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. 2008 Sep 2;76(11):5139–5148. doi: 10.1128/IAI.00895-08

Pertussis Toxin Inhibits Early Chemokine Production To Delay Neutrophil Recruitment in Response to Bordetella pertussis Respiratory Tract Infection in Mice▿

Charlotte Andreasen 1,†, Nicholas H Carbonetti 1,*
PMCID: PMC2573337  PMID: 18765723

Abstract

Pertussis is an acute respiratory disease of humans caused by the bacterium Bordetella pertussis. Pertussis toxin (PT) plays a major role in the virulence of this pathogen, including important effects that it has soon after inoculation. Studies in our laboratory and other laboratories have indicated that PT inhibits early neutrophil influx to the lungs and airways in response to B. pertussis respiratory tract infection in mice. Previous in vitro and in vivo studies have shown that PT can affect neutrophils directly by ADP ribosylating Gi proteins associated with surface chemokine receptors, thereby inhibiting neutrophil migration in response to chemokines. However, in this study, by comparing responses to wild-type (WT) and PT-deficient strains, we found that PT has an indirect inhibitory effect on neutrophil recruitment to the airways in response to infection. Analysis of lung chemokine expression indicated that PT suppresses early neutrophil recruitment by inhibiting chemokine upregulation in alveolar macrophages and other lung cells in response to B. pertussis infection. Enhancement of early neutrophil recruitment to the airways in response to WT infection by addition of exogenous keratinocyte-derived chemokine, one of the dominant neutrophil-attracting chemokines in mice, further revealed an indirect effect of PT on neutrophil chemotaxis. Additionally, we showed that intranasal administration of PT inhibits lipopolysaccharide-induced chemokine gene expression and neutrophil recruitment to the airways, presumably by modulation of signaling through Toll-like receptor 4. Collectively, these results demonstrate how PT inhibits early inflammatory responses in the respiratory tract, which reduces neutrophil influx in response to B. pertussis infection, potentially providing an advantage to the pathogen in this interaction.

Bordetella pertussis is a small gram-negative bacterium that infects the human respiratory tract and causes the disease pertussis, also known as whooping cough. B. pertussis binds to ciliated cells and proliferates within the upper and lower respiratory tract, where several toxins are released (11, 17, 30, 47). Pertussis toxin (PT) is produced exclusively by B. pertussis and is thought to play a major role in the development of the infection (8, 12). PT is an exotoxin with an AB5 structure that ADP-ribosylates heterotrimeric Gi proteins in mammalian cells, leading to disruption of downstream cell signaling events (28). Several systemic symptoms have been attributed to PT activity, and although PT is considered to be essential for virulence, the role for PT during infection is still under investigation (4). PT has been shown to have inhibitory effects on several parts of the immune response, including macrophage function, serum antibody production, and the expression of surface molecules on dendritic cells (6, 8, 24).

Previous data from our lab demonstrated that PT has a role in establishing B. pertussis infection in the mouse respiratory tract (7). The bacterial loads of a wild-type (WT) strain of B. pertussis were significantly higher than those of a PT-deficient mutant strain (ΔPT), even as soon as 1 to 2 days after inoculation. Interestingly, intranasal pretreatment of mice with purified PT up to 14 days before inoculation with bacteria, but not treatment 1 day postinoculation, increased the level of infection of the ΔPT strain to that of the WT strain. These results, as well as results of coinfection experiments, suggest that PT plays an early role in establishing B. pertussis infection in the respiratory tract; however, PT is not an adherence factor (7).

One potential target for PT activity that is consistent with its apparent early role is the innate immune system. Purified PT has been found to affect several cell types involved in innate immune responses, including macrophages and neutrophils (18, 40, 51). Neutrophils are key players in the innate immune response and have been shown to be essential for protection against several lung bacterial pathogens, including Pseudomonas aeruginosa (45), Legionella pneumophila (42), and Klebsiella pneumoniae (26). In vitro studies have shown that human neutrophils are able to phagocytose and kill B. pertussis (22), but the role of neutrophils during B. pertussis infection has yet to be determined. However, data from our lab and other labs indicate that PT may inhibit early neutrophil recruitment to the airways in response to B. pertussis infection in mice (5, 7, 20).

Several studies have investigated the effect of PT on neutrophil chemotaxis. Intravenous injection of PT can inhibit neutrophil chemotaxis to the peritoneum in response to lipopolysaccharide (LPS) and other inflammatory mediators (3), and in vitro studies have shown that direct activity of PT on neutrophils can prevent neutrophil chemotaxis, presumably by inhibiting signaling through G-protein-coupled chemokine receptors on neutrophils by ADP-ribosylation of Gi proteins (39, 40). Inhibition of neutrophil recruitment by PT in response to infection with B. pertussis in a mouse model has also been described by other workers (20). These workers concluded that inhibition of neutrophil recruitment to the airways was due to direct intoxication of neutrophils by PT and was not caused by the effect of PT on chemokine production. However, only in vitro experiments were performed to assay chemokine production, and only one time point was selected; therefore, it is possible that critical time points showing inhibition of chemokine production by PT were missed (20).

We reasoned that it is unlikely that, during infection, PT is able to diffuse through the lung tissues, enter the bloodstream, and directly intoxicate neutrophils before they respond to chemokines released by resident airway cells. Furthermore, PT has a long-lived (more than 2 weeks) enhancing effect on B. pertussis infection (7), while neutrophils are short-lived cells with a half-life of approximately 8 h in vivo (29). As an alternative explanation for the early inhibition of neutrophil recruitment to the airways, we hypothesized that PT inhibits the release of neutrophil-attracting chemokines by targeting resident cells in the lung tissue, such as alveolar macrophages and epithelial cells.

The production of chemokines by alveolar macrophages and other cells in the lung is essential for the recruitment and activation of neutrophils, macrophages, NK cells, and certain types of T cells (35). There are four different subgroups of chemokines, two of which have been shown to play a role in lung immune responses. One group is the ELR+ CXC chemokines, which have a three-amino-acid ELR motif (glutamate, leucine, arginine) before the first cysteine close to the N terminus and a CXC motif with the two cysteines closest to the N terminus separated by one amino acid (35). The key functions of ELR+ CXC chemokines are recruitment and activation of neutrophils. Some important murine neutrophil-attracting chemokines are keratinocyte-derived chemokine (KC) and macrophage inflammatory protein 2 (MIP-2), which are the murine functional equivalents of human interleukin 8 in the lung, as well as LPS-induced CXC chemokine (LIX), which is the human CXCL5/ENA-78 homolog (41). In this report we show that PT delays neutrophil recruitment to the airways in response to B. pertussis infection by inhibiting the early transcription of KC, LIX, and MIP-2 by alveolar macrophages, as well as by other cells present in the lung tissue. We also show that PT is able to inhibit chemokine production and neutrophil recruitment in response to intranasal administration of LPS.

MATERIALS AND METHODS

Bacterial strains.

The B. pertussis strains used in this study were streptomycin- and nalidixic acid-resistant derivatives of Tohama I and were produced as previously described (7). The PT-deficient mutant strain (ΔPT) contained an in-frame deletion of PT genes, and the WT strain was the parental strain that produced native PT. The PT* strain of B. pertussis produced PT with two amino acid substitutions in the S1 subunit, which rendered the toxin enzymatically inactive and thereby unable to ADP-ribosylate target Gi proteins. B. pertussis strains were grown on Bordet-Gengou (BG) agar plates containing 10% defibrinated sheep blood and 400 μg/ml of streptomycin.

Mouse infection.

Six-week-old female BALB/c mice (Charles River Laboratories) were used in our studies. Inocula were prepared by plating bacterial strains from frozen cultures on BG blood agar plates with streptomycin. After growth for 3 days at 37°C, bacteria were transferred to new plates and allowed to grow for an additional 2 days. The bacterial strains were resuspended, and appropriate dilutions were prepared using sterile phosphate-buffered saline (PBS). Mice were anesthetized by inhalation of either Metofane (Medical Developments Australia) or isoflurane (Baxter) and were inoculated intranasally with 50 μl of an inoculum. In addition, the inoculum was diluted and plated on BG blood agar plates with streptomycin to determine viable counts. Mice were euthanized by carbon dioxide inhalation at specified time points, and the lungs and trachea were removed and homogenized in 2 ml of sterile PBS. Appropriate dilutions were plated on BG blood agar plates with streptomycin, and the bacteria were counted after 4 days of incubation at 37°C to determine the number of CFU per respiratory tract. A minimum of three mice per group were used. A two-tailed t test was used for statistical analysis.

Intranasal administration of PT and LPS.

The purified PT used in the intranasal administration experiment was previously prepared in our lab from B. pertussis cultures by using fetuin-agarose affinity chromatography as described by Kimura et al. (19) and was stored at −80°C until use. Mice were anesthetized and inoculated intranasally with 50 μl of the appropriate concentration of PT or Escherichia coli LPS (generously provided by Stefanie Vogel, University of Maryland, Baltimore [UMB]) in a PBS solution. Inoculation with 50 μl of sterile PBS was used as a control.

BAL.

Mice were euthanized by carbon dioxide inhalation, and dissection was performed to expose the trachea and lungs. A 20-gauge blunt-end needle was inserted into a small incision toward the top of the trachea and tied in place with surgical thread. Bronchoalveolar lavage (BAL) was performed by flushing the lungs two times with 0.7 ml of sterile PBS (this was performed twice using a total of 1.4 ml of PBS). Aliquots of BAL fluid were used for cell counting with a hemocytometer and for cytospin centrifugation. Cytospin centrifugation was performed at 600 rpm for 5 min, and the slides were stained with modified Wright's stain (Hema 3 stain set; Fisher) used according to the manufacturer's protocol to identify different cell types. Approximately 100 cells from several microscope fields (five or six fields) were counted and identified for each sample.

Lung perfusion and cell preparation.

Mice were euthanized by carbon dioxide inhalation, and the thoracic cavity was exposed. The aorta was nicked to allow blood to drain, and 10 ml of PBS was injected into the right ventricle of the heart, resulting in removal of blood from the lung tissue. The lung tissue was finely cut with scissors and treated with collagenase type 4-DNase I for 45 min at 37°C. The digested tissue was passed through a cell strainer (BD Falcon) using the plunger from a 20-ml syringe, and the cells were resuspended in sterile PBS. Cell counting and cytospin centrifugation were performed as described above.

Preparation of lung tissue for gene expression studies.

Mice were euthanized by carbon dioxide inhalation, and the thoracic cavity was exposed. The two left lobes of the lung were removed, snap frozen in a dry ice-ethanol bath, and stored at −80°C until they were used.

RNA preparation.

RNA was prepared from either whole lung tissue, BAL fluid cells, or cultured MH-S cells (mouse airway macrophage cell line; ATCC CRL-2019) using the phenol-chloroform method. For whole lung tissue, the samples were homogenized in 1 ml of RNA Stat-60 (Tel-Test, Inc.). BAL fluid and MH-S cells were resuspended in 1 ml of RNA Stat-60. Subsequently, 200 μl of chloroform was added to each preparation, and the sample was centrifuged at 13,000 × g for 15 min at 4°C. The aqueous phase was transferred to a 1.5-ml tube containing 500 μl of isopropanol, and the samples were stored at −20°C overnight. The samples were centrifuged at 13,000 × g for 15 min at 4°C, and the supernatants were removed from the RNA pellets. The RNA pellets were washed twice with 80% ethanol and centrifuged, and ethanol was removed from the pellets. The samples were dried with a DNA Speedvac (Savant) at a low temperature for 15 min until the pellets became transparent. Each pellet was resuspended in 50 μl of nuclease-free H2O and placed in a 65°C water bath for 30 min. Samples were placed on ice, and RNA was quantified using an RNA spectrophotometer or an ND-1000 NanoDrop spectrophotometer at 260 nm. A260/A280 values were used to determine the purity of the RNA.

cDNA synthesis.

cDNA was synthesized with a reverse transcription system kit (Promega) used according to the manufacturer's protocol with random primers. One microgram of RNA was used for each sample. The reaction mixture was incubated at room temperature for 10 min, and reverse transcription was performed with a thermal cycler at 42°C for 15 min and at 95°C for 5 min. Samples were placed on ice for 5 min to stop the reaction and diluted 1:10 in nuclease-free H2O.

Real-time PCR.

Real-time PCR was performed using the SYBR green system (ABI). Primer sequences were generously provided by Stefanie Vogel (UMB) or were designed using the Primer Express software. The sequences of the primers used are as follows: hypoxanthine phosphoribosyltransferase (HPRT) forward primer, 5′-GCTGACCTGCTGGATTACATTAA-3′; HPRT reverse primer, 5′-TGATCATTACAGTAGCTCTTCAGTCTGA-3′; KC forward primer, 5′-GCTTGAAGGTGTTGCCCTCA-3′; KC reverse primer, 5′-GTGGCTATGACTTCGGTTTGG-3′; LIX forward primer, 5′-AGCTGCCCCTTCCTCAGTC-3′; LIX reverse primer, 5′-TCCACCTCCAAATTAGCGATCAAT-3′; MIP-2 forward primer, 5′-ACCAACCACCAGGCTACAGG-3′; MIP-2 reverse primer, 5′-CAGGCATTGACAGCGCAGT-3′; tumor necrosis factor alpha (TNF-α) forward primer, 5′-GACCCTCACACTCAGATCATCTTCT-3′; TNF-α reverse primer, 5′-CCACTTGGTGGTTTGCTACGA-3′; gamma interferon (IFN-γ) forward primer, 5′-CTGCCACGGCACAGTCATTG-3′; and IFN-γ reverse primer, 5′-TGCATCCTTTTTCGCCTTGC-3′.

A master mixture was prepared by combining 12.5 μl of 2× SYBR green master mixture, 0.75-μl portions of the appropriate forward and reverse primers (stock concentration, 10 μM), and 6 μl of H2O for each sample. Five microliters (approximately 30 ng) of cDNA and 20 μl of the master mixture were added to each well of a 96-well optical reaction plate (Applied Biosystems). All samples, including a H2O control, were run in duplicate. The data were analyzed using ΔΔCT calculations, and expression of all genes was normalized by using the mouse HPRT housekeeping gene. The results were expressed as increases compared with the values for PBS-treated mice or cells.

Cytokine ELISAs.

Mice were euthanized by carbon dioxide inhalation, and the thoracic cavity was exposed. The two left lobes of the lung were removed and homogenized in 1.5 ml of Tris-HCl (pH 7.4) containing 1 μg of pepstatin (Roche) and one Complete Mini protease inhibitor cocktail tablet (Roche) per 10 ml of solution. The samples were centrifuged at 13,000 × g for 5 min, and the supernatants were transferred to 1.5-ml Eppendorf tubes. This process was repeated two times to remove residual cells. The supernatants were stored at −80°C, and enzyme-linked immunosorbent assays (ELISAs) were performed at the UMB Cytokine Core Laboratory.

Infection of MH-S cells.

The MH-S (ATCC CRL-2019) murine alveolar macrophage cell line was also used in experiments. MH-S cells were seeded in six-well plates at a concentration of 5 × 105 cells per well and incubated overnight at 37°C in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum. WT or ΔPT bacteria were added to the wells at a multiplicity of infection (MOI) of 1 or 10, and the cells were harvested at the 4- and 6-h time points by removing the medium, adding 1 ml of RNA Stat-60 (Tel-Test, Inc.) to each well, scraping cells using a cell scraper, and transferring the cells to 1.5-ml Eppendorf tubes for RNA preparation.

Statistical analysis.

All statistical analyses were performed using SigmaStat (Systat Software Inc.). Statistical comparisons of gene expression and ELISA experiment results were performed using a one-way analysis of variance allowing comparison of three sets of data per time point. A Tukey post hoc test was used for pairwise comparison of data sets.

RESULTS

Early kinetics of neutrophil recruitment to the lungs and airways in response to B. pertussis.

Previously, we showed that PT inhibits early (day 1 to 2) neutrophil recruitment to the airways by comparing the responses to B. pertussis WT and ΔPT infection of BALB/c mice (5, 7). To determine the kinetics of neutrophil recruitment to the lung tissue and the airways in response to WT and ΔPT infection of mice, we inoculated groups (n = 4) of BALB/c mice intranasally with 5 × 105 CFU, a dose at which ΔPT has a significant infection defect (5, 7). On days 1 and 2 postinfection we performed lung perfusion and digestion of lung tissue and BAL in order to determine the number of neutrophils present in the lung tissue and in the airways, respectively. Figure 1A shows that there was an early (day 1) delay in neutrophil recruitment to the lung tissue in response to WT infection compared to the response to ΔPT. However, by day 2 there were significantly more neutrophils in the lungs of mice infected with the WT strain than in the lungs infected with ΔPT. In the airways (BAL), the delay in neutrophil influx in response to the WT strain (compared to the response to ΔPT) persisted through day 2 (Fig. 1B). This indicates that PT has an inhibitory effect on early neutrophil recruitment to the lung tissue, as well as a more prolonged inhibitory effect on neutrophil transmigration from the lung tissue to the airways.

FIG. 1.

FIG. 1.

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Effect of PT on early neutrophil recruitment to the lung tissue and airways in response to B. pertussis infection. The numbers of neutrophils in lung tissue (A) or BAL fluid (B) on days 1 and 2 after infection with 5 × 105 CFU of the B. pertussis WT or ΔPT strain (three mice/group) were determined. The asterisks indicate P values for comparisons of the WT and ΔPT groups (*, P < 0.02; **, P < 0.001). The data are representative of the results of two similar experiments. D1, day 1; D2, day 2.

Neutrophil recruitment in mice pretreated with PT.

Due to the early effect of PT on neutrophil recruitment to the lungs in response to B. pertussis infection, as well as evidence from our laboratory showing that PT has a long-lived enhancing effect on B. pertussis infection (even though neutrophils are very short-lived), we reasoned that it is unlikely that early neutrophil chemotaxis is inhibited directly by PT intoxication of neutrophils. To investigate whether PT inhibits neutrophil recruitment by having a direct or indirect inhibitory effect on neutrophils early after B. pertussis infection, groups of mice were treated intranasally with either 10 or 100 ng of purified PT 5 days prior to inoculation with 5 × 105 CFU of ΔPT. Mice pretreated with PBS and inoculated with 5 × 105 CFU of the WT or ΔPT strain were used as controls. Neutrophil recruitment to the airways was examined on days 1 and 2 postinfection, and the bacterial loads were evaluated on day 2 postinfection. As observed previously, there were significantly more neutrophils in the airways of mice in response to ΔPT than there were in response to the WT strain on days 1 and 2 postinfection (with a PBS-pretreated control group) (Fig. 2A), and the bacterial loads of the WT strain were significantly higher than those of ΔPT by day 2 postinfection (Fig. 2B) (previously we have shown that on day 1 after infection with this dose, the higher WT loads are not quite significantly different than the ΔPT loads [5, 7]). Mice that were pretreated with 10 or 100 ng of PT and subsequently infected with ΔPT showed inhibition of neutrophil recruitment at both time points compared to the ΔPT control group (Fig. 2A). The number of neutrophils in the airways of mice pretreated with either dose of PT was comparable to the number of neutrophils in the airways of mice that were infected with the WT strain (pretreated with PBS). Figure 2B confirms that pretreatment of mice with PT increased the bacterial loads after subsequent ΔPT infection to WT levels, as we have previously observed (7). These data suggest that PT has a long-lived inhibitory effect on neutrophil recruitment to the airways in response to B. pertussis infection in mice. Since neutrophils are short-lived cells (29), it is unlikely that PT inhibits neutrophil chemotaxis to the airways by direct intoxication of neutrophils. Instead, the data are more consistent with the hypothesis that PT acts on longer-lived cells residing in the airways, thereby indirectly delaying neutrophil recruitment in response to infection.

FIG. 2.

FIG. 2.

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Effect of PT pretreatment on recruitment of neutrophils to the airways in response to B. pertussis ΔPT infection. (A) Number of neutrophils in BAL fluid on days 1 and 2 after infection with 5 × 105 CFU of the B. pertussis WT or ΔPT strain with or without intranasal PT treatment (at the indicated dose) 5 days earlier. (B) Bacterial loads in the lower respiratory tract on day 2 after infection with 5 × 105 CFU of the B. pertussis WT or ΔPT strain with or without intranasal PT treatment (at the indicated dose) 5 days earlier (four mice/group). The asterisks indicate P values for comparisons with the ΔPT group that was not pretreated (*, P < 0.05; **, P < 0.005). The data are representative of the results of two similar experiments. D1, day 1; D2, day 2.

Production of chemokines and cytokines in response to infection.

We investigated the early expression of three murine chemokines (KC, LIX, and MIP-2) important for neutrophil recruitment by real-time PCR analysis of RNA isolated from lung samples of mice inoculated with PBS, the WT strain, or ΔPT (5 × 105 CFU). The lungs were harvested and analyzed at several time points in order to identify transient differences in gene expression in response to infection with the two strains. All samples were normalized to values for the HPRT housekeeping gene and increases were determined by comparison to PBS-inoculated control mouse samples. Compared to control mice, there was a small (three- to fourfold) increase in KC mRNA expression in response to infection with either bacterial strain at 3 h postinfection (Fig. 3A). At 6 h postinfection, the expression of KC was increased approximately sevenfold (compared with controls) in response to WT infection; however, the KC mRNA expression in response to ΔPT was approximately 22-fold greater than that in control mice and 3-fold greater than that in response to the WT strain (P < 0.001). At 12, 24, and 48 h there was three- to fivefold induction of KC mRNA expression in response to the WT and ΔPT strains (compared with controls), and there was not a significant difference in KC expression between the responses to the two bacterial strains. This indicates that PT suppresses the steady-state levels of KC mRNA at 6 h after infection with B. pertussis (likely by inhibiting transcription), which correlates with our previous data showing that PT inhibits early neutrophil recruitment in response to infection. Similar expression patterns were seen for MIP-2 (Fig. 3B) and LIX (Fig. 3C); there was three- to fourfold inhibition of gene expression by PT in response to infection at the 6-h time point for both chemokines, whereas no significant differences were seen in response to the WT and ΔPT strains at the other time points. A group of mice infected with the same dose of PT*, a B. pertussis strain producing an enzymatically inactive form of PT, was used as an additional control at the 6-h time point to verify that inhibition of chemokine production was dependent on the enzymatic activity of the toxin. There was no significant difference in expression of any of the three chemokines in response to ΔPT and PT* (Fig. 3A, 3B, and 3C). These data indicate that PT inhibits the expression of neutrophil-attracting chemokine genes in the lungs at 6 h after infection with B. pertussis and that this inhibition is dependent on the ADP-ribosylation activity of the toxin. To verify that the differences that we observed in chemokine gene expression in response to the WT and ΔPT strains represented differences in protein levels, the concentrations of KC in response to the two strains were determined at selected time points using an ELISA. Allowing for a delay in protein expression and for protein accumulation in the tissue, we selected 8 h postinfection as a time point likely to reflect the gene expression differences that we observed at the 6-h time point. A group of mice inoculated with PBS and dissected at the 8-h time point was used as a control. At the 8-h time point, the concentration of KC in response to the WT strain was threefold lower than the concentration of KC in response to ΔPT (P < 0.01) (Fig. 3D). Importantly, there was no significant increase in the KC protein level in response to the WT strain compared with the PBS control. No significant differences in protein levels in response to the WT and ΔPT strains were detected at the 24- and 48-h time points. This confirms that the gene expression data correspond with protein levels in the lung tissue.

FIG. 3.

FIG. 3.

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Early chemokine and cytokine expression in response to B. pertussis WT and ΔPT infection. (A to C, E, and F) Kinetics of gene expression (measured by real-time reverse transcription-PCR) of the indicated chemokines and cytokines for 48 h after infection with 5 × 105 CFU of the B. pertussis WT or ΔPT strain (or after infection with PT* at selected time points). The data are mean increases relative to samples from PBS control-treated mice. (D) Levels of KC protein (measured by ELISA) in lung homogenates of mice for 48 h after infection with 5 × 105 CFU of the B. pertussis WT or ΔPT strain (or PBS control-treated mice). A double dagger indicates that there was a significant increase compared with the PBS control group (four mice/group). An asterisk indicates that the P value for a comparison with the ΔPT group was <0.02.

Infection with B. pertussis, as well as vaccination against pertussis, has been shown to induce Th1 responses in humans (1, 25, 49) and in mice (2, 21). In order to determine if PT induces early increases in the levels of inflammatory cytokines associated with Th1 responses, the expression of IFN-γ and TNF-α genes was examined. No significant increases in expression of genes encoding these cytokines were observed in response to WT or ΔPT infection compared to controls from the time of inoculation through day 2 postinfection (Fig. 3E and F). These results suggest that at the dose used B. pertussis does not induce early production of IFN-γ or TNF-α in the lungs and further support our hypothesis that PT inhibits early neutrophil recruitment to the airways in response to infection by inhibiting the production of neutrophil-attracting chemokines.

Exogenous addition of KC.

In order to confirm that inhibition of chemokine production by PT caused the reduced neutrophil influx and to demonstrate further that PT delays neutrophil recruitment indirectly by inhibiting the release of neutrophil-attracting chemokines rather than by direct intoxication of neutrophils, we inoculated mice with 5 × 105 CFU of the WT or ΔPT strain and administered 1 μg of KC (or PBS to control mice) intranasally after 6 h. BAL was performed on days 1 and 2 postinfection to assay neutrophil recruitment to the airways. As observed previously, neutrophil recruitment was inhibited in response to infection with the WT strain compared to infection with ΔPT on both days 1 and 2 (Fig. 4). Exogenous addition of KC significantly increased the recruitment of neutrophils to the airways in response to WT infection to levels that were the same as or greater than the levels of neutrophil recruitment in response to ΔPT alone. This finding further supports our hypothesis that PT delays neutrophil recruitment in response to B. pertussis infection by inhibiting the production of neutrophil-attracting chemokines rather than by ADP-ribosylating Gi proteins within neutrophils, thereby blocking their ability to respond to chemokines.

FIG. 4.

FIG. 4.

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Effect of addition of exogenous KC on recruitment of neutrophils to the airways in response to B. pertussis infection. The numbers of neutrophils in BAL fluid on days 1 and 2 after infection with 5 × 105 CFU of the B. pertussis WT or ΔPT strain with intranasal administration of 1 μg KC (or PBS in control mice) 6 h postinfection (three mice/group) were determined. The asterisks indicate P values for comparisons with the ΔPT control group (*, P < 0.05; **, P < 0.01). The data are representative of the results of two similar experiments. D1, day 1; D2, day 2.

Gene expression in alveolar macrophages.

Our data indicated that PT inhibits neutrophil recruitment to the lungs in response to B. pertussis infection by inhibiting chemokine production by cells residing in the lung. However, the main target cell(s) for this PT activity is not known yet. Alveolar macrophages are the first cells to respond to pulmonary infections and are potent secretors of chemokines (35). By using cytospin centrifugation, differential staining, and microscopy, we determined that 95 to 100% of the cells recovered by BAL at early time points (up to at least 12 h) after infection with B. pertussis were macrophages (data not shown). Consequently, it was possible to obtain a nearly pure population of alveolar macrophages by performing BAL at 6 h postinfection for analysis of gene expression. However, extracting a sufficient number of macrophages for RNA preparation and gene expression analysis proved to be a considerable challenge. BAL cells from at least 10 mice per group inoculated with PBS or with the WT or ΔPT strain were combined, resulting in only one pooled sample per group. In addition, whole lung tissue was obtained from three mice per group inoculated with PBS or the WT or ΔPT strain for gene expression analysis to confirm our previous observation that PT inhibits chemokine gene expression 6 h postinfection. BALB/c mice were inoculated with 5 × 105 CFU of the WT or ΔPT strain, and the relative levels of expression of the KC, LIX, and MIP-2 genes in lung tissue and pooled alveolar macrophages were analyzed. Although a statistical analysis of BAL samples was not performed because there was only one pooled BAL sample per group, our results indicated that the relative expression of the KC gene by alveolar macrophages in response to infection mirrored that of whole lung tissue (Fig. 5A). Furthermore, PT appeared to inhibit KC expression in alveolar macrophages to the same extent that it inhibited it in whole lung tissue, suggesting that macrophages, as well as other cell types present in the lung tissue (most likely epithelial cells), produce this chemokine and are targets for PT during B. pertussis infection. Alveolar macrophages did not appear to be strong producers of LIX (Fig. 5B). While the level of LIX expression by alveolar macrophages in response to WT infection was lower than the level of expression in response to ΔPT infection, the responses to both strains were approximately fivefold lower than the responses of whole lung tissue, suggesting that other cell types (likely epithelial cells) have an important role in LIX production. In contrast, MIP-2 was expressed by alveolar macrophages at levels that were 5- to 10-fold higher than the levels of expression by whole lung tissue (Fig. 5C), indicating that macrophages are the main producers of MIP-2. However, the inhibitory effect of PT was less in alveolar macrophages than in whole lung tissue, suggesting that other cell types may be involved in production of MIP-2 as well. Epithelial cells in the lungs are potent producers of proinflammatory chemokines and cytokines (43), and it has been shown that PT is able to ADP-ribosylate target Gi proteins in pulmonary epithelial cells in vitro (36), suggesting that these cells are possible target cells for PT activity during B. pertussis infection.

FIG. 5.

FIG. 5.

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Effect of PT on expression of chemokine genes by alveolar macrophages (BAL cells) in response to B. pertussis infection in mice. Levels of gene expression (measured by real-time reverse transcription-PCR) of the indicated chemokines at 6 h after infection with 5 × 105 CFU of the B. pertussis WT or ΔPT strain in whole lung tissue or BAL cells were determined. The levels of expression of KC, LIX, and MIP-2 were all greater than the control levels in lung tissues in response to WT and ΔPT infection (P < 0.05). The data are mean increases (whole lung tissue; n = 4) or increases for pooled samples from 10 mice (BAL cells) compared to samples from PBS control-treated mice. An asterisk indicates that the P value for a comparison with the ΔPT group was <0.02.

Gene expression in cultured MH-S cells.

Rather than using large numbers of mice to reproduce these findings, we used a murine alveolar macrophage cell line (MH-S cells). MH-S cells (5 × 105 cells per well) were seeded in six-well plates and incubated overnight at 37°C. WT or ΔPT bacteria were added to the wells at an MOI of 1 or 10, and the cells were harvested at the 4- and 6-h time points. At an MOI of 1, the levels of expression of the genes encoding all three chemokines were approximately two- to threefold higher in response to ΔPT than in response to the WT strain at both time points (Fig. 6A to C), indicating that PT has a role in inhibiting chemokine gene expression by alveolar macrophages in response to B. pertussis infection. The levels of chemokine induction in MH-S cells in response to bacteria were higher than the levels of chemokine induction observed in BAL macrophages (Fig. 5); however, the same expression pattern was observed, with LIX expressed at relatively low levels (Fig. 6B) and MIP-2 expressed at high levels in response to infection (Fig. 6C) compared to the expression by control cells treated with PBS. The overall higher levels of expression of chemokine genes in response to B. pertussis infection by MH-S cells than by BAL macrophages could have been due to the MOI used or could have been an artifact of using the MH-S cell line.

FIG. 6.

FIG. 6.

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Expression of chemokine genes by MH-S cells in response to WT and ΔPT infection. The levels of KC (A), LIX (B), and MIP-2 (C) mRNA expression were significantly lower in response to WT infection than in response to ΔPT infection at an MOI of 1 at 4 and 6 h postinfection. However, at an MOI of 10, KC (D), LIX (E), and MIP-2 (F) gene expression in response to B. pertussis infection was not inhibited by PT at 4 and 6 h postinfection, suggesting that high bacterial loads may overcome the inhibitory effect of PT. A diamond indicates that the level of gene expression was not significantly greater than the control level (three wells/treatment group). The data are the results of one of two separate experiments. *, P < 0.05; **, P < 0.01.

The chemokine gene expression by MH-S cells was strongly induced in response to WT and ΔPT infection at an MOI of 10, and we observed no significant differences between the responses to the two strains for any of the chemokines at the 4-h time point (Fig. 6D to F). The level of MIP-2 gene expression was again the highest level (approximately 2,000-fold greater than control levels by 4 h after infection with the WT and ΔPT strains) (Fig. 6F), and interestingly, the level of MIP-2 gene expression was significantly lower in response to ΔPT infection than in response to WT infection at the 6-h time point. These data suggest that alveolar macrophages may be important targets for PT inhibition of early chemokine production (Fig. 6A to C), but they also indicate that high levels of infection can overcome the inhibitory effect of PT on chemokine production (Fig. 6D to F), which is consistent with data from our laboratory showing that inoculation of mice with a high dose (5 × 106 CFU) of WT or ΔPT bacteria leads to early recruitment of neutrophils to the airways (day 1) that is not inhibited by PT (data not shown).

PT inhibition of responses to LPS.

One of the main inducers of proinflammatory responses to gram-negative bacteria is LPS, which signals primarily through Toll-like receptor 4 (TLR4) and induces the production of inflammatory chemokines and cytokines. There is evidence from in vitro studies that PT can modulate LPS-induced signaling through TLR4 (50, 51); however, data obtained by Mann et al. indicate that TLR4 is not critical for limiting B. pertussis bacterial loads during the first 3 days of infection in mice (23). To investigate if PT acts as an inhibitor of neutrophil recruitment and chemokine gene expression in response to LPS in vivo, we inoculated groups of BALB/c mice intranasally with 1, 10, or 100 ng of E. coli LPS with or without 100 ng of PT. Mice were sacrificed on day 1 postinoculation, and both neutrophil recruitment to the airways and KC gene expression in the lungs were examined. LPS induced strong neutrophil recruitment, but coadministration of PT with LPS inhibited neutrophil recruitment to the airways approximately sevenfold in response to 1 ng of LPS (P < 0.001) and fourfold in response to 10 ng of LPS (P < 0.001) (Fig. 7A). However, PT did not inhibit neutrophil recruitment significantly in response to 100 ng of LPS (P = 0.10), suggesting that administration of a sufficient quantity of LPS can overcome the inhibitory effect on neutrophil recruitment by PT.

FIG. 7.

FIG. 7.

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Effect of PT on neutrophil recruitment and KC gene expression in response to LPS treatment in mice. (A) Number of neutrophils in BAL fluid in mice 24 h after intranasal administration of 1, 10, and 100 ng of E. coli LPS with or without 100 ng PT. (B) KC gene expression in lungs of mice 24 h after intranasal administration of 1, 10, and 100 ng of E. coli LPS with or without 100 ng PT. A diamond indicates that the level of gene expression was not significantly greater than the control level (four mice/treatment group). The asterisks indicate P values for comparisons with the group that received only LPS (*, P < 0.03; **, P < 0.001).

LPS induced a fourfold increase in KC gene expression, but in response to 1 and 10 ng of LPS the increase was inhibited significantly by coadministration of PT (P < 0.03 and P < 0.02, respectively) (Fig. 7B). PT significantly inhibited KC gene expression in response to 100 ng of LPS as well (P < 0.01); however, at this dose of LPS, the inhibition by PT was less than twofold. These data are consistent with our hypothesis that the inhibitory effect of PT on chemokine production and neutrophil recruitment in a mouse model can be reduced or overcome by strong proinflammatory stimuli. Additional experiments performed in our laboratory indicated that intranasal administration of 100 ng of purified PT (in the absence of other factors) had no stimulatory effect on chemokine gene expression or neutrophil recruitment to the airways for up to 1 week postadministration (data not shown).

DISCUSSION

While PT has been studied extensively both in vitro (9, 18, 31, 33, 34, 38, 48, 51) and in vivo (3, 5-8, 10, 20, 40, 44), the role(s) of PT during B. pertussis infection is not yet fully understood. Preliminary studies in our laboratory suggested that PT inhibits neutrophil influx to the airways in response to B. pertussis infection in BALB/c mice (7). In the present study we confirmed that PT inhibits early (day 1 or 2) neutrophil recruitment to the airways in response to infection with 5 × 105 CFU of B. pertussis, and we observed that the inhibition of neutrophil influx to the airways was more prolonged than the inhibition of neutrophil recruitment to the lung tissue. These findings indicate that PT may affect at least two distinct steps in neutrophil recruitment to the airways in mice: neutrophil chemotaxis from the circulation to the lung tissue and transmigration of neutrophils across the epithelium into the airways.

As mentioned above, PT can affect neutrophils directly by ADP-ribosylating the Gi proteins associated with chemokine receptors on their surface, thereby inhibiting neutrophil migration in response to chemokine production (3, 20, 40). However, the previous studies either were performed in vitro or did not involve B. pertussis infection. Since neutrophils are short-lived cells and PT was shown to have a long-term inhibitory effect on neutrophil recruitment to the airways in response to infection (Fig. 2), it seems unlikely that PT achieves this by a direct effect on neutrophils. Instead, we hypothesized that the inhibitory effect of PT on neutrophil recruitment in response to B. pertussis infection is due to inhibition of early chemokine production, and this hypothesis was supported by our gene expression and ELISA data (Fig. 3). The increase in neutrophil recruitment to the airways in response to WT infection after addition of exogenous KC provided additional evidence that PT inhibits early neutrophil chemotaxis indirectly in response to B. pertussis infection by inhibiting the production of neutrophil-attracting chemokines in the airways and that neutrophils are still capable of responding to sufficient concentrations of chemokines. This conclusion is in contrast to that reached by Kirimanjeswara et al. (20) in their study using a different mouse strain (C57BL/6), in which they concluded that PT directly intoxicates neutrophils and inhibits their chemotactic response to B. pertussis infection. However, the study of these workers also showed that cultured murine alveolar macrophage cells produced lower levels of the chemokine KC after 12 h of exposure to a B. pertussis WT strain than after 12 h of exposure to ΔPT, consistent with our findings.

Our analysis of chemokine gene expression in alveolar macrophages derived from BAL fluid 6 h after inoculation with B. pertussis revealed that PT targets alveolar macrophages, inhibiting their production of chemokines in response to infection. However, the relative levels of gene expression in whole lung tissue and alveolar macrophages varied for different chemokines, indicating that other cells in the lungs (likely epithelial cells) also make important contributions to the chemokine response and that PT also targets other cell types in the airways. Epithelial cells make up the lining of the lung in contact with the airways and are important producers of inflammatory chemokines and cytokines in response to other lung pathogens (43), making it likely that they are also involved in the chemokine response to B. pertussis. Further investigation of responses of murine epithelial cells in vitro to B. pertussis infection is necessary to confirm this conclusion. Nevertheless, our in vitro studies using the murine alveolar macrophage cell line MH-S infected with B. pertussis at an MOI of 1 revealed responses similar to those seen in BAL macrophages after infection, supporting the conclusion that alveolar macrophages are targets for PT in inhibition of inflammatory responses.

LPS is clearly a major stimulus for the innate immune system in B. pertussis infection (21). There is some evidence from in vitro studies that PT can modulate LPS-induced signaling through TLR4 (50, 51), and while TLR4 is not a G-protein-coupled receptor, PT-sensitive heterotrimeric Gi proteins may be involved in the LPS signaling response through TLR4 (14, 50). Our data showed that administration of 100 ng of PT inhibits LPS-induced KC gene expression in the lungs, as well as neutrophil recruitment to the airways, in a dose-dependent manner. However, at high doses of LPS (≥100 ng), the stimulatory effect of LPS is able to overcome the inhibitory effect of PT to some extent (Fig. 7). This is consistent with observations from in vivo experiments performed in our laboratory suggesting that high doses of WT B. pertussis induce a substantial and rapid neutrophil influx (>100,000 neutrophils) to the airways (data not shown). Furthermore, while PT had an inhibitory effect on expression of chemokine genes by MH-S cells in response to B. pertussis added at an MOI of 1, addition of B. pertussis to MH-S cells at an MOI of 10 resulted in strong induction of chemokine gene expression that was not inhibited by the presence of PT. Nevertheless, one role for PT soon after B. pertussis infection may be to modulate inflammatory responses (including chemokine upregulation and neutrophil recruitment) mediated by LPS-TLR4 signaling. Although we used E. coli LPS in this study rather than B. pertussis LPS, both molecules are known to signal through TLR4, and a recent report demonstrated that E. coli LPS was a more potent stimulator of several proinflammatory responses than B. pertussis LPS (13), underscoring the inhibitory capacity of PT in this signaling pathway. We are currently attempting to dissect the TLR4 signaling pathway to determine how PT-sensitive heterotrimeric Gi proteins may be involved in generating these responses.

Overall, our results indicate that in the mouse model used, PT inhibits early chemokine gene expression in alveolar macrophages (and other cells in the airways) in response to B. pertussis infection to delay neutrophil recruitment, possibly in order to allow the bacteria to establish early colonization. However, neutrophil depletion studies indicated that this role of PT may be effective in promoting infection only in immune mice and not in naïve mice (20; our unpublished data). Our previous study indicated that a more important role for PT in naïve mice may be to inhibit the protective functions (other than neutrophil recruitment) of resident airway macrophages (8), and we are currently investigating the specific macrophage functions targeted by PT. Whether PT plays the same roles in human infection is still unclear, since we know nothing about the early events after B. pertussis acquisition in humans. However, in the mouse model, although overt symptomatic disease is not elicited, several characteristics of the human infection are reproduced, such as limitation of infection to the respiratory tract, multiplication and clearance of the bacteria, increased severity of infection in young animals, and various PT-associated systemic physiological changes (16, 32, 37, 46). Furthermore, studies have shown that this may also be a useful model for the preclinical assessment of acellular pertussis vaccine efficacy (15, 27). We may be able to gain some insight into PT activities in human infection by similar analysis of responses of human airway cells in culture, but until human volunteer studies are permissible, the mouse model may provide valuable information to increase our understanding of this host-pathogen interaction.

Acknowledgments

This study was supported by Public Health Service grant R01 AI063080 from the National Institute of Allergy and Infectious Diseases.

We are grateful to our colleague Stefanie Vogel and members of her lab for the gift of E. coli LPS and for determining PCR primer sequences, as well as technical advice, and to members of the Carbonetti lab for many helpful discussions.

Editor: R. P. Morrison

Footnotes

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Published ahead of print on 2 September 2008.

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