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Abstract
Δ9-Tetrahydrocannabinol (Δ9-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 Δ9-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 Δ9-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 Δ9-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 Δ9-THC treatment. Enumeration of inflammatory cell types in bronchoalveolar lavage fluid showed an attenuation of macrophages and CD4+ and CD8+ T cells in Δ9-THC-treated mice compared with controls. Likewise, the magnitude of inflammation and virus-induced mucous cell metaplasia, as assessed by histopathology, was reduced in Δ9-THC-treated mice by 10 dpi. Collectively, these results suggest that Δ9-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.
Influenza viruses are common human respiratory pathogens throughout the world. Aerosol droplets released from infected individuals spread influenza virus person-to-person. In immunocompetent human hosts, the virus infects the airway epithelium lining the respiratory tract (Van Reeth, 2000), eliciting an immune response in which both innate and acquired immunity play a critical role in viral clearance. Aside from the lytic activity of the virus alone, infected epithelial cells are also destroyed by the actions of CD8+ cytotoxic T cells (Topham et al., 1997). In addition, there is growing support for the role of CD4+ T cells as contributing immune effectors in the protection against influenza (Brown et al., 2004, 2006; Swain et al., 2006). CD4+ T cells carry out this role through the promotion of long-lasting CD8+ T memory cells, mediating the clearance of virus in an IFN-γ-dependent mechanism, by direct cytolytic effects on infected cells via Fas-Fas ligand interactions, or by a combination of these functions (Brown et al., 2006). The aforementioned findings illustrate the complex, as well as redundant, mechanisms used by the immune system to defend the host against infectious pathogens.
Δ9-Tetrahydrocannabinol (Δ9-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 Δ9-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 Δ9-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 Δ9-THC exposure as a potential determinant of susceptibility.
The objective of the current study was to evaluate the effect of Δ9-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 Δ9-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 Δ9-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 Δ9-THC treatment and the diminished immune response.
Materials and Methods
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 Δ9-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 Δ9-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 Δ9-THC control group administered 75 mg/kg Δ9-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 Δ9-THC. In addition, as would be anticipated, values for the 75 mg/kg Δ9-THC control were not different from those for the corn oil control.
Δ9-Tetrahydrocannabinol. Δ9-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 CT value. The amount of target gene normalized to its endogenous reference was calculated by subtracting the endogenous reference CT from the target gene CT (ΔCT). Relative mRNA expression was calculated by subtracting the mean ΔCT of the control samples from the ΔCT of the treated samples (ΔΔCT). 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 — ΔΔCT.
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-γ, TNF-α, 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-γ, TNF-α, 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 Δ9-THC was evaluated by using a Student's t test. The differences between groups treated with PR8 in the presence or absence of Δ9-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
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 Δ9-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 Δ9-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 Δ9-THC and were significantly elevated in mice administered 75 mg/kg Δ9-THC compared with those in PR8-challenged mice in the absence of Δ9-THC.
Measurements of Injury, Immune Cell Recruitment, and Soluble Chemical Mediators in BALF. As a measure of alveolar/capillary membrane integrity, total protein in BALF was assayed. There were marked increases in BALF-associated total protein in the influenza group at 7 dpi compared with the saline control, but there were no differences observed in the protein concentration recovered in BALF from mice treated with any dose of Δ9-THC (data not shown).
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 Δ9-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 Δ9-THC at all dose levels. In addition, treatment of mice with Δ9-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). Δ9-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 Δ9-THC compared with the influenza group in the absence of Δ9-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-α, IFN-γ, IL-6, MCP-1, and IL-10 at 7 dpi compared with those for the saline control (Table 3). In mice treated with Δ9-THC, an increase in MCP-1 at a dose of 50 mg/kg was observed. Δ9-THC treatment had no effect on BALF-associated concentrations of TNF-α, IFN-γ, 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 Δ9-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 Δ9-THC resulted in no observed decreases in inflammation at 7 dpi for each Δ9-THC dose. At 10 dpi, treatment with Δ9-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 Δ9-THC.
Inflammation Scores from Histopathology. The magnitude and severity of inflammation observed in histologic sections of lung isolated from the right and left lobes were independently scored, as described under Materials and Methods, and compared between treatment groups at 7 dpi (Fig. 4A) and 10 dpi (Fig. 4B). There was no observed inflammation in the lungs of mice intranasally instilled with saline. In contrast, a marked increase in the inflammation score was noted for lungs in the influenza-alone treatment group, representing a moderate to severe inflammatory response. The inflammation scores for sections from mice challenged with PR8 and administered Δ9-THC were significantly attenuated at 10 dpi, representing mild to moderate levels of inflammation.
Caspase-3 mRNA Expression Levels and G5 Main Axial Airway Numeric Cell Densities. There were markedly higher CAS-3 mRNA levels at 7 dpi in total lung homogenates from mice treated with influenza alone compared with the saline control, suggesting an increase in apoptotic cell death in response to PR8 infection (Fig. 5A). There was also an apparent trend toward increasing levels of CAS-3 mRNA with increasing doses of Δ9-THC; however, these increases were not statistically significant. In addition, immunohistochemical staining for CAS-3 yielded marked increases in the number of bronchiolar epithelial cells immunoreactive to CAS-3 with influenza treatment alone at 7 dpi (Fig. 5B). There was a decrease (p = 0.056) in CAS-3 immunoreactive epithelial cells at 7 dpi with 25 mg/kg Δ9-THC treatment (Fig. 5B). There were no significant differences observed in numeric cell densities for CAS-3 with any of the treatments at 10 dpi (Fig. 5C).
MUC5ACmRNA Expression Levels and G5 Main Axial Airway Numeric Cell Densities. The levels of MUC5AC mRNA were increased in mice challenged with influenza alone (Fig. 6A). In mice challenged with PR8 and treated with 25 mg/kg Δ9-THC, a 4-fold increase in MUC5AC mRNA levels was observed compared with the influenza-alone treatment group. There were also marked increases in the number of epithelial cells staining for Alcian Blue (acidic mucosubstances) with influenza treatment alone by 10 dpi but not at 7 dpi (Fig. 6, B and C). Δ9-THC treatment did not affect the number of Alcian Blue-stained epithelial cells observed along the main axial airway at 7 dpi (Fig. 6B) but did attenuate the number of Alcian Blue-positive cells observed at 10 dpi in mice receiving 75 mg/kg Δ9-THC (Fig. 6C) compared with mice challenged with PR8 alone.
Discussion
The objective of the present investigation was to characterize the effects of Δ9-THC on the intrapulmonary immune response and associated changes in the morphology of the bronchiolar epithelium after challenge with a nonlethal dose of PR8. In the current study we demonstrated that oral administration of Δ9-THC decreased immune competence evidenced by an increase in influenza viral load, as assessed by H1 mRNA levels, and decreased lymphocytic and monocytic recruitment into the lungs. Oral administration was chosen because it is a relevant route of Δ9-THC exposure, as for its synthetic therapeutic form, also known as Marinol. More importantly, Δ9-THC is highly lipophilic, requiring a non-aqueous diluent for delivery, which in itself would have the potential for inducing irritation and damage to the airways. In contrast, oral administration of Δ9-THC in corn oil, as used in this study, is well tolerated, albeit, absorption from the gastrointestinal tract is poor, resulting in modest blood concentrations of Δ9-THC and its metabolites. Preliminary studies revealed that oral administration of 75 mg/kg Δ9-THC for 5 consecutive days led to serum concentrations of 66.2 ng/ml of the parent compound (unpublished observation). These levels correlate well with a previous report by Azorlosa et al. in 1992, in which human plasma levels ranged from 57 to 268 ng/ml. In the current study, PR8 was administered by intranasal instillation in saline on day 3, with Δ9-THC coadministration surrounding the day of infection. The rationale for this dosing paradigm was to investigate the effects of Δ9-THC treatment on the immune response to PR8 during the early stages of infection.
Δ9-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 Δ9-THC treatment were dose-dependent albeit without an effect on mortality, suggesting that Δ9-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 Δ9-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). Δ9-THC had no effect on BALF total protein, suggesting that Δ9-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 Δ9-THC treatment. Despite trends toward decreased total leukocytes in BALF with Δ9-THC treatment, differential cell counts indicated that Δ9-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 Δ9-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 Δ9-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 Δ9-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-α, IFN-γ, MCP-1, and IL-10 by 7 dpi in BALF. Collectively, no clear profile of activity emerged concerning the effects of Δ9-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 Δ9-THC effects on cytokines and chemokines in BALF, several inflammatory mediators evaluated in this study exhibited dose-dependent modulation in response to Δ9-THC treatment. Specifically, treatment with Δ9-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 Δ9-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 Δ9-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 Δ9-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 Δ9-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 Δ9-THC treatment, suggesting that Δ9-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 Δ9-THC treatment on MUC5AC mRNA at 7 dpi and Alcian Blue staining at 10 dpi, suggesting that the effects of Δ9-THC on MUC5AC occur at the level of gene transcription. It is unclear whether Δ9-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, Δ9-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 Δ9-THC administration (Klein et al., 2000). As in the present study with PR8, Klein and coworkers similarly showed that Δ9-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 Δ9-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 Δ9-THC-mediated effects observed.
Source, Graphs and Figures: Modulation of Airway Responses to Influenza A/PR/8/34 by
Δ9-Tetrahydrocannabinol (Δ9-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 Δ9-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 Δ9-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 Δ9-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 Δ9-THC treatment. Enumeration of inflammatory cell types in bronchoalveolar lavage fluid showed an attenuation of macrophages and CD4+ and CD8+ T cells in Δ9-THC-treated mice compared with controls. Likewise, the magnitude of inflammation and virus-induced mucous cell metaplasia, as assessed by histopathology, was reduced in Δ9-THC-treated mice by 10 dpi. Collectively, these results suggest that Δ9-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.
Influenza viruses are common human respiratory pathogens throughout the world. Aerosol droplets released from infected individuals spread influenza virus person-to-person. In immunocompetent human hosts, the virus infects the airway epithelium lining the respiratory tract (Van Reeth, 2000), eliciting an immune response in which both innate and acquired immunity play a critical role in viral clearance. Aside from the lytic activity of the virus alone, infected epithelial cells are also destroyed by the actions of CD8+ cytotoxic T cells (Topham et al., 1997). In addition, there is growing support for the role of CD4+ T cells as contributing immune effectors in the protection against influenza (Brown et al., 2004, 2006; Swain et al., 2006). CD4+ T cells carry out this role through the promotion of long-lasting CD8+ T memory cells, mediating the clearance of virus in an IFN-γ-dependent mechanism, by direct cytolytic effects on infected cells via Fas-Fas ligand interactions, or by a combination of these functions (Brown et al., 2006). The aforementioned findings illustrate the complex, as well as redundant, mechanisms used by the immune system to defend the host against infectious pathogens.
Δ9-Tetrahydrocannabinol (Δ9-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 Δ9-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 Δ9-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 Δ9-THC exposure as a potential determinant of susceptibility.
The objective of the current study was to evaluate the effect of Δ9-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 Δ9-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 Δ9-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 Δ9-THC treatment and the diminished immune response.
Materials and Methods
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 Δ9-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 Δ9-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 Δ9-THC control group administered 75 mg/kg Δ9-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 Δ9-THC. In addition, as would be anticipated, values for the 75 mg/kg Δ9-THC control were not different from those for the corn oil control.
Δ9-Tetrahydrocannabinol. Δ9-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 CT value. The amount of target gene normalized to its endogenous reference was calculated by subtracting the endogenous reference CT from the target gene CT (ΔCT). Relative mRNA expression was calculated by subtracting the mean ΔCT of the control samples from the ΔCT of the treated samples (ΔΔCT). 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 — ΔΔCT.
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-γ, TNF-α, 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-γ, TNF-α, 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 Δ9-THC was evaluated by using a Student's t test. The differences between groups treated with PR8 in the presence or absence of Δ9-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
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 Δ9-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 Δ9-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 Δ9-THC and were significantly elevated in mice administered 75 mg/kg Δ9-THC compared with those in PR8-challenged mice in the absence of Δ9-THC.
Measurements of Injury, Immune Cell Recruitment, and Soluble Chemical Mediators in BALF. As a measure of alveolar/capillary membrane integrity, total protein in BALF was assayed. There were marked increases in BALF-associated total protein in the influenza group at 7 dpi compared with the saline control, but there were no differences observed in the protein concentration recovered in BALF from mice treated with any dose of Δ9-THC (data not shown).
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 Δ9-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 Δ9-THC at all dose levels. In addition, treatment of mice with Δ9-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). Δ9-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 Δ9-THC compared with the influenza group in the absence of Δ9-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-α, IFN-γ, IL-6, MCP-1, and IL-10 at 7 dpi compared with those for the saline control (Table 3). In mice treated with Δ9-THC, an increase in MCP-1 at a dose of 50 mg/kg was observed. Δ9-THC treatment had no effect on BALF-associated concentrations of TNF-α, IFN-γ, 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 Δ9-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 Δ9-THC resulted in no observed decreases in inflammation at 7 dpi for each Δ9-THC dose. At 10 dpi, treatment with Δ9-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 Δ9-THC.
Inflammation Scores from Histopathology. The magnitude and severity of inflammation observed in histologic sections of lung isolated from the right and left lobes were independently scored, as described under Materials and Methods, and compared between treatment groups at 7 dpi (Fig. 4A) and 10 dpi (Fig. 4B). There was no observed inflammation in the lungs of mice intranasally instilled with saline. In contrast, a marked increase in the inflammation score was noted for lungs in the influenza-alone treatment group, representing a moderate to severe inflammatory response. The inflammation scores for sections from mice challenged with PR8 and administered Δ9-THC were significantly attenuated at 10 dpi, representing mild to moderate levels of inflammation.
Caspase-3 mRNA Expression Levels and G5 Main Axial Airway Numeric Cell Densities. There were markedly higher CAS-3 mRNA levels at 7 dpi in total lung homogenates from mice treated with influenza alone compared with the saline control, suggesting an increase in apoptotic cell death in response to PR8 infection (Fig. 5A). There was also an apparent trend toward increasing levels of CAS-3 mRNA with increasing doses of Δ9-THC; however, these increases were not statistically significant. In addition, immunohistochemical staining for CAS-3 yielded marked increases in the number of bronchiolar epithelial cells immunoreactive to CAS-3 with influenza treatment alone at 7 dpi (Fig. 5B). There was a decrease (p = 0.056) in CAS-3 immunoreactive epithelial cells at 7 dpi with 25 mg/kg Δ9-THC treatment (Fig. 5B). There were no significant differences observed in numeric cell densities for CAS-3 with any of the treatments at 10 dpi (Fig. 5C).
MUC5ACmRNA Expression Levels and G5 Main Axial Airway Numeric Cell Densities. The levels of MUC5AC mRNA were increased in mice challenged with influenza alone (Fig. 6A). In mice challenged with PR8 and treated with 25 mg/kg Δ9-THC, a 4-fold increase in MUC5AC mRNA levels was observed compared with the influenza-alone treatment group. There were also marked increases in the number of epithelial cells staining for Alcian Blue (acidic mucosubstances) with influenza treatment alone by 10 dpi but not at 7 dpi (Fig. 6, B and C). Δ9-THC treatment did not affect the number of Alcian Blue-stained epithelial cells observed along the main axial airway at 7 dpi (Fig. 6B) but did attenuate the number of Alcian Blue-positive cells observed at 10 dpi in mice receiving 75 mg/kg Δ9-THC (Fig. 6C) compared with mice challenged with PR8 alone.
Discussion
The objective of the present investigation was to characterize the effects of Δ9-THC on the intrapulmonary immune response and associated changes in the morphology of the bronchiolar epithelium after challenge with a nonlethal dose of PR8. In the current study we demonstrated that oral administration of Δ9-THC decreased immune competence evidenced by an increase in influenza viral load, as assessed by H1 mRNA levels, and decreased lymphocytic and monocytic recruitment into the lungs. Oral administration was chosen because it is a relevant route of Δ9-THC exposure, as for its synthetic therapeutic form, also known as Marinol. More importantly, Δ9-THC is highly lipophilic, requiring a non-aqueous diluent for delivery, which in itself would have the potential for inducing irritation and damage to the airways. In contrast, oral administration of Δ9-THC in corn oil, as used in this study, is well tolerated, albeit, absorption from the gastrointestinal tract is poor, resulting in modest blood concentrations of Δ9-THC and its metabolites. Preliminary studies revealed that oral administration of 75 mg/kg Δ9-THC for 5 consecutive days led to serum concentrations of 66.2 ng/ml of the parent compound (unpublished observation). These levels correlate well with a previous report by Azorlosa et al. in 1992, in which human plasma levels ranged from 57 to 268 ng/ml. In the current study, PR8 was administered by intranasal instillation in saline on day 3, with Δ9-THC coadministration surrounding the day of infection. The rationale for this dosing paradigm was to investigate the effects of Δ9-THC treatment on the immune response to PR8 during the early stages of infection.
Δ9-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 Δ9-THC treatment were dose-dependent albeit without an effect on mortality, suggesting that Δ9-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 Δ9-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). Δ9-THC had no effect on BALF total protein, suggesting that Δ9-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 Δ9-THC treatment. Despite trends toward decreased total leukocytes in BALF with Δ9-THC treatment, differential cell counts indicated that Δ9-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 Δ9-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 Δ9-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 Δ9-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-α, IFN-γ, MCP-1, and IL-10 by 7 dpi in BALF. Collectively, no clear profile of activity emerged concerning the effects of Δ9-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 Δ9-THC effects on cytokines and chemokines in BALF, several inflammatory mediators evaluated in this study exhibited dose-dependent modulation in response to Δ9-THC treatment. Specifically, treatment with Δ9-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 Δ9-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 Δ9-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 Δ9-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 Δ9-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 Δ9-THC treatment, suggesting that Δ9-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 Δ9-THC treatment on MUC5AC mRNA at 7 dpi and Alcian Blue staining at 10 dpi, suggesting that the effects of Δ9-THC on MUC5AC occur at the level of gene transcription. It is unclear whether Δ9-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, Δ9-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 Δ9-THC administration (Klein et al., 2000). As in the present study with PR8, Klein and coworkers similarly showed that Δ9-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 Δ9-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 Δ9-THC-mediated effects observed.
Source, Graphs and Figures: Modulation of Airway Responses to Influenza A/PR/8/34 by
