Abstract
This study examines innate immunity to oral Salmonella during primary infection and after secondary challenge of immune mice. Splenic NK and NKT cells plummeted early after primary infection, while neutrophils and macrophages (Mφ) 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-γ were, unexpectedly, neutrophils and Mφ, the former having intracellular stores of IFN-γ that were released during infection. IFN-γ-producing phagocytes greatly outnumbered IFN-γ-producing NK cells, NKT cells, and T cells during the primary response. TNF-α production was also dominated by neutrophils and Mφ, which vastly outnumbered NKT cells producing this cytokine. Neither T cells nor NK cells produced TNF-α early during primary infection. The TNF-α response was reduced in a secondary response, but remained dominated by neutrophils and Mφ. Moreover, no significant IFN-γ production by Mφ was associated with the secondary response. Indeed, only NK1.1+ cells and T cells produced IFN-γ in these mice. These studies provide a coherent view of innate immunity to oral Salmonella infection, reveal novel sources of IFN-γ, and demonstrate that immune status influences the nature of the innate response.
The innate immune system has the basic function of identifying and eradicating microbial invaders and alerting the adaptive immune system to their presence (1, 2, 3). The innate response to a bacterial challenge involves recognition of bacterial components, such as LPS and DNA (3) and activation of cells as a result of such an encounter (1, 2). The consequent release of inflammatory mediators results in infiltration of various cell types to the site of infection and amplification of the response. Bacterial uptake and destruction by phagocytic cells also facilitates host protection. Innate immune processes are conducted by cells relatively unrestricted in pathogen specificity, including NK cells, NKT cells, neutrophils, and macrophages (Mφ)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 Mφ 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, Mφ appear to function in a primarily protective capacity, rather than acting as APC. The induction of acquired immunity is not significantly compromised in Mφ-depleted mice infected with Salmonella, whereas Mφ 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-α (19, 20, 21, 22, 23, 24, 25), IFN-γ (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-γ results in increased bacterial replication (20, 24) and host susceptibility (20), but does not influence Ab production (28). Neutralization of TNF-α 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-α and IFN-γ 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-γ early during infection (20).
Thus, neutrophils, Mφ, 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.
Materials and Methods
Mice
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 χ4550 and χ4666 ΔasdA1 derivatives carrying the asd+ plasmid pYA3259 encoding OVA were used in these studies (32, 33). χ4550-OVA is attenuated (Δcya-1 Δcrp-1 ΔasdA1) and has a 50% lethal dose of >1010 bacteria by the oral route. χ4666-OVA (ΔasdA1) has a 50% lethal dose of <105 bacteria by the oral route, with doses from 105 up to 1010 χ4666-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 χ4666 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 × 108 to 1 × 1010 bacteria/mouse. No observable dose-related differences in experimental outcome were observed within this dose range. Oral administration of χ4666 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 × 106 χ4550 i.p. or 1 × 109 χ4550 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 χ4550 gave a very mild, short-term infection. For examination of secondary innate responses, mice were left for 10–18 wk following immunization with χ4550. At this stage mice were either sacrificed as immune controls or were challenged orally with 1 × 109 χ4666.
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-CD8α), 145.2C11 (anti-CD3), N418 (anti-CD11c), 2.4G2 (anti-FcγRII/III), RA3.6B2 (anti-B220), F4/80 (anti-Mφ), M5/114 (anti-MHC-II), and C17.8 (anti-IL-12p40) (34) were used. H57-597 (anti-TCR β-chain), XMG1.2 (anti-IFN-γ), RB6-8C5 (anti-Ly6G/Gr-1), and PK136 (anti-NK1.1) were purchased from BD PharMingen (San Diego, CA). XT22 (anti-TNF-α) 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-FcγRII/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 Mφ, 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.
Results
Differential modulation of splenic lymphoid cell populations during primary Salmonella infection
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 χ4666 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 × 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+TCRαβ−, and NKT cells, defined as NK1.1+TCRαβ+, were dramatically diminished as early as days 2–3 postinfection, with significant reductions in these populations being apparent on day 5 (Fig. 1⇓a 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 TCRαβ+NK1.1− cells, was also significantly decreased 5 days postinfection. In contrast, the absolute number of splenic B220+TCRαβ− B cells remained relatively stable during this period (Fig. 1⇓a 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.
Kinetics of cell population changes during primary Salmonella infection. The relative quantity of cell populations within total splenocytes from naive mice (N) or mice challenged 2–5 days previously with Salmonella are shown. Results are expressed as the mean relative population level compared with mean values from naive mice and were calculated based on the absolute number of each cell type as described in Materials and Methods. a, The relative level of T cells (TCRαβ+NK1.1−; ▪), B cells (B220+TCRαβ−; □), NK cells (TCRαβ−NK1.1+; •), and NKT cells (TCRαβ+NK1.1+; ○) 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 Mφ (CD68highF4/80int-highGr-1low-int; ▵) 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.
Absolute number of splenic cell populations early during primary Salmonella infection
Neutrophil and Mφ populations dramatically increase during primary Salmonella infection
The absolute number of splenic neutrophils and Mφ 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⇓). Mφ were identified as a nonoverlapping CD68highF4/80int-high population expressing lower levels of Gr-1 (Fig. 2⇓).
Splenic neutrophil and Mφ populations during primary Salmonella infection. Viable (7AAD−) splenocytes were gated in phenotypic flow cytometric analyses. Neutrophils and Mφ were identified as nonoverlapping populations based on the expression of CD68, F4/80, and Gr-1, as shown in the left dot plot. Neutrophils were defined as CD68lowF4/80low-int (R1) and Gr-1high (R3). Mφ were defined as CD68highF4/80int-high (R2) and subsequently as Gr-1low-int (R4). All axes represent log fluorescence intensity. Plots are representative of spleen samples from naive mice or mice infected 5 days previously with Salmonella as indicated.
Primary Salmonella infection resulted in qualitative and quantitative changes in splenic neutrophil and Mφ 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 Mφ 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 Mφ. MHC-II, which was expressed to some extent by <60% of CD68highF4/80int-high cells (Mφ) 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 Mφ populations early during primary Salmonella infection. Neutrophils were fewer in number than Mφ in the spleens of naive mice and increased rapidly and steadily throughout the primary infection, becoming at least equivalent in number to Mφ by day 5 (Fig. 1⇑b and Table I⇑). This involved a 10-fold increase in neutrophil number compared with a tripling in the absolute number of Mφ, the latter increasing slowly in the spleens of infected mice (Fig. 1⇑b).
Thus, both qualitative and quantitative changes occur in splenic Mφ 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 Mφ dominate the TNF-α 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 Mφ were defined as nonoverlapping CD68lowGR1high and CD68highGR1low-int populations, respectively (Fig. 3⇓a). Parallel stainings analyzing F4/80 expression on cells from the same animals demonstrated that the populations analyzed for cytokine production were also neutrophils or Mφ 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.
Salmonella-induced TNF-α production by splenic cell populations. a, For intracellular cytokine analysis, viable (7AAD−) neutrophils and Mφ were defined as nonoverlapping CD68lowGr-1high (R1) and CD68highGr-1low-int (R2) populations, respectively. These populations were subsequently examined for neutrophil and Mφ TNF-α expression (R3 and R4, respectively). All axes represent log fluorescence intensity. b, The absolute number of TNF-α-positive cells within the indicated splenic cell population during primary Salmonella infection is shown. □, Naive mice; ▪ and ▨, 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, TCRαβ+NK1.1− T cells; NK1.1, NK1.1+ cells; Neut, CD68lowGr-1high neutrophils; Mφ, CD68highGr-1low-int Mφ.
In the spleens of animals undergoing a primary infection with virulent Salmonella, few, if any, TNF-α-producing TCRαβ+NK1.1− T cells or NK1.1+ cells were detected (Fig. 3⇑b). As discussed above, it was difficult to accurately quantify TNF-α-producing NK and NKT cells separately, particularly in animals on day 4 or 5 of infection. However, a TNF-α+ 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-α-producing NKT cells was low in infected mice, TNF-α+ 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-α response to primary Salmonella infection.
TNF-α and IFN-γ production by splenic NK and NKT cells during Salmonella infection. Splenocytes from naive mice or mice infected 3 days earlier with Salmonella were analyzed for intracellular TNF-α or IFN-γ expression. Viable (7AAD−) NK1.1+ cells were gated and subsequently analyzed for expression of TCRαβ and cytokine. Representative dot plots are shown. Six mice were analyzed in each group in two separate experiments. All axes represent log fluorescence intensity.
In contrast to lymphoid cell populations, both neutrophils and Mφ demonstrated dramatically increased numbers of cells producing TNF-α on day 5 of infection (Fig. 3⇑b). At this time, ∼15% of cells within the neutrophil and Mφ populations produced TNF-α. Numerically, TNF-α-producing phagocytes were >50 times more frequent than splenic lymphocytes making this cytokine on day 5. Thus, nonlymphocyte populations completely dominate the TNF-α response early during primary Salmonella infection.
Significant Mφ IFN-γ production during primary Salmonella infection
The production of IFN-γ by cell populations involved in the early response to Salmonella was also addressed by intracellular cytokine staining (Figs. 4⇑ and 5⇓). Neutrophils and Mφ were defined as described for TNF-α staining, although neutrophil expression of IFN-γ was examined as a distinct phenomenon, which is described below. Within splenic cell populations during a primary infection, increased numbers of IFN-γ-producing TCRαβ+ cells were apparent only on day 5 (Fig. 5⇓b). Splenic IFN-γ-producing NK1.1+ cells began to increase in number 2 days after oral infection and were further increased on day 5 (Fig. 5⇓b). Production of IFN-γ was observed in both the TCRαβ+ and TCRαβ− 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-γ response during primary infection was dominated by the Mφ population, which exhibited significantly increased numbers of IFN-γ-positive cells as early as day 2 after infection that further increased on day 5 (Fig. 5⇓b). In some individuals up to 30% of all splenic Mφ expressed IFN-γ at this later time point. Thus, although there is a significant involvement of nonphagocytic cells in the IFN-γ response on day 5 of primary Salmonella infection, the largest number of IFN-γ-expressing cells is within the Mφ population during the early response.
Salmonella-induced splenic IFN-γ production. a, Viable (7AAD−) populations within the spleen were defined by surface phenotype and subsequently analyzed for IFN-γ expression. Representative examples of IFN-γ production by splenic NK1.1+ cells (R1; upper dot plots) and Mφ (R2; lower dot plots) in naive animals and on d 5 of Salmonella infection are shown. Mφ were defined as in Fig. 3⇑. All axes represent log fluorescence intensity. b, The absolute number of IFN-γ+ cells in defined splenic cell populations during primary Salmonella infection. □, Naive mice; ▪ and ▨, mice infected 2 or 5 days earlier with Salmonella, respectively. Bars indicate the mean ± 1 SD. Six to 12 mice were analyzed for both naive and infected mice at each time point in at least two separate experiments. Significant p values, calculated vs the value from the relevant control group using Student’s t test, are indicated. T, TCRαβ+NK1.1− T cells; NK1.1, NK1.1+ cells; Mφ, CD68highGr-1low-int Mφ.
Splenic neutrophils constitutively express intracellular IFN-γ
The contribution of neutrophils to the IFN-γ response in Salmonella infection was examined by intracellular cytokine staining. Neutrophils in spleen of naive mice unexpectedly stained positive for IFN-γ (Fig. 6⇓a). IFN-γ-positive neutrophils were also detected in primary Salmonella-infected mice (Fig. 6⇓a). However, animals examined on days 3–5 following primary infection had a neutrophil population containing a higher proportion of IFN-γ-negative cells than that observed in naive mice or mice examined 1–2 days following primary infection (our unpublished observations).
Splenic neutrophils express intracellular IFN-γ in naive and Salmonella-infected mice. a, Neutrophils, defined within viable splenocytes as in Fig. 3⇑, were stained using anti-IFN-γ or an isotype-matched control mAb as indicated. The samples shown are from a naive mouse or a mouse infected 3 days previously with Salmonella as indicated. All axes represent log fluorescence intensity. b, Splenocytes were incubated with (thin line) or without (thick line) BFA for 5 h before staining for surface molecules and intracellular IFN-γ. Histograms show gated neutrophils (CD68lowGr-1high) from a representative individual naive mouse and an individual taken on day 3 of Salmonella infection. M1, IFN-γ-negative neutrophils. The y-axes represent cell number, and the x-axes represent log fluorescence intensity. Staining for intracellular IFN-γ or using isotype control mAb was performed on a total of 48 mice (nine naive and 39 Salmonella-infected) in at least five independent experiments. For the intracellular IFN-γ stainings, BFA was included in samples from all nine control mice, while it was omitted from parallel samples from six of these animals. Likewise, BFA was included in samples from all 39 infected animals and was omitted from parallel samples from 21 of the infected mice.
The specificity of the IFN-γ 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-γ Ab (Fig. 6⇑a). Second, staining with anti-TNF-α, which is the same isotype as the anti-IFN-γ mAb, did not stain neutrophils in naive mice (Fig. 3⇑a). Third, no specific IFN-γ staining was observed in cells not subjected to permeabilization (our unpublished observations). Finally, the anti-IFN-γ 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 Mφ from primary infected animals (Figs. 4⇑ and 5⇑a).
To further analyze the nature of neutrophil IFN-γ 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-γ resulted in a reduced number of IFN-γ-positive neutrophils and a concomitant appearance of an IFN-γ-negative population (Fig. 6⇑b). 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. 6⇑b). The function of BFA was controlled in two ways. First, omitting BFA in parallel samples stained for intracellular TNF-α resulted in the loss of TNF-α-positive neutrophils in Salmonella-infected mice (our unpublished observations; see Fig. 3⇑a). Moreover, omitting BFA from intracellular IFN-γ 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-γ 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 χ4550 and orally challenged with virulent Salmonella χ4666 were used to examine quantitative and qualitative aspects of cell populations early after a secondary bacterial challenge. Parallel groups of mice given χ4550, but not challenged with χ4666 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 χ4666 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⇓).
Absolute number of splenic cell populations during secondary Salmonella infection
As in a primary response, secondary Salmonella infection resulted in a reduction of absolute T cell, but not B cell, number in the spleen on day 5 compared with immune control mice (Table II⇑). 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 Mφ 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 Mφ on day 5 (Fig. 1⇑b and Table I⇑). Furthermore, the response to secondary Salmonella challenge was associated with only a 1.5-fold increase in splenic Mφ number 5 days after secondary challenge (Table II⇑), a less dramatic rise compared with the tripling of splenic Mφ 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 Mφ is observed during the early stages of a response to a secondary challenge.
Reduced TNF-α and negligible IFN-γ expression by innate cell populations during the early response to secondary Salmonella challenge
Production of TNF-α and IFN-γ 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-α were observed on day 5 after secondary infection, revealing only a minimal contribution of these populations to TNF-α 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-α response by phagocytes. That is, increased numbers of TNF-α-producing neutrophils and Mφ were present in infected mice compared with immune controls (Table III⇓). However, the absolute number of TNF-α-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. 3⇑b and Table III⇓). This is not only due to the presence of fewer neutrophils and Mφ 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-α. Thus, on day 5 of secondary challenge, 2–4% of phagocytes produce TNF-α compared with ∼15% at the same time point following primary infection. This proportional reduction in TNF-α expression was apparent in both neutrophil and Mφ populations.
Absolute number of cytokine-positive cells in splenic populations during secondary Salmonella infection
The absolute number of IFN-γ-producing TCRαβ+ 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-γ-producing NK1.1+ cells on day 5 (Fig. 5⇑b and Table III⇑). Furthermore, the number of splenic Mφ expressing IFN-γ was not significantly increased in mice receiving a second infection of Salmonella compared with immune controls (Table III⇑). Intracellular IFN-γ 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-α and IFN-γ responses. Moreover, the relative contributions of defined cell populations in these responses are altered compared with those observed following a primary challenge.
Discussion
The relevance of individual components of the innate immune system to host survival against primary bacterial infection is beyond doubt; the protective contribution of neutrophils, Mφ, 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 Mφ 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 Mφ. 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-α and IFN-γ (19, 20, 23, 24, 25). The abundance of neutrophils and Mφ producing TNF-α underscore their importance as the dominate source of this cytokine early during infection.
Our data also show that IFN-γ, 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-γ, such as NK cells, NKT cells, and TCRαβ T cells, our results revealed two surprising IFN-γ-positive populations in Salmonella-infected mice, Mφ and neutrophils. Quantitatively, Mφ were the most abundant splenic cell population producing this cytokine 2–5 days after primary infection, while NK1.1+ cells and TCRαβ T cells also contained significant numbers of IFN-γ-producing cells on day 5. It has been shown in vitro that Mφ can secrete IFN-γ following stimulation with IL-12 and IL-18 (37, 38) or IL-12 and mycobacteria (39). Moreover, Mφ can secrete IFN-γ during the course of mycobacterial infection (40). However, the relative contribution of Mφ-derived IFN-γ to the overall IFN-γ response during infection has not previously been quantified. Both the capacity of Mφ to quickly produce IFN-γ 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. Mφ-derived IFN-γ 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-γ that can be released upon Salmonella infection. This raises the possibility that these abundant cells may also be important sources of IFN-γ early during infection. IFN-γ expression by murine neutrophils has only recently been reported (57), and human placental neutrophils can produce IFN-γ (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. Mφ, 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-γ (48) and, as observed here in response to Salmonella infection, TNF-α. 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 α-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-γ-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 1α and 1β (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-α-producing neutrophils exceeded that of Mφ producing this cytokine, the converse was true following secondary challenge of immune mice. The surprising absence of the dominant IFN-γ-producing Mφ population observed in the primary response together with the diminished number of NK1.1+ cells producing IFN-γ show that the overall IFN-γ 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 Mφ in infected immune hosts was reduced compared with a primary response. Furthermore, substantially fewer TNF-α-producing phagocytes and approximately one-fifth as many IFN-γ-producing NK1.1+ cells were also present 5 days after secondary Salmonella challenge compared with day 5 following primary infection. Moreover, an increase in Mφ producing IFN-γ, which was a striking feature of the primary response, was lacking in the secondary response. Thus, despite the significant influx of neutrophils and Mφ 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 Mφ producing IFN-γ, 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 Mφ 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-γ expression by murine neutrophils has also recently been reported in a pulmonary infection model (57).
Footnotes
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↵1 This work was supported by funds from the Swedish Research Council (Project 621-201-1720, K2001-16X-14005); The Kochs, Österlunds, and Crafoord Foundations; and the Lund University Medical Faculty.
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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
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↵3 Abbreviations used in this paper: Mφ, macrophage; 7AAD, 7-aminoactinomycin D; BFA, brefeldin A; PP, Peyer’s patches.
- Received April 25, 2002.
- Accepted August 12, 2002.
- Copyright © 2002 by The American Association of Immunologists