Protection against Lethal Challenge with Streptococcus pneumoniae ...

INFECTION AND IMMUNITY, Oct. 2006, p. 5679–5686 0019-9567/06/$08.00⫹0 doi:10.1128/IAI.00837-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Vol. 74, No. 10

Protection against Lethal Challenge with Streptococcus pneumoniae Is Conferred by Aryl Hydrocarbon Receptor Activation but Is Not Associated with an Enhanced Inflammatory Response Beth A. Vorderstrasse and B. Paige Lawrence* Department of Pharmaceutical Sciences, Washington State University, Pullman, Washington 99164-6534 Received 24 May 2006/Returned for modification 7 July 2006/Accepted 20 July 2006

Streptococcus pneumoniae is a common respiratory pathogen and a major cause of morbidity and mortality in humans, particularly in the elderly and young children. The pulmonary immune response to S. pneumoniae is initiated very rapidly, and, ideally, innate immune responses are able to contain bacterial colonization. In the studies presented here, we sought to determine whether activation of the aryl hydrocarbon receptor (AhR) would protect mice from an otherwise lethal infection with S. pneumoniae. The rationale for this hypothesis is that, although most AhR agonists are potent immunosuppressants, AhR activation enhances the inflammatory response to pathogenic and nonpathogenic stimuli. Specifically, neutrophil numbers and levels of inflammatory cytokines are often increased in mice treated with the potent AhR agonist 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD). To test the hypothesis, vehicle control- or TCDD-treated mice were intranasally infected with S. pneumoniae. Mortality, pulmonary bacterial burden, cytokine/chemokine levels, and influx of immune cells to the lung were analyzed at various times postinfection. As predicted, survival was substantially improved in the mice treated with TCDD, and the pulmonary bacterial burden was decreased. Surprisingly, however, there was no evidence suggesting that protection resulted from an enhanced inflammatory response. In fact, neutrophil numbers and inflammatory chemokines and cytokines were all decreased in the TCDD-treated mice relative to vehicle control-treated mice. This suggests that the protective effect of AhR activation is not the result of altered immune function but instead may reflect a direct effect on the response of lung cells to infection.

Streptococcus pneumoniae is a common human pathogen and a major source of serious disease, particularly in the elderly and in young children. While the bacteria are commonly found colonizing the nasopharynxes of asymptomatic individuals, their presence in the lower respiratory tract and extrapulmonary sites causes numerous diseases, including pneumonia, otitis media, and meningitis (32). When the bacteria reach the lung, they trigger a rapid inflammatory response, characterized by an influx of neutrophils and release of numerous cytokines and chemokines such as tumor necrosis factor alpha (TNF-␣), interleukin-1 (IL-1), monocyte chemoattractant protein (MCP), and macrophage inflammatory protein-1 (MIP-1) (8, 9, 23, 26). In general, the inflammatory response initiated by infection is host protective, although under certain circumstances it can be detrimental to the host. Inflammatory mediators triggered by the presence of pathogens activate the release of antimicrobial substances, recruit phagocytes that contribute to pathogen clearance, and aid in the priming of the adaptive immune response (1, 17, 38). However, excessive or sustained inflammation causes destruction of tissue, which, particularly when occurring in the lung, contributes to morbidity and mortality (19, 33, 47) An example of this dichotomy between beneficial and detrimental effects of inflammation in S. pneumoniae in-

fection is illustrated in a study by Dallaire et al. (8), in which infected mice that were visibly sick had greater numbers of neutrophils in their lungs and higher levels of inflammatory mediators than did animals that appeared healthy. Furthermore, mice that appeared sick inevitably died. Thus, an enhanced inflammatory response was associated with a poor prognosis. However, resistance to a lethal bacterial challenge was substantially augmented if the mice were given an exogenous inflammatory stimulus at the time of infection. These observations suggest that an enhanced inflammatory response is beneficial if it occurs during the early stages of the infection but may be detrimental when it occurs later or persists too long. The aryl hydrocarbon receptor (AhR) is an orphan nuclear receptor whose biological function is an active area of study (10, 30). The AhR belongs to the PAS (Per-Arnt-Sim) family of transcriptional regulators, which control a number of physiological processes, including circadian rhythms, hormone signaling, and response to hypoxia (15, 36). Currently the AhR is best characterized for its activation by exogenous compounds, particularly by environmental contaminants such as polyhalogenated aromatic hydrocarbons and polyaromatic hydrocarbons (11). Activation of the AhR by these compounds induces the expression of numerous metabolic enzymes, which facilitates the clearance of many of these chemicals from the body. In addition to a role in the metabolism of exogenous compounds, there is growing evidence that the AhR plays a role in normal physiological functions and likely has endogenous ligands. For example, it is evident that the AhR is involved in

* Corresponding author. Present address: Dept. of Environmental Medicine, Box 850, University of Rochester School of Medicine, 575 Elmwood Avenue, Rochester, NY 14642. Phone: (585) 275-1974. Fax: (585) 276-0239. E-mail: [email protected]. 5679

5680

VORDERSTRASSE AND LAWRENCE

normal fetal development, as AhR null mice display certain physiological abnormalities, including reduced liver size and patent hepatic shunt (45). Importantly, the defective formation of the hepatic shunt can be corrected in AhR hypomorphs if the receptor is activated exogenously, suggesting that endogenous activation of the receptor plays a role in normal fetal development (45). Specific endogenous ligands identified to date include tryptophan metabolites, heme degradation products, and arachidonic acid metabolites (11). Taken together, such information has invigorated interest in further defining the role that the receptor plays in physiological processes. Activation of the AhR with exogenous agonists, particularly the polyhalogenated aromatic hydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (dioxin), which is the highest-affinity ligand identified to date, affects many organs and deregulates numerous biological processes (2, 35, 42). The immune response is considered one of the most sensitive targets of AhR agonists, and suppression of the adaptive immune response in TCDD-treated mice is well characterized (20). Interestingly, and concomitant with impaired adaptive immunity, certain elements of the innate immune response are enhanced by TCDD exposure. For example, TCDD treatment causes increased neutrophil numbers and enhanced expression of inflammatory mediators such as TNF, IL-1, and gamma interferon (IFN-␥) in mice challenged with antigens such as influenza virus, allogeneic tumor cells, or sheep red blood cells (7, 12, 22, 29, 34, 48, 50). Studies conducted with AhR null mice have demonstrated that both suppressed adaptive immunity and enhanced inflammation are mediated by the AhR (41, 44). Given that exogenous augmentation of the inflammatory response has been shown to protect mice from a lethal challenge of S. pneumoniae, we hypothesized that AhR activation by TCDD would likewise protect against infection by enhancing inflammation. Consistent with our hypothesis, survival was substantially improved in the animals upon activation of the AhR. Surprisingly, however, the evidence did not support the idea that protection resulted from an enhanced inflammatory response. Instead, it is possible that AhR activation causes changes within the lung itself to reduce the infection. MATERIALS AND METHODS Animal care and TCDD treatment. Female C57BL/6 mice were purchased from NCI Charles River (Frederick, MD). AhR null mice (13) were originally obtained from the National Cancer Institute and subsequently bred as homozygotes at Washington State University. TCDD (ⱖ99% purity; Cambridge Isotope Laboratories, Woburn, MA) was dissolved in anisole and diluted in peanut oil. Mice were gavaged with TCDD (10 ␮g/kg of body weight) or vehicle control (peanut oil with 0.1% anisole) 1 day prior to infection. Animal treatments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee. Infection. Mice were infected with 104 CFU of Streptococcus pneumoniae prepared as previously described (24). Briefly, S. pneumoniae (type 3; ATCC 6303) from a blood agar plate was grown overnight at 37°C in brain heart infusion medium (BHI). The following day, the culture was diluted 1:50 in BHI containing 10% horse serum (Colorado Serum Company, Denver, CO) and grown to mid-logarithmic phase. Bacteria were pelleted, washed with phosphatebuffered saline (PBS), and diluted to the appropriate concentration for infecting. The concentration was verified by plating on blood agar. Mice were anesthetized with avertin (2,2,2-tribromoethanol; Aldrich, Milwaukee, WI), given an intranasal infection of 104 CFU in 30 ␮l, and held upright for 1 min after inoculation. Bacterial burden. Lungs were homogenized in PBS. Blood was collected by cardiac puncture. A series of 10-fold dilutions was prepared in PBS and 50 ␮l

INFECT. IMMUN. plated per quadrant on a blood agar plate. Colonies were counted after overnight incubation. In general, it was possible to count colonies in quadrants containing fewer than 100 colonies; a value of 100 was assigned when the colonies were too numerous to count. Analysis of BAL cells and fluid. Lungs were perfused with three sequential washes with RPMI (48). The first wash was retained as bronchoalveolar lavage (BAL) fluid. Cells from all three washes were pooled, counted, and stained for flow cytometric analysis. Immunophenotypic analysis. Cells were incubated with rat immunoglobulin G (IgG) block, followed by addition of fluorochrome-conjugated Gr-1 and F4/80 antibodies to identify neutrophils and macrophages. Appropriately labeled isotype control antibodies were used to determine nonspecific fluorescence. Listmode data were collected on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and analyzed using WinList (Verity Software, Topsham, ME). Cytokines/chemokines. Cytokine and chemokine proteins were measured using a fluorescent-microsphere-based multiplex assay (performed by Linco Systems, St. Charles, MO). S. pneumoniae-specific antibody enzyme-linked immunosorbent assay. Coating antigen was prepared by heat inactivating (1 h at 60°C) and sonicating a logphase culture of S. pneumoniae. Plates were coated with 5 mg/ml of antigen. A dilution series of plasma, ranging from 1:25 to 1:25,600, was added to the plate. Biotinylatated detection antibodies for mouse IgM, IgG, and IgA (Southern Biotech, Birmingham, AL) were used to assess the relative amount of each antibody isotype. Growth rate of S. pneumoniae in liquid culture. S. pneumoniae from a blood agar plate was cultured overnight in BHI at 37°C. The following morning, subcultures were grown in BHI (with 10% horse serum) containing either 10⫺9 M TCDD (four subcultures) or vehicle control (0.1% dimethyl sulfoxide [DMSO]) (four subcultures). Growth was monitored by measuring the optical density at 600 nm (OD600). Western blotting for CYP 1A1. Lungs were homogenized in PBS and protein extracted by incubating in buffer containing 0.02 M HEPES, 0.05 M NaCl, 0.001 M EGTA, 1% Triton X, 0.01% sodium dodecyl sulfate (SDS), and protease inhibitors. Debris was separated by centrifugation. The protein concentration was determined by the Pierce bicinchoninic acid assay and samples prepared by boiling in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Fifteen micrograms of protein was subjected to SDS-PAGE and transferred to nitrocellulose. Cytochrome P450 (CYP) 1A1 protein was visualized by blotting with goat anti-rat serum (Gentest, Woburn, MA). Bands were visualized using ECL reagents (Amersham Pharmacia, Piscataway, NJ). Statistics. t tests were performed using StatView statistical software (SAS, Cary, NC). A two-sided P value of ⱕ0.05 was considered significant.

RESULTS Activation of the AhR improves survival of mice infected with S. pneumoniae. Exposure to AhR ligands typically causes immune suppression but has also been reported to enhance inflammation. To test the hypothesis that AhR activation by TCDD improves survival of S. pneumoniae-challenged mice by enhancing early inflammatory events, animals were intranasally infected with 104 CFU of S. pneumoniae 1 day after treatment with vehicle or TCDD. Survival was monitored daily. As predicted, TCDD treatment was host protective in mice infected with S. pneumoniae (Fig. 1). In fact, while only 35% of the vehicle-treated animals survived infection, exposure to TCDD improved survival to 75%. Bacterial burden is diminished in mice treated with TCDD. The bacterial burden in the lung was assessed in separate cohorts of mice that were sacrificed on days 1 to 4 following infection (Fig. 2A). In the vehicle control-treated mice, bacteria were present in the lung homogenates of every animal at all time points examined (Fig. 2A and B). In contrast, and consistent with the decreased mortality observed in the host resistance study, treatment with TCDD substantially decreased the pulmonary bacterial burden. This decrease was evident as early as day 1 and was statistically significant at all time points

VOL. 74, 2006

AhR ACTIVATION IN S. PNEUMONIAE INFECTION

5681

FIG. 1. Survival following challenge with S. pneumoniae is improved by exposure to TCDD. C57BL/6 mice (n ⫽ 20 per treatment) were given an oral dose of TCDD or peanut oil vehicle 1 day prior to intranasal infection with 104 CFU of S. pneumoniae. Survival was monitored daily for 16 days, at which time all remaining animals appeared healthy. This result was repeated in two additional experiments with the following results: Experiment 2, survival ⫽ 45% (vehicle) versus 75% (TCDD) (n ⫽ 20 mice per treatment group); experiment 3, survival ⫽ 30% (vehicle) versus 70% (TCDD) (n ⫽ 10 mice per treatment group).

examined (Fig. 2A). Furthermore, at each time point there were some TCDD-treated mice that had no detectable bacterial burden. Specifically, among the eight TCDD-treated mice sacrificed at each time point, lung homogenates from only two to five animals had detectable bacteria (Fig. 2B). Bacteremia

FIG. 3. Pulmonary neutrophilia is reduced in TCDD-treated mice. Vehicle- and TCDD-treated mice (n ⫽ 6 to 8 per treatment group per day) were infected with 104 CFU of S. pneumoniae. The numbers of neutrophils (A), macrophages (B), and total cells (C) recovered by BAL were determined by immunophenotypic analysis. Day 0 values reflect cellular recovery from airways of mice that were not infected (NI) (n ⫽ 2). Error bars indicate standard errors of the means. ⴱ, significant difference between vehicle- and TCDD-treated infected mice on a given day (P ⱕ 0.05 in a two-sided t test).

FIG. 2. AhR activation is associated with diminished pulmonary bacterial burden. (A) Vehicle (open bars)- and TCDD (filled bars)treated mice were infected with 104 CFU of S. pneumoniae and sacrificed on days 1 to 4 postinfection (n ⫽ 8 per treatment group per day). Serial dilutions of lung homogenates were plated on blood agar. Bars represent the average number of colonies (⫾ standard error of the mean) in 50 ␮l of a 10⫺2 dilution. No colonies were detected in lung homogenates from mice that were not infected with bacteria (data not shown). ⴱ, significant difference from vehicle control (P ⱕ 0.05 in a two-sided t test). #, P ⫽ 0.06. (B) Number of mice depicted in panel A that had detectable bacteria in the lung homogenate.

was also assessed in a separate time course study, and TCDD had the same protective effect that was observed in the lung. Specifically, on day 4 following infection, S. pneumoniae was detected in blood samples from four of four vehicle-treated mice, whereas only one of four TCDD-treated animals had bacteria in the blood. No bacteria were detected in the blood of any mouse on days 1 to 3 following infection (data not shown). Activation of the AhR reduces the inflammatory response in the lungs of S. pneumoniae-infected mice. Enhanced inflammation, characterized by increased numbers of neutrophils and

5682


excessive cytokine production, is one reported consequence of AhR activation in other model systems and provides a plausible explanation for the increased bacterial clearance and improved survival in the TCDD-treated animals. To determine whether an enhanced inflammatory response was present in the TCDD-treated mice, lung lavage cells and levels of pulmonary cytokines and chemokines were evaluated on days 1 to 4 following infection. While infection with S. pneumoniae caused a marked increase in the number of neutrophils recovered from the lungs of vehicle-treated mice (Fig. 3A), we were surprised to find that treatment with TCDD reduced this influx. Specifically, there were approximately 90% fewer neutrophils recovered from airways of TCDD-treated mice than from those of vehicle control-treated mice on days 2 and 3 following infection (Fig. 3A). This did not reflect a global decrease in inflammatory cells, as there was no effect of TCDD treatment on the number of macrophages recovered from the lung (Fig. 3B). The kinetics and effect of AhR activation on the levels of pulmonary cytokines and chemokines were similar to those observed for the neutrophil numbers. Specifically, in the vehicle-treated mice, infection generally caused a substantial induction of these proteins, especially on days 3 and 4 (Fig. 4). In particular, S. pneumoniae infection increased levels of IFN-␥, TNF-␣, IL-6, IL-1␣, IL-1␤, IL-10, MIP-1, MCP, keratinocytederived chemokine (KC), granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, RANTES, 10-kDa IFN-␥inducible protein (IP-10), and IL-17 in lung lavage fluid. However, the infection-associated increase in each of these factors was suppressed in the mice treated with TCDD. This was particularly evident on day 3 postinfection, at which time the levels of most of the proteins were 80 to 90% lower in lavage fluid from TCDD-treated mice than in that from vehicle control-treated mice (Fig. 4). Exposure to TCDD does not directly impair bacterial growth. Given that AhR activation decreased the number of neutrophils and pulmonary cytokines and chemokines, enhanced inflammation is an unlikely mechanism for the host protection observed in TCDD-treated mice. In fact, it is reasonable to conclude that the decreased inflammation was a downstream effect of an earlier event that altered the host response to S. pneumoniae infection. One explanation that could account for the increased host resistance and decreased inflammation is that TCDD acts directly on the bacteria to impair their growth. This possibility was formally tested in two ways: (i) by assessing the effects of TCDD on growth of bacteria in liquid culture and (ii) by determining whether TCDD is still protective in mice lacking the AhR. To test the possibility that TCDD directly impairs growth of S. pneumoniae, bacteria were grown in liquid cultures containing vehicle or 10⫺9 M TCDD. Growth was monitored by measuring the turbidity of the cultures (OD600). As shown in Fig. 5, the absorbance curves assessed over time were identical in cultures containing TCDD or DMSO vehicle, indicating that TCDD does not directly impair bacterial growth. However, it remained possible that TCDD could interfere with bacterial growth within the lung or with some other bacterial function required for a productive infection in vivo. To directly determine whether TCDD interferes with bacterial growth in vivo, we evaluated the survival of infected AhR

INFECT. IMMUN.

null mice, which are insensitive to TCDD. Specifically, we expected that if the decreased bacterial growth and improved survival of TCDD-treated mice is due to effects of TCDD on the host (as opposed to direct effects on the bacteria), TCDD treatment would not be host protective in AhR null mice. The outcome of this experiment (Fig. 6A) was consistent with this idea, as TCDD exposure was not protective in the AhR null mice. Together with the lack of effect of TCDD on bacterial growth in culture, these results demonstrate that the protective effect of AhR activation against S. pneumoniae infection is due to a direct effect of TCDD within the mouse. Moreover, it is possible that protection is conferred by an AhR-dependent event within cells of the lung. Two observations support this idea. First, there is not an increase in the number of inflammatory cells or the level of inflammatory cytokines in lungs of infected mice treated with TCDD, suggesting that immune targets are not the cause of altered bacterial growth or host resistance. Second, CYP 1A1, a hallmark indicator of AhR activation, was induced in the lung on day 1 following infection (Fig. 6B). This result underscores that the lung is a direct target for AhR ligands and that AhR activation induces a rapid biological response. Exposure to TCDD perturbs the adaptive immune response to S. pneumoniae. Suppression of the adaptive immune response is a hallmark indicator of immune toxicity induced by exposure to AhR agonists. Therefore, we were interested in characterizing the effects of TCDD exposure on the antibody response in S. pneumoniae-infected mice. Levels of S. pneumoniae-specific IgA, IgG, and IgM in plasma samples from vehicle- and TCDD-treated mice were measured on day 16 following infection (Fig. 7). TCDD treatment increased the IgA response but suppressed the production of IgG and IgM isotypes, consistent with results of other studies examining the effects of TCDD on the antibody response (16, 21, 39, 48). Although not germane to the enhanced protection observed in the TCDD-treated mice, the altered antibody response against S. pneumoniae infection indicates that AhR activation is still exerting its classic effects on the adaptive immune response. Together with the observation of suppressed inflammation in the TCDD-treated mice, the diminished adaptive immune response strongly suggests that protective effects against S. pneumoniae infection do not result from enhanced activity of the immune system per se. DISCUSSION The protective effect of AhR activation reported here correlated with reduced bacterial burden, diminished numbers of neutrophils, and lower levels of cytokines in the lungs of S. pneumoniae-infected mice, suggesting that enhanced inflammation is an unlikely mechanism for the observed protection. This raises an interesting question about the role of inflammation in host resistance to infection. Specifically, did the mice have a reduced bacterial burden and improved survival because AhR activation decreased the inflammatory response to infection, or does AhR activation alter the environment in the lung such that it is more difficult for the bacteria to colonize? In other words, does the attenuated inflammatory response reflect a reduction in the ability of the bacteria to colonize or multiply in the respiratory tract?

VOL. 74, 2006


5683

FIG. 4. Induction of many cytokines and chemokines is suppressed by AhR activation. BAL fluid was recovered from mice treated as described in Fig. 3. Protein levels of (A) IFN-␥, (B) TNF-␣, (C) IL-6, (D) IL-1␣, (E) IL-1␤, (F) IL-10, (G) MIP-1, (H) MCP, (I) KC, (J) GM-CSF, (K) G-CSF, (L) RANTES, (M) IP-10, and (N) IL-17 were analyzed using a fluorescent-microsphere-based multiplex assay performed by Linco Systems (St. Charles, MO). The limit of detection was typically ⱕ16 pg/ml. Error bars indicate standard errors of the means. ⴱ, significant difference between vehicle- and TCDD-treated infected mice (P ⱕ 0.05 in a two-sided t test). In addition to the infection-associated increases in the 14 cytokines and chemokines reported here, the multiplex assay tested for IL-2, -4, -5, -7, -9, -12, -13, and -15; none of these were present at detectable levels in any sample (data not shown).

5684


INFECT. IMMUN.

FIG. 5. S. pneumoniae growth is not directly impaired by TCDD exposure in vitro. Subcultures (n ⫽ 4 per treatment) of S. pneumoniae were grown in medium containing 10⫺9 M TCDD or vehicle control (0.1% DMSO). Growth was monitored by measuring absorbance (OD600) over time. This concentration of DMSO did not impair growth of the bacteria relative to a control without DMSO control (data not shown).

The timing of the observed decrease in neutrophil numbers and inflammatory mediators argues against a causal link between decreased inflammation and host protection. Specifically, AhR activation reduced the pulmonary bacterial burden as early as the first day after infection, whereas the decreased inflammatory response was not apparent until day 3. In addition to this discrepancy in timing, there is substantial evidence that the recruitment of neutrophils and release of proinflammatory mediators are protective against S. pneumoniae-induced mortality. For example, inhibition of neutrophil migration to the lung with agents such as morphine and anti-CD97 decreased host resistance in infected mice (28, 46). Furthermore, when the immune responses to S. pneumoniae-susceptible and -resistant mice are compared, infection of S. pneumoniae-resistant mice results in a greater number of neutrophils and increased levels of inflammatory cytokines than in lungs of susceptible strains of mice (14, 23). Finally, provision of an exogenous inflammatory stimulus early during the course of S. pneumoniae infection increased pulmonary neutrophils and inflammatory cytokines, conferring improved protection (8). Taken together, this evidence suggests that suppression of the pulmonary inflammatory response to infection does not underlie the reduced bacterial burden and improved survival in TCDD-treated mice. Instead, our data suggest that the decreased inflammation results from a reduction in the ability of the bacteria to colonize or multiply in the respiratory tract. In other words, the observed diminution in the inflammatory response following infection is a downstream consequence of an earlier event that alters the host’s response to S. pneumoniae. Two possible explanations that could account for this are that (i) AhR activation alters the environment in the host such that bacterial growth is impaired or (ii) TCDD acts directly on the bacteria. Our studies do not support the latter explanation, since TCDD did not directly affect bacterial growth. Thus, instead of acting directly on the bacteria, our findings suggest that AhR activation by TCDD alters the environment within the lung, rendering it less permissive to bacterial growth.

FIG. 6. Protection against S. pneumoniae-induced mortality results from a direct effect of TCDD within the mouse. (A) TCDD treatment is not host protective in mice lacking the AhR. C57BL/6 (wild type [WT]) or AhR null mice (n ⫽ 5 to 13 per treatment group) were given an oral dose of TCDD or peanut oil vehicle 1 day prior to intranasal infection with 104 CFU of S. pneumoniae. Survival was monitored daily for 16 days, at which time all remaining animals appeared healthy. Difficulties in breeding the AhR null mice limited the number of animals available for use in this study; however this experiment was repeated in a separate study with similar results. (B) Cytochrome P450 1A1 is induced in the lungs of TCDD-treated mice. Mice were treated as described in the legend to Fig. 2. Lung homogenates were prepared from vehicle- or TCDD-treated mice sacrificed 1 day following infection with S. pneumoniae. Fifteen milligrams of prepared homogenate was subjected to SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to CYP 1A1. Note that it is established that TCDD treatment does not induce CYP 1A1 expression in AhR null mice (31).

The lung is often overlooked as a target of AhR agonists, particularly in terms of host resistance. The AhR is expressed in lung epithelial cells, Clara cells, and ciliated cells (31, 43; our unpublished observations). Moreover, exposure of mice to TCDD induced CYP 1A1, a hallmark indicator of AhR activation, in endothelial cells of capillaries and larger vessels in the lung, type II pneumocytes, Clara cells, and bronchoepithelial cells (3, 27). In mice infected with S. pneumoniae, TCDD exposure induced pulmonary CYP 1A1 protein levels on the first day following infection, demonstrating an early and direct biological effect of AhR activation on the lung. Given that cells in the lung are directly responsive to AhR activation, pulmonary mediators of the host’s response to infection are potential targets of AhR-mediated alterations. Many lung-derived pro-

VOL. 74, 2006


5685

viewed in reference 20). In fact, mice and rats treated with TCDD demonstrate increased susceptibility to infection with numerous pathogens, including influenza virus, herpesviruses, Salmonella spp., Listeria monocytogenes, and Plasmodium yoelli (20, 48). A previous report also described suppressed host resistance to intraperitoneal challenge with S. pneumoniae (49); however, in the context of respiratory infection with S. pneumoniae, we observed a protective effect of AhR activation. Our findings do not necessarily contradict the previous report, because differences in the mouse strains, in bacterial serotypes, and particularly in the route of exposure (intranasal versus intraperitoneal), make direct comparison difficult. In summary, we have found that activation of the AhR protects mice from lethal challenge with S. pneumoniae, reducing bacterial growth and the pathophysiology associated with infection. This protection is due to AhR-dependent changes within the host but does not result from an enhanced inflammatory response. Instead, it is likely that other changes within the host, possibly within the lung itself, underlie the increased host protection. These novel findings underscore the idea that AhR agonists are potent modulators of the response of the lung to infection. Elucidating the mechanism responsible for TCDD-mediated host protection against S. pneumoniae infection may lead to the creation of improved therapeutic approaches for those infected with this common human pathogen. ACKNOWLEDGMENTS We thank Sylvia Knapp (Medical University of Vienna, Austria) for methodological advice and helpful discussion. We also thank Jennifer Cundiff, Haley Neff-LaFord, and Sabine Teske for technical assistance. These studies were supported by National Institute of Environmental Health Sciences grant R01 ES-10619 (awarded to B.P.L.). The authors have no financial or commercial conflict of interest. REFERENCES

FIG. 7. Exposure to TCDD alters the generation of antibodies in mice infected with S. pneumoniae. Plasma was collected from mice that survived the infection shown in Fig. 1 (day 16 following infection; n ⫽ 7 vehicle- and 15 TCDD-treated mice). Antibodies specific for S. pneumoniae were analyzed by stacking enzyme-linked immunosorbent assay. Graphs depict the relative levels of S. pneumoniae-specific (A) IgA, (B) IgG, and (C) IgM detected in a dilution series of plasma. NI, not infected. Error bars indicate standard errors of the means. ⴱ, significant difference between plasma samples from vehicle- and TCDD-treated infected mice (P ⱕ 0.05 in a two-sided t test).

teins and innate immune factors are important in controlling infection with S. pneumoniae. For example, alterations in the expression or activity of platelet-activating factor receptor, Toll-like receptors, surfactant proteins, C-reactive protein, or complement could underlie the observed changes in host protection (4–6, 18, 25, 37, 40). Exposure to AhR agonists is typically associated with suppressed adaptive immunity and decreased host resistance (re-

1. Bals, R., and P. S. Hiemstra. 2004. Innate immunity in the lung: how epithelial cells fight against respiratory pathogens. Eur. Respir. J. 23:327– 333. 2. Birnbaum, L. S., and J. Tuomisto. 2000. Non-carcinogenic effects of TCDD in animals. Food Addit. Contam. 17:275–288. 3. Bohn, A. A., K. S. Harrod, S. Teske, and B. P. Lawrence. 2005. Increased mortality associated with TCDD exposure in mice infected with influenza A virus is not due to severity of lung injury or alterations in Clara cell protein content. Chem. Biol. Interact. 155:181–190. 4. Branger, J., S. Knapp, S. Weijer, J. C. Leemans, J. M. Pater, P. Speelman, S. Florquin, and T. van der Poll. 2004. Role of Toll-like receptor 4 in gram-positive and gram-negative pneumonia in mice. Infect. Immun. 72: 788–794. 5. Cabellos, C., D. E. MacIntyre, M. Forrest, M. Burroughs, S. Prasad, and E. Tuomanen. 1992. Differing roles for platelet-activating factor during inflammation of the lung and subarachnoid space. The special case of Streptococcus pneumoniae. J. Clin. Investig. 90:612–618. 6. Casal, J., and D. Tarrago. 2003. Immunity to Streptococcus pneumoniae: factors affecting production and efficacy. Curr. Opin. Infect. Dis. 16:219–224. 7. Choi, J. Y., J. A. Oughton, and N. I. Kerkvliet. 2003. Functional alterations in CD11b(⫹)Gr-1(⫹) cells in mice injected with allogeneic tumor cells and treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Int. Immunopharmacol. 3:553–570. 8. Dallaire, F., N. Ouellet, Y. Bergeron, V. Turmel, M. C. Gauthier, M. Simard, and M. G. Bergeron. 2001. Microbiological and inflammatory factors associated with the development of pneumococcal pneumonia. J Infect. Dis. 184:292–300. 9. Dallaire, F., N. Ouellet, M. Simard, Y. Bergeron, and M. G. Bergeron. 2001. Efficacy of recombinant human granulocyte colony-stimulating factor in a murine model of pneumococcal pneumonia: effects of lung inflammation and timing of treatment. J. Infect. Dis. 183:70–77. 10. Denison, M. S., and S. HeathPagliuso. 1998. The Ah receptor: a regulator of the biochemical and toxicological actions of structurally diverse chemicals. Bull. Environ. Contam. Toxicol. 61:557–568.

5686


11. Denison, M. S., and S. R. Nagy. 2003. Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu. Rev. Pharmacol. Toxicol. 43:309–334. 12. Fan, F., B. Yan, G. Wood, M. Viluksela, and K. K. Rozman. 1997. Cytokines (IL-1 beta and TNF alpha) in relation to biochemical and immunologincal effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats. Toxicology 116:9–16. 13. Fernandez-Salguero, P. M., T. Pineau, D. M. Hilbert, T. McPhail, S. S. T. Lee, S. Kimura, D. W. Nebert, S. Rudikoff, J. M. Ward, and F. J. Gonzalez. 1995. Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science 268:722–726. 14. Gingles, N. A., J. E. Alexander, A. Kadioglu, P. W. Andrew, A. Kerr, T. J. Mitchell, E. Hopes, P. Denny, S. Brown, H. B. Jones, S. Little, G. C. Booth, and W. L. McPheat. 2001. Role of genetic resistance in invasive pneumococcal infection: identification and study of susceptibility and resistance in inbred mouse strains. Infect. Immun. 69:426–434. 15. Gu, Y. Z., J. B. Hogenesch, and C. A. Bradfield. 2000. The PAS superfamily: sensors of environmental and developmental signals. Annu. Rev. Pharmacol. Toxicol. 40:519–561. 16. House, R. V., L. D. Lauer, M. J. Murray, P. T. Thomas, J. P. Erlich, G. R. Burleson, and J. H. Dean. 1990. Examination of immune parameters and host resistance mechanisms in B6C3F1 mice following adult exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Toxicol. Environ. Health 31:203–215. 17. Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu. Rev. Immunol. 20:197–216. 18. Kadioglu, A., and P. W. Andrew. 2004. The innate immune response to pneumococcal lung infection: the untold story. Trends Immunol. 25:143–149. 19. Karupiah, G., J. H. Chen, S. Mahalingam, C. F. Nathan, and J. D. MacMicking. 1998. Rapid interferon gamma-dependent clearance of influenza A virus and protection from consolidating pneumonitis in nitric oxide synthase 2-deficient mice. J. Exp. Med. 188:1541–1546. 20. Kerkvliet, N. I. 1994. Immunotoxicology of dioxins and related chemicals, p. 199–225. In A. Schecter (ed.), Dioxins and health. Plenum Press, New York, N.Y. 21. Kerkvliet, N. I., L. Baecher-Steppan, D. M. Shepherd, J. A. Oughton, B. A. Vorderstrasse, and G. K. Dekrey. 1996. Inhibition of TC-1 cytokine production, effector cytotoxic T lymphocyte development and alloantibody production by 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Immunol. 157:2310–2319. 22. Kerkvliet, N. I., and J. A. Oughton. 1993. Acute inflammatory response to sheep red blood cell challenge in mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): phenotypic and functional analysis of peritoneal exudate cells. Toxicol. Appl. Pharmacol. 119:248–257. 23. Kerr, A. R., J. J. Irvine, J. J. Search, N. A. Gingles, A. Kadioglu, P. W. Andrew, W. L. McPheat, C. G. Booth, and T. J. Mitchell. 2002. Role of inflammatory mediators in resistance and susceptibility to pneumococcal infection. Infect. Immun. 70:1547–1557. 24. Knapp, S., L. Hareng, A. W. Rijneveld, P. Bresser, J. S. van der Zee, S. Florquin, T. Hartung, and T. van der Poll. 2004. Activation of neutrophils and inhibition of the proinflammatory cytokine response by endogenous granulocyte colony-stimulating factor in murine pneumococcal pneumonia. J. Infect. Dis. 189:1506–1515. 25. Knapp, S., C. W. Wieland, C. van ’t Veer, O. Takeuchi, S. Akira, S. Florquin, and T. van der Poll. 2004. Toll-like receptor 2 plays a role in the early inflammatory response to murine pneumococcal pneumonia but does not contribute to antibacterial defense. J. Immunol. 172:3132–3138. 26. Kobrynski, L. J., A. O. Sousa, A. J. Nahmias, and F. K. Lee. 2005. Antibody production to pneumococcal polysaccharides requires CD1 molecules and CD8⫹ T cells. J. Immunol. 174:1787–1790. 27. Lang, D. S., S. Becker, R. B. Devlin, and H. S. Koren. 1998. Cell-specific differences in the susceptibility of potential cellular targets of human origin derived from blood and lung following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cell Biol. Toxicol. 14:23–38. 28. Leemans, J. C., A. A. te Velde, S. Florquin, R. J. Bennink, K. de Bruin, R. A. van Lier, T. van der Poll, and J. Hamann. 2004. The epidermal growth factor-seven transmembrane (EGF-TM7) receptor CD97 is required for neutrophil migration and host defense. J. Immunol. 172:1125–1131. 29. Luebke, R. W., C. B. Copeland, L. R. Bishop, M. J. Daniels, and M. I. Gilmour. 2002. Mortality in dioxin-exposed mice infected with influenza:

Editor: J. N. Weiser

INFECT. IMMUN.

30. 31.

32. 33.

34.

35. 36. 37.

38. 39. 40. 41.

42.

43. 44.

45. 46. 47. 48.

49. 50.

mitochondrial toxicity (Reye’s-like syndrome) versus enhanced inflammation as the mode of action. Toxicol. Sci. 69:109–116. Marlowe, J. L., and A. Puga. 2005. Aryl hydrocarbon receptor, cell cycle regulation, toxicity, and tumorigenesis. J. Cell Biochem. 96:1174–1184. Martinez, J. M., C. A. Afshari, P. R. Bushel, A. Masuda, T. Takahashi, and N. J. Walker. 2002. Differential toxicogenomic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin in malignant and nonmalignant human airway epithelial cells. Toxicol. Sci. 69:409–423. Mitchell, T. J. 2003. The pathogenesis of streptococcal infections: from tooth decay to meningitis. Nat. Rev. Microbiol. 1:219–230. Mizrachi-Nebenzahl, Y., S. Lifshitz, R. Teitelbaum, S. Novick, A. Levi, D. Benharroch, E. Ling, and R. Dagan. 2003. Differential activation of the immune system by virulent Streptococcus pneumoniae strains determines recovery or death of the host. Clin. Exp. Immunol. 134:23–31. Moos, A. B., L. Baecher-Steppan, and N. I. Kerkvliet. 1994. Acute inflammatory response to sheep red blood cells in mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin: the role of proinflammatory cytokines, IL-1 and TNF. Toxicol. Appl. Pharmacol. 127:331–335. Pohjanvirta, R., and J. Tuomisto. 1994. Short-term toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals: effects, mechanisms, and animal models. Pharmacol. Rev. 46:483–549. Puga, A., C. R. Tomlinson, and Y. Xia. 2005. Ah receptor signals cross-talk with multiple developmental pathways. Biochem. Pharmacol. 69:199–207. Rijneveld, A. W., S. Weijer, S. Florquin, P. Speelman, T. Shimizu, S. Ishii, and T. van der Poll. 2004. Improved host defense against pneumococcal pneumonia in platelet-activating factor receptor-deficient mice. J. Infect. Dis. 189:711–716. Schluger, N. W., and W. N. Rom. 1997. Early responses to infection: chemokines as mediators of inflammation. Curr. Opin. Immunol. 9:504–508. Shepherd, D. M., E. D. Dearstyne, and N. I. Kerkvliet. 2000. The effects of TCDD on the activation of ovalbumin (OVA)-specific DO11.10 transgenic T cells in adoptively-transferred mice. Toxicol. Sci. 56:340–350. Takeda, K., and S. Akira. 2004. TLR signaling pathways. Semin. Immunol. 16:3–9. Teske, S., A. A. Bohn, J. F. Regal, J. J. Neumiller, and B. P. Lawrence. 2005. Activation of the aryl hydrocarbon receptor increases pulmonary neutrophilia and diminishes host resistance to influenza A virus. Am. J. Physiol. Lung Cell Mol. Physiol. 289:L111–124. Theobald, H. M., and R. E. Peterson. 1997. In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-rho-dioxin: effects on development of the male and female reproductive system of the mouse. Toxicol. Appl. Pharmacol. 145:124–135. Tritscher, A. M., J. Mahler, C. J. Portier, G. W. Lucier, and N. J. Walker. 2000. Induction of lung lesions in female rats following chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Pathol. 28:761–769. Vorderstrasse, B. A., L. B. Steppan, A. E. Silverstone, and N. I. Kerkvliet. 2001. Aryl hydrocarbon receptor-deficient mice generate normal immune responses to model antigens and are resistant to TCDD-induced immune suppression. Toxicol. Appl. Pharmacol. 171:157–164. Walisser, J. A., M. K. Bunger, E. Glover, and C. A. Bradfield. 2004. Gestational exposure of Ahr and Arnt hypomorphs to dioxin rescues vascular development. Proc. Natl. Acad. Sci. USA 101:16677–16682. Wang, J., R. A. Barke, R. Charboneau, and S. Roy. 2005. Morphine impairs host innate immune response and increases susceptibility to Streptococcus pneumoniae lung infection. J. Immunol. 174:426–434. Wang, S. Z., and K. D. Forsyth. 2000. The interaction of neutrophils with respiratory epithelial cells in viral infection. Respirology 5:1–10. Warren, T. K., K. A. Mitchell, and B. P. Lawrence. 2000. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses the humoral and cell-mediated immune responses to influenza A virus without affecting cytolytic activity in the lung. Toxicol. Sci. 56:114–123. White, K. L., Jr., H. H. Lysy, J. A. McCay, and A. C. Anderson. 1986. Modulation of serum complement levels following exposure to polychlorinated dibenzo-p-dioxins. Toxicol. Appl. Pharmacol. 84:209–219. Yin, H., Y. Li, and T. R. Sutter. 1994. Dioxin-enhanced expression of interleukin-1 beta in human epidermal keratinocytes: potential role in the modulation of immune and inflammatory responses. Exp. Clin. Immunogenet. 11:128–135.