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Department of Cell and Molecular Biology, Section for Immunology, Lund University, Lund, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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)
increased 10- and 3-fold, respectively. In contrast, immune animals had
only a modest reduction in NK cells, no loss of NKT cells, and a slight
increase in phagocytes following secondary challenge. During primary
infection, the dominant sources of IFN-
were, unexpectedly,
neutrophils and M, the former having intracellular stores of IFN-
that were released during infection. IFN--producing phagocytes
greatly outnumbered IFN--producing NK cells, NKT cells, and T cells
during the primary response. TNF-
production was also dominated by
neutrophils and M, which vastly outnumbered NKT cells producing this
cytokine. Neither T cells nor NK cells produced TNF- early during
primary infection. The TNF- response was reduced in a secondary
response, but remained dominated by neutrophils and M. Moreover, no
significant IFN- production by M was associated with the
secondary response. Indeed, only NK1.1+ cells and T cells
produced IFN- in these mice. These studies provide a coherent view
of innate immunity to oral Salmonella infection, reveal
novel sources of IFN-, and demonstrate that immune status influences
the nature of the innate response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)3 (1, 2). The primary response to Salmonella enterica serovar Typhimurium (S. typhimurium), a Gram-negative bacterium that serves as a model of human typhoid fever pathogenesis, is thought to involve each of these innate cell populations (4). Salmonella naturally infects a host by the oral route, traversing the gut barrier by penetrating specialized epithelial cells overlying Peyer’s patches (PP) (5) and possibly by other mechanisms (6, 7). Organs targeted as sites of infection include spleen, liver, mesenteric lymph nodes, and PP (4). Within target organs Salmonella resides in host cells, including CD18+ phagocytes (8). Salmonella-specific CD4+ T cell responses begin to be detectable 7 days postinfection (9), although the precise kinetics of the adaptive response may vary with the strain of Salmonella encountered. Thus, it is before this time that the functions of the innate immune system are critical after primary exposure to Salmonella.
Depletion experiments have shown that both neutrophils and M are
important for host survival during the primary response to
Salmonella infection (10, 11, 12, 13, 14, 15), most likely
through the control of bacterial replication (12, 16).
Indeed, electron microscopy studies have visualized the destruction of
Salmonella by these cells (17, 18). Moreover,
M appear to function in a primarily protective capacity, rather than
acting as APC. The induction of acquired immunity is not significantly
compromised in M-depleted mice infected with Salmonella,
whereas M depletion from immune animals decreases survival against a
secondary Salmonella challenge (13).
Experiments studying host survival in mice with genetic deficiencies
for either a specific cytokine or its receptor as well as Ab-mediated
cytokine neutralization studies have shown that TNF-
(19, 20, 21, 22, 23, 24, 25), IFN- (20, 24, 26, 27, 28), IL-12
(29, 30, 31), and IL-18 (27, 29) are important
early in the primary response to Salmonella. A lack of
IFN- results in increased bacterial replication (20, 24) and host susceptibility (20), but does not
influence Ab production (28). Neutralization of TNF- in
vivo can also prevent the host from mounting a protective response, and
the animals succumb to the infection (20, 21, 22, 25). Despite
their importance, the cellular sources of TNF- and IFN- produced
early during primary Salmonella infection have not been
defined. However, it has been shown that cells other than T cells are
responsible for protective IFN- early during infection
(20).
Thus, neutrophils, M, NK, and NKT cells capable of producing a
variety of cytokines are likely to be involved early during
Salmonella infection. However, the relative involvement and
function of each innate cell population during a primary response to
bacterial infection remain undefined. Furthermore, the effect of
previous exposure to a pathogen on the nature of the innate immune
response to a secondary challenge has not been established. Thus, the
present study examines the population dynamics of, and cytokine
production by, cells of the innate immune system in the spleen during
the early response to oral Salmonella challenge in both
naive and immune animals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice were used between 7 and 10 wk of age. Age-matched naive controls were included as stated. All mice were bred and housed in the animal facilities at Lund University and were provided with food and water ad libitum.
Bacteria and immunization procedures
S. typhimurium 
4550 and 4666
asdA1 derivatives carrying the
asd+ plasmid pYA3259 encoding OVA were used
in these studies (32, 33). 4550-OVA is attenuated
(cya-1 crp-1 asdA1) and has a
50% lethal dose of >1010 bacteria by the oral
route. 4666-OVA (asdA1) has a 50% lethal dose of
<105 bacteria by the oral route, with doses from
105 up to 1010 4666-OVA
resulting in death on days 7–10 in naive animals (our unpublished
observations). Both strains are SR11 derivatives. Overnight cultures of
bacteria were grown in Luria-Bertoni broth and incubated at 37°C with
shaking. The concentration of bacteria was determined
spectrophotometrically, and bacteria were washed, resuspended at the
appropriate concentration in sterile PBS, and used to infect mice. The
actual bacterial doses administered were determined by counting
colonies following plating of serial dilutions of the inoculum on
Luria-Bertoni agar plates.
To examine the innate response to primary Salmonella
infection, a single oral dose of 4666 was used. For oral
immunizations, mice were treated intragastrically with 0.1 ml 1%
sodium bicarbonate, followed 5–10 min later by intragastric
inoculation of bacteria in a volume of 0.2 ml. The oral bacterial dose
given ranged from 5 x 108 to 1 x
1010 bacteria/mouse. No observable dose-related
differences in experimental outcome were observed within this dose
range. Oral administration of 4666 resulted in an acute infection in
naive animals. The infection was not lethal up to 5 days after
administration, and mice showed no overt signs of illness during this
period. Spleens from infected mice showed signs of enlargement compared
with naive mice only at 5 or more days of infection.
To examine the innate response to a secondary Salmonella
challenge, Salmonella immune mice were generated by
immunizing naive animals with 1 x 106
4550 i.p. or 1 x 109 4550 orally. The
use of either of these routes to generate immune mice resulted in no
detectable differences upon evaluation of the innate response after
secondary challenge. Immunization with 4550 gave a very mild,
short-term infection. For examination of secondary innate responses,
mice were left for 10–18 wk following immunization with 4550. At
this stage mice were either sacrificed as immune controls or were
challenged orally with 1 x 109 4666.
Spleens were removed aseptically at the time of sacrifice, and the bacterial load in each organ was determined by plating serial dilutions of single-cell suspensions on Luria-Bertoni agar plates. The total number of bacteria per organ was calculated based on the total cell count for each organ.
mAbs
The mAbs from the hybridomas GK1.5 (anti-CD4), YTS169.4
(anti-CD8), 145.2C11 (anti-CD3), N418 (anti-CD11c),
2.4G2 (anti-FcRII/III), RA3.6B2 (anti-B220), F4/80
(anti-M), M5/114 (anti-MHC-II), and C17.8
(anti-IL-12p40) (34) were used. H57-597 (anti-TCR
-chain), XMG1.2 (anti-IFN-), RB6-8C5 (anti-Ly6G/Gr-1),
and PK136 (anti-NK1.1) were purchased from BD PharMingen (San
Diego, CA). XT22 (anti-TNF-) was purchased from Nordic BioSite
(Stockholm, Sweden). FA-11 (anti-CD68/macrosialin) was obtained
from Serotec (Oxford, U.K.). The Abs R3-34 (rat IgG1), R35-95 (rat
IgG2a), and A95-1 (rat IgG2b; all from BD PharMingen) were used as
isotype controls. Abs used in flow cytometry were either directly
conjugated with PE, FITC, or allophycocyanin or were used biotinylated
as described below.
Cell preparation
Single-cell suspensions of spleens were prepared by mashing the organs through a cell filter (BD Biosciences, Le Pont de Claix, France). Cell preparations were washed with HBSS and resuspended in RPMI containing 10% FCS. A fraction was removed for calculating total viable cell number in each organ by trypan blue exclusion. The absolute number of each defined cell population was then determined using the total splenocyte count and flow cytometric analysis. To calculate relative population levels, the absolute number of a specific cell population in infected mice was divided by that in naive mice, with naive animals having a relative level of 1 for each population. A further fraction of the cell preparation was used to determine the bacterial load, as described above. Approximately two-thirds of the remainder was then transferred to Ultra-Low Cluster 24-well tissue culture plates (Costar Corning, Cambridge, MA), and brefeldin A (BFA; Sigma-Aldrich, St. Louis, MO) was added to a final concentration of 5 µg/ml. Plates were incubated for 5 h at 37°C before flow cytometric analysis of intracellular cytokine expression. Where indicated, BFA was omitted, and cells were cultured in medium alone for 5 h before staining. The remainder of each cell preparation was used immediately for flow cytometric analysis of surface markers as described below.
Flow cytometry
Single-cell suspensions for surface phenotype analyses were
washed in wash buffer (HBSS containing 3% FCS, 1 mM EDTA, and 10 mM
HEPES), and all subsequent steps were conducted in this buffer. Samples
were first blocked with anti-FcRII/III mAb for 15 min on ice.
Cells were washed once, and mAbs were applied for 30 min on ice.
7-Aminoactinomycin D (7AAD; Sigma-Aldrich) was included in all
stainings to define viable cells. In the majority of cases, all mAbs
were used directly conjugated. Where a biotinylated mAb was used, cells
were washed and incubated with streptavidin-allophycocyanin (BD
PharMingen) for 30 min on ice. CD68, which was used as one of the
markers to define M, is expressed weakly at the cell surface, but
strongly intracellularly. Therefore, samples examined for CD68
expression were first fixed in 2% paraformaldehyde in PBS for 20 min
at room temperature and then washed in permeabilization buffer (HBSS
containing 0.5% saponin and 0.5% BSA (Sigma-Aldrich)). Anti-CD68 was
applied in permeabilization buffer for 30 min at room temperature.
Cells were washed in permeabilization buffer and resuspended in wash
buffer. All samples were analyzed by four-color flow cytometry.
For the detection of intracellular cytokines, cells were washed and stained to detect surface molecules as described above. Cells were then fixed with 2% paraformaldehyde, and intracellular cytokines were detected by addition of biotinylated mAbs, followed by streptavidin-allophycocyanin diluted in permeabilization buffer for 30 min at room temperature. After final washes, cells were resuspended in wash buffer and analyzed by four-color flow cytometry.
All samples were acquired using a FACSCalibur flow cytometer (BD Biosciences) and were analyzed using CellQuest software (BD Biosciences). Total viable cells were acquired and stored in each case to allow the accurate determination of the proportions of each specific phenotype.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To quantitate changes within specific cell populations during the
early stages of Salmonella infection, spleens from naive
mice or from mice given a single oral dose of virulent
Salmonella 4666 were examined by flow cytometry. Mice
were sacrificed 2, 3, 4, or 5 days postinfection, and bacterial loads
were determined. On day 2 of infection mice had a mean of
<103 total bacteria/spleen (n =
9), which increased to 1.4 x 105 total
bacteria/organ on day 5 (n = 9). The lethal bacterial
load for Salmonella is 
108
organisms in the spleen (4). Thus, the animals were
examined during the progression of the infection and not during the
final overwhelming stages of disease.
Kinetic analysis of primary infection revealed that
Salmonella differentially influenced several cell
populations during the early course of infection, and dramatic changes
in relative cell number were evident as early as 2–3 days
postinfection (Fig. 1
). For example,
numbers of splenic NK cells, defined as
NK1.1+TCR-, and NKT
cells, defined as
NK1.1+TCR+, were
dramatically diminished as early as days 2–3 postinfection, with
significant reductions in these populations being apparent on day 5
(Fig. 1a and Table I). NK and
NKT cells were reduced by 62 and 88%, respectively, on day 5 after
primary infection. The number of splenic T lymphocytes, defined as
TCR+NK1.1- cells,
was also significantly decreased 5 days postinfection. In contrast, the
absolute number of splenic
B220+TCR- B cells
remained relatively stable during this period (Fig. 1a and
Table I). Thus, significant quantitative changes are apparent in
splenic cell populations of both the innate and adaptive immune systems
within the first 5 days of primary Salmonella infection.
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populations dramatically increase during
primary Salmonella infection
The absolute number of splenic neutrophils and M was determined
over the first 5 days following oral Salmonella infection.
These populations were identified by flow cytometry based on
differential surface expression of F4/80, CD68, and Gr-1 (35, 36) (Fig. 2). Neutrophils were
defined as cells expressing the highest level of Gr-1 within the
population expressing negative to low levels of CD68 (Fig. 2). M
were identified as a nonoverlapping
CD68highF4/80int-high
population expressing lower levels of Gr-1 (Fig. 2).
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populations during
the first 5 days of infection (Figs. 1
b and 2). First, some
up-regulation of F4/80 and Gr-1 expression was apparent within the M
population during infection (Fig. 2). Salmonella infection
did not, however, significantly influence surface expression of F4/80
or Gr-1 on neutrophils (Fig. 2). In addition, Salmonella
infection altered the surface expression of MHC-II on both neutrophils
and M. MHC-II, which was expressed to some extent by <60% of
CD68highF4/80int-high cells
(M) in the spleen of naive animals, was expressed by >95% of these
cells on day 5 of infection (n = 6; our
unpublished observations). Likewise, MHC-II expression was increased on
neutrophils in response to Salmonella. While only 5% of
CD68lowGr-1high cells from
naive mice or mice on day 2 after Salmonella challenge were
MHC-II+, 30% of neutrophils expressed surface
MHC-II 5 days postinfection (n = 6; our
unpublished observations).
Quantitative changes were also apparent within neutrophil and M
populations early during primary Salmonella infection.
Neutrophils were fewer in number than M in the spleens of naive mice
and increased rapidly and steadily throughout the primary infection,
becoming at least equivalent in number to M by day 5 (Fig. 1b and Table I). This involved a 10-fold increase in
neutrophil number compared with a tripling in the absolute number of
M, the latter increasing slowly in the spleens of infected mice
(Fig. 1b).
Thus, both qualitative and quantitative changes occur in splenic M
and neutrophil populations as a result of oral Salmonella
infection. The observed increase in total splenic cellularity 5 days
after Salmonella infection (Table I) can largely be
attributed to increases in these two phagocytic cell populations.
Furthermore, the dramatic increase in neutrophil number makes them the
predominant phagocytic population in this organ during primary
Salmonella infection.
Neutrophils and M dominate the TNF- response to primary
Salmonella infection
Functional aspects of the primary innate immune response to
Salmonella were addressed by ex vivo flow cytometric
analysis of intracellular cytokine expression in cells from
Salmonella-infected animals. In these experiments
neutrophils and M were defined as nonoverlapping
CD68lowGR1high and
CD68highGR1low-int
populations, respectively (Fig. 3a). Parallel stainings
analyzing F4/80 expression on cells from the same animals demonstrated
that the populations analyzed for cytokine production were also
neutrophils or M according to the previous definition
(F4/80low and
F4/80int-high, respectively; Fig. 2 and data not
shown). Furthermore, due to the limited number of NK and NKT cells at
later times postinfection (Fig. 1), it was not always possible to
accurately distinguish between these populations. Therefore, total
NK1.1+ cells, which include both NK and NKT
cells, were analyzed as a single population for cytokine analyses
unless stated otherwise.
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-producing
TCR+NK1.1- T cells
or NK1.1+ cells were detected (Fig. 3b). As discussed above, it was difficult to
accurately quantify TNF--producing NK and NKT cells separately,
particularly in animals on day 4 or 5 of infection. However, a
TNF-+ population was observed in splenic NKT
cells, but not in NK cells, in mice examined 3 days after primary
Salmonella infection (Fig. 4).
Although the number of TNF--producing NKT cells was low in infected
mice, TNF-+ NKT cells were present at a
greater level than in naive mice in six of nine animals on day 2, three
of four animals on day 3, four of four animals on day 4, and five of
six animals on day 5. Thus, NKT, but not NK, cells contribute to the
splenic TNF- response to primary Salmonella
infection.
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demonstrated dramatically increased numbers of cells producing TNF-
on day 5 of infection (Fig. 3b). At this time, 15% of
cells within the neutrophil and M populations produced TNF-.
Numerically, TNF--producing phagocytes were >50 times more frequent
than splenic lymphocytes making this cytokine on day 5. Thus,
nonlymphocyte populations completely dominate the TNF- response
early during primary Salmonella infection.
Significant M IFN- production during primary
Salmonella infection
The production of IFN- by cell populations involved in the
early response to Salmonella was also addressed by
intracellular cytokine staining (Figs. 4 and 5). Neutrophils and M were defined as
described for TNF- staining, although neutrophil expression of
IFN- was examined as a distinct phenomenon, which is described
below. Within splenic cell populations during a primary infection,
increased numbers of IFN--producing TCR+
cells were apparent only on day 5 (Fig. 5b). Splenic
IFN--producing NK1.1+ cells began to increase
in number 2 days after oral infection and were further increased on day
5 (Fig. 5b). Production of IFN- was observed in both the
TCR+ and TCR-
fractions of NK1.1+ cells in animals in which a
sufficient number of both populations remained to allow their separate
assessment (Fig. 4). Surprisingly, the splenic IFN- response during
primary infection was dominated by the M population, which exhibited
significantly increased numbers of IFN--positive cells as early as
day 2 after infection that further increased on day 5 (Fig. 5b). In some individuals up to 30% of all splenic M
expressed IFN- at this later time point. Thus, although there is a
significant involvement of nonphagocytic cells in the IFN- response
on day 5 of primary Salmonella infection, the largest number
of IFN--expressing cells is within the M population during the
early response.
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The contribution of neutrophils to the IFN- response in
Salmonella infection was examined by intracellular cytokine
staining. Neutrophils in spleen of naive mice unexpectedly stained
positive for IFN- (Fig. 6a). IFN--positive
neutrophils were also detected in primary
Salmonella-infected mice (Fig. 6a). However,
animals examined on days 3–5 following primary infection had a
neutrophil population containing a higher proportion of
IFN--negative cells than that observed in naive mice or mice
examined 1–2 days following primary infection (our unpublished
observations).
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staining within the neutrophil
population from both naive and Salmonella-challenged animals
was confirmed based on several observations. First, staining with
isotype-matched control Abs demonstrated that the background level of
staining for the bulk neutrophil population was well below that
observed with the anti-IFN- Ab (Fig. 6a). Second,
staining with anti-TNF-, which is the same isotype as the
anti-IFN- mAb, did not stain neutrophils in naive mice (Fig. 3a). Third, no specific IFN- staining was observed in
cells not subjected to permeabilization (our unpublished
observations). Finally, the anti-IFN- Ab functioned as expected,
giving low background staining in a number of cell types in naive
animals and specific staining in cells such as
NK1.1+ cells and M from primary infected
animals (Figs. 4 and 5a).
To further analyze the nature of neutrophil IFN- during the primary
response to Salmonella, parallel spleen samples were
incubated either with or without BFA during the 5-h incubation period
before intracellular staining. Omitting BFA when neutrophils from mice
infected with Salmonella 3, 4, or 5 days previously were
stained for intracellular IFN- resulted in a reduced number of
IFN--positive neutrophils and a concomitant appearance of an
IFN--negative population (Fig. 6b). In naive animals or
animals infected 2 days earlier with Salmonella, neither of
these changes was apparent when BFA was omitted from the intracellular
staining protocol (Fig. 6b). The function of BFA was
controlled in two ways. First, omitting BFA in parallel samples stained
for intracellular TNF- resulted in the loss of TNF--positive
neutrophils in Salmonella-infected mice (our
unpublished observations; see Fig. 3a). Moreover, omitting
BFA from intracellular IFN- staining of splenic T cells from mice
infected 14 days earlier and restimulated with Salmonella
lysate in vitro resulted in a profound reduction of cytokine-positive
cells (our unpublished observations). Together these data show
that neutrophils from naive mice have an intracellular pool of IFN-
that is a potential source of this cytokine during
Salmonella infection.
Quantitative splenic responses in Salmonella-challenged immune mice differ from those occurring during a primary infection
Animals immunized 10–18 wk previously with avirulent
Salmonella 4550 and orally challenged with virulent
Salmonella 4666 were used to examine quantitative and
qualitative aspects of cell populations early after a secondary
bacterial challenge. Parallel groups of mice given 4550, but not
challenged with 4666 were used as age-matched immune control mice.
No bacteria were recovered from the spleen of the immune control mice
in any instance (n = 11), while mice sacrificed 5 days
after 4666 challenge had 104 total
bacteria/spleen (n = 12). In contrast to splenomegaly
and increased total spleen cellularity in mice 5 days after primary
Salmonella infection (Table I), total spleen cell number was
not significantly altered 5 days after secondary infection of
Salmonella-immune mice relative to immune control
animals (Table II).
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). The
extent of the reduction in T cell number, 25%, was similar 5 days
after primary or secondary infection. In contrast to the changes
observed during primary infection, where a dramatic reduction of NK and
NKT cells was apparent (Table I), only the absolute number of splenic
NK cells, but not NKT cells, was significantly affected on day 5 of
secondary Salmonella challenge (Table II). Moreover, the
influence of secondary challenge on NK cells was limited compared with
that after a primary infection. While over 65% of splenic NK cells
remained at day 5 of secondary infection, only 35% of these cells
remained 5 days after primary infection.
Quantitative analysis of phagocytic cell populations demonstrated that
splenic neutrophil number increased <3 times by day 5 of secondary
Salmonella infection compared with immune control mice,
remaining less abundant than M in this organ. This is in contrast to
the 10-fold increase in the splenic neutrophils during primary
Salmonella infection, which were more abundant than M on
day 5 (Fig. 1b and Table I). Furthermore, the response to
secondary Salmonella challenge was associated with only a
1.5-fold increase in splenic M number 5 days after secondary
challenge (Table II), a less dramatic rise compared with the tripling
of splenic M 5 days after primary infection (Table I).
Some differences in the absolute number of specific cell populations,
such as NKT cells and neutrophils, were apparent in the spleen of
immune control mice compared with naive animals (Tables I and II). This
may be a result of the immunization or of the age of the immune control
animals used in the secondary infection experiments, which were 10–18
wk older than the naive controls used in primary infection experiments.
However, it is unlikely that the observed differences in absolute cell
numbers in the spleen of the naive controls vs immune controls
influenced the results obtained when analyzing the responses occurring
after a secondary Salmonella challenge. This is underscored
by data obtained in experiments, run in parallel with those examining
the secondary response, where age-matched (18- to 24-wk-old) naive
animals were given a primary oral challenge with Salmonella.
In these experiments the older infected mice had a similar bacterial
load (105/spleen), changes in the absolute
number of innate cell populations (Table II) and phagocyte-dominated
cytokine response following the infection (see below) to those
described for younger animals. Thus, a significant, but much less
pronounced, influx of neutrophils and M is observed during the early
stages of a response to a secondary challenge.
Reduced TNF- and negligible IFN- expression by innate cell
populations during the early response to secondary
Salmonella challenge
Production of TNF- and IFN- was assessed in cell populations
during the first few days following a secondary challenge of immune
animals with virulent Salmonella. Few T cells or
NK1.1+ cells producing TNF- were observed on
day 5 after secondary infection, revealing only a minimal contribution
of these populations to TNF- production in challenged, immune mice
early during the secondary response (Table III). Similar to the primary response,
Salmonella challenge of immune animals resulted in the
dominance of the splenic TNF- response by phagocytes. That is,
increased numbers of TNF--producing neutrophils and M were
present in infected mice compared with immune controls (Table III).
However, the absolute number of TNF--positive splenic phagocytes was
10-fold fewer 5 days after secondary challenge of immune animals
relative to that observed 5 days after primary infection (Fig. 3b and Table III). This is not only due to the presence of
fewer neutrophils and M in the spleen of challenged immune mice
compared with after primary infection (Table II), but also a reduction
in the proportion of phagocytes producing TNF-. Thus, on day 5 of
secondary challenge, 2–4% of phagocytes produce TNF- compared with
15% at the same time point following primary infection. This
proportional reduction in TNF- expression was apparent in both
neutrophil and M populations.
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-producing
TCR+ cells in immune/challenged mice was
elevated compared with the number in immune control animals (Table III). However, in contrast to the primary response, challenge of
animals previously immunized with Salmonella resulted in
only small numbers of IFN--producing NK1.1+
cells on day 5 (Fig. 5b and Table III). Furthermore, the
number of splenic M expressing IFN- was not significantly
increased in mice receiving a second infection of Salmonella
compared with immune controls (Table III). Intracellular IFN-
expression was also observed by splenic neutrophils from both immune
control (n = 11) and secondary-infected animals
(n = 13; our unpublished observations). Together
these data demonstrate that challenge of immune hosts results in
considerably limited splenic TNF- and IFN- responses. Moreover,
the relative contributions of defined cell populations in these
responses are altered compared with those observed following a primary
challenge. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, NK cells, and various
cytokines has been demonstrated in a number of infections, including
Salmonella. However, an integrated picture of the innate
response to a pathogen has not been established. The current study
addressed this by characterizing quantitative and functional changes in
defined cell populations in the first few days following oral
Salmonella infection of naive mice as well as within the
framework of previously acquired specific immunity.
The earliest changes in splenic cell populations detected were a rapid
and dramatic reduction in the absolute number of NK and NKT cells and a
concomitant increase in splenic M and neutrophils. The 10-fold
increase in neutrophil number that occurred after primary infection
transformed this minor splenocyte population to the most abundant
phagocytic cell type in the spleen, outnumbering even M. The cell
populations undergoing changes in absolute number during the early
stages of infection also contributed to production of cytokines
important for surviving Salmonella infection, such as
TNF- and IFN- (19, 20, 23, 24, 25). The abundance of
neutrophils and M producing TNF- underscore their importance as
the dominate source of this cytokine early during infection.
Our data also show that IFN-, another cytokine critical for host
survival to Salmonella (20, 24, 26), is
produced by both lymphoid and myeloid cells early during infection. In
addition to populations predicted to be early sources of IFN-, such
as NK cells, NKT cells, and TCR T cells, our results revealed two
surprising IFN--positive populations in
Salmonella-infected mice, M and neutrophils.
Quantitatively, M were the most abundant splenic cell population
producing this cytokine 2–5 days after primary infection, while
NK1.1+ cells and TCR T cells also contained
significant numbers of IFN--producing cells on day 5. It has been
shown in vitro that M can secrete IFN- following stimulation with
IL-12 and IL-18 (37, 38) or IL-12 and mycobacteria
(39). Moreover, M can secrete IFN- during the course
of mycobacterial infection (40). However, the relative
contribution of M-derived IFN- to the overall IFN- response
during infection has not previously been quantified. Both the capacity
of M to quickly produce IFN- during Salmonella
infection and their abundance in the spleen of infected mice suggest an
additional role of these phagocytes in both the innate and adaptive
responses to primary infection. M-derived IFN- may serve to
enhance the bactericidal capacity of phagocytes, facilitate Ag
presentation, and/or influence the Th polarization of the immune
response (41). In addition, the data show that neutrophils
contain intracellular stores of IFN- that can be released upon
Salmonella infection. This raises the possibility that these
abundant cells may also be important sources of IFN- early during
infection. IFN- expression by murine neutrophils has only recently
been reported (57), and human placental neutrophils can
produce IFN- (42). Furthermore, murine neutrophils
contain intracellular stores of IL-12 (43, 44). Thus, in
addition to their function as phagocytes, neutrophils may be a
previously unappreciated source of cytokines that contribute to
antimicrobial immunity.
The mechanisms underlying the early quantitative and functional changes
in innate cell populations and whether they require direct bacterial
contact or are mediated by soluble factors are currently not known.
However, the mild bacterial load during early infection suggests that
relatively few cells will have directly encountered
Salmonella. Further cognate pathogen recognition may occur,
for example, through the interaction of Salmonella-derived
LPS and Toll-like receptor 4 (45). Pathogen-derived
stimuli combined with host-derived factors, such as cytokines and
chemokines, may recruit and activate additional cells. This would
result in a second wave of anti-microbial defense mechanisms. M,
neutrophils, as well as NKT cells may be part of the first wave of
direct responders, since these cells are a bacterial target during
early infection (8, 46).
Cognate interaction may also be involved in the observed changes in NKT
cells during Salmonella infection. Although
Salmonella-derived NKT cell-specific ligands remain to be
defined, NKT cells rapidly respond to bacterially derived,
nonproteinaceous, cell wall components (47). Once
activated, NKT cells produce high levels of cytokines, including
IFN- (48) and, as observed here in response to
Salmonella infection, TNF-. These NKT-derived cytokines
may subsequently activate NK cells (49, 50) and other
bystanders (48). Activated NKT cells up-regulate the
expression of Fas and undergo rapid apoptosis (51), and in
vivo activation of hepatic NKT cells by -GalCer results in a nearly
complete (>98%) temporary depletion of these cells (52).
Thus, the rapid loss of NKT cells from the spleen following primary
challenge with Salmonella may result from activation-induced
apoptosis of these cells and is a strong indication that these
cells are among the first responders to Salmonella
infection.
Salmonella infection not only influenced cells of the innate
immune system during the early course of infection, but also splenic T,
but not B, lymphocytes. The observed reduction in splenic T cells
during primary Salmonella infection could be due to cell
death or recruitment to the circulation and periphery. Although T cells
proximal to infectious foci undergo apoptosis during the early stages
of Listeria infection (53), it is currently not
known whether Salmonella directly affects T cell viability
in vivo. However, splenic T cells are broadly activated when examined
later during Salmonella infection (33), and
IFN--producing T cells are significantly elevated 5 days after
primary infection. These data suggest that activation-induced death of
splenic T cells could contribute to the observed T cell loss during
primary Salmonella infection. The induced release of splenic
T cells into the periphery, as a result of inflammation at other sites,
may also contribute to this phenomenon. Alternatively, the expression
of homeostatic site chemokines, such as secondary lymphoid tissue
chemokine and stromal-derived factor-1, within the spleen may be
replaced by inflamed site chemokines, including RANTES and macrophage
inflammatory proteins 1 and 1 (54), during
Salmonella infection. This may result in an inability of
circulating naive T cells to traffic back into the spleen and an
overall decrease in splenic T cell number.
Analysis of innate cell populations in immune mice orally challenged
with Salmonella revealed some differences relative to those
observed after primary infection. In sharp contrast to the primary
response, no change in the absolute number of splenic NKT cells was
apparent 5 days following secondary Salmonella challenge.
This suggests that NKT cells may not be involved in the initiation of
the innate or acquired response to Salmonella in an immune
host. In addition, splenic NK cell depletion was less dramatic in
previously immune mice. The cytokine-producing populations also
differed in primary vs secondary infection. Whereas in primary
infection, the number of TNF--producing neutrophils exceeded that of
M producing this cytokine, the converse was true following secondary
challenge of immune mice. The surprising absence of the dominant
IFN--producing M population observed in the primary response
together with the diminished number of NK1.1+
cells producing IFN- show that the overall IFN- response is less
5 days after secondary infection relative to the same time following
primary infection. Together these data suggest that alternate
initiating populations and mechanisms may be employed during the innate
response during primary and secondary bacterial challenges.
Phagocyte influx into the spleen remained the dominant cellular feature
of secondary Salmonella infection in immune hosts, in which
these cells can comprise 10% of total splenocytes on day 5. However,
the relative increase and absolute number of neutrophils and M in
infected immune hosts was reduced compared with a primary response.
Furthermore, substantially fewer TNF--producing phagocytes and
approximately one-fifth as many IFN--producing
NK1.1+ cells were also present 5 days after
secondary Salmonella challenge compared with day 5 following
primary infection. Moreover, an increase in M producing IFN-,
which was a striking feature of the primary response, was lacking in
the secondary response. Thus, despite the significant influx of
neutrophils and M into the spleen in response to secondary
infection, the magnitude of the phagocyte cytokine response is severely
restricted.
Access to bacteria influences phagocyte responses. More bacteria were
recovered from the spleen on day 5 of primary infection than at the
same time after secondary challenge of immune mice. In contrast, fewer
bacteria were recovered from the spleen on day 2 after primary
infection relative to the number on day 5 after challenge of immune
mice. Despite this, a significant increase in M producing IFN-,
for example, was detected only during the primary response. Thus, a
background of specific acquired immunity rather than splenic bacterial
load appears to influence the function of the innate system. The lower
quantity of bacteria recovered from the spleen of immune/challenged
mice may result from reduced intestinal penetration due to the actions
of secretory IgA. However, identical results were obtained when immune
mice were generated using either i.p. or oral immunization routes,
making it unlikely that specific secretory IgA accounts for the
observed differences. Alternatively, Ab-mediated opsonic killing could
reduce the capacity of viable bacteria to reach the spleen or enhance
bacterial killing within this organ. Such functions of Abs present in
immune mice could, in turn, diminish or alter the response of splenic
innate cell populations. Indeed, B cells have been shown to play a role
in protecting against oral challenge with virulent
Salmonella (55, 56), possibly by influencing
the establishment of T cell memory. It is also possible that M and
neutrophils receive additional, possibly cognate, signals as well as an
altered cytokine milieu during a secondary compared with a primary
response. Thus, interplay between the innate and adaptive immune
systems that influence the innate response in immune hosts occurs, but
the mechanisms underlying this cross-talk remain to be elucidated.
Note added in proof.
IFN- expression by murine neutrophils has also recently
been reported in a pulmonary infection model (57).
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mary Jo Wick, Department of Clinical Immunology, University of Goteborg, Guldhedsgatan 10, SE 413 46 Goteborg, Sweden. E-mail address: mary-jo.wick{at}immuno.gu.se
3 Abbreviations used in this paper: M, macrophage; 7AAD, 7-aminoactinomycin D; BFA, brefeldin A; PP, Peyer’s patches.
Received for publication April 25, 2002. Accepted for publication August 12, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), B cells
(B220+TCR
), NK cells
(TCR
) in the spleen 2, 3, 4,
or 5 days after Salmonella infection are shown.
b, The relative level of neutrophils
(CD68lowF4/80low-intGr-1high; 
)
and M
) in the spleen 2, 3, 4, or 5 days after Salmonella
infection are shown. For all time points, five to nine mice were
examined in at least two separate experiments. The data represent the
mean value of all mice analyzed in each group.
, mice infected 2 or 5 days earlier
with Salmonella. Bars indicate the mean ± 1 SD.
Six to 12 individuals were examined at each time point in at least two
separate experiments. Significant p values, calculated
vs the value from the respective control group using Student’s
t test, are indicated. T,
TCR
