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The Journal of Immunology, 2007, 179, 6952 -6962
Copyright © 2007 by The American Association of Immunologists, Inc.
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The Aryl Hydrocarbon Receptor Is Required for Optimal Resistance to Listeria monocytogenes Infection in Mice1

Lewis Zhichang Shi, Nancy G. Faith, Yumi Nakayama, Makulasiddappa Suresh, Howard Steinberg and Charles J. Czuprynski2,*,{dagger}

* Department of Pathobiological Sciences, and {dagger} Food Research Institute, College of Agriculture and Life Sciences, and the Molecular and Environmental Toxicology Center, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The aryl hydrocarbon receptor (AhR) is part of a powerful signaling system that is triggered by xenobiotic agents such as polychlorinated hydrocarbons and polycyclic aromatic hydrocarbons. Although activation of the AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin or certain polycyclic aromatic hydrocarbons can lead to immunosuppression, there is also increasing evidence that the AhR regulates certain normal developmental processes. In this study, we asked whether the AhR plays a role in host resistance using murine listeriosis as an experimental system. Our data clearly demonstrate that AhR null C57BL/6J mice (AhR–/–) are more susceptible to listeriosis than AhR heterozygous (AhR+/–) littermates when inoculated i.v. with log-phase Listeria monocytogenes. AhR–/– mice exhibited greater numbers of CFU of L. monocytogenes in the spleen and liver, and greater histopathological changes in the liver than AhR+/– mice. Serum levels of IL-6, MCP-1, IFN-{gamma}, and TNF-{alpha} were comparable between L. monocytogenes-infected AhR–/– and AhR+/– mice. Increased levels of IL-12 and IL-10 were observed in L. monocytogenes-infected AhR–/– mice. No significant difference was found between AhR+/– and AhR–/– macrophages ex vivo with regard to their ability to ingest and inhibit intracellular growth of L. monocytogenes. Intracellular cytokine staining of CD4+ and CD8+ splenocytes for IFN-{gamma} and TNF-{alpha} revealed comparable T cell-mediated responses in AhR–/– and AhR+/– mice. Previously infected AhR–/– and AhR+/– mice both exhibited enhanced resistance to reinfection with L. monocytogenes. These data provide the first evidence that AhR is required for optimal resistance but is not essential for adaptive immune response to L. monocytogenes infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The aryl hydrocarbon receptor (AhR)3 is a ligand-activated member of the basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) family of transcriptional regulators (1, 2, 3). Together with its bHLH-PAS nuclear partner, the aryl hydrocarbon receptor nuclear transporter (ARNT), it provides a powerful signaling system that plays critical roles in response to several environmental toxins, most notably 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). There is also increasing evidence for the importance of the AhR in normal development, circadian rhythm, response to hypoxia, and hormone signaling (4, 5, 6). In the absence of ligands, AhR is inactive and remains in the cytoplasm complexed with a dimer of HSP90 (heat shock protein 90), a src protein kinase, and an immunophilin XAP2 (hepatitis B virus X-associated protein 2) (7, 8, 9). Once ligands bind and activate the AhR, these complexes change their conformation and translocate to the nucleus. The cytoplasmic accessory proteins then dissociate from the ligand-AhR complex and ARNT binds to the AhR, forming a heterodimer. This heterodimer binds to xenobiotic response elements (XRE, whose core motif is 5'-GCGTG-3') present in the upstream regulatory region of target genes (10). A battery of genes (cytochromes P450, NAD(P)H quinine oxidoreductase, UDP-glucuronosyltransferase-6, AhR repressor, Bax, DNA polymerase {kappa}, and Fas) are up-regulated by the AhR signaling pathway (3, 10, 11). Proinflammatory cytokines constitute another important class of genes regulated by the AhR. TCDD treatment causes increased expression of TNF-{alpha}, IL-1, IFN-{gamma}, IL-8, IL-6, and CCL1 (12, 13, 14, 15, 16), suggesting a role for the AhR in inflammation and immunity.

The immune system is a sensitive target for the toxicity of TCDD. Many polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene (DMBA), also act in part via AhR activation. Early studies showed that exposure of mice to TCDD led to bone marrow hypocellularity with depressed colony formation of macrophage-granulocyte progenitor cells and pluripotent stem cells (17, 18). Similar suppressive effects on the immune response were observed in DMBA-exposed mice (19). Treatment of mice with TCDD suppressed the production of Abs and the generation of CTLs (20, 21), which consequently altered the host resistance to many diseases (22). More recently, it was reported that TCDD treatment led to premature emigration and proliferation arrest of triple negative thymocytes, possibly via an induced ecotopic expression of Kruppel-like factor-2 (KLF2) in these cells (23). AhR–/– mice exhibited decreased accumulation of lymphocytes in the spleen and lymph nodes (24). Conversely, TCDD treatment of mice increased the numbers of peritoneal neutrophils and macrophages in response to SRBC injection (25). These studies suggest a role for the AhR in regulating immune responses.

The cellular and molecular mechanisms involved in PAH- or TCDD-related immunotoxicity remain incompletely understood. T cells are a possible direct target for TCDD, as evidenced by the presence of the AhR in T cells (26), and inhibition of T cell growth by the expression of a constitutively active AhR mutant in AhR-null Jurkat T cells (27) or following TCDD treatment (28, 29). Exposure to PAHs such as benzo[a]pyrene, DMBA, and 3-methylcholanthrene (3-MC) inhibited differentiation of human monocytes into macrophages (30) as well as differentiation and maturation of human monocyte-derived dendritic cells (DCs) (31) in an AhR-dependent manner. Kerkvliet and colleagues showed that expression of AhR in both CD4+ and CD8+ T cells is required for a full suppression of an allospecific CTL response by TCDD (32), indicating a direct role for AhR in these TCDD-induced immunosuppressive effects. More recently, they identified a group of T regulatory cells (CD4+CD25+) after TCDD treatment (33). By adoptively transferring AhR–/– CD8+ cells into AhR+/+ mice, Lawrence and colleagues demonstrated that CD8+ T cells are not directly affected by TCDD. Rather TCDD appears to mediate its effects by altering hemopoiesis (34).

Listeria monocytogenes is a Gram-positive, facultative intracellular bacterium that causes food-borne disease (listeriosis) that results in considerable morbidity and a relatively high mortality rate (35). It is estimated that L. monocytogenes causes ~2500 cases of serious illness and as many as 500 deaths per year in the United States (36). Risk factors for listeriosis include age (>65 years), pregnancy, HIV infection, immunosuppressive therapy, diabetes, kidney disease, and cancer (37). L. monocytogenes is also widely studied as a model intracellular pathogen, which can multiply to great numbers within infected cells (i.e., hepatocytes and macrophages) and pass directly from one cell to the next (38). Because activation of the AhR by TCDD or PAHs frequently causes immunosuppression, one might expect that AhR activation would increase susceptibility to infection with L. monocytogenes. However, prior studies have yielded inconsistent results (17, 18, 19, 39, 40). These disparate findings could reflect differences in the AhR agonists used or their routes of administration. There have been no reports on the constitutive role of the AhR in resistance to L. monocytogenes or other intracellular bacterial pathogens.

In this study, we compared the susceptibility of AhR–/– and AhR+/– mice to i.v. challenge with L. monocytogenes. Our data convincingly demonstrate that AhR–/– mice are more susceptible than AhR+/– mice to a primary L. monocytogenes infection. However, AhR–/– mice exhibit no defect in acquired resistance to re-infection, nor significant differences in T cell-mediated production of cytokines. These findings demonstrate a heretofore unrecognized role for the AhR in host defense against listeriosis in mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

RPMI 1640 medium, L-glutamine, penicillin/streptomycin, HBSS with or without calcium and magnesium, DMEM (high glucose) medium, and nonessential amino acids were purchased from Mediatech; FBS was obtained from Atlanta Biologicals; ammonium chloride (NH4Cl), sodium azide, trypan blue working solution, EDTA, proteose peptone, and paraformaldehyde were obtained from Sigma-Aldrich; BSA was obtained from Calbiochem; recombinant mouse (rm) M-CSF was obtained from R&D Systems; 10 x PBS stock was from Fisher Bioreagents; listerial peptides (LLO190, LLO253, LLO296) were synthesized by Biosynthesis; Golgi plug, IL-2, anti-CD8-APC, anti-CD4-PE, anti-CD44-FITC, anti-IFN-{gamma} -FITC, anti-TNF-PeCy7, cytofix/cytoperm solution, perm/wash buffer, and mouse inflammation kit were purchased from BD Biosciences.

Animal care and breeding

C57BL/6J-AhR-deficient (AhR–/–) mice, created by deletion of exon 2 of the AhR gene (3), were obtained from Dr. C. Bradfield (Madison, WI) and maintained as a breeding colony in the animal care facility at the University of Wisconsin School of Veterinary Medicine. The AhR–/– mice could not be bred efficiently by homozygous mating. Instead, AhR+/+, AhR+/–, and AhR–/– littermates were generated by breeding AhR+/– females with AhR–/– males or AhR+/– females with AhR+/– males. The offspring were genotyped by PCR. All mice were provided either regular chow or breeder chow (Harlan Teklad 8604 rodent diet) and water ad libitum. All animal handling and experimental processes were performed in accordance with an IACUC approved protocol.

Infection of mice with L. monocytogenes (EGD)

Frozen overnight cultures of L. monocytogenes EGD (a serotype 1/2a strain) were diluted 1/50 in brain heart infusion broth and grown to mid-logarithmic phase (4.5–7 h at 37°C on a rotating platform). The bacterial cells were pelleted by centrifugation at 1500 x g for 10 min, washed twice with sterile PBS, and diluted in PBS to the appropriate concentration for inoculation. The number of viable L. monocytogenes in the inoculum was confirmed by plating serial dilutions on blood agar. Seven- to 12-wk-old mice were placed in a cage on a heating pad for ~2 min and then put in a plastic mouse restrainer. One of the lateral tail veins was chosen for i.v. injection of L. monocytogenes at the dose indicated in each experiment.

Bacterial burden in tissues

The listerial burden in each tissue was determined as described previously (41). In brief, mice were euthanized with carbon dioxide overdose at the specified times. The abdominal cavity was aseptically opened, and the gallbladder and portions of the spleen and liver were removed, weighed in sterile weigh boats, and placed in separate sterile tissue grinders containing 1 ml of cold PBS. The tissues were homogenized, serially diluted in sterile saline, and plated onto blood agar. Similarly, the cecum and duodenum from each mouse were removed to separate tissue grinders, homogenized, serially diluted, and plated on modified Oxoid agar plates. The plates were incubated at 37°C and the colonies were counted after 24–48 h. The plates with CFU counts in the range of 30–300 were selected.

Cytometric bead assay (CBA)

Serum cytokine levels were quantified using a CBA assay kit purchased from BD Biosciences, which is capable of detecting IL-6, IL-10, MCP-1, IFN-{gamma}, TNF, and IL-12p70. The samples were read using a FACSCalibur flow cytometer at the University of Wisconsin-Madison Comprehensive Cancer Center. The results were then analyzed with CBA software from BD Biosciences.

Infection of peritoneal and bone marrow-derived macrophages

Peritoneal exudate macrophages were prepared as described previously (42). In brief, age-matched AhR+/– and AhR–/– mice were injected i.p. with 1 ml of 10% sterile proteose peptone. At 72 h after injection, the mice were euthanized and peritoneal exudate cells were recovered by flushing the peritoneal cavity with HBSS (Ca- and Mg-free) containing 0.01 M EDTA. Cells were washed twice with HBSS containing Ca2+ and Mg2+ and resuspended in 1 ml of DMEM containing 10% FBS, glucose, sodium pyruvate, HEPES, and penicillin/streptomycin. Cells were quantified and seeded onto a 24-well plate at an initial concentration of 6.0 x 105 cells/well at 37°C in a 5% CO2 incubator. Nonadherent cells were removed 2 h later by washing three times with serum-free DMEM (no antibiotics). Macrophage monolayers were infected the next day with L. monocytogenes as described below.

To obtain bone marrow-derived macrophages, age-matched AhR+/– and AhR–/– mice were euthanized, their femurs were removed and flushed with 3 ml of RPMI 1640 containing L-glutamine. Bone marrow cells were washed twice with DMEM and resuspended in DMEM containing 10% FBS, glucose, sodium pyruvate, HEPES, and penicillin/streptomycin. Cells (5.0 x 106 cells/ml) were placed in a T-25 flask with 10 ng/ml rm-M-CSF for 24 h at 37° C in a 5% CO2 incubator. Nonadherent progenitor cells were transferred to a new T-75 flask containing rm-M-CSF and incubated for 4 days, at which point the medium with floating cells were removed and replaced with fresh medium with rm-M-CSF (no antibiotics). The cells were incubated at 37°C with 5% CO2 for 3 days, and confirmed to be macrophages by esterase staining (>99% positive). Macrophages were harvested and 1.0 x 105 cells were seeded into each well of a 24-well plate. The following day, the cells were infected as described below.

To infect peritoneal and bone marrow-derived macrophages, cell monolayers were incubated with 1 x 106 log phase L. monocytogenes for 2 h at 37°C. The uningested listerial cells were removed by washing 3x with DMEM, and fresh culture medium with 5 µg/ml gentamicin was added to kill extracellular L. monocytogenes. After an additional 6 h of incubation at 37°C, the cells were washed three times with DMEM to remove gentamicin and lysed with sterile double-distilled H2O. The lysates were diluted in PBS and plated on blood agar. The plates were incubated for 24–48 h at 37°C. The colonies were counted to determine CFU of L. monocytogenes.

Surface staining of CD44

To monitor the activation and expansion of CD4+ and CD8+ T cells following infection with L. monocytogenes, freshly isolated splenocytes were stained for 1 h on ice in the dark with combinations of mAbs purchased from BD Pharmingen: PE-labeled anti-CD4, allophycocyanin-labeled anti-CD8, and FITC-labeled anti-CD44 (43). For all samples, data were collected from >200,000 events using a BD FACSCalibur flow cytometer and then analyzed using FlowJo software by gating on viable splenocytes.

Quantitation of L. monocytogenes-specific T cells by intracellular cytokine staining

Intracellular cytokine staining to detect Ag-specific CD8 or CD4 T cells was done as previously described (44). In brief, single cell suspensions of erythrocyte-depleted splenocytes were cultured with MHC I- or MHC II-restricted listerial epitope peptides (LLO296 or LLO190 and LLO253, respectively) for 5 h in the presence of brefeldin A (1 µl/ml) and IL-2 (50 U/ml). After culture, cells were stained for cell surface CD8 or CD4, and intracellular IFN-{gamma} and TNF-{alpha}, using the cytofix/cytoperm kit (BD Pharmingen). The number of IFN-{gamma}- and TNF-{alpha}-producing CD4 or CD8 T cells was analyzed by flow cytometry.

Histopathology

Portions of the liver were removed and fixed in 10% formalin. The fixed samples were thin sectioned and stained with H&E or a tissue gram stain at the histopathology laboratory in the University of Wisconsin School of Veterinary Medicine. Photomicrographs of representative sections were taken with an Olympus microscope equipped with a built-in digital camera.

Statistical methods

All statistical analyses were performed using Prism 4.0 (GraphPad Software). Significant differences were determined by a one-way ANOVA followed by the Tukey posttest or the Student t test. Differences were considered statistically significant when p was <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Wild-type AhR+/+ and heterozygous AhR+/– mice exhibit similar resistance to listeriosis

We first determined whether wild-type AhR+/+ and heterozygous AhR+/– mice differed in their resistance to listeriosis. AhR+/+ and AhR+/– littermates (5 mice in each group) were injected i.v. with 6.7 x 104 CFU L. monocytogenes via a lateral tail vein. At the peak of the infection (3 days following inoculation), mice were euthanized and tissue burdens of L. monocytogenes in liver, spleen, blood, and gallbladder were determined as described in Materials and Methods. As shown in Fig. 1, there was no significant difference in the listerial burdens between AhR+/+ and AhR+/– mice. As a result and because of the lack of AhR+/+ mice resulting from our mating system (AhR+/– x AhR–/–), we used AhR+/– mice as controls in all subsequent experiments.


Figure 1
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  		FIGURE 1. There is no difference in susceptibility to listeriosis between AhR+/– (Figure 1) and AhR+/+ ({blacksquare}) mice. Mice were infected by i.v. injection of 6.7 x 104 CFU L. monocytogenes. Three days later, mice were euthanized and the CFU of L. monocytogenes was determined in the liver (per gram), spleen (per gram), blood (per ml), and gallbladder (whole organ). Data represent the mean ± SEM (n = 5 mice per group).

 
AhR–/– mice are more susceptible than AhR+/– mice to listeriosis

We next compared the susceptibility of AhR–/– and AhR+/– mice to listeriosis. Mice (5 to 8 mice per group) were injected i.v. with 3.0 x 105 CFU L. monocytogenes via a tail vein. Three days later, mice were euthanized, and their liver, spleen, blood, and gallbladder were processed and plated on blood agar. Our results show that AhR–/– mice are significantly less resistant to a primary listerial infection than AhR+/– mice (Fig. 2A). Portions of the livers from infected mice were also subjected to histopathology. There were few histopathological changes in the livers (Fig. 2B) or hepatocytes (Fig. 2D) from L. monocytogenes-infected AhR+/– mice. Granulomas were rarely observed and, when present (Fig. 2F), were smaller than in AhR–/– mice (Fig. 2G). Nor were Gram-positive rods commonly detected in hepatocytes from AhR+/– mice (Fig. 2H). In contrast, the livers from L. monocytogenes-infected AhR–/– mice exhibited a large area of necrosis and granulomas (Fig. 2, C and G). Their hepatocytes displayed a foamy appearance (Fig. 2E), and Gram-positive rods were readily seen within hepatocytes (Fig. 2I).


Figure 2
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  		FIGURE 2. AhR–/– ({square}) mice are more susceptible than AhR+/– (Figure 2) mice to a primary L. monocytogenes infection. Mice were inoculated i.v. with 3.0 x 105 CFU L. monocytogenes and euthanized 3 days later. A, The CFU of L. monocytogenes was determined in the liver (per gram), spleen (per gram), blood (per ml), and gallbladder (whole organ). Data represent the mean ± SEM (n = 5 to 8 mice per group). *, p < 0.05 in comparison to AhR+/– mice. Panels B–I show representative histopathological changes for AhR+/– (B, D, and F) and AhR–/– mice (C, E, and G) (H and E staining). E, Note the large mass of necrosis (arrows in C) and the foamy appearance of hepatocytes from AhR–/– mice ({blacktriangledown}). Gram staining (H and I) revealed Gram-positive rods in hepatocytes of AhR–/– (H) but not AhR+/– mice (I) (Figure 2).

 
A similar difference between AhR–/– and AhR+/– mice was observed when they were inoculated with a lower challenge dose of log-phase L. monocytogenes (7.8 x 102 CFU, i.v.). Here too significantly more CFU of L. monocytogenes were recovered from the spleens of AhR–/– than AhR+/– mice at 3 days postinoculation (data not shown). Furthermore, the effect of AhR absence on resistance to L. monocytogenes infection was seen in both male and female mice (data not shown).

To further examine the role of the AhR in resistance to L. monocytogenes infection, a time course experiment was performed in which AhR+/– and AhR–/– mice were inoculated i.v. with 7.8 x 103 CFU and euthanized at 1, 3, and 5 days after injection (5 mice per group at each time point). As shown in Fig. 3, there was no significant difference between AhR+/– and AhR–/– mice on day 1. However, there were significantly greater CFU recovered from the spleens and livers of AhR–/– than AhR+/– mice at 3 and 5 days after inoculation. Greater numbers of CFU were also recovered from the gallbladders of AhR–/– mice on day 5.


Figure 3
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  		FIGURE 3. Time-course of L monocytogenes infection in AhR–/– ({square}) and AhR+/– (Figure 3) mice. Mice were infected i.v. with 7.8 x 103 CFU L. monocytogenes. On days 1, 3, and 5, five mice of each genotype (AhR–/– or AhR+/–) were euthanized and the CFU of L. monocytogenes in the liver (A), spleen (B), and gallbladder (C) were determined. Data represent the mean ± SEM of 5 mice per group. *, p < 0.05 as compared with AhR+/– mice at the same time point.

 
AhR–/– mice exhibit a delayed ability to clear a primary L. monocytogenes infection

The preceding data showed that AhR–/– mice harbored greater bacterial burden than AhR+/– mice at the peak time of the infection. We next asked whether they were also impaired in their ability to clear the infection. To do so, AhR–/– and AhR+/– mice (Fig. 4) were injected i.v. with a low dose of L. monocytogenes (8.2 x 102 CFU/mouse). All mice (both AhR–/– and AhR+/–) survived the infection and did not demonstrate outward signs of clinical illness. On days 7 and 14 postinoculation, mice were euthanized and portions of their liver, spleen, blood, and whole gallbladder were processed to estimate CFU of L. monocytogenes. The duodenum and cecum from each mouse were also removed to quantify the colonization of L. monocytogenes in the gastrointestinal tract. AhR–/– mice displayed a delayed ability to clear a primary listerial infection, as evidenced by significantly greater numbers of L. monocytogenes in the liver, gallbladder, and spleen on day 7. By day 14, all mice (both AhR–/– and AhR+/–) had cleared L. monocytogenes from their tissues (data not shown). At necropsy, we noted that some L. monocytogenes-infected AhR–/– mice had gallbladders that were firm and white in gross appearance at 14 days after inoculation. This observation occurred in the absence of recovery of viable L. monocytogenes from these organs, suggesting that the gross changes were the result of prolonged inflammation. Overall, these data indicate that AhR–/– mice are delayed, but not unable, to clear a primary listerial infection within a 14-day period.


Figure 4
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  		FIGURE 4. AhR–/– mice exhibited delayed clearance of L. monocytogenes. AhR–/– ({blacksquare}) and AhR+/– (•) mice were injected i.v. with 8.2 x 102 CFU L. monocytogenes. On day 7 postinoculation, groups of 4 to 6 mice were euthanized and the numbers of viable L. monocytogenes in the indicated tissues were determined. Symbols represent log10 CFU/g for the liver and spleen, log10 CFU/ml for blood, and log10CFU/whole organ for the gallbladder, duodenum, and cecum in individual mice. The horizontal bars indicate the mean values for groups of mice. p < 0.05 for AhR–/– as compared with AhR+/– mice in the liver, spleen, and gallbladder. On day 14, L. monocytogenes was not recovered from any tissue of any mice (data not shown).

 
Cytokine production is not impaired in AhR–/– mice

We then examined whether AhR–/– mice were impaired in their ability to produce several inflammatory cytokines in response to L. monocytogenes infection. To elicit a vigorous inflammatory response, mice were injected i.v. with 4.0 x 105 CFU L. monocytogenes (high challenge dose) and euthanized at 24 h postinoculation (early phase of infection). The numbers of viable L. monocytogenes in the liver, spleen, and gallbladder were determined. Serum was prepared from whole blood obtained by cardiac puncture at the time of euthanasia. Cytokine levels (IL-6, IL-10, MCP-1, IFN-{gamma}, TNF-{alpha}, and IL-12) in the sera were determined using the CBA mouse inflammation kit from BD Biosciences. Fig. 5A illustrates representative flow diagrams of the CBA assay for control (injected with sterile PBS) and L. monocytogenes-infected mice. Fig. 5B confirms that greater numbers of L. monocytogenes were recovered from AhR–/– than AhR+/– mice in this experiment. Fig. 5C demonstrates that comparable levels of inflammatory cytokines were present in the sera from AhR+/– and AhR–/– mice. The exceptions were IL-12 and IL-10, for which significantly greater amounts were detected in the sera of AhR–/– than AhR+/– mice. Perhaps this observation reflects the more severe infection in AhR–/– than AhR+/– mice.


Figure 5
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  		FIGURE 5. Production of cytokines is not impaired in AhR–/– mice. AhR–/– and AhR+/– mice were injected i.v. with 4.0 x 105 CFU L. monocytogenes. Control animals were injected i.v. with sterile PBS. Mice were euthanized 24 h later. A, Representative dot plots of distinct cytometric beads used to specifically detect IL-12, TNF-{alpha}, IFN-{gamma}, MCP-1, IL-10, or IL-6. B, CFU of L. monocytogenes recovered from the liver (per gram), spleen (per gram), and gallbladder (per whole organ). C, Serum cytokine levels as measured by the CBA assay. Data represent the mean ± SEM of 4 mice per group. *, p < 0.05 as compared with the AhR+/– mice.

 
AhR–/– mice generate normal T cell-mediated responses to a primary L. monocytogenes infection

We next compared the ability of AhR–/– and AhR+/– mice to mount T cell responses to L. monocytogenes infection. On day 7 postinoculation, we quantified the numbers of L. monocytogenes-specific CD8+ and CD4+ T cells in the spleen by intracellular staining for IFN-{gamma} and TNF-{alpha} following incubation with LLO peptides ex vivo. As shown in Fig. 6, the frequencies and total numbers of LLO190-specific CD4 T cells (A and B) or LLO-296-specific CD8 T cells (A and C) were comparable between AhR+/– and AhR–/– mice. Likewise, data obtained on day 14 after inoculation showed no significant differences in CD4+IFN-{gamma} + and CD4+TNF-{alpha}+ cells, nor CD8+IFN-{gamma}+ and CD8+TNF-{alpha}+ cells, between the AhR+/– and AhR–/– mice (data not shown).


Figure 6
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  		FIGURE 6. Activation of CD4+ and CD8+ T cells is similar in AhR–/– and AhR+/– mice on day 7 postinoculation. The numbers of L. monocytogenes-specific CD4+ and CD8+ T cells were quantified by intracellular cytokine staining. Splenocytes were stimulated with the MHC II-restricted listerial epitope peptide LLO190 or MHC I-restricted epitope peptide LLO-296 and the numbers of IFN-{gamma}- or TNF-producing CD4 or CD8 T cells respectively were determined by flow cytometry. Dot plots in A are gated on total splenocytes and the numbers are the percentage of IFN-{gamma}- or TNF-producing CD8/CD4 T cells among splenocytes. Data in B and C are total numbers of L. monocytogenes-specific CD4+ and CD8+ T cells, respectively, from 4 to 5 mice per group.

 
AhR–/– mice develop enhanced resistance to reinfection with L. monocytogenes

We then asked whether AhR–/– mice could develop enhanced resistance to reinfection following clearance of a primary L. monocytogenes infection. To do so, age-matched AhR+/– and AhR–/– mice were immunized by i.v. injection of a low dose of L. monocytogenes (8.5 x 102 CFU). Nonimmunized AhR+/– and AhR–/– mice (injected with sterile PBS) were included as naive control groups and housed in the same animal room with the immunized animals. Fifteen days later, immunized AhR+/– and AhR–/– mice were re-infected i.v. with 3.0 x 104 CFU L. monocytogenes/mouse. Nonimmunized control AhR+/– and AhR–/– mice were injected i.v. with a lower inoculum of L. monocytogenes (3.0 x 103 CFU/mouse) to prevent death of these immunologically naive mice. Three days later, all mice were euthanized and their tissues removed for bacteriological and histopathological evaluation, as described above. As expected, far fewer CFU were recovered from immunized than naive mice. There was no significant difference between immunized AhR–/– and AhR+/– mice in the CFU of L. monocytogenes recovered from the liver or spleen (Fig. 7, A and B). Likewise, the numbers of viable L. monocytogenes recovered from the blood or gallbladders were not significantly different between immunized AhR–/– and AhR+/– mice, (data not shown). Taken as a whole, these data show that AhR–/– mice are capable of developing an adaptive immune response that protects them against reinfection with L. monocytogenes.


Figure 7
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  		FIGURE 7. Immunized AhR–/– mice are protected against a second challenge with L. monocytogenes. Mice were injected i.v. with 8.5 x 102 CFU L. monocytogenes (immunized mice) or with PBS (naive control mice). Fifteen days later, immunized and naive mice were challenged i.v. with 3.0 x 104 and 3.0 x 103 CFU L. monocytogenes, respectively. The lower challenge dose for the naive mice was chosen to reduce deaths in the AhR–/– mice. Three days later, all mice were euthanized and the CFU of L. monocytogenes in the liver (A) and spleen (B) were determined. Data represent the mean ± SEM of 4 mice per group. a, Values of p < 0.05 in comparison to AhR+/– mice. b, p < 0.05 as compared with the corresponding phenotype of naive mice. Groups sharing the same letter (a or b) are not significantly different from each other. C, Representative histopathological changes in the livers of immunized and naive mice. Necrotic foci (indicated by the black arrow) were greater in size and number in AhR–/– naive mice. Larger inflammatory cell aggregates, surrounding necrotic debris (yellow arrow) were seen in immunized AhR–/– mice as compared with immunized AhR+/– mice.

 
Histopathological analyses of the livers from these mice revealed relatively normal liver structure, with occasional small foci of necrosis or inflammatory cell aggregates in L. monocytogenes-infected naive AhR+/– mice (Fig. 7C). Larger areas of necrosis and inflammatory foci that were surrounded by a mixture of inflammatory cells were evident in L. monocytogenes-infected naive AhR–/– mice. Consistent with the reduced bacterial burden illustrated in Fig. 6, the histopathological changes were less in the livers of immunized mice. Occasional inflammatory foci were found in the livers of immunized AhR+/– mice. Larger inflammatory foci, which sometimes contained necrotic debris, were observed in some immunized AhR–/– mice. Histopathological changes in the spleens were minor and did not differ substantially between AhR+/– and AhR–/– mice (data not shown).

We also investigated the effects of AhR on T cell responses from the spleens of naive and immunized mice. As indicated in Fig. 8, reinfection resulted in a 3- to 4-fold increase in the frequencies and total numbers of CD4+CD44high (A and B) and CD8+CD44high (A and C) spleen cells from immunized AhR–/– and AhR+/– mice, as compared with naive mice of the same genotype. The numbers of CD4+CD44high cells did not differ between immunized AhR+/– and AhR–/– mice. However, fewer CD8+CD44high cells were seen in the immunized AhR+/– than AhR–/– mice.


Figure 8
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  		FIGURE 8. Comparable secondary T cell responses to reinfection with L. monocytogenes in AhR–/– and AhR+/– mice. Immunized or naive mice were challenged with 3 x 104 and 3 x 103 CFU L. monocytogenes, respectively. Three days later, splenocytes from these mice were prepared and stained with CD4, CD8, and CD44 to quantitative activated memory T cells. Zebra plots (FlowJo) in A are gated on total splenocytes and the numbers are the percentage of CD44 CD8/CD4 T cells among splenocytes. Data in B and C are total numbers of L. monocytogenes-specific CD4+CD44high and CD8+CD44high T cells, respectively, from 4 to 5 mice per group. **, p < 0.01 as compared with the corresponding genotype of naive mice.

 
We also evaluated intracellular cytokine expression by spleen cells following ex vivo stimulation with the immunodominant CD4+ T cell-specific peptide LLO190. Similar to the results above, there were significantly increased numbers of LLO190-specific IFN-{gamma} -producing (Fig. 9B) and TNF-{alpha}-producing (Fig. 9C) CD4 T cells in the spleens of immunized AhR–/– and AhR+/– mice, as compared with naive mice of the same genotype. We also recovered significantly more L. monocytogenes-specific TNF-{alpha} -producing CD4 T cells from the spleens of immunized AhR–/– than AhR+/– mice (Fig. 9C).


Figure 9
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  		FIGURE 9. Secondary Ag-specific T cell responses in AhR–/– and AhR+/– mice. The numbers of L. monocytogenes-specific CD4 T cells in the spleens of immunized and naive mice were quantified by intracellular cytokine staining for IFN-{gamma} or TNF-{alpha}. Splenocytes were stimulated with the MHC II-restricted listerial epitope peptide LLO190 and the numbers of IFN-{gamma}- or TNF-producing CD4 T cells respectively were determined by flow cytometry. Dot plots in A are gated on total splenocytes and the numbers are the percentage of IFN-{gamma}- or TNF-producing CD4+ T cells among splenocytes. Data in B and C are total numbers of L. monocytogenes-specific IFN-{gamma}+ (B) and TNF-{alpha}+ (C) CD4+ T cells, respectively, from 4 to 5 mice per group. **, p < 0.01 as compared with the corresponding genotype of naive mice.

 
AhR–/– and AhR+/– peritoneal and bone marrow-derived macrophages do not differ in their ability to ingest and restrict the intracellular growth of L. monocytogenes

Mononuclear phagocytes are important in controlling the early stage of L. monocytogenes infection. We compared the ability of AhR+/– and AhR–/– macrophages to ingest and inhibit intracellular listerial growth. Fig. 10A shows that ingestion and intracellular growth of L. monocytogenes were similar for peritoneal exudate macrophages from AhR+/– and AhR–/– mice. Pretreatment with 100 U/ml IFN-{gamma} (24 h) augmented the ability of macrophages of both genotypes to control the intracellular growth of L. monocytogenes; AhR+/– and AhR–/– macrophages did not differ in this regard. We performed similar experiments with bone marrow-derived macrophages. As depicted in Fig. 10B, AhR+/– and AhR–/– bone marrow-derived macrophages activated by pretreatment with IFN-{gamma} did not differ in their ability to ingest or restrict the intracellular growth of L. monocytogenes. Thus, we observed no significant differences between AhR+/– and AhR–/– macrophages with regard to their ability to inhibit intracellular L. monocytogenes.


Figure 10
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  		FIGURE 10. AhR–/– and AhR+/– peritoneal and bone marrow-derived macrophages do not differ in their ability to ingest and restrict the intracellular growth of L. monocytogenes. A, Peritoneal macrophages were infected with 106 log-phase L. monocytogenes for 2 h (ingestion) and then further incubated for 6 h in the presence of 5 µg/ml gentamicin. Macrophages were washed, lysed, and the lysates plated on blood agar to quantify the CFU of L. monocytogenes. Some macrophages were pretreated with IFN-{gamma} (100 U/ml) for 24 h before addition of L. monocytogenes. Results are the mean ± SEM of a representative experiment from four experiments that were performed. *, p < 0.05, in comparison to macrophages without IFN-{gamma} treatment. In B, bone marrow-derived macrophages were pretreated with IFN-{gamma} (100 U/ml, 24 h) and then similarly infected with L. monocytogenes as described in A. Results are the mean ± SEM of one representative experiment.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study presents the first evidence that AhR–/– mice are more susceptible to L. monocytogenes infection than wild type or heterozygous AhR+/–mice, as quantified by the recovery of viable L. monocytogenes from the spleen, liver and other tissues (Fig. 2–5, 7). Although our experiments were designed to evaluate bacterial burden in internal organs, rather than survival, in preliminary experiments we observed that AhR–/– mice did not survive L. monocytogenes infection when the inoculation dose was ≥104 CFU (i.v.) (data not shown). This is a challenge dose at which deaths would not ordinarily occur in AhR+/+ or AhR+/– mice. Moreover, AhR–/– mice exhibited a delayed ability to clear a sublethal infection with L. monocytogenes (Figs. 3 and 4). Besides harboring a greater burden of L. monocytogenes in their tissues, AhR–/– mice also exhibited more extensive histopathological damage in their livers. Granulomas were greater in number and size (Fig. 2G) and necrotic foci were more evident in AhR–/– mice (Figs. 2C and 7C). Hepatocytes in AhR–/– mice exhibited a foamy appearance (Fig. 2E) and Gram-positive L. monocytogenes were readily seen (Fig. 2I). In contrast, relatively few histopathological changes were seen in the livers of L. monocytogenes-infected AhR+/– mice.

The AhR has been shown to play important roles in regulating the expression of several cytokines. For example, exposure of rats to TCDD led to up-regulation of IL-1β and TNF-{alpha} in the liver (12). Interestingly, although TCDD suppressed the production of IFN-{gamma} by mediastinal lymph node cells, there was a 10-fold increase in the IFN-{gamma} level in the lungs of TCDD-treated mice (16). A general suppression of IL-12 was seen in both mediastinal lymph node cells and the lung in the same study (16). IL-6 (13), CCL1 (14) and IL-8 (15) have also been identified as altered by the AhR signaling pathway. Several cytokines interacting with the AhR signaling pathway (12, 16, 45) (e.g., IL-1, TNF-{alpha}, IFN-{gamma}, IL-12) are critically important for innate immunity to listeriosis (46).

Resistance to experimental listeriosis has been shown to be dependent on production of several inflammatory cytokines (i.e., IL-12, TNF-{alpha}, and IFN-{gamma}). Therefore, we asked whether differential production of inflammatory cytokines by AhR+/– and AhR–/– mice in response to listerial infection was associated with the increased susceptibility of AhR–/– mice to listeriosis. Using a cytometry bead assay, we detected no significant difference in serum levels of TNF-{alpha}, IFN-{gamma}, MCP-1, and IL-6 between AhR+/– and AhR–/– mice at 24 h after L. monocytogenes inoculation. Somewhat to our surprise, serum levels of IL-12 and IL-10 were significantly higher in the AhR–/– mice. When interpreting these data, it should be noted that these mice received a higher challenge dose (4.0 x 105 CFU per mouse i.v.) than in the earlier experiments, to elicit a robust cytokine response at 24 h after inoculation. As a result significantly greater numbers of CFU were recovered from the spleens and livers of AhR–/– than AhR+/– mice at 24 h postinoculation. Therefore, the increased production of IL-12 and IL-10 in AhR–/– mice could simply reflect the greater bacterial burden in these mice. IL-12 is critical for the expression of a protective Th1 immune response during L. monocytogenes infection (47). In contrast, IL-10 is generally considered to be anti-inflammatory and an inhibitor of Th1 cellular immunity, as reflected in IL-10–/– mice being more resistant to listeriosis (48, 49). Female mice also produce more IL-10, which correlates with their greater susceptibility to listeriosis than male mice (50). We were surprised to find no difference in serum levels of IFN-{gamma} and TNF-{alpha} between L. monocytogenes-infected AhR+/– and AhR–/– mice. These cytokines are essential to controlling listeriosis, especially in the early phase of infection (51). Consistent with these data, we also did not observe any significant difference in serum TNF-{alpha} levels between AhR+/– and AhR–/– mice at 3 days postinoculation as determined by ELISA (data not shown). Overall, these data indicate that production of inflammatory cytokines is not impaired in AhR–/– mice when infected with L. monocytogenes.

The greater susceptibility of AhR–/– mice to listeriosis, despite comparable cytokine responses and numbers of activated T cells, suggests diminished innate immunity. Because we observed no defect in production of inflammatory cytokines in AhR–/– mice, we speculated that there might be quantitative and/or qualitative differences of the cellular components of innate immunity to listeriosis (for instances, neutrophils, macrophages, and NK cells) between the AhR–/– and AhR+/– mice. However, using peritoneal exudate macrophages and bone marrow-derived macrophages, we observed no differences between AhR+/– and AhR–/– macrophages in their ingestion or antimicrobial activity against L. monocytogenes. Nor did we see quantitative difference in the numbers of peritoneal exudate cells, or relative percentages of macrophages, recovered from AhR+/– and AhR–/– mice (data not shown). Transmission electron microscropy examination revealed comparable fusion of lysosomes and phagosomes, and cytoplasmic escape of intracellular listerial cells in AhR+/– and AhR–/– macrophages (data not shown). Interestingly, the number of bone marrow cells recovered from AhR–/– mice was only ~50% that obtained from age-matched AhR+/– mice (our observations), which might reflect in part the fact that AhR–/– mice are smaller than age-matched AhR+/– mice. Although our experiments provide no direct evidence for the importance of macrophages in the difference in resistance of AhR+/– and AhR–/– mice, we cannot exclude the possibility that interactions of macrophages with other cells or mediators in vivo contributes in some way to the reduced resistance of AhR–/– mice.

Complete elimination of L. monocytogenes is dependent on an adaptive immune response that involves CD8+ and CD4+ T cells (52). Using intracellular cytokine staining, we found equal or greater numbers of IFN-{gamma} - and TNF-{alpha} -producing L. monocytogenes-specific CD4 and CD8 cells in AhR–/– mice as compared with AhR+/– mice at 7 and 14 days after the primary infection. These data indicate that AhR–/– mice develop a normal and protective T cell response to L. monocytogenes infection. AhR–/– mice also exhibited a normal contraction of Ag specific CD4 and CD8 cells between 7 and 14 days after inoculation. These findings are consistent with the clearance of L. monocytogenes from AhR–/– mice by 14 days after inoculation. In agreement with these observations, it was reported that AhR–/– mice mount normal humoral and cellular immune responses to injection of SRBC and P815 tumor cells, as compared with AhR+/+ mice (53).

When AhR–/– and AhR+/– mice were immunized with a sublethal challenge does of L. monocytogenes, they both exhibited acquired resistance to reinfection with L. monocytogenes. As expected, there were significant increases in CD4+CD44high and CD8+CD44high T cells in the immunized mice as compared with naive mice of both genotypes. There was no significant difference in the frequency of CD44high T cells in AhR–/– and AhR+/– mice, thus indicating that AhR is not required for the expansion of memory T cells following L. monocytogenes infection. On the contrary, there were more CD8+CD44high cells in the AhR–/– than AhR+/– mice. This could indicate that more memory CD8 cells were produced in response to the greater listerial burden in AhR–/– mice during primary infection with L. monocytogenes. An alternative possibility is that increased production of IL-10 in L. monocytogenes-infected AhR–/– mice results in a stronger clonal expansion of CD8+ T cells, since IL-10 is required for optimal CD8+ T cell memory following L. monocytogenes infection (54). There were significantly more IFN-{gamma} and TNF-{alpha} producing L. monocytogenes-specific CD4 T cells in immunized than non-immunized AhR–/– and AhR+/– mice. However, there was no significant difference in numbers of L. monocytogenes-specific CD8+IFN-{gamma}+ and CD8+TNF-{alpha}+ cells between immunized and AhR–/– and AhR+/– mice in (data not shown). This observation is consistent with adoptive transfer studies using L. monocytogenes-specific CD4+ and CD8+ T cells, which showed that CD4+ T cell-mediated protective immunity is IFN-{gamma}-dependent, whereas CD8+ T cells can mediate IFN-{gamma} - independent protection (55, 56). Overall, these data indicate no deficiency in the T cell response of AhR–/– mice, suggesting that the AhR is not required for the development of acquired cellular immunity to L. monocytogenes infection.

Although the immunosuppressive effect of TCDD via the AhR signaling pathway is well recognized, we demonstrate here for the first time that constitutive AhR expression is required for optimal resistance to L. monocytogenes infection in mice. A similar beneficial role for the AhR in resistance to Streptococcus pneumoniae infection in mice was recently reported (57). In that study, the authors found that activation of AhR by TCDD protected mice from an otherwise lethal challenge with that extracellular bacterial pathogen. It is curious that, as in the present study, the mechanism responsible for the role of the AhR in resistance to S. pneumoniae infection was not obvious. In that study neither the inflammatory response nor the numbers of neutrophils that accumulated appeared to be responsible for the increased resistance to S. pneumoniae (57). Thus, the AhR appears to play a significant role in host defense against extracellular (S. pneumoniae) and intracellular (L. monocytogenes) bacterial infection by a mechanism as yet to be determined.

In summary, we present here the first evidence that AhR is constitutively required for optimal resistance to murine listeriosis. The AhR was not required for a normal inflammatory cytokine or adaptive T cell response to L. monocytogenes infection, although our data suggest that AhR–/– mice might differ somewhat in the magnitude of the protective adaptive immune response to L. monocytogenes infection. We infer from these results that the AhR may play a novel constitutive role in innate immunity to L. monocytogenes infection in mice, although the underlying mechanisms remain to be elucidated.


   Acknowledgments
 
We thank Drs. C. Bradfield and C. Jefcoate for valuable suggestions and critical reading of this manuscript and Lyndsey Berryman for assistance in establishing bone marrow-derived macrophages.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by funds from the National Public Health Sciences (National Institutes of Health Grant R21AI059656), the United States Department of Agriculture National Research Institute (2005-35201-15313), the National Alliance for Food Safety and Security (58-1935-4450), and the Walter and Martha Renk Endowed Laboratory for Food Safety. Back

2 Address correspondence and reprint requests to Dr. Charles J. Czuprynski, Department of Pathobiological Sciences, 2015 Linden Drive, Madison, WI 53706. E-mail address: czuprync{at}svm.vetmed.wisc.edu Back

3 Abbreviations used in this paper: AhR, aryl hydrocarbon receptor; bHLH-PAS, basic helix-loop-helix Per-Arnt-Sim; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; ARNT, aryl hydrocarbon receptor nuclear transporter; DMBA, 7,12-dimethylbenz[a]anthracene; PAH, polycyclic aromatic hydrocarbon; CBA, cytometric bead assay. Back

Received for publication April 24, 2007. Accepted for publication September 4, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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