvasive bacteria could,
in principle, also regulate epithelial cell responses to
cytokine stimulation. In support of this concept, it has
been shown that infection of T84 epithelial cells with live
S. typhimurium increases mRNA and cell surface expression of the IL-7 receptor [4], although the functional
consequences of this event have not been established.
2.2. Monocytes/macrophages

Monocytes/macrophages play a key role in controlling

and clearing Salmonella infection from the host. Like
neutrophils, macrophages can destroy the pathogen, but
in contrast to neutrophils, the interactions between Salmonella and macrophages are often prolonged and characterized by a delicate balance between macrophage effector mechanisms to kill the pathogen and bacterial defenses
to resist killing. For this reason, macrophages are in a
prime position to sense the presence of bacteria in the host
and send cytokine signals to neighboring cells which can
contribute to activating appropriate host defenses.
Exposure of monocytes/macrophages to Salmonella or
several of its products increases mRNA expression and
secretion for a wide range of cytokines, as shown in detail
in table II. These include the prototypic proinflammatory
cytokines, IL-1, TNF and IL-6, as well as several chemokines (MIP-1, MIP-1, MIP-2, KC) and hematopoietic
growth and survival factors (GM-CSF). Interestingly, this
spectrum of cytokines resembles that induced in intestinal
epithelial cells after Salmonella infection (compare tables
I and II). As for epithelial cells, these cytokines can function to recruit phagocytic cells (e.g., neutrophils, additional macrophages) to the site of infection, and to induce
other events associated with acute inflammation (e.g.,
induction of cell adhesion molecules on neighboring
endothelial cells).
In addition, monocytes/macrophages express two cytokines, IL-12 and IL-18, that are not expressed, or known to
be actively secreted, by intestinal epithelial cells. Secretion
of the p40 subunit of IL-12 increases markedly after Salmonella infection of macrophages, while that of the bioactive
IL-12p70 heterodimer increases only minimally after infection [5]. Furthermore, mRNA expression and secretion of
IL-18 is suppressed in Salmonella-infected macrophages at
648 h after infection [6], although early after infection
there may be a transient increase in IL-18 secretion [7].
Microbes and Infection
2001, 1191-0

Cytokine family

Cytokine

Target cells of
cytokine

Functions of
cytokine

Bacterial
stimulus

Epithelial
cells used

Epithelial response Direction of

to stimulus
cytokine secretion

CXC chemokines

IL-8

neutrophils

chemoattraction

T84, Caco-2

[12, 40, 41]

neutrophils

chemoattraction

not known

[42]

GRO
(MIP-2)
GRO
ENA-78

neutrophils

chemoattraction

S. dublin

HT-29

mRNA ,
secretion
mRNA ,
secretion
mRNA

basolateral

GRO

S. dublin,
S. typhimurium
S. dublin

not known

[39]

neutrophils
neutrophils

chemoattraction
chemoattraction

S. dublin
S. dublin

HT-29, Caco-2
HT-29

not known
not known

[42]
[42]

IP-10

CD4+ Th1
memory T cells

chemoattraction

S. dublin

HT-29

mRNA
mRNA ,
secretion
secretion

Mig

CD4+ Th1
memory T cells

chemoattraction

S. dublin

HT-29

I-TAC

CD4+ Th1
memory T cells

chemoattraction

S. dublin

HT-29

MCP-1

macrophages,
T cells
macrophages,
T cells
immature dendritic
cells, CD45RO+
T cells
T cells,
eosinophils,
macrophages
neutrophils

chemoattraction

S. dublin

chemoattraction

S. dublin

HT-29, Caco-2,
T84
HT-29, Caco-2

secretion in
synergy with
IFN-
secretion in
synergy with
IFN-
mRNA ,
secretion
mRNA

chemoattraction

S. dublin

HT-29, T84

secretion

chemoattraction

S. dublin

HT-29, Caco-2

chemoattraction

S. typhimurium

proliferation,
activation
proliferation,
activation
acute phase
reactions,
proliferation,
differentiation
cytokine release

CC chemokines

MIP-1
MIP-3
RANTES
Other
chemoattractants
Other cytokines

PEEC
G-CSF
GM-CSF
IL-6

1193

, increased

macrophages,
epithelial cells,
fibroblasts, T cells

not known,
basolateral after
IL-1 + IFN-
not known,
basolateral after
IL-1 + IFN-
not known,
basolateral after
IL-1 + IFN-
not known

[1, 42]
[1]
[1]
[40, 42]

not known

[42]
[2]

mRNA

not known,
basolateral after
IL-1
not known

[42]

T84

secretion

apical

[3]

S. dublin

HT-29

not known

[39]

S. dublin

HT-29

not known

[40]

S. typhi

Int407, IEC-6,
Caco-2, freshly
isolated cells

mRNA ,
Secretion
mRNA ,
Secretion
mRNA ,
Secretion

not known

[43]

S. dublin

HT-29, T84

not known

[40]

mRNA ,
Secretion

Forum in Immunology

TNF

neutrophils and
their precursors
neutrophils,
macrophages
macrophages, B
cells, epithelial
cells, fibroblasts

HT-29, Caco-2

Reference

Cytokines in host defense against Salmonella

Microbes and Infection

2001, 1191-0

Table I. Cytokine responses of cultured intestinal epithelial cells to Salmonella infection.

Forum in Immunology

1194

Table II. Cytokine responses of monocytes/macrophages to Salmonella.

Target cells of
cytokine

Functions of cytokine

Bacterial stimulus

Cells used

Cellular response to stimulus

IL-1

most cells

proinflammatory

most cells

proinflammatory

TNF

most cells

IL-6
IL-12p70

macrophages, B
cells, epithelial
cells, fibroblasts
T cells, NK cells

induction of proinflammatory S. typhimurium,

cytokine secretion,
S. typhimurium LPS,
many others
S. typhimurium porins,
S. typhi flagella,
S. enteritidis flagellin
acute phase reactions,
S. typhimurium + CyD,
proliferation, differentiation S. typhimurium porins,
S. typhimurium LPS
induction of IFN- secretion S. dublin

human peripheral blood

monocytes
mouse RAW macrophages,
human peripheral blood
monocytes, mouse peritoneal
macrophages
mouse RAW macrophages,
human peripheral blood
monocytes, human U937
monocytic cells

secretion

IL-1

S. typhimurium porins,
S. typhimurium LPS
S. typhimurium,
S. typhimurium LPS,
S. typhi flagella

IL-18

T cells, NK cells

induction of IFN- secretion

in synergy with IL-12

S. dublin, S. typhimurium

mouse peritoneal
macrophages, human
peripheral blood monocytes
mouse peritoneal
macrophages
mouse peritoneal and splenic
macrophages

MIP-1

T cells,
macrophages
T cells,
macrophages

chemoattraction

MIP-2

neutrophils

chemoattraction

S. typhimurium,
S. typhimurium LPS
S. typhimurium,
S. typhimurium LPS,
S. typhimurium + CyD
S. typhimurium,
S. typhimurium LPS

KC

neutrophils

chemoattraction

S. typhimurium + CyD

GM-CSF

neutrophils,
macrophages

proliferation, activation

S. typhimurium + CyD,
S. typhimurium flagella,

MIP-1

chemoattraction

Microbes and Infection

2001, 1191-0

, increased; , decreased; CyD, cytochalasin D.

mouse RAW macrophages

mouse RAW macrophages,
mouse peritoneal
macrophages
mouse RAW macrophages,
mouse peritoneal
macrophages
mouse peritoneal
macrophages
mouse peritoneal
macrophages

Reference
[10]

mRNA , secretion ,
intracellular production

[8, 9, 44]

mRNA , secretion ,
intracellular production

[811]

mRNA , secretion

[10, 44]

IL-12p40 secretion ,
IL-12p70 secretion
mRNA at 624 h, and
secretion at 2448 h;
secretion at 1 h
mRNA , secretion

[6, 7]

mRNA

[8, 44]

mRNA

[8, 44]

mRNA

[44]

mRNA

[44]

[5]

[8]

Eckmann and Kagnoff

Cytokine

Cytokines in host defense against Salmonella

Given that IL-12 and IL-18 are important for the induction
of IFN-, and the latter cytokine is central for successful
host defense against Salmonella (see below), these cell
culture data could suggest that Salmonella-induced suppression of IL-18 expression, combined with the lack of
substantial IL-12p70 secretion, could act as a virulence
principle to delay IFN- induction.
Induction of an array of cytokines occurs in response to
infection of macrophages with live Salmonella, but also to
a similar degree after exposure of the cells to various
Salmonella products, including LPS, flagellin and porins
[811]. This conclusion is underlined by a recent study in
which gene expression arrays were used to assess mRNA
expression in macrophages for > 500 genes, since the
changes in gene expression after infection with live S. typhimurium were similar to those induced by LPS stimulation [8]. Although one would predict, a priori, that the
prolonged presence of live bacteria inside macrophages
would be accompanied by cellular cytokine responses
that differ from those induced by exposure of the macrophages to Salmonella products, present data looking at
different profiles of cytokine expression and secretion
suggest that such differences are not readily apparent,
although they may require longer times to become manifest than usually encompassed in cell culture experiments.
This situation differs from that in intestinal epithelial cells,
which are unresponsive or, at least, hyporesponsive to
bacterial products (e.g., LPS) when compared to infection
with live Salmonella [12]. Although the underlying mechanisms for the respective sensitivities of macrophages and
intestinal epithelial cells to bacterial products remain to be
fully elucidated, one could speculate that such a differential responsiveness has adaptive advantages. Thus, the
apical side of intestinal epithelium is physiologically
exposed to bacterial products in the intestinal lumen, and
this does not represent an infectious threat to the host,
whereas the systemic presence of bacterial products, as
detected by macrophages, indicates such a threat and the
need for an adequate host response.
2.3. Other cell types

Neutrophils and dendritic cells are known to play, or

are likely to play, a role in determining the course of
infection with Salmonella. Several studies have characterized cytokine release after interactions between these cells
and Salmonella. For example, exposure of freshly isolated
human neutrophils to heat-killed opsonized S. typhimurium increased mRNA expression and secretion of the
chemokines IL-8 and MIP-1 [13]. Infection of purified
CD11c+ dendritic cells with live S. dublin increased mRNA
expression and secretion of IL-1, IL-6 and IL-12p40, as
well as secretion of IL-12p75 and TNF, while IL-10
secretion was minimally affected by infection [14]. These
studies also showed that Salmonella invaded CD11c+
dendritic cells in vivo after systemic infection with this
pathogen [14], suggesting that the direct cellular interactions between Salmonella and dendritic cells play a role in
the pathogenesis of Salmonella infections. In another study,
S. typhimurium infection of embryonic fibroblasts
increased mRNA expression and the release of IFN-1,
which may increase resistance of these cells to further
Microbes and Infection
2001, 1191-0

Forum in Immunology

Salmonella infection [15]. Furthermore, infection of osteoblasts with live S. dublin upregulated mRNA levels and
secretion of G-CSF and GM-CSF [16], analogous to the
findings in intestinal epithelial cells (table I). Although it is
not clear if fibroblasts or osteoblasts are invaded by Salmonella during the natural course of infection, these data
may suggest a mechanistic link between Salmonella infection and bone and joint disease which can be associated
with the infection.

3. Animal studies
Studies on the role of specific cytokines in controlling
host defense against Salmonella have largely focused on
mouse models of infection, since immunologic reagents
are widely available for mice and mouse models allow
defined functional manipulations of the host immune and
inflammatory response. This section will focus on results
from those studies. Relatively few studies have been
reported on cytokine involvement in Salmonella infection
in humans, and findings from selected clinical studies are
highlighted in a subsequent section.
Mouse infection studies, generally, have addressed two
sets of goals: 1) to characterize the expression of specific
cytokines in various tissues during the course of infection,
and 2) to define the consequences of altering cytokine
levels for the course and severity of infection. Cytokine
levels in vivo can be decreased through genetic means
(e.g., knock-out mutations) or pharmacological means
(e.g., treatment with neutralizing antibodies), or increased
by treatment with the respective cytokine. Results from
such studies are summarized in table III for specific cytokines. The findings are discussed in greater detail in the
following paragraphs. For ease of discussion, cytokines
are grouped into several groups, according to their known
functions and/or functional interdependence.
3.1. Classical proinflammatory cytokines: IL-1 and TNF

IL-1 and TNF are expressed by a wide range of cells

and have multiple functions associated with host inflammatory responses. Salmonella infection is typically accompanied by intense inflammation. Accordingly, IL-1 and
TNF expression is increased during Salmonella infection
in all organs that have been studied to date [17, 18].
Neutralization of TNF by genetic or pharmacological
approaches increases the severity of Salmonella infection
and decreases survival of the host, if the latter was used as
an endpoint in the respective studies [19, 20]. Furthermore, mice that lack the TNF p55 receptor show increased
susceptibility to Salmonella infection [21]. Treatment with
IL-1 or TNF increases survival of the host after Salmonella infection [22]. Together, these data indicate that IL-1
and TNF play important roles in host defense against
Salmonella. Although both cytokines have similar functions in many experimental systems, their actions in Salmonella host defense may not be fully overlapping since
they show synergistic effects in enhancing survival after
Salmonella infection [22]. Given that many, if not most,
cells express receptors for IL-1 and TNF, it is not clear
how these cytokines exert their functions in Salmonella
host defense, but it appears likely that their effects on
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Importance in disease pathogenesis

Cytokine group

Cytokine

Classical
IL-1
proinflammatory
TNF
IFN- axis

Antiinflammatory

IFN-

Expression levels in tissues

during infection

S. typhimurium,
S. dublin
S. typhimurium,
S. dublin
S. typhimurium,
S. enteritidis,
S. dublin

mRNA in intestine,
spleen, and liver
mRNA in intestine,
spleen, and liver
mRNA in intestinal wall,
Peyers patches, mesenteric
lymph nodes, spleen, and
liver
IL-12p40 mRNA in Peyers
patches, mesenteric lymph
nodes, spleen, and liver;
IL-12p35 mRNA in
Peyers patches and
mesenteric lymph nodes;
IL-12p70 secretion in
mesenteric lymph nodes
mRNA in Peyers patches
and mesenteric lymph nodes
at 6-24 h;
secretion in Peyers patches
and spleen at 3 and 7 days
mRNA in spleen and liver,
mRNA in peritoneal T cells

IL-12

S. dublin

IL-18

S. typhimurium,
S. dublin

IL-4

S. typhimurium,
S. dublin,
S. choleraesuis
S. typhimurium,
S. choleraesuis,
S. dublin

IL-10

TGF1

S. typhimurium,
S. dublin

IL-2
IL-6
IL-15

S. dublin
S. dublin,
S. typhimurium
S. choleraesuis

GM-CSF

S. dublin

, increased; , decreased; , unchanged.

mRNA in intestinal tract,

spleen and liver

mRNA in spleen and liver

in resistant mice, mRNA in
spleen at 2 and 5 days in
susceptible mice
mRNA in spleen
mRNA in intestinal tract,
spleen and liver
mRNA and secretion in
peritoneal exudate cells
mRNA in spleen and liver

Knock-out mice

CFU/spleen ,
CFU/liver

Neutralizing antibody
treatment

Cytokine treatment

References

Survival

[17, 18, 22]

CFU/spleen at 5 and
6 days, Survival
CFU/spleen at 5 and
6 days; survival

Survival

[17, 19, 20, 22]

CFU/spleen at 4 days;
survival

survival

CFU/spleen ,
CFU/liver ,
survival

CFU/spleen and CFU/liver CFU/spleen

at 3 days; survival
and CFU/liver ;
survival
delayed death, no
difference in LD50

CFU in peritoneal cavity at number of liver

3 days
abscesses
CFU/spleen: at 1, 3, 5
days in susceptible mice;
CFU/liver and peritoneal
cavity at 6,10 days in
susceptible mice infected
with an attenuated strain

[17, 19, 20, 2325]

[17, 27, 28]

[6, 7, 30]

[17, 31, 32]

[17, 18, 33, 34]

CFU/spleen and liver

, survival

[17, 35]

[17]
[17, 18]
CFU/spleen and CFU/liver
at 6 days

[36]
[17]

Eckmann and Kagnoff

Microbes and Infection

2001, 1191-0

Other

Bacteria used

Forum in Immunology

1196

Table III. Role of cytokines in host defense against Salmonella in animal models.

Cytokines in host defense against Salmonella

macrophage functions, which critically determine the outcome of infection, are important in this regard. However, it
also conceivable that indirect functions of these cytokines, i.e. functions such as upregulation of endothelial
adhesion molecules that are not targeted directly towards
Salmonella-harboring cells, may be equally important for
successful host defense against Salmonella.
3.2. Cytokines of the IFN- axis: IFN-, IL-12 and IL-18

IFN- is probably the most studied cytokine in host

defense against Salmonella. It is expressed by only a few
cell types in the body (T cells, NK cells), but can affect a
wide range of cells and cellular functions. IFN- expression is rapidly upregulated in the intestinal mucosa, Peyers patches, mesenteric lymph nodes, spleen and liver in
response to Salmonella infection, and increased levels of
circulating IFN- are readily detectable in infected mice
[17, 23]. Neutralization of IFN- functions with antibodies
or in knock-out mice increases bacterial numbers in the
spleen and liver and decreases survival of the host [19, 20,
24]. Conversely, IFN- treatment of infected mice
decreases bacterial counts and increases host survival
[25]. These data show that IFN- plays a central role in
controlling Salmonella infection. Although IFN- can affect
many functions in phagocytic and non-phagocytic cells,
the most likely mechanism by which IFN- exerts its
functions in host defense against Salmonella is by activating the ability of macrophages to kill Salmonella [26].
T cells and NK cells that produce IFN- most likely have
little direct contact with Salmonella during the course of
infection, suggesting that IFN- production by these cells
is largely induced through communication with other cells
that do have direct contact with the bacteria. Two cytokines that have IFN--inducing properties are IL-12 and
IL-18, both of which are produced by macrophages. Bioactive IL-12 is a heterodimer of p35 and p40 subunits.
IL-12p35 is ubiquitously expressed, while IL-12p40 is
highly inducible in macrophages and dendritic cells. Consistent with this, Salmonella infection leads to increased
IL-12p40 expression in Peyers patches, mesenteric lymph
nodes, spleen and liver, while IL-12p35 expression is not
affected [17, 27]. Neutralization of IL-12 increases bacterial counts in the spleen and decreases host survival, while
IL-12 treatment increases host survival [28]. This indicates
that IL-12 plays a protective role in host defense against
Salmonella. This role is likely to be mediated by IFN-,
since neutralization of IL-12 in Salmonella-infected mice
is accompanied by a decrease in splenic IFN- mRNA
expression and serum IFN- levels compared to infected
mice not treated with anti-IL-12 [29]. Furthermore, IFN-
treatment largely reverses the effects of IL-12 neutralization on splenic bacterial load [29].
IL-18, like IL-12, has IFN--inducing properties. The
regulation of IL-18 expression during the course of Salmonella has been studied in two reports, but with apparently
conflicting results. In one study, IL-18 levels increased in
homogenates of Peyers patches and spleens at 12 h and 1,
3 and 7 days after oral infection with an attenuated Salmonella strain [30], while in another report IL-18 mRNA
expression decreased in Peyers patches and mesenteric
lymph nodes at 6, 12 and 24 h after oral infection with a
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2001, 1191-0

Forum in Immunology

wild-type S. dublin [6]. It is possible that these differences

are related to the use of wild-type versus attenuated bacterial strains, or that the time of analysis is important, with
reduced levels early after infection and increased levels at
later times. In any case, neutralization of IL-18 leads to
increased bacterial numbers in spleen and liver and
decreased host survival, while IL-18 treatment decreases
bacterial counts in spleen and liver and increases host
survival [7, 30]. This shows that IL-18 plays an important
role in host defense against Salmonella. This effect is likely
to be mediated by IFN-, since serum IFN- levels were
significantly lower after IL-18 neutralization, and IL-18
injection into mice deficient for IFN- receptors had no
beneficial effects towards reducing bacterial load [7]. Furthermore, IL-18 appears to interact with IL-12 for the
induction of IFN- production, since IL-18 neutralization
decreased splenic IFN- levels and survival in wild-type
mice infected with Salmonella, but had no effect on splenic
IFN- levels or survival in IL-12p40 knock-out mice [30].
3.4. Anti-inflammatory cytokines: IL-4, IL-10 and TGF1

The cytokines in this group have similar functions in

that they downregulate inflammatory responses, although
they also have functions unique to each cytokine (e.g.,
TGF1 affects growth and differentiation of many cell
types, while IL-4 has a role in regulating IgE responses).
Expression of IL-4 mRNA was little affected in spleen and
liver by Salmonella infection [17]. Expression appeared to
increase in peritoneal T cells after i.p. infection with a
Salmonella strain of low virulence, although the latter
seemed to be related to influx of IL-4 producing NK1.1+
T cells into the peritoneal cavity, rather than upregulation
of IL-4 expression by other T cells [31]. Neutralization of
IL-4 functions reduced the number of bacteria in the
peritoneal cavity after i.p. infection, which was accompanied by increased serum levels for IFN- and IL-12 [31]. In
another study using IL-4 knock-out mice, lack of IL-4 was
associated with delayed death after Salmonella infection
and reduced liver abscess formation, although the LD50
did not differ between knock-out mice and littermate
controls [32]. Together, these data indicate that a lack of
IL-4 is protective against Salmonella infection, suggesting
that IL-4 downregulates crucial defenses against this pathogen. A possible underlying mechanism is IL-4-mediated
inhibition of IFN- production by T cells.
In contrast to IL-4, expression of IL-10 is strongly
increased in the intestinal tract, spleen, and liver during
the course of Salmonella infection [17, 18, 33]. Neutralization of IL-10 had little effect on bacterial growth in the
spleen of susceptible mice infected i.v. with a wild-type
Salmonella strain [33]. In contrast, anti-IL-10 treatment
reduced bacterial numbers in the peritoneal cavity and
spleen in mice infected i.p. with an attenuated Salmonella
strain [34]. The apparent discrepancy in outcomes may be
related to differences in the routes used for infection vs
antibody treatment (i.e., an effect was seen when both
bacteria and antibodies were given i.p., but not when
bacteria were given i.v. and antibodies i.p.), or the virulence of the infecting bacteria relative to the genetic susceptibility of the host (e.g., virulent Salmonella may not be
controllable by a susceptible host regardless of other host
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factors). Nonetheless, these studies could suggest that IL-10,

like IL-4, is anti-protective against Salmonella, although
additional studies are required, using genetically resistant
hosts and wild-type Salmonella, to fully assess the functions of IL-10 in controlling infection with this pathogen.
TGF1, among other functions, downregulates inflammatory responses in different organs, since mice deficient
for TGF1 are characterized by severe multiple-organ
inflammation. Based on this property, one might predict
that TGF1 would have functions in Salmonella pathogenesis that parallel those of IL-4 and IL-10. However, that
does not appear to be the case. Expression of TGF1
mRNA changed little in spleen and liver after Salmonella
infection in resistant mice [17], although levels increased
in the spleen of susceptible mice in another study [35].
However, TGF1 mRNA levels may not fully represent
TGF1 levels in the tissues, since levels of bioactive TGF1
are controlled by multiple post-translational factors,
including activation of latent TGF1 in the extracellular
space. Neutralization studies of TGF1 have not been
reported, but one study assessed the consequences of
TGF1 treatment on Salmonella infection [35]. Administration of TGF1 decreased bacterial counts in the spleens
and livers of Salmonella-infected mice, and increased host
survival [35]. This was accompanied by increased splenic
IFN- and iNOS mRNA expression, and increased serum
IFN- levels and NO release from splenic macrophages,
although the Salmonella-induced increase in IL-6 mRNA
levels was suppressed by TGF1 treatment [35]. Therefore, TGF1 has protective functions in the host defense
against Salmonella, which is very different from IL-4 and
IL-10. Although TGF1 would be expected to suppress
some aspects of the host inflammatory response during
infection (e.g., IL-6) and, therefore, be detrimental to host
defense, other functions of TGF1, such as direct induction of iNOS in macrophages, are apparently more important in determining the overall effects of TGF1 on host
defense against Salmonella.
3.5. Other cytokines

Several other cytokines have been studied in the pathogenesis of Salmonella infection, although most of these
have not been explored to the same degree as the ones
mentioned above. Several additional cytokines are upregulated after Salmonella infection, which include IL-2, IL-6
and GM-CSF [17, 18], but no information has been
reported on the functional roles of these cytokines in host
defense against Salmonella. Expression of IL-15, a growth
factor for T cells and NK cells, is increased in peritoneal
macrophages after i.p. infection with an attenuated Salmonella strain, and neutralization of this cytokine by antibody treatment increased bacterial counts in the spleen,
liver and peritoneal cavity [36]. Anti-IL-15 antibody treatment inhibited the influx of NK cells into the peritoneal
cavity and decreased serum IFN- levels [36], suggesting
that IFN- production by NK cells is important for controlling Salmonella infection.

4. Clinical studies
Only a few clinical studies have been reported to date on
the physiological role of specific cytokines in host defense
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against Salmonella in humans. Particularly informative in

this regard are studies on patients with genetic deficiencies
that render the patients more susceptible to Salmonella
infections. In one study analyzing the molecular basis for
the occurrence of recurrent severe mycobacterial and Salmonella infections it was found that the affected patients
had inactivating mutations of the 1 chain of the IL-12
receptor [37]. Consequently, cells from the patients were
deficient in IL-12 receptor signaling and IL-12-dependent
IFN- production [37]. In another study, a child with recurrent mycobacterial and Salmonella infections was found to
be defective in IL-12p70 production due to a homozygous
deletion within the IL-12p40 subunit [38]. Consistent with
the IFN--inducing properties of IL-12, the child showed
impaired IFN- production by peripheral blood lymphocytes. Interestingly, IFN- therapy led to marked symptomatic improvement, which suggests that the cellular functions downstream of IFN- were intact in this patient [38].
Together the two studies show that IL-12 and its receptor are
required for efficient host defense against Salmonella in
humans. This conclusion parallels the observations in
mouse models of infection, and underlines that the findings
in mouse models have clinical relevance.

5. Outlook
To date, expression of 25 cytokines has been characterized in detail in cultured intestinal epithelial cells,
macrophages and other cells. In animal models of infection, expression studies have been performed for 15
cytokines, of which 10 were further characterized in
regard to their physiologic functions using neutralizing
antibodies, knock-out mice, and administration of recombinant cytokines. The number of cytokines coded for in
the human or mouse genome is not known at this time, but
is likely to be at least a hundred. Therefore, the analysis of
cytokine involvement in Salmonella pathogenesis is likely
to continue for some time.
New technologies for parallel analysis of gene expression for a large number of genes are becoming widely
available, and the first studies using such technologies
have been reported for Salmonella interactions with cultured host cells [8, 39]. Such studies using gene microarrays and cultured host cells will ultimately need to be
expanded to encompass all human or mouse genes. Moreover, mRNA studies with gene arrays will need to be
complemented by studies of the respective proteins to
confirm and extend the results. Currently, this is more
difficult to achieve since tools for the parallel analysis of
large numbers of proteins (proteomics) are not as developed and available as those for mRNA analysis. Furthermore, it will be important to conduct mRNA expression
surveys in animal models of infection to define the genes
that are up- or down-regulated during the course of Salmonella infection in vivo. For such studies, it will be
necessary both to confirm the mRNA findings at the protein level, and to achieve single-cell resolution of mRNA
expression analysis, since tissues, in contrast to cell culture models, consist of complex mixtures of cell types.
Gene expression analysis at the single-cell level can be
performed by in situ hybridization (although this contin
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2001, 1191-0

Cytokines in host defense against Salmonella

ues to be technically challenging, and may not be sufficiently sensitive for many genes) or by laser capture microdissection techniques, which allow one to cut out specific
cells from tissue sections and analyze mRNA levels for
single genes in extracts of those cells.
Beyond analysis of cytokine gene expression, it will be
essential to determine the physiologic functions of specific
cytokines in Salmonella pathogenesis. This can be
achieved, as pointed out above, by characterizing the
consequences of altering levels of the target cytokine in
vivo, either through administration of neutralizing antibodies or recombinant cytokines by using genetically
engineered knock-out mice, or possibly transgenic mice
that over-express the gene of interest. The basic technologies for these studies are generally available. It will be
important to continue to apply these approaches to newly
discovered cytokines that are possibly relevant to Salmonella pathogenesis as suitable reagents or mice are generated. Once a specific cytokine is shown to have a physiologic function in host defense against Salmonella, an
important next step will be to determine which cells
mediate the functions of that cytokine. This involves two
sets of questions. First, what are the relevant cells that
secrete the specific cytokine that mediates the physiologic
function? Second, what cells are the relevant targets of that
cytokine? Analysis of cell-type specific functions is currently best achieved by genetic approaches in which
specific target genes are inactivated in defined cell populations using conditional knock-out mice (e.g., Cre-loxPmediated recombination). At present, this technology is
complex and usually requires support by specialized core
facilities that have experience with ES cell techniques and
the generation of transgenic mice. Nonetheless, conditional knock-out technology in mice will be a powerful
tool to apply to the characterization of cytokine functions
in host defense against Salmonella.

Acknowledgments
Work in the authors laboratory was supported by NIH
grants DK35108 and DK8960 and a research grant from
the Crohns and Colitis Foundation of America.

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