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The Journal of Immunology, 2007, 179: 247-255.
Copyright © 2007 by The American Association of Immunologists, Inc.
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Aryl Hydrocarbon Receptor Activation during Influenza Virus Infection Unveils a Novel Pathway of IFN-{gamma} Production by Phagocytic Cells1

Haley Neff-LaFord*, Sabine Teske*, Timothy P. Bushnell{dagger} and B. Paige Lawrence2,*,{ddagger}

* Department of Pharmaceutical Sciences and Pharmacology/Toxicology Graduate Program, College of Pharmacy, Washington State University, Pullman, WA 99164; {dagger} Department of Pediatrics and {ddagger} Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The contribution of environmental factors is important as we consider reasons that underlie differential susceptibility to influenza virus. Aryl hydrocarbon receptor (AhR) activation by the pollutant dioxin during influenza virus infection decreases survival, which correlates with a 4-fold increase in pulmonary IFN-{gamma} levels. We report here that the majority of IFN-{gamma}-producing cells in the lung are neutrophils and macrophages not lymphocytes, and elevated IFN-{gamma} is associated with increased pulmonary inducible NO synthase (iNOS) levels. Moreover, we show that even in the absence of dioxin, infection with influenza virus elicits IFN-{gamma} production by B cells, {gamma}{delta} T cells, CD11c+ cells, macrophages and neutrophils, as well as CD3+ and NK1.1+ cells in the lung. Bone marrow chimeric mice reveal that AhR-mediated events external to hemopoietic cells direct dioxin-enhanced IFN-{gamma} production. We also show that AhR-mediated increases in IFN-{gamma} are dependent upon iNOS, but elevated iNOS in lung epithelial cells is not driven by AhR-dependent signals from bone marrow-derived cells. Thus, the lung contains important targets of AhR regulation, which likely influence a novel iNOS-mediated mechanism that controls IFN-{gamma} production by phagocytic cells. This suggests that AhR activation changes the response of lung parenchymal cells, such that regulatory pathways in the lung are cued to respond inappropriately during infection. These findings also imply that environmental factors may contribute to differential susceptibility to influenza virus and other respiratory pathogens.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The cytokine IFN-{gamma} influences both innate and adaptive immunity and has been long considered an important first line of defense during viral infection. Although some IFN-{gamma} is important for antiviral immunity, excessive IFN-{gamma} production leads to downstream pathology characterized by enhanced inflammation and severe tissue damage in many organ systems, including the lung, liver, CNS and eye (1, 2, 3, 4, 5). Although much is known about both the downstream beneficial and detrimental consequences of IFN-{gamma}, the factors that regulate the production of this cytokine are less understood. For example, until very recently, the production of IFN-{gamma} was thought to be restricted to NK cells and activated CD4+ and CD8+ T cells. However, several additional cellular sources of IFN-{gamma} have been reported, including {gamma}{partial}T cells, NKT cells, macrophages, dendritic cells, neutrophils, and B cells (6, 7, 8, 9, 10, 11, 12, 13, 14). The production of IFN-{gamma} by T cells and NK cells is regulated by many cytokines, in particular IL-2, IL-12, IL-18, and IFN-{alpha}beta (15, 16, 17, 18). In addition to cytokines, IFN-{gamma} expression in T cells is controlled by multiple transcriptional regulators, including NF-{kappa}B, NFAT, and ATF2 (19, 20, 21). In contrast to IFN-{gamma} derived from T cells and NK cells, very little is known about the regulation of IFN-{gamma} production by other immune cell types.

We have discovered that during influenza virus infection activation of the aryl hydrocarbon receptor (AhR)3 by the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) results in 4-fold higher IFN-{gamma} levels in the lung and prolongs the duration of this elevated IFN-{gamma} by ~48 h. Thus, maximal IFN-{gamma} levels occur 7 instead of 5 days after infection (22). In addition to these effects on pulmonary IFN-{gamma} levels, exposure to TCDD impairs survival following influenza virus infection (22, 23, 24). Interestingly, decreased survival does not appear to be caused by impaired viral clearance, but by elevated pulmonary inflammation (22, 23, 24, 25). These observations are of interest because the AhR is a ligand-activated transcription factor expressed in cells of the immune system and the lung (26, 27). The AhR is generally considered an orphan receptor because it has been difficult to identify an endogenous ligand. However, in addition to TCDD, numerous other environmental contaminants activate the AhR. For instance, coplanar polychlorinated biphenyls and polyaromatic hydrocarbons such as benzo[a]pyrene and 7,12-dimethylbenzanthracene, which are found in cigarette smoke and diesel exhaust, are AhR ligands (28, 29). Also, many plant-derived natural compounds and tryptophan metabolites bind the AhR (28, 29). Consequently, human exposure to a variety of AhR ligands occurs daily through ingestion and inhalation.

It is not clear why some individuals infected with influenza virus have significant pathology, whereas others present relatively mild symptoms. It has been suggested that environmental factors contribute to differential outcomes among infected populations; however, how differences in host environment affect the pathology associated with respiratory diseases remains unclear. Our data suggest that via activation of the AhR, environmental factors may influence disease severity by altering host resistance mechanisms. Epidemiological evidence supports this idea, since exposure to pollutants containing AhR agonists correlates with diminished host resistance, altered immune function, and an increased incidence of influenza and other respiratory infections (30, 31, 32, 33, 34). In the present study, we sought to improve our understanding of the mechanistic relationship between exposure to AhR agonists and altered immune responsiveness to influenza virus infection. To do this, we characterized the cellular sources of IFN-{gamma} in the lung and examined possible mechanisms that could drive AhR-mediated enhancement of IFN-{gamma} levels. While conducting these studies, we have identified a novel cellular source of IFN-{gamma} during influenza virus infection and propose an additional mechanism for the regulation of IFN-{gamma} production in the infected lung.


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

Four- to 6-wk-old female C57BL/6 (CD45.2+ phenotype), B6-LY5.2/Cr-congenic mice (CD45.1+ phenotype), and iNOS-deficient (B6.129P2-Nos2tm1Lau/J) mice were purchased from The Jackson Laboratory or the National Cancer Institute-Frederick (Frederick, MD). AhR-deficient (B6.129-Ahrtm1Bra/J) mice are maintained at Washington State University and bred as previously described (23). Animals were housed three to five per cage under pathogen-free conditions on a 12/12-h light cycle with food and water available ad libitum. All treatment was in accordance with protocols approved by the Institutional Animal Care and Use Committee. TCDD (≥99% purity; Cambridge Isotope Laboratories) was dissolved in anisole and diluted in peanut oil. Mice were gavaged with TCDD (10 µg/kg body weight) or peanut oil vehicle 1 day before infection. Oral exposure was selected because the major route of human exposure to dioxin-like compounds is via the food chain (35). Animals were anesthetized with Avertin (2,2,2-tribromoethanol) before intranasal infection with 120 hemagglutinating units of influenza virus, strain A/HKx31 (H3N2; x31), diluted in 25 µl of sterile PBS. In vehicle-treated mice, this dose of virus does not typically cause mortality (22, 24). Mock-infected mice received 25 µl of sterile endotoxin-tested PBS and were included as nonimmune controls for the infection. Animals were sacrificed at various times relative to infection by asphyxiation with CO2.

Collection of immune cells

Cells in the airways were collected by bronchoalveolar lavage (BAL) as previously described (22). Medium from the first wash was retained as BAL fluid. Cells recovered from all washes were pooled and enumerated. Total lung-derived immune cells were obtained by digesting intact lungs (i.e., unlavaged) with collagenase (RPMI 1640 medium containing 0.7 mg/ml collagenase A (Worthington Biochemical), 30 µg/ml DNase I (Sigma-Aldrich), 2.5% FBS, and 10 mM HEPES). Immune cells from interstitial spaces of the lung were obtained by removing airway cells by lavage and then digesting lungs with collagenase-containing medium. After 25 min of incubation at 37°C in 5% CO2, lung cell suspensions were layered over Lympholyte-M (Cedarlane Laboratories) and centrifuged at room temperature for 20 min at 1000 x g. The leukocyte-containing fraction was collected and washed. More than 99% of the cells in this fraction were CD45+ (our unpublished observations). Gr1+ cells were enriched from collagenase-digested whole lung using a MACS column (Miltenyi Biotec). Briefly, following centrifugation through Lympholyte M, CD45+ cells from the lung were labeled with a PE-conjugated anti-Gr1 Ab (eBioscience) and incubated with anti-PE-coated microbeads (Miltenyi Biotec). Gr1+ cells were collected following retention and elution from the MACS column. The purity of the Gr1+ fraction was ~65%.

Immunophenotypic analyses and imaging cytometry

Cells were incubated with the following fluorescent-conjugated mAbs directed against: NK1.1 (FITC or PE), CD3 (PE), CD8{alpha} (FITC or tri-color), F4/80 (FITC or tri-color), CD11c (FITC), TCR (FITC), CD11b (FITC or PE-Cy5), CD4 (FITC), and Gr1 (PE-Cy5) (Invitrogen Life Technologies, eBioscience, or BD Biosciences). Appropriately labeled, isotype-matched mAbs were used to determine nonspecific fluorescence. For all experiments, listmode data were collected on 10,000–50,000 stained cells using a FACScan or FACSort flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star). To identify IFN-{gamma}-producing cells, cells were incubated in 24-well plates at 37°C in 5% CO2 for 5 h in the presence of 12.5 U/ml recombinant mouse IL-2, 1 µM influenza virus nucleoprotein peptide (NP366–374 ASNENMETM), and 10 µg/ml brefeldin A (12, 14). Where noted, IL-2 and NP366–372 were omitted from the in vitro restimulation medium for specific experiments. Cells were stained with mAbs specific for cell surface molecules, fixed with 2% formalin, permeabilized with 1% saponin, and incubated with an allophycocyanin-labeled anti-IFN-{gamma} mAb (BD Biosciences or eBioscience). Incubation of permeabilized cells with excess unlabeled anti-IFN-{gamma} mAb before the addition of allophycocyanin-labeled anti-IFN-{gamma} substantially reduced the percentage of IFN-{gamma}+ cells detected.

For imaging cytometry, nuclei of Ab-labeled cells were counterstained with 50 µg/ml 7-aminoactinomycin D (Invitrogen Life Technologies), and images were acquired on the ImageStream imaging flow cytometer (Amnis) using the manufacturer’s acquisition software. Spectral compensation was performed as previously described (36, 37) and data analysis was performed using the ImageStream Data Exploration and Analysis Software (IDEAS; Amnis). Briefly, debris and cell aggregates were excluded and individual cells were identified by gating on nuclear intensity and aspect ratio (ratio of length to width, a measure of circularity). Individual cell populations were identified by gating on cells expressing surface makers and confirmed by visual inspection of the pattern of fluorescence of the images.

ELISA and ELISPOT

IFN-{gamma} levels in BAL fluid were measured using an IFN-{gamma}-specific sandwich ELISA according to the manufacturer’s recommended protocol (BD Biosciences). The lower limit of detection for this assay is 250 pg/ml. The number of IFN-{gamma}-producing cells was determined by ELISPOT analysis. Sterile nitrocellulose-bottomed multiscreen-IP 96-well plates (Millipore) were coated overnight at 4°C with an anti-IFN-{gamma} mAb (clone AN-18; Mabtech). In some experiments, cells from five to six mice per group were pooled and used in the ELISPOT assay before or following immunomagnetic depletion of NK1.1+ and CD8+ cells (Invitrogen Life Technologies). Depletion efficiency was 77% (CD8+ cells) and 60% (NK1.1+ cells). Three-fold dilutions of cells were added in RPMI 1640 with 2.5% FBS. Cells were incubated for 20 h at 37°C in 5% CO2 in the presence of 12.5 U/ml recombinant mouse IL-2 and 1 µM viral peptide. Plates were washed and incubated with a biotinylated anti-IFN-{gamma} Ab (clone R4-6A2; Mabtech) followed by avidin peroxidase. IFN-{gamma}-producing cells were visualized using 3-amino-9-ethylcarbazole (Sigma-Aldrich). Spots were analyzed in blinded samples by ZellNet Consulting using a high-resolution automated ELISPOT reader system from Zeiss with KS ELISPOT software, version 4.3.

Generation of bone marrow chimeric mice

Four-week-old female C57BL/6 mice (CD45.2+ phenotype) and B6-Ly5.2/Cr-congenic mice (CD45.1+ phenotype) received sterile-filtered, acidified water (pH 3.0) supplemented with 1 mg/ml oxytetracycline HCl (terramycin) and were fed irradiated food beginning 1 wk before irradiation. Mice were maintained on this regimen throughout the study. B6-CD45.1+-congenic mice were irradiated with two separate doses of 600 rad with 3.5 h between doses (Philips SL-15 linear accelerator; Radiology Department, Washington State University College of Veterinary Medicine; Ref. 38). One hour after the second radiation treatment, 1.5 x 106 bone marrow cells from either B6-CD45.2+AhR+/+ or B6-CD45.2 AhR–/– donor mice were i.v. injected into irradiated B6-CD45.1+AhR+/+ recipient mice. Irradiated B6-CD45.1+AhR+/+ mice that did not receive donor bone marrow cells served as controls for the irradiation and died within 2 wk of radiation exposure.

Four weeks after irradiation and bone marrow reconstitution, chimerism was confirmed via flow cytometry, which showed that >95% of the bone marrow cells were CD45.2+ (i.e., donor derived), regardless of the AhR status of the donor (data not shown). Importantly, bone marrow chimeric mice were immunocompetent regardless of the AhR status of the immune system, because both AhR–/–->AhR+/+ and AhR+/+->AhR+/+ mice survived challenge with influenza virus (120 hemagglutinin units, x31; data not shown). To determine whether AhR-mediated events within bone marrow-derived cells drive IFN-{gamma} production directly, chimeric mice were treated with vehicle or TCDD (10 µg/kg) 1 day before infection with x31 as described above.

Immunoblotting and immunohistochemistry

Whole lungs from individual animals were thawed in cold homogenization buffer (10 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.6% Nonidet P-40, 10 µg/ml aprotinin and leupeptin, and 20 µg/ml PMSF) and homogenized using a Tissue Tearor (Biospec Products). Protein concentrations were determined using a bicinchoninic acid protein assay (Pierce Endogen). Homogenates (50 µg) were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and blocked overnight at 4°C with 5% nonfat dry milk in TBS. Membranes were probed with a rabbit polyclonal anti-iNOS IgG at a 1/500 dilution (Cayman Chemical) followed by a HRP-conjugated goat anti-rabbit Ab (Sigma-Aldrich, 1/20,000 dilution). Ab complexes were visualized using chemiluminescent reagents (SuperSignal West Dura Extended Duration Substrate; Pierce Endogen). Lungs were perfused with 10% formalin, embedded in paraffin, sliced, and mounted (Washington Diseases and Diagnostic Laboratory; Washington State University). Tissue slices were deparaffinized using xylenes and rehydrated before incubation in PBS, with 1% saponin. Slides were incubated with 3% H2O2 for 5 min and nonspecific binding was blocked using 3% normal goat serum. Sections were exposed to a polyclonal rabbit anti-murine iNOS IgG (1/50; Upstate Cell Signaling Solutions) overnight at 4°C followed by a HRP-conjugated goat anti-rabbit IgG F(ab')2 (1/100, Jackson ImmunoResearch Laboratories). Cells expressing iNOS were identified using either 3-amino-9-ethylcarbazole or 3,3'-diaminobenzidine substrate and were counterstained with hematoxylin (Vector Laboratories).

AhR response element (AhRE) identification

Putative AhRE, defined as the 5-mer core sequence 5'-GCGTG-3' were identified using a manual search of the annotated gene sequence, Genomatix software (www.genomatix.de) and Vector NTI (Invitrogen Life Technologies). Results obtained from all three methods were comparable. Sequences and transcription start sites (TSS) were defined using ENSEMBL (www.ensembl.org). The search for putative AhRE encompassed sequences from –3000 to +3000, relative to the TSS.

Statistics

Statistical analyses were performed with StatView statistical software (SAS) or Prism (GraphPad software). Differences between independent variables were compared over time and between each treatment group using one-way ANOVA, followed by post hoc Fisher’s protected least-significant difference tests. Data collected at a single point in time were analyzed using a one-tailed Student’s t test. A value of p ≤ 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
AhR activation increases the percentage of IFN-{gamma}-producing cells in the infected lung, the majority of which are Gr1+CD11b+

Exposure to TCDD significantly elevates infection-associated production of IFN-{gamma} in the lung, with the peak levels occurring 7 days after infection (22, 24). However, the mechanism responsible for this increase in IFN-{gamma} in the lung is unclear. It is unlikely that an elevation in systemic IFN-{gamma} levels is responsible for exacerbated pulmonary levels because no IFN-{gamma} is detected in the blood (data not shown), and IFN-{gamma} levels in mediastinal lymph nodes are decreased following exposure to TCDD (22, 39). Instead, we found that exposure to TCDD raises pulmonary IFN-{gamma} levels via an AhR-mediated increase in the frequency of IFN-{gamma}+ cells in the lung (Figs. 1, A and B). This increase is triggered by infection, because there is no IFN-{gamma} in lavage fluid nor are there IFN-{gamma}+ cells in lungs from naive or mock-infected mice, regardless of treatment with TCDD (data not shown). To identify the cellular sources of this excess IFN-{gamma} during infection, we first examined whether AhR activation increases IFN-{gamma} production by T cells (CD3+) and NK cells (NK1.1+). In Fig. 1C, we show that although both of these cell types produce IFN-{gamma} 7 days after infection, neither CD3+ cells nor NK1.1+ cells account for the majority of IFN-{gamma}-producing cells in the lung. Interestingly, this was observed in lung-derived immune cells from both vehicle- and TCDD-treated, influenza virus-infected mice. Moreover, when we enumerated IFN-{gamma}-producing cells by ELISPOT, immune cells isolated from the lungs of vehicle- and TCDD-treated, infected mice produced IFN-{gamma} even when both CD8+ T cells and NK1.1+ cells had been depleted (Fig. 1D). Taken together, these findings suggest that another cell type is responsible for the majority of the IFN-{gamma} produced in the lungs during infection with influenza virus.


Figure 1
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  		FIGURE 1. AhR activation increases the percentage of IFN-{gamma}-producing cells in the lungs of infected mice, but the majority is neither CD3+ nor NK1.1+. C57BL/6 (AhR+/+) or AhR–/– mice were given peanut oil vehicle or TCDD (10 µg/kg) orally 1 day before intranasal infection with influenza virus strain A/HKx31 (x31). A, Seven days after infection, IFN-{gamma} levels in BAL fluid of AhR+/+ and AhR–/– mice were compared. B, Representative dot plots show the percentage of IFN-{gamma}+ cells in lungs of vehicle- and TCDD-treated AhR+/+ mice 7 days after infection. Numbers in the upper right corner indicate the average percentage (±SEM) of IFN-{gamma}+ cells in each treatment group. C, The average percentage of IFN-{gamma}+ cells that express CD3 or NK1.1 are shown. The gray portion of the bar indicates the average number of IFN-{gamma}+ cells that were neither CD3+ nor NK1.1+. D, The average number of IFN-{gamma}-producing cells in an ELISPOT assay before and after CD8+ and NK1.1+ cell depletion is shown. All error bars, SEM. *, A significant difference from vehicle-treated mice (p ≤ 0.05). These data are representative of at least three separate experiments (four to six mice per treatment group per day).

 
We therefore sought to further characterize the phenotype of IFN-{gamma}+ cells in lungs of mice infected with influenza virus. We found that a small proportion of the IFN-{gamma}+ cells were CD3+, NK1.1+, CD19+, {gamma}{delta}TCR+, CD11b+ or CD11c+, whereas the majority of IFN-{gamma}+ cells were Gr1+ (Fig. 2A). Because Gr1+ cells have not been previously reported to produce IFN-{gamma} during influenza virus infection, we characterized these cells further using several different approaches. First, we found that the majority of the Gr1+ IFN-{gamma}-producing cells in lungs are CD11b+, but are generally not CD3+, NK1.1+, CD11c+, or {gamma}{delta}TCR+ (Fig. 2B), and are CD8– (Fig. 3A). Additionally, these IFN-{gamma}-producing Gr1+ and CD11b+ cells are present in the airways and interstitium of lungs from vehicle- and TCDD-treated infected mice (Fig. 2C), and AhR activation greatly elevates IFN-{gamma} production by Gr1+ cells, regardless of CD11b status. There is also a small population of IFN-{gamma}+CD11b+Gr1– cells, which in separate experiments were identified and enumerated using F4/80, a mAb that binds macrophages (see Fig. 4). Given that phagocytic cells often express very high levels of FcR, we also determined whether the IFN-{gamma} staining was intracellular or was due to nonspecific binding of anti-IFN-{gamma} Abs to the cell surface. Using imaging cytometry, it is clear that the IFN-{gamma} staining of both Gr1+ and CD8+ cells is intracellular (Fig. 3A). Furthermore, when enriched using immunomagnetic beads and examined using light microscopy, the Gr1+ cells have the characteristic appearance of neutrophils (Fig. 3B). Finally, Gr1+ cells continue to produce IFN-{gamma} in vitro even in the absence of exogenous viral peptide and IL-2, which is in direct contrast to CD8+ T cells (Fig. 3C). Collectively, these findings suggest that the majority of IFN-{gamma} present in the lung 7 days after infection is produced by nonlymphocyte sources and that AhR activation during infection further elevates IFN-{gamma} production by neutrophils and macrophages.


Figure 2
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  		FIGURE 2. The majority of the IFN-{gamma}-producing cells in the lungs of TCDD-treated mice are Gr1+ and CD11b+. C57BL/6 mice (four to six per group) were treated as described in Fig. 1. A, The phenotype of IFN-{gamma}+ cells from vehicle ({square})- and TCDD-treated mice ({blacksquare}) was determined 7 days after infection using flow cytometry, and the percentage of lung cells that expressed IFN-{gamma} and the indicated cell surface markers are shown. B, Representative dot plots depict the percentage of IFN-{gamma}+ Gr1+ cells that coexpress CD11b, CD3, NK1.1, CD11c and {gamma}{delta}TCR in the lungs of vehicle-(top row) and TCDD-treated (bottom row) mice. C, Graphs show the average number and phenotype of IFN-{gamma}+ cells isolated from the lung airways or interstitial spaces of vehicle ({square})- and TCDD-treated ({blacksquare}) mice. Note: these graphs depict the phenotype of plastic-adherent cells, thus lymphocyte populations are not included in these graphs. *, A statistically significant difference compared with the vehicle-treated controls (p ≤ 0.05). Error bars, SEM. Experiments were repeated three times with comparable results.

 

Figure 3
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  		FIGURE 3. Gr1+ cells make IFN-{gamma} and do not require restimulation with exogenous IL-2 or viral peptide. A, Representative imagery of IFN-{gamma}+Gr1+ and IFN-{gamma}+CD8+ cells shows IFN-{gamma} staining is localized within the cell. Note that the pixel value intensity range (the gain) for the IFN-{gamma} stain in the CD8+ cells is more restricted (increased gain) when compared with the IFN-{gamma} staining in the Gr1+ cells. This is a result of the lower overall staining intensity of IFN-{gamma} in the CD8+ cells. B, H&E staining shows that the majority of the Gr1+ cells are morphologically neutrophils (arrows; original magnification, x100). C, Representative dot plots depict the frequency of IFN-{gamma}+CD8+ cells and IFN-{gamma}+Gr1+ cells when in vitro restimulation conditions do and do not include exogenous IL-2 and a peptide fragment of influenza virus NP (aa 366–372). The numbers in the upper right quadrant on each dot plot indicate the average percentage (±SEM) of IFN-{gamma}+ cells of each phenotype in each treatment group. *, A significant difference compared with vehicle-treated mice (n = 4/group p ≤ 0.05).

 

Figure 4
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  		FIGURE 4. An AhR-mediated signal from the lung drives aberrant IFN-{gamma} production by macrophages and neutrophils. Bone marrow chimeric mice were produced by reconstituting irradiated B6.CD45.1+ (AhR+/+)-congenic mice with bone marrow cells derived from either CD45.2+AhR+/+ (AhR+/+->AhR+/+) or CD45.2+AhR–/– (AhR–/–->AhR+/+) mice. Four weeks after bone marrow cell transfer, the chimeric mice were treated with either vehicle or TCDD (10 µg/kg) 1 day before infection with x31. Mice were sacrificed 7 days after infection. A, Bars represent the average levels of IFN-{gamma} in BAL fluid as determined by ELISA. B, The number of IFN-{gamma}+CD45.2+ (i.e., donor-derived) cells in the lung was determined by flow cytometry. C, The phenotype of IFN-{gamma}+ cells in the lungs of chimeric mice was examined by flow cytometry, which shows that >95% of the IFN-{gamma}+ cells were from donor mice (i.e., CD45.2+). Further analysis of the CD45.2+IFN-{gamma}+ cells shows that TCDD treatment increases the percentage of IFN-{gamma}+ phagocytic cells even when donor-derived cells are AhR-null. These results are representative of three separate experiments (five to six animals per treatment group were used for each of these studies). Error bars, SEM; *, A statistically significant difference (p ≤ 0.05) compared with vehicle-treated mice of the same chimeric group.

 
Neither known IFN-{gamma}-stimulating cytokines nor direct AhR:AhRE binding in the IFN-{gamma} gene likely account for increased IFN-{gamma} production by phagocytic cells

When activated, ligand-bound AhR binds to AhRE found in the upstream regulatory regions of Ah-responsive genes. To evaluate whether enhanced IFN-{gamma} levels in lungs of TCDD-treated mice results from direct AhR:AhRE binding, we searched for AhRE binding sites 3000 bp upstream and downstream of the IFN-{gamma} gene transcriptional start site using three different approaches (see Materials and Methods for additional details). None of these search strategies identified a putative AhRE in the IFN-{gamma} gene. Additionally, we searched for the newly described AhRE II binding site (CATG(N6)CTATG) (40). As with the AhRE, we did not identify putative AhRE II binding sites in the IFN-{gamma} gene. Thus, it is unlikely that direct AhR-AhRE interaction in the promoter region of the IFN-{gamma} gene is responsible for its elevated production.

Potential inducers of IFN-{gamma} gene expression include cytokines. For instance, many studies have demonstrated that IFN-{gamma} production by lymphocytes is stimulated by IL-12, IL-18, IFN{alpha}beta, and IL-2 (15, 16, 17, 18). We have previously shown that AhR activation by TCDD suppresses pulmonary IL-12 levels (22, 24) and does not alter IFN-{alpha}beta, TNF, IL-1, or IL-18 levels in the lung during infection (25). We also observe a significant diminution in IL-2 levels in TCDD-treated, infected animals (22). Collectively, these findings suggest that cytokines known to stimulate IFN-{gamma} production by T cells and NK cells probably do not drive IFN-{gamma} overproduction by phagocytic cells in the lungs of TCDD-treated, influenza virus-infected mice.

An AhR-mediated signal extrinsic to hemopoietic cells increases IFN-{gamma} production by phagocytic cells

Although the excess IFN-{gamma} in the lungs of TCDD-treated mice is dependent on AhR activation (Fig. 1A), we have been unable to link the elevated IFN-{gamma} levels to an increase in IFN-{gamma}-inducing cytokines or direct AhR:AhRE binding. Thus, these data suggest that the overproduction of IFN-{gamma} by macrophages and neutrophils may not occur through an AhR-mediated event within the phagocytic cells themselves. Using chimeric mice, we tested whether AhR activation within bone marrow-derived cells is directly responsible for elevated IFN-{gamma} production by macrophages and neutrophils. To do so, lethally irradiated congenic mice (B6-CD45.1+AhR+/+) received donor bone marrow cells isolated from B6-CD45.2+AhR–/– AhR-deficient mice or B6-CD45.2+AhR+/+ wild-type mice. As expected, TCDD-treated wild-type chimeric (AhR+/+->AhR+/+) mice had at least 6-fold higher levels of IFN-{gamma} in their lungs compared with vehicle-treated wild-type chimeric controls (Fig. 4A). Interestingly, TCDD-treated mice that received AhR-deficient immune cells (AhR–/–->AhR+/+) also had increased IFN-{gamma} levels relative to vehicle-treated infected mice, and which were similar to the levels observed in TCDD-treated AhR+/+->AhR+/+ chimeric mice (Fig. 4A). The number of IFN-{gamma}-producing cells was also elevated in the lungs of the TCDD-treated AhR–/–->AhR+/+ mice (Fig. 4B) to levels similar to those in the TCDD-exposed AhR+/+->AhR+/+ controls. Importantly, we show that even in the AhR–/–->AhR+/+ mice, >95% of the IFN-{gamma}-producing cells in the lung are CD45.2+ (i.e., donor-derived, AhR-null). Most of the IFN-{gamma}+ cells express Gr1 or F4/80 cell surface Ags (Fig. 4C). Taken together, these data suggest that activation of the AhR in cells extrinsic to bone marrow-derived cells drives the excess IFN-{gamma} production by phagocytic cells in the lungs of virus-infected mice.

Elevated iNOS levels may drive the excessive IFN-{gamma} production in the lungs of TCDD-treated mice

In addition to elevating pulmonary IFN-{gamma} levels during influenza virus infection, AhR activation augments iNOS levels in the infected lung (Fig. 5A). This AhR-mediated increase in iNOS in infected mice is apparent 5 days after infection, peaks on day 7, and stems from the overinduction of iNOS in lung epithelial cells and alveolar macrophages (Fig. 5B). Although IFN-{gamma} is a well-characterized stimulator of iNOS, some studies have suggested that iNOS can induce IFN-{gamma} production (41, 42, 43), although the molecular mechanism for iNOS-regulated IFN-{gamma} expression is not yet known. In this study, we used two different experimental approaches to determine whether an AhR-mediated increase in iNOS expression could drive elevated IFN-{gamma} production by Gr1+ cells.


Figure 5
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  		FIGURE 5. Activation of the AhR increases iNOS levels in the lung during influenza virus infection. C57BL/6 AhR+/+ or AhR–/– mice were treated as described in Fig. 1 and sacrificed on the indicated days after infection. A, iNOS and beta-actin levels in lung homogenates were examined by Western blotting. The top row of each group depicts relative iNOS (130-kDa) levels in the lungs of two vehicle (V)- and TCDD (T)-treated mice at each point in time, and are representative of six mice in each treatment group (two to three for AhR–/–). Lung homogenates from mock-infected mice were included as controls (d0). B, iNOS in the lungs of vehicle- and TCDD-treated mice was examined 0 (a and b) and 7 days (c and d) relative to infection. Photomicrographs are representative of six samples per treatment group. Epithelial cells are denoted with closed arrows, alveolar macrophages with open arrows. Micrographs in a and b are at x40, whereas c and d are x100 magnification.

 
As shown in Fig. 5A, similar to exacerbated IFN-{gamma} production, enhanced iNOS levels in lungs of TCDD-treated infected mice are AhR dependent. Using AhR–/–->AhR+/+ chimeric mice, we found that exposure to TCDD increased iNOS in lung epithelial cells regardless of the AhR status of hemopoietic cells (Fig. 6). Interestingly, exposure to TCDD elevated iNOS even in macrophages that lacked AhR expression (Fig. 6, F and H). These findings suggest that signals from AhR-containing tissues (e.g., the lung) drive not only excessive IFN-{gamma} production by phagocytic cells but also up-regulate iNOS levels in macrophages.


Figure 6
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  		FIGURE 6. Activation of the AhR in the lung, but not in the immune system, induces iNOS expression by epithelial cells and alveolar macrophages. Chimeric mice were generated and treated as described in Fig. 4. Seven days after infection, mice were sacrificed and iNOS expression in the lungs of vehicle- and TCDD-treated mice was examined. Alveolar macrophages are depicted with arrows. Photographs are representative of at least five fields of view from three to five samples per treatment group (A, C, E, and G are at x40 and B, D, F, and H are at x100 magnification).

 
To further examine a possible regulatory relationship between iNOS and IFN-{gamma} during infection and AhR activation, we measured IFN-{gamma} levels in lung lavage fluid of iNOS-deficient mice. In contrast to iNOS+/+ mice treated with TCDD, infected iNOS–/– mice did not demonstrate elevated pulmonary IFN-{gamma} levels upon infection with influenza virus (Fig. 7). This finding suggests that an iNOS-dependent pathway drives the production of excess IFN-{gamma} in the lungs of TCDD-treated mice. However, because IFN-{gamma} levels in lung lavage fluid were similar between iNOS+/+ and iNOS–/– mice in the absence of AhR activation, these data also suggest that infection-induced IFN-{gamma} production in the lung is not solely regulated by iNOS. Thus, it is likely that additional mechanisms influence IFN-{gamma} production by phagocytic cells during influenza virus infection.


Figure 7
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  		FIGURE 7. Pulmonary IFN-{gamma} levels are not elevated in TCDD-treated iNOS-deficient infected mice. C57BL/6 (iNOS+/+) and iNOS–/– mice were treated as described in Fig. 1. Mice were sacrificed 7 days after infection and lung lavage fluid was collected. IFN-{gamma} levels in lung lavage fluid from wild-type and iNOS–/– mice were determined by ELISA. Error bars, SEM. Data are representative of two separate experiments (three to six animals per treatment group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The same immune mechanisms that protect the host from invading microorganisms are capable of causing tissue injury and disease, especially when regulatory pathways are inappropriately up- or down-regulated. While studying how exposure to the pollutant dioxin disrupts the immune response to influenza virus, we discovered that infected lungs of TCDD-treated mice have considerably more IFN-{gamma} than lungs from infected mice in the control treatment group. Furthermore, TCDD-treated mice die from an otherwise sublethal infection (22, 24). However, the relationship between dioxin exposure and deregulated IFN-{gamma} production during respiratory viral infection has not been well defined. Thus, the present studies were conducted to address this, and the findings reported here provide several new pieces of information. First, we show here that influenza virus infection alone stimulates IFN-{gamma} production by macrophages and neutrophils in the lung. Second, we show that via the AhR, exposure to TCDD enhances the number of these cells producing IFN-{gamma}. Finally, we found that an AhR-mediated signal external to bone marrow-derived cells drives the excess production of IFN-{gamma}, and this signal is likely downstream of an AhR-dependent increase in iNOS.

During the process of characterizing the cellular source of the excess IFN-{gamma}, we found that many cell types in the infected lung appear to produce IFN-{gamma}, including T cells, NK cells, {gamma}{partial}T cells, B cells, and CD11c+ cells. Interestingly, the majority of the pulmonary IFN-{gamma}+ cells were neutrophils and alveolar macrophages, which are nontypical sources. Although the production of IFN-{gamma} by B cells and neutrophils has been reported previously (12, 13, 18), to our knowledge, this is the first study documenting IFN-{gamma} production by these cell types in vivo in the context of influenza virus infection. Furthermore, the findings of this study are interesting because we show that the majority of the IFN-{gamma}+ cells in the lung at this time point during infection are not lymphocytes.

The mechanisms that drive IFN-{gamma} production by phagocytic cells have not been well characterized. In the context of influenza virus infection, the production of excess IFN-{gamma} by macrophages and neutrophils in the lung does not appear to be driven by IL-2, IL-18, or IFN-{alpha}beta (25, 44). Likewise, although IL-12 levels in lung lavage fluid rise steadily during influenza virus infection (22, 45); IL-12 may not play an essential role in the regulation of pulmonary IFN-{gamma} production during this immune response (46). The observation that there are IFN-{gamma}+ phagocytic cells in the infected vehicle control group, even in the absence of detectable IL-2, IL-18, TNF, or IFN-{alpha}beta suggests that factors in addition to these cytokines probably regulate IFN-{gamma} expression by phagocytic cells during influenza virus infection. By examining how AhR activation stimulates neutrophils and macrophages to further increase their production of IFN-{gamma} during infection, we have gained insight into a potential mechanism for regulating IFN-{gamma} in these cells. The changes in IFN-{gamma} observed in TCDD-treated mice were not observed in lungs of naive or mock-infected mice treated with TCDD, nor were they observed in AhR-null mice treated with TCDD. Thus, our findings suggest that the regulatory pathways involved in the host’s response to viral infection are cued to respond inappropriately when the AhR is activated. Importantly, our data also indicate that activation of the AhR targets selected immunoregulatory pathways during infection, because although the levels of some mediators were disrupted, the infection-associated changes in many other regulatory molecules were not affected by TCDD (23, 25). Finally, our investigation of the underlying mechanism suggests that activation of the AhR during respiratory viral infection has direct effects on lung epithelial cells, not phagocytic cells. This idea is consistent with the results of several other studies demonstrating that TCDD directly affects various types of epithelial cells, including lung airway epithelial cells (47, 48, 49). Although AhR expression in specific subpopulations within the lung are only now being evaluated, rodent and human lungs contain the AhR, and exposure to AhR agonists induces metabolic enzymes in these tissues, which are well-characterized AhR-regulated genes (Refs. 50, 51, 52 , and our unpublished observations).

We present here the idea that AhR-mediated elevation of IFN-{gamma} levels occurs via an AhR-dependent increase in iNOS in lung tissue. In contrast to the ifng gene, we have identified four putative AhRE the inos enhancer/promoter, at the following sites relative to the TSS: –2133–2129, –415–411, +319–323, and +859–863. In addition to possible direct AhR-AhRE interaction, the AhR has been reported to interact with the p65 subunit of NF-{kappa}B (53, 54, 55), which is an important regulator of iNOS expression (56, 57). The AhR also associates with other regulatory factors that may influence iNOS expression and the lung’s response to infection. For instance, there is an AhRE in the C/EBPbeta promoter, and exposure to TCDD affects C/EBPbeta transcription and binding to its response element (58, 59). Therefore, either singly or in combination with other transcriptional regulators, AhR activation may influence iNOS levels in the lung.

Although IFN-{gamma}-mediated induction of iNOS has been well characterized, there is little known about the mechanisms by which pathways downstream of iNOS specifically regulate IFN-{gamma} production. However, it has been shown that iNOS plays a role in the production of IFN-{gamma} by NK cells (41, 42). Thus, it is likely that factors downstream of elevated iNOS, such as reactive nitrogen species and nitrosylated amino acids, may influence IFN-{gamma} expression, and that aberrant iNOS induction can have pathological consequences in many systems, including influenza virus-induced pneumonia (60).

The reasons why some individuals have relatively mild symptoms following influenza virus infection, but others have significant pathology is unclear. Our results suggest that activation of the AhR by pollutants deregulates IFN-{gamma} production during viral infection via a novel iNOS-mediated pathway, which may provide a mechanism for differences in the severity of infection. Additionally, other studies have shown that overproduction of IFN-{gamma} leads to hyperinflammation in the lungs of both humans and experimental animals with chronic obstructive pulmonary disease (61, 62, 63), and that elevated levels of IFN-{gamma} and iNOS correlate with an increased severity of disease-associated pathology in mice with acute respiratory distress syndrome. Furthermore, both respiratory viral infections (including influenza virus) and AhR activation have been separately implicated in the etiology and increased severity of these diseases (64, 65, 66). Thus, our results indicate that inappropriate AhR activation during infection may influence the development or severity of these complex inflammatory disorders of the lung.


   Acknowledgments
 
We thank Dr. Allen Silverstone (State University of New York Upstate Medical University, Syracuse, NY) and Dr. David Shepherd (University of Montana, Missoula, MT) for their helpful advice with the bone marrow chimeric mice and Dr. Janean Fidel and Robert Houston for irradiation (Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University). We are also grateful to Mark Bauter, Jennifer Cundiff, and Kevin Kipp for technical assistance with these experiments and to Drs. Beth A. Vorderstrasse (Washington State University) and David L. Woodland (Trudeau Institute) for their thoughtful discussion and comments on this manuscript.


   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 These studies were supported by grants from the National Institutes of Environmental Health Sciences (R01ES10619 and K02ES012409, to B.P.L.). Back

2 Address correspondence and reprint requests to Dr. B. Paige Lawrence, Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, NY 14642. E-mail address: paige_lawrence{at}urmc.rochester.edu Back

3 Abbreviations used in this paper: AhR, aryl hydrocarbon receptor; AhRE, AhR response element; BAL, bronchoalveolar lavage; iNOS, inducible NO synthase; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TSS, transcription start site; NP, nucleoprotein. Back

Received for publication December 8, 2006. Accepted for publication April 13, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
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