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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

Modulation of Airway Responses to Influenza A/PR/8/34 by ⁹-Tetrahydrocannabinol in C57BL/6 Mice

Departments of Pharmacology and Toxicology (J.P.B., N.E.K) and Pathobiology and Diagnostic Investigation (J.R.H., K.J.W.) and Center for Integrative Toxicology (J.P.B., P.W.F.K., J.R.H, N.E.K.), Michigan State University, East Lansing, Michigan

Received April 20, 2007; accepted August 27, 2007.

	Abstract

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⁹-Tetrahydrocannabinol (⁹-THC) has been widely established as a modulator of host immune responses. Accordingly, the objective of the present study was to examine the effects of ⁹-THC on the immune response within the lungs and associated changes in the morphology of the bronchiolar epithelium after one challenge with a nonlethal dose of the influenza virus A/PR/8 (PR8). C57BL/6 mice were treated by oral gavage with ⁹-THC and/or vehicle (corn oil) for 5 consecutive days. On day 3, mice were instilled intranasally with 50 plaque-forming units of PR8 and/or vehicle (saline) 4 h before ⁹-THC exposure. Mice were subsequently killed 7 and 10 days postinfection (dpi). Viral hemagglutinin 1 (H1) mRNA levels in the lungs were increased in a dose-dependent manner with ⁹-THC treatment. Enumeration of inflammatory cell types in bronchoalveolar lavage fluid showed an attenuation of macrophages and CD4⁺ and CD8⁺ T cells in ⁹-THC-treated mice compared with controls. Likewise, the magnitude of inflammation and virus-induced mucous cell metaplasia, as assessed by histopathology, was reduced in ⁹-THC-treated mice by 10 dpi. Collectively, these results suggest that ⁹-THC treatment increased viral load, as assessed by H1 mRNA levels, through a decrease in recruitment of macrophages and lymphocytes, particularly CD4⁺ and CD8⁺ T cells, to the lung.

⁹-Tetrahydrocannabinol (⁹-THC) is the primary psychoactive component in marijuana and is one of more than 60 structurally related cannabinoids identified in the plant, Cannabis sativa (Dewey, 1986). Exposure to ⁹-THC occurs either through inhalation as a part of a complex mixture of chemicals including cannabinoids and pyrolysis products of smoked marijuana (e.g., recreational use) or via oral consumption as a synthetically derived therapeutic agent for the treatment of symptoms such as AIDS-induced wasting or chemotherapy-induced emesis (e.g., dronabinol). For the latter immunocompromised groups, cancer patients, and those suffering from AIDS, there is concern over their use of potentially immunosuppressive cannabinoids. Cannabinoids, including ⁹-THC, are widely established as immunomodulators, affecting innate, humoral, and cell-mediated immune responses in a variety of animal and cell-based models (reviewed by Berdyshev, 2000). Accordingly, decreased host resistance to opportunistic pathogens (Morahan et al., 1979; Cabral et al., 1986; Specter et al., 1991; Klein et al., 2000) has rendered ⁹-THC exposure as a potential determinant of susceptibility.

The objective of the current study was to evaluate the effect of ⁹-THC exposure on the primary immune response to influenza infection (Buchweitz et al., 2007) and affected airways. In particular, we sought to characterize the effects of ⁹-THC on the levels of viral H1 expressed in the lung and on immune cell recruitment and soluble chemokines/cytokines in the bronchoalveolar lavage fluid (BALF). Morphologic changes in the surface epithelium lining the intrapulmonary conducting airway that occur as a consequence of viral infection and the associated immune response were also assessed. The results of this study suggest that ⁹-THC increased viral H1 load, as measured by mRNA levels, through diminished recruitment of CD4⁺ and CD8⁺ T lymphocytes. Additionally, there were significant decreases in airway epithelial cell apoptosis and in mucous cell metaplasia associated with ⁹-THC treatment and the diminished immune response.

	Materials and Methods

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Animals. Pathogen- and respiratory disease-free female C57BL/6 mice, 8 to 10 weeks old (Charles River Breeding Laboratories, Portage, MI), were randomly assigned to and housed in plastic cages containing sawdust bedding (five mice per cage). Mice were quarantined for 1 week upon arrival and then used in accordance with guidelines set forth by the Institutional Animal Care and Use Committee at Michigan State University. Food (Purina Certified Laboratory Chow) and water were provided ad libitum and mice were not used for experimentation until their body weight was 17 to 20 g. Animal holding rooms were maintained at 21 to 24°C and 40 to 60% relative humidity with a 12-h light/dark cycle.

Experimental Design. Mice, five per treatment group, were administered ⁹-THC (25, 50, or 75 mg/kg) and/or vehicle (corn oil) by oral gavage for 5 consecutive days (Fig. 1). On the 3rd day of treatment, mice were instilled intranasally with influenza virus 4 h before ⁹-THC treatment. Mice were killed at 7 and 10 dpi. Day 7 postinfection was previously established as the transition day between neutrophils and lymphocytes infiltrating the airways in response to PR8 infection and the peak day for the detection of immune mediators released by leukocytes in the airways. Day 10 postinfection was previously established as the day that marked the transition from epithelial cell death (7 dpi) to epithelial regeneration and the onset of mucous cell metaplasia (Buchweitz et al., 2007). Accordingly, two separate experiments were conducted for the measurements of mRNA and BALF at 7 dpi and a third experiment was conducted for routine histopathology and immunohistochemistry at both 7 and 10 dpi. Experimental values obtained for the ⁹-THC control group administered 75 mg/kg ⁹-THC and intranasally instilled with saline were removed from graphs and tables so that unwarranted comparisons were not made between this control and influenza A/PR/8/34 cotreatment groups receiving different dosage levels of ⁹-THC. In addition, as would be anticipated, values for the 75 mg/kg ⁹-THC control were not different from those for the corn oil control.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Experimental time course. Time course of 9-THC () administration in relationship to influenza virus (*) challenge and times of sacrifice (#).

⁹-Tetrahydrocannabinol. ⁹-THC was provided by the National Institute on Drug Abuse (Bethesda, MD) as a resin, which was solubilized in corn oil.

Influenza A/PR/8/34 Instillation. Influenza A/PR/8/34 (PR8) was generously supplied by the laboratory of Dr. Alan Harmsen (Montana State University, Bozeman, MT). Mice were anesthetized with 4% isoflurane in oxygen, and 50 µl of PR8 in pyrogen-free saline was instilled as 25 µl/nare at a total dose of 50 plaque-forming units.

Necropsy, Lavage Collection, and Tissue Preparation. Mice were anesthetized by an i.p. injection of 0.1 ml of 12% pentobarbital solution, a midline laparotomy was performed, and mice were exsanguinated by cutting the abdominal aorta. Immediately after death, the trachea was exposed and then cannulated; the heart and lung were excised en bloc. In the first experiment, lung lobes were immersed in TRI reagent and stored at –80°C until RNA isolation. In the second experiment, 1 ml of sterile saline was instilled through the tracheal cannula and withdrawn to recover BALF. A second saline lavage was performed, and fluids recovered were combined with those from the first lavage. In the third experiment, the left and right lung lobes were inflated under constant pressure (30 cm H₂O) with 10% neutral buffered formalin (Sigma-Aldrich, St. Louis, MO) for 1 h. The tracheal airway was ligated, and the inflated lobes were stored in a large volume of the same fixative for at least 24 h until further processing for histopathology.

RNA Isolation. Total RNA was isolated from the lung lobes by using the TRI reagent method (Sigma-Aldrich). Relative expression levels of H1 and MUC5AC mRNA were determined using TaqMan real-time multiplex reverse transcriptase-PCR with custom designed TaqMan primers and probe to the target gene and the manufacturer's predeveloped primers and probes to 18S (Applied Biosystems, Foster City, CA). Measurements of caspase-3 (CAS-3) were determined similarly by using the manufacturer's predeveloped primers and probe for CAS-3. The primers and probe to both the target gene and endogenous reference gene were specifically designed to exclude detection of genomic DNA. In brief, aliquots of isolated tissue RNA (1 mg of total RNA) were converted to cDNA using random primers (Invitrogen, Carlsbad, CA). The resultant cDNA (2 µl) was added to a reaction mixture that consisted of the target gene primers and probe, endogenous reference primers and probe (18S ribosomal RNA), and TaqMan Universal Master Mix. After PCR, amplification plots (change in dye fluorescence versus cycle number) were examined, and a dye fluorescence threshold within the exponential phase of the reaction was set separately for the target gene and the endogenous reference (18S). The cycle number at which each amplified product crosses the set threshold represents the C_T value. The amount of target gene normalized to its endogenous reference was calculated by subtracting the endogenous reference C_T from the target gene C_T (C_T). Relative mRNA expression was calculated by subtracting the mean C_T of the control samples from the C_T of the treated samples (C_T). The amount of target mRNA, normalized to the endogenous reference and relative to the calibrator (i.e., RNA from control), is calculated by using the formula 2 – C_T.

Total Protein. Total protein was quantified in BALF using the bicinchoninic acid method as provided by the manufacturer (Pierce Chemical, Rockford, IL). In brief, BALF samples were centrifuged at 300g for 5 min to pellet cellular debris. Supernatants were removed and stored at –20°C until testing was performed. BALF supernatant (25 µl) or bovine serum albumin protein standard (25–2000 µg/ml) was incubated in a 96-well microplate with 200 µl of a 50:1 mixture of reagents A (sodium carbonate, sodium bicarbonate, bicinchoninic acid, and sodium tartrate in 0.1 M sodium hydroxide) and B (4% cupric sulfate) for 30 min at 37°C. Samples were read on a Bio-Tek microplate reader at 562 nm.

Bronchoalveolar Lavage Cellularity. The total number of leukocytes in BALF was counted with a hemacytometer. In brief, 10 µl of BALF supernatant was added to a hemacytometer, and the numbers of leukocytes in the four etched corner quadrants were counted and multiplied by 2500 to yield the number of leukocytes per milliliter of sample. The percentage of total leukocytes consisting of eosinophils, lymphocytes, macrophages, and neutrophils were determined from counts of 200 cells in a cytospin sample stained with Diff-Quick (Dade Behring, Inc., Newark, DE). The percentages of eosinophils, lymphocytes, macrophages, and neutrophils were multiplied by the total number of leukocytes determined from the hemacytometer to yield the respective number of each cell type per milliliter of sample.

Secreted Inflammatory Cytokines. BALF samples were centrifuged at 300g for 5 min to pellet cellular debris. Supernatants were removed and stored at –20°C until testing was performed. The cytokines IL-6, IL-10, MCP-1, IFN-, TNF-, and IL-12p70 were detected simultaneously by using the cytometric bead array mouse inflammation kit (BD PharMingen, San Diego, CA). In brief, 50 µlof BALF from each sample was incubated individually with a mixture of capture beads and 50 µl of phycoerythrin (PE) detection reagent consisting of PE-conjugated anti-mouse IL-6, IL-10, MCP-1, IFN-, TNF-, and IL-12p70. The samples were incubated at room temperature for 2 h in the dark. After incubation, samples were washed once and resuspended in 300 µl of wash buffer before acquisition on a BD FACSCalibur flow cytometer. Data were analyzed using cytometric bead array software (BD PharMingen). Standard curves were generated for each cytokine using the mixed cytokine standard provided with the kit. The concentration of each cytokine was determined by interpolation from the corresponding standard curve. The range of detection was 20 to 5000 pg/ml for each cytokine measured.

Analysis of BALF-Associated T Cells by Flow Cytometry. After enumerating the retrieved cells in BALF, the cells were pelleted by centrifugation and reconstituted in 150 ml of flow cytometer buffer [phosphate-buffered saline supplemented with 2% (w/v) bovine serum albumin and 0.09% (w/v) sodium azide] with purified anti-mouse CD16/CD32 (Fcg III/II receptor) antibody (BD Biosciences, San Jose, CA) to block for 30 min. The samples were then split into two groups of equal volume, washed, and reconstituted in flow cytometer buffer. One group received antibodies for CD3 (APC anti-mouse CD3e), CD4 [PE anti-mouse CD4(L3T4)], and CD8 [fluorescein isothiocyanate anti-mouse CD8a(Ly-2)], whereas the other group received the cognate isotype control antibodies. Samples were allowed to incubate for 1 h at 4°C, washed twice, and then fixed for 10 min with Cytofix. Samples were then washed again and reconstituted to a volume of 300 µl for analysis on the BD FACSCalibur flow cytometer. The total number of events taken per each sample was 10,000.

Microdissection and Routine Histopathology. The intrapulmonary airways of the fixed left lung lobe from each rodent were microdissected according to a modified version of the technique of Plopper et al. (1983), which is fully described in a previous publication (Harkema and Hotchkiss, 1992). Beginning at the lobar bronchus, airways are split down the long axis of the main axial airway through the 12th airway generation. Two transverse tissue blocks were excised at the level of the 5th (proximal) and 11th (distal) airway generation. Transverse tissue blocks were also excised from the middle of the four right lung lobes perpendicular to the largest airway branch entering each lobe. Tissue blocks from the left and right lung lobes were embedded in paraffin, sectioned at a thickness of 5 µm, and then stained with hematoxylin and eosin for light microscopic examination. Other paraffin sections were stained with Alcian Blue (pH 2.5)/hematoxylin to identify acidic intraepithelial mucosubstances.

Immunocytochemistry. Hydrated paraffin sections (5 µm thick) from formalin-fixed lung tissues were treated with 0.05% proteinase K for 2 min and washed with 1 N HCI for 1 h. Sections were then treated with 3% H₂O₂ (in methanol) to block endogenous peroxide and incubated with a polyclonal antibody to CAS-3 (Abcam, Inc., Cambridge, MA) at 20 µg/ml for 1 h. Immunoreactive CAS-3 was visualized with the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA) using 3',3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) as a chromogen.

Histopathology Scores for Inflammation. A histopathologic score was established on the basis of the numbers and distribution of inflammatory cells within the tissues, as well as noninflammatory changes such as evidence of bronchiolar epithelial injury and repair. The scores assigned were as follows: 0, no inflammation; 1, mild, inflammatory cell infiltrate of the perivascular/peribronchiolar compartment; 2, moderate, inflammatory cell infiltrate of the perivascular/peribronchiolar space with modest extension into the alveolar parenchyma; and 3. severe, inflammatory cell infiltrate of the perivascular/peribronchiolar space with a greater magnitude of inflammatory foci found in the alveolar parenchyma. A board-certified pathologist scored each lung section independently without prior knowledge of the treatment groups. A mean score with S.E.M. was calculated for each treatment group.

CAS-3 and Alcian Blue Numeric Cell Densities. Slides of lung sections either stained immunohistochemically for CAS-3 or stained for Alcian Blue (acidic mucosubstances) were examined. Numeric cell densities were determined for epithelial cells immunohistochemically reactive to CAS-3 via light microscopy by counting the number of nuclear profiles of these immunoreactive epithelial cells lining the bronchiolar epithelium at generation 5 and dividing by the length of the underlying basal lamina. Numeric cell densities for CAS-3 were expressed as the number of immunoreactive cells per millimeter of basal lamina. In a similar manner, numeric cell densities were determined for epithelial cells staining with Alcian Blue (acidic mucosubstances). The numeric cell density of epithelial cells staining for Alcian Blue (acidic mucosubstances) was expressed as the number of Alcian Blue-reactive epithelial cells per millimeter of basal lamina.

Statistical Analysis. Data are expressed as means ± S.E.M. Outliers were identified and removed from sample sets using Grubb's test. The difference between saline controls and mice treated with PR8 in the absence of ⁹-THC was evaluated by using a Student's t test. The differences between groups treated with PR8 in the presence or absence of ⁹-THC were determined by one-way analysis of variance (ANOVA) and multiple comparisons by the Student-Newman-Keuls post hoc test. For data that were not normally distributed, a Kruskall-Wallis ANOVA on ranks with Dunn's post hoc test was used. Statistical analysis was performed with SigmaStat software (version 2.03; Jandel Scientific, San Rafael, CA). The criterion for significance was taken to be p < 0.05.

	Results

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Expression of H1 in Mouse Lungs Challenged with PR8. Hemagglutinin 1 is an influenza virus surface protein that is a specific antigen target for B cell-derived immunoglobulin. H1 mRNA levels in the lungs of mice challenged with influenza in the absence of ⁹-THC were significantly elevated at 7 dpi compared with those for the saline control (Fig. 2). The H1 mRNA levels in the lungs of mice treated with PR8 and ⁹-THC at 25 mg/kg were mildly attenuated compared with those of mice challenged with PR8 alone. Viral H1 mRNA levels increased with increasing doses of ⁹-THC and were significantly elevated in mice administered 75 mg/kg ⁹-THC compared with those in PR8-challenged mice in the absence of ⁹-THC.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Pulmonary hemagglutinin 1 mRNA levels. Effects of 9-THC on hemagglutinin 1 mRNA levels after influenza challenge in whole lung homogenates at 7 dpi. Data represent means ± S.E.M., presented as the fold change in gene expression of hemagglutinin 1 normalized to mice instilled with saline. *, significant difference between corn oil-treated mice infected with PR8 and respective controls instilled with saline. #, significant effect elicited by 9-THC-treated mice infected with PR8 compared with corn oil-treated mice infected with PR8 by ANOVA.

Consistent with an immune response to viral challenge, the total number of leukocytes in BALF was significantly elevated in mice receiving only influenza at 7 dpi (Table 1). There were apparent trends indicating dose-dependent effects of ⁹-THC on total cell counts. Despite this finding, there were marked decreases observed in the number of lymphocytes retrieved in BALF from mice treated with ⁹-THC at all dose levels. In addition, treatment of mice with ⁹-THC at doses of 25 and 50 mg/kg led to decreases in the number of macrophages retrieved in BALF. Flow cytometry was performed to evaluate differences in T lymphocyte subsets in BALF. The absolute numbers of CD4⁺ and CD8⁺ T cells were determined (Table 2). ⁹-THC treatment significantly decreased the number of CD8⁺ T cells at all dose levels with respect to influenza alone; a similar effect on the number of CD4⁺ T cells was found in mice administered 50 and 75 mg/kg ⁹-THC compared with the influenza group in the absence of ⁹-THC.

Secreted inflammatory chemokines and cytokines were measured as an indicator of immune cell function in response to PR8 challenge. Mice challenged with PR8 had significantly increased BALF concentrations of TNF-, IFN-, IL-6, MCP-1, and IL-10 at 7 dpi compared with those for the saline control (Table 3). In mice treated with ⁹-THC, an increase in MCP-1 at a dose of 50 mg/kg was observed. ⁹-THC treatment had no effect on BALF-associated concentrations of TNF-, IFN-, IL-6, IL-10, or IL-12p70 at any dose compared with mice treated with PR8 alone.

Descriptive Pulmonary Histopathology. Exposure of mice to the corn oil vehicle or ⁹-THC alone did not result in significant histologic changes within the control mice (Fig. 3A). Infection of mice with influenza induced a significant cellular and inflammatory reaction at 7 dpi in all lung regions examined. The inflammatory infiltrate was centered on the bronchioloalveolar duct junction and extended out into the surrounding alveolar parenchyma. The inflammatory cells were a mix of predominantly lymphocytes and neutrophils, with fewer macrophages and plasma cells. The lymphocytic and neutrophilic population often filled the alveoli, and there was moderate alveolar interstitial infiltration with similar inflammatory cells. The lymphocytes often formed well organized perivascular and peribronchiolar aggregates. Acute epithelial necrosis was present in small numbers of bronchioles, and the remaining epithelial cells were moderately attenuated. At 10 dpi with influenza the inflammation was more severe and often obscured the alveolar parenchyma (Fig. 3B). The inflammatory cells at 10 dpi were primarily lymphocytes, with smaller numbers of neutrophils. The bronchiolar epithelium at 10 dpi was moderately hyperplastic and hypertrophied, and there were scattered foci of alveolar bronchiolarization (extension of bronchiolar epithelium into the adjacent alveolar spaces). Treatment of influenza-challenged mice with ⁹-THC resulted in no observed decreases in inflammation at 7 dpi for each ⁹-THC dose. At 10 dpi, treatment with ⁹-THC at all dose levels resulted in a mild to moderate decrease in histologically apparent inflammation within the lungs (Fig. 3C). The inflammation within the mice was not uniformly distributed throughout all lung regions, as found in the control PR8-infected mice. The decreases in inflammation included decreases in both inflammatory cell numbers and the extent of distribution within the tissue. The bronchiolar epithelial changes noted above were still present at 10 dpi in influenza-infected mice treated with ⁹-THC.

View larger version (99K): [in this window] [in a new window]   Fig. 3. Histopathology of the inflammatory response to influenza challenge at generation 5. Effects of 9-THC on the inflammatory response observed at generation 5 of the main axial airway on day 10 postinfection. Light photomicrographs were taken for lung sections at generation 5 of the main axial airway (MAA) to include the bronchiolar artery (a) and alveolar parenchyma (p). A, lung section from a mouse receiving a corn oil gavage surrounding a saline instillation. There was no evidence of inflammation in the perivascular/peribronchiolar submucosal compartment (sm) or alveolar parenchyma. B, lung section from a mouse receiving a corn oil gavage surrounding an influenza instillation. There was severe inflammation of the perivascular/peribronchiolar submucosal compartment with marked extension into the alveolar parenchyma. C, lung section from a mouse receiving a 9-THC (75 mg/kg) gavage surrounding an influenza instillation. There was severe inflammation of the perivascular/peribronchiolar submucosal compartment with modest extension into the alveolar parenchyma. Scale bar, 100 µm.

View larger version (36K): [in this window] [in a new window]   Fig. 4. Combined histopathology-based inflammation scores for the right and left lung lobes. Effects of 9-THC on the inflammatory response gathered within the subepithelial interstitium and alveolar parenchyma after influenza challenge. Inflammation scores were recorded from lung sections taken at 7 (A) and 10 dpi (B). Scores were tabulated and averaged for sections of the four right lobes and two sections taken from generations 5 and 11 of the left lung lobe. Scores: 0, no inflammation; 1, mild, inflammatory cell infiltrate of the perivascular/peribronchiolar compartment; 2, moderate, inflammatory cell infiltrate of the perivascular/peribronchiolar space with modest extension into the alveolar parenchyma; and 3, severe, inflammatory cell infiltrate of the perivascular/peribronchiolar space with a greater magnitude of inflammatory foci found in the alveolar parenchyma. Data represent means ± S.E.M., presented as an inflammation score. *, significant difference between corn oil-treated mice infected with PR8 and respective controls instilled with saline. #, significant effect elicited by 9-THC-treated mice infected with PR8 compared with corn oil-treated mice infected with PR8 by ANOVA. N.D., inflammation not detectable.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Quantification of pulmonary apoptotic cell death by caspase-3 mRNA expression and numeric cell densities of caspase-3-immunopositive cells. The effects of 9-THC on apoptotic cell death in response to influenza challenge were measured by the expression of CAS-3 mRNA (A) and immunohistochemical staining of CAS-3 in the epithelium lining the main axial airway at generation 5 of the left lung lobe at 7 dpi (B) and 10 dpi (C). In A data represent means ± S.E.M., presented as the fold change in gene expression of caspase-3 normalized to mice instilled with saline. In B and C data represent the number of caspase-3-positive epithelial cells per millimeter of basal lamina. *, significant difference between corn oil-treated mice infected with PR8 and respective controls instilled with saline. N.D., caspase-3 not detectable.

View larger version (29K): [in this window] [in a new window]   Fig. 6. Quantification of bronchiolar epithelial mucous cell metaplasia by MUC5AC mRNA levels and Alcian Blue numeric cell densities. The effects of 9-THC on the development of mucous cell metaplasia in the epithelium lining the main axial airway after influenza challenge and subsequent inflammatory cell responses is shown. Expression levels of MUC5AC mRNA (A) and generation 5 main axial airway numeric cell densities of Alcian Blue were determined at 7 dpi (B) and 10 dpi (C). In A data represent the mean ± S.E.M., presented as the fold change in gene expression of MUC5AC normalized to mice instilled with saline. In B and C data represent the number of Alcian Blue-positive epithelial cells per millimeter of basal lamina. *, significant difference between corn oil-treated mice infected with PR8 and respective controls instilled with saline. #, significant effect elicited by 9-THC-treated mice infected with PR8 compared with corn oil-treated mice infected with PR8 by ANOVA.

	Discussion

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⁹-THC-treated mice exhibited higher levels of lung-associated H1 viral mRNA than corn oil-treated mice infected with PR8. In the current study, viral H1 mRNA was measured by real-time PCR, which allowed rapid, sensitive, and quantitative analysis of numerous tissue samples while yielding results similar to those typically obtained with more conventional methods (Spackman et al., 2002; Jaspers et al., 2005). The increased H1 mRNA levels induced by ⁹-THC treatment were dose-dependent albeit without an effect on mortality, suggesting that ⁹-THC administration impaired immune effectors involved in PR8 clearance. By 10 dpi H1 mRNA levels approached the level of detection (data not shown) in all groups, suggesting that viral clearance had occurred. The timeline of viral clearance is consistent with that for uncomplicated infections in humans (Hayden et al., 1998).

The effects of ⁹-THC on immune cell function were assessed by the measurement of edema, immune cell recruitment, and cytokine production in response to PR8 challenge. Epithelial cell death in response to PR8 infection begins as early as 3 dpi and continues through 7 dpi when the epithelium has begun to regenerate (Buchweitz et al., 2007). ⁹-THC had no effect on BALF total protein, suggesting that ⁹-THC did not significantly affect the kinetics of inflammatory mediators released by immune cells accruing in alveolar tissue that increase vascular permeability and leakage at the alveolar/capillary interface. Likewise, there was no difference in the total number of leukocytes retrieved in BALF between mice challenged with PR8 in the presence or absence of ⁹-THC treatment. Despite trends toward decreased total leukocytes in BALF with ⁹-THC treatment, differential cell counts indicated that ⁹-THC treatment decreased, in a dose-related manner, the number of macrophages and lymphocytes in the airways. Dose-dependent modulation of immune cell recruitment to the lungs by ⁹-THC during inflammation has been reported previously (Berdyshev et al., 1998). Lymphocytes infiltrating the airways were composed of CD4⁺ and CD8⁺ T cells. One of the most significant findings in this study was that ⁹-THC administration markedly attenuated both CD4⁺ and CD8⁺ T cell counts in BALF. Given the marked lymphocytic and monocytic infiltration of the airway submucosa, these data were considered in conjunction with histopathology, which suggests that ⁹-THC modulated the immune response to PR8 through an influence on leukocyte migration. These findings are consistent with cannabinoid effects on lymphocyte and monocyte chemotaxis reported by others (Stefano et al., 1998; Joseph et al., 2004; Sacerdote et al., 2000, 2005).

As previously reported (Buchweitz et al., 2007), the immune response to PR8 challenge included marked increases in IL-6, TNF-, IFN-, MCP-1, and IL-10 by 7 dpi in BALF. Collectively, no clear profile of activity emerged concerning the effects of ⁹-THC on PR8 cytokine or chemokine levels in BALF. This is not altogether surprising as each cytokine and chemokine is regulated in its own unique manner with its own distinct kinetics. In addition, molecular mechanisms by which certain cytokines and chemokines are modulated by cannabinoids are only partially understood. In spite of a clear profile of ⁹-THC effects on cytokines and chemokines in BALF, several inflammatory mediators evaluated in this study exhibited dose-dependent modulation in response to ⁹-THC treatment. Specifically, treatment with ⁹-THC at 50 and 75 mg/kg led to increased levels of MCP-1 and IL-6 in BALF, whereas IL-10 concentrations were decreased at these doses. The modulation of these cytokines by cannabinoid treatment is consistent with previous findings in other experimental models (Molina-Holgado et al., 1998; Sacerdote et al., 2005).

Histopathology revealed that ⁹-THC, at all dose levels, attenuated the magnitude of inflammation at 10 dpi. The inflammatory response in the lungs of mice challenged with influenza alone extended beyond the perivascular/peribronchiolar submucosal compartment and into the alveolar parenchyma. With ⁹-THC treatment, the inflammatory response was centered on the submucosal compartment. The histopathology supports our finding of decreased numbers of macrophages and lymphocytes observed in BALF from ⁹-THC-treated mice. Because clearance of influenza virus is critically dependent on both CD4⁺ and CD8⁺ T cells, a decrease in their numbers is consistent with the increase in H1 mRNA observed with ⁹-THC treatment.

Examination of epithelial cell changes in airways showed that PR8 treatment alone induced CAS-3, a well known marker for committed activation of apoptosis (Fischer et al., 2003), as evidenced by mRNA levels and by tissue staining. Because apoptosis can be initiated either directly by the virus in infected host cells (Takizawa et al., 1999; Wurzer et al., 2003) or by effector cells, specifically cytotoxic T cells and/or natural killer cells, it is presently unclear which mechanism(s) are predominantly responsible for the observed increase in CAS-3. Interestingly, a dose-related decrease in CAS-3 tissue staining, but not in mRNA levels was observed with ⁹-THC treatment, suggesting that ⁹-THC may interfere in part with PR8-induced translation of CAS-3 mRNA or at the level of protein synthesis.

After bronchiolar epithelial shedding at 3 and 7 dpi in response to PR8 infection and concomitant with the cell-mediated immune response, the regenerative airway epithelium undergoes metaplastic changes, resulting in increased numbers of mucous goblet cells at 10 dpi (Buchweitz et al., 2007). Mucous cell metaplasia is an adaptive response of the epithelium brought about by soluble mediators of inflammation (Jamil et al., 1997; Dabbagh et al., 1999; Shim et al., 2001; Justice et al., 2002; Kawano et al., 2002; Reader et al., 2003). An early indicator of increased mucin production and possibly of mucous cell metaplasia is the expression of MUC5AC mRNA that encodes for the goblet cell-derived mucin MUC5AC. Corn oil-treated mice challenged with PR8 exhibited an increase in both the levels of MUC5AC mRNA and Alcian Blue staining, as shown previously (Buchweitz et al., 2007). Interestingly, there was a correlative trend in the dose-dependent effects of ⁹-THC treatment on MUC5AC mRNA at 7 dpi and Alcian Blue staining at 10 dpi, suggesting that the effects of ⁹-THC on MUC5AC occur at the level of gene transcription. It is unclear whether ⁹-THC treatment can directly interfere with the up-regulation of MUC5AC gene transcription or whether the effect is mediated indirectly through the suppression of the inflammatory response that induces MUC5AC transcription.

In conclusion, ⁹-THC administration modulated the host immune response to PR8 as evidenced by an increased viral load and a decreased magnitude of macrophage and lymphocyte (CD4⁺ and CD8⁺) recruitment. These findings were supported by histopathology and are consistent with similar studies that demonstrated increased susceptibility to the respiratory pathogen, Legionella pneumophila, after ⁹-THC administration (Klein et al., 2000). As in the present study with PR8, Klein and coworkers similarly showed that ⁹-THC affected T cell and macrophage function as suggested by altered regulation of T cell-derived cytokines and a suppression of T cell activation to Legionella by attenuation of the expression of costimulatory and polarizing molecules on the antigen-presenting dendritic cells (Klein et al., 2000; Lu et al., 2006). Further studies will need to be conducted to determine the effects of ⁹-THC on the later stages of the immune response to PR8, as well as to ascertain the role of cannabinoid receptors, CB1 and CB2, on the ⁹-THC-mediated effects observed.

	Acknowledgements

 
We extend our thanks to Dr. James Klaunig at Indiana University-Purdue University Indianapolis for providing analytical support in the measurement of ⁹-THC and its metabolites and Dr. Alan Harmsen for PR8. We also thank Robert Crawford and Lori Bramble for providing technical assistance with flow cytometry, and Kim Hambleton for assistance in the submission of this manuscript.

	Footnotes

 
This work was supported by a Michigan State University Foundation strategic partnership grant, National Institutes of Health Grant DA07908, and the National Institute of Environmental Health Sciences Training Grant T32 ES07255.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.124719.

ABBREVIATIONS: IFN, interferon; ⁹-THC, ⁹-tetrahydrocannabinol; BALF, bronchoalveolar lavage fluid; H1, hemagglutinin 1; dpi, days postinfection; PR8, influenza A/PR/8/34; PCR, polymerase chain reaction; CAS-3, caspase-3; IL, interleukin; MCP, monocyte chemoattractant protein; TNF, tumor necrosis factor; PE, phycoerythrin; ANOVA, analysis of variance.

Address correspondence to: Dr. Norbert E. Kaminski, 315 National Food Safety and Toxicology Building, Michigan State University, East Lansing, MI 48824-1317. E-mail address: kamins11{at}msu.edu

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