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Abstract
We have previously reported that Δ-9-tetrahydrocannabinol (Δ9-THC)-treated mice challenged with influenza virus A/PR/8/34 (PR8) developed increased viral hemagglutinin 1 (H1) mRNA levels and decreased monocyte and lymphocyte recruitment to the pulmonary airways when compared with mice challenged with PR8 alone. The objective of the present study was to examine the role of cannabinoid (CB1/CB2) receptors in mediating the effects of Δ9-THC on immune and epithelial cell responses to PR8. In the current study, Δ9-THC-treated CB1/CB2 receptor null (CB1−/−/CB2−/−) and wild-type mice infected with PR8 had marked increases in viral H1 mRNA when compared with CB1−/−/CB2−/− and wild-type mice challenged with PR8 alone. However, the magnitude of the H1 mRNA levels was greatly reduced in CB1−/−/CB2−/− mice as compared with wild-type mice. In addition, Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 had increased CD4+ T cells and IFN-γ in bronchoalveolar lavage fluid with greater pulmonary inflammation when compared with Δ9-THC-treated wild-type mice infected with PR8. Δ9-THC treatment of CB1−/−/CB2−/− mice in the presence or absence of PR8 challenge also developed greater amounts of mucous cell metaplasia in the affected bronchiolar epithelium. Collectively, the immune and airway epithelial cell responses to PR8 challenge in Δ9-THC-treated CB1−/−/CB2−/− and wild-type mice indicated the involvement of CB1/CB2 receptor-dependent and -independent mechanisms.
INTRODUCTION
In humans, the primary routes of exposure to Δ-9-tetrahydrocannabinol (Δ9-THC) occur through inhalation as a complex mixture of chemicals, including cannabinoid (CB) compounds and pyrolysis products of smoked marijuana or via oral consumption, which can include a synthetically derived therapeutic for the treatment of AIDS-induced wasting or chemotherapy-induced emesis (e.g., dronabinol). Despite its highly debated therapeutic use, it is well established that the plant-derived CB Δ9-THC has immunosuppressive properties. CBs, such as Δ9-THC, have been found to increase host susceptibility to pathogens in a number of in vivo experimental models [1], including HSV [2 , 3], Listeria monocytogenes [2], Legionella pneumophila [4], and more recently, influenza virus A/PR/8/34 (PR8) [5]. The mechanism for increased susceptibility to pathogens appears to be correlated with the suppression of cell-mediated immune responses by Δ9-THC, which suppresses immune cell function by decreasing cytokine secretion and expression of costimulatory molecules by APC [6], suppressing the migration of monocytes and T cell subsets (CD4+ and CD8+ T cells) into the infected tissue [5], altering the cytoplasmic polarization of cytolytic T cells toward pathogen-infected cells [3], and suppressing the release of cytokines, such as IL-12 and IFN-γ, which are essential for competent, cell-mediated immunity regulated by TH1 cells [7]. Additionally, in a mouse model of allergic airway disease, Δ9-THC attenuated the TH2 cytokine response to an inhaled allergen, decreasing mRNA expression of the cytokines IL-2, IL-4, IL-5, and IL-13 elicited by OVA challenge [8]. Production of IL-2 is especially crucial to T cell-dependent responses via its role in T cell clonal expansion. Accordingly, Δ9-THC and other CB compounds have been shown to suppress lymphocyte proliferation in vitro [9] as well as markedly suppress IL-2 expression, in part, through inhibition of the NFAT [10 , 11]. The precise mechanism by which Δ9-THC modulates APC and T cell function as well as the role of CB1 and CB2 have remained elusive.
We have recently reported [5] that Δ9-THC increased host susceptibility to influenza virus PR8, a hemagluttinin 1, neuraminadase 1 (H1N1) influenza strain, in the C57BL/6 mouse. Influenza virus is a common respiratory pathogen that infects epithelial cells lining the respiratory tract, as well as mononuclear cells [12]. During the course of infection, inflammatory cells comprised primarily of lymphocytes and neutrophils infiltrate the pulmonary airways [13]. Viral clearance is achieved through immunocompetent, virus-specific T cell responses. We have recently reported that treatment of mice with increasing doses of Δ9-THC led to a dose-dependent decrease in recruitment of monocytes and lymphocytes (CD4+ and CD8+ T cells) with a concomitant increase in viral load at 7 days postinfection (dpi) [5]. These results suggest that Δ9-THC disrupted cell-mediated immune responses that are critically involved in viral clearance and recovery from influenza infection.
Two subtypes of CB receptors have been characterized: CB1 (brain receptor) and CB2 (peripheral receptor). The CB2 receptor is predominantly expressed in cells of the immune system, including neutrophils, monocytes, NK cells, T cells, and B cells [14 , 15], and has been thought to mediate many of the immunomodulatory effects produced by CBs. In particular, the CB-induced inhibition of antigen processing and presentation in macrophage [16 , 17] and antitumor activity in a murine model of lung cancer [18] has been shown to be mediated by the CB2 receptor. There is, however, emerging evidence for CB-mediated effects on cytokine production pertinent to T-dependent humoral and cell-mediated immune responses that occur independently of CB1/CB2 receptors. Specifically, the suppression of IL-2 production by a variety of immunosuppressive CBs is not attenuated by the CB receptor antagonists SR141716A and SR144528 [19]. Likewise, the endogenous CB (endoCB), 2-arachidonyl glycerol, suppressed PMA/ionomycin-elicited IFN-γ expression similarly in splenocytes obtained from CB receptor null mice (CB1−/−/CB2−/−) as compared with splenocytes from wild-type mice [20]. Additional evidence for CB receptor-independent effects of CBs is illustrated by the plant-derived CB, cannabidiol, which possesses weak affinity for the CB receptors CB1 and CB2, yet exhibits potent anti-inflammatory activity including the reduced production of PGE2, NO and other oxygen-derived free radicals in response to carrageenan-induced inflammation [21], inhibition of the release of IL-1, TNF-α, and IFN-γ by PBMC [22], and suppressed MIP-1α, MIP-1β, and IL-8 in the human T cell lymphotropic virus 1 B cell line [23]. Collectively, these studies illustrate different aspects of host immunity that are CB receptor (CB1 and CB2)-dependent and -independent.
The objective of the present study was to evaluate the role of CB1 and CB2 receptors in the suppression of cell-mediated immunity in mice infected with PR8 by Δ9-THC. Accordingly, CB1−/−/CB2−/− mice and background strain C57BL/6 mice were compared by using a mouse model of host resistance to infection with murine-adapted PR8. Measurements of viral load and immune cell recruitment and secretion of cytokines in the airways were determined. In addition, previously characterized, morphologic changes in airway epithelium, including apoptosis and mucous cell metaplasia (MCM), were quantified as a measure of viral and immune cell-mediated, pathologic outcomes [5]. Results from this study suggest that Δ9-THC is modulating immunological and pulmonary airway responses to influenza virus through CB receptor (CB1 and CB2)-dependent and -independent mechanisms.
MATERIALS AND METHODS
Animals
Two independent experiments were conducted as a part of this study. In the first experiment, 32 female C57BL/6 mice (8—10 weeks old, from Charles River, Portage, MI, USA) and 32 female CB1−/−/CB2−/− mice (8—16 weeks old), bred at Michigan State University (MSU; East Lansing, MI, USA) from breeding pairs provided by Dr. Andreas Zimmer [24], were randomly assigned to one of four experimental groups (n=8 per group) by genotype. The mice were transferred to plastic cages containing sawdust bedding (four mice per cage) and quarantined for 1 week in an animal holding room that was maintained at 21—24°C and 40—60% relative humidity with a 12-h light/dark cycle. The mice were provided food (Purina Certified Laboratory Chow) and water ad libitum and not used for experimentation until their body weight was 17—20 g. In the second experiment, 30 female C57BL/6 mice (8—10 weeks old) and 34 female CB1−/−/CB2−/− mice (8—16 weeks old) were randomly assigned to one of four experimental groups (n=5—14 per group) by genotype. CB1−/−/CB2−/− mice were subjected to an extensive battery of serological screens prior to breeding, as well as designated offspring sentinels after breeding. Tests for all pathogens assayed were negative. All mice used in this study were kept free of pathogens and respiratory disease and used in accordance with guidelines set forth by the Institutional Animal Care and Use Committee at MSU.
Δ9-THC
Δ9-THC was provided by the National Institute on Drug Abuse (Bethesda, MD, USA) as a resin, which was solubilized in corn oil at concentrations that yielded a dose of 75 mg/kg for administration to mice by oral gavage in a volume of 100 μl.
Influenza PR8 instillation
The laboratory of Dr. Alan Harmsen (Montana State University, Bozeman, MT, USA) generously supplied influenza PR8. Mice were anesthetized with 4% isoflurane in oxygen, and 50 μl PR8 in pyrogen-free saline was instilled as 25 μl per nare at a total dose of 50 pfu.
Experimental design
In both experiments, mice were orally administered Δ9-THC for 5 consecutive days at a dose of 75 mg/kg. On the 3rd day of dosing, mice were instilled intranasally with influenza virus (50 pfu) 4 h prior to Δ9-THC administration. All mice were killed at 7 dpi. In the first experiment, the right lung lobes were ligated and removed for RNA analysis, and the left lung lobe was lavaged with saline for cellular and biochemical measurements and then fixed in 10% neutral-buffered formalin for immunohistochemical analysis as described below. In the second experiment, the entire lung was immersed in TRI reagent (Sigma Chemical Co, St. Louis, MO, USA) for RNA isolation and analysis.
These experiments required the breeding of a sufficient number of female mice to support a large n value per treatment group. As breeding was a limiting factor in the design of these experiments, it was deemed necessary in the initial experiment to divide the right and left lung lobes to perform mRNA, bronchoalveolar lavage fluid (BALF), and immunohistochemical measurements. The assumption was made that the virus and associated immune response would be equally distributed between the right and left lung lobes and that there would be uniform and most importantly, discernible differences between treatment with Δ9-THC or vehicle. The trends in the results were similar to those observed previously [5]; however, a second experiment was performed to provide clarity in our measurement of viral hemagglutinin 1 (H1) mRNA for whole lung homogenates.
Necropsy, lavage collection, and tissue preparation
Mice were first anesthetized by an i.p. injection of 0.1 ml 12% pentobarbital solution, and then a midline laparotomy was performed, and mice were exsanguinated by cutting the abdominal aorta. Immediately after death, the trachea was exposed and cannulated, and the heart and lung were excised en bloc. The right lung lobes were ligated, removed, and immersed in TRI reagent and stored at −80°C until RNA was isolated. The left lung lobe was lavaged with 1 ml sterile saline, instilled through the tracheal cannula, and withdrawn to recover BALF. The left lung lobe was then inflated under constant pressure (30 cm H2O) with 10% neutral-buffered formalin (Sigma Chemical Co.) 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 the second study, the entire lung was immersed in TRI reagent and stored at −80°C until RNA was isolated.
RNA isolation
Total RNA was isolated from the lung using TRI reagent (Sigma Chemical Co.). The evaluation of the relative levels of H1 mRNA were determined using the TaqMan real-time multiplex RT-PCR with custom-designed TaqMan primers and probe to the target gene and the manufacturer's predeveloped primers and probe to 18S (Applied Biosystems, Foster City, CA, USA). The primers and probe to the target gene and endogenous reference gene were designed specifically to exclude detection of genomic DNA. Aliquots of isolated tissue RNA (1 μg total RNA) were converted to cDNA using random hexamers (Invitrogen, Carlsbad, CA, USA). 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 rRNA), and Taqman Universal Master Mix to a final volume of 30 μl. Following PCR, amplification plots (change in dye fluorescence vs. 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 comparative threshold cycle (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.
BALF cellularity
The total number of leukocytes in BALF was counted with a hemacytometer. In brief, 10 μl BALF supernatant was added to a hemacytometer, and the number of leukocytes in the four-etched corner quadrants was counted and multiplied by 2500 to yield the number of leukocytes per ml of sample. The percent of total leukocytes consisting of eosinophils, lymphocytes, monocytes, and neutrophils was determined from counts of 200 cells in a cytospin sample stained with Diff-Quick (Dade Behring, Newark, DE, USA). The percentage of eosinophils, lymphocytes, monocytes, and neutrophils was multiplied by the total number of leukocytes determined from the hemacytometer to yield the respective number of each cell type per ml of sample.
Total protein
Total protein was quantified in BALF using the bicinchoninic acid (BCA) method as provided by the manufacturer (Pierce, Rockford, IL, USA). In brief, BALF samples were centrifuged at 300 g for 5 min to pellet cellular debris. Supernatants were removed and stored at —20°C until testing was performed. BALF supernatant (25 μl) or BSA 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, BCA, and sodium tartrate in 0.1 M sodium hydroxide) and B (4% cupric sulfate) for 30 min at 37ºC. Samples were read on a Biotek microplate reader at 562 nm.
Inflammatory and TH1/TH2 cytokines
BALF samples were centrifuged at 300 g for 5 min to pellet cellular debris. BALF supernatants were 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 (CBA) mouse inflammation kit (BD PharMingen, San Diego, CA, USA), and the TH1/TH2 cytokines IL-2, IL-4, IL-5, IFN-γ, and TNF-α were detected simultaneously by using the mouse TH1/TH2 kit. In brief, 50 μl BALF supernatant from each sample was incubated individually with a mixture of capture beads and 50 μl PE detection reagent consisting of PE-conjugated anti-mouse IL-6, IL-10, MCP-1, IFN-γ, TNF-α, and IL-12p70 or 50 μl PE detection reagent consisting of PE-conjugated anti-mouse IL-2, IL-4, IL-5, IFN-γ, and TNF-α. The samples were incubated at room temperature for 2 h in the dark. After incubation, samples were washed once and resuspended in 300 μl wash buffer before analysis on a BD FACSCalibur flow cytometer. Data were analyzed using CBA software (BD PharMingen). Standard curves were generated for each cytokine using the mixed cytokine standard provided with the respective kits. The concentration of each cytokine was determined by interpolation from the corresponding standard curve that spanned concentrations ranging from 20 to 5000 pg/ml for each cytokine measured.
T-lymphocyte flow cytometry
After counting the number of cells retrieved in BALF, cells were pelleted by centrifugation at 300 g for 5 min, supernatants were removed, and cells were reconstituted in 150 μl flow cytometer (FCM) buffer [PBS supplemented with 2% (w/v) BSA and 0.09% (w/v) sodium azide] with purified anti-mouse CD16/CD32 (FcγRIII/II) antibody (BD Biosciences, San Jose, CA, USA) to block for 30 min. The samples were then divided into two groups of equal volume, washed, and reconstituted in FCM buffer. One group received antibodies directed against CD3 (APC anti-mouse CD3ε), CD4 [PE anti-mouse CD4 (L3T4)], and CD8 [FITC anti-mouse CD8a (Ly-2)], and the other group received the isotype control antibodies. Samples were allowed to incubate 1 h at 4°C, washed twice, and then fixed for 10 min with Cytofix (BD Biosciences). 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 3000.
Microdissection
The intrapulmonary airways of the fixed left lung lobe from each mouse were microdissected according to a modified version of the technique of Plopper et al. [25]. Beginning at the lobar bronchus, airways were 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. Tissue blocks were embedded in paraffin, sectioned at a thickness of 5 μm, and then stained with H&E for light microscopic examination. Other paraffin sections were stained with alcian blue (pH=2.5)/periodic acid Schiff (AB/PAS) to detect neutral and acidic mucosubstances in the respiratory epithelium lining the intrapulmonary-conducting airways (large and small diameter bronchioles).
Immunocytochemistry
Hydrated paraffin sections (5—6 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 peroxidase and then incubated with a polyclonal antibody to caspase-3 (CAS-3; Abcam, Cambridge, MA, USA) at 20 μg/ml for 1 h. Immunoreactive CAS-3 was visualized with the Vectastain Elite ABC kit (Vectastain Laboratories Inc., Burlingame, CA, USA) using 3′,3′-diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) as a chromagen.
Histopathology scores for inflammation
A histopathology score was established based on a previously established scoring system [5]. In brief, the scores assigned were: 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 large inflammatory foci found in the alveolar parenchyma. A board-certified pathologist scored the two left lung sections (generations 5 and 11) independently and without prior knowledge of treatment groups. A mean score with the SEM was calculated for each treatment group.
CAS-3 and mucosubstance numeric cell densities
Slides of lung sections stained immunohistochemically for CAS-3 or stained for mucosubstances (AB/PAS) 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 CAS-3-positive epithelial cells per mm basal lamina. In a similar manner, numeric cell densities were determined for epithelial cells staining with AB/PAS. The numeric cell density of epithelial cells staining for mucosubstances was expressed as the number of AB/PAS-positive epithelial cells per mm basal lamina.
Statistical analysis
Data are expressed as mean ± SEM. Outliers were identified and excluded from sample sets by using Grubb's test. The differences in outcomes between treatment groups within each genotype were analyzed by two-way ANOVA with multiple comparisons made by the Student Newman Keuls post hoc test using SigmaStat software, Version 2.03, from Jandel Scientific (San Rafael, CA, USA). Differences between treatment groups and genotypes were made using a Student's t-test. The criterion for significance was taken to be P < 0.05.
RESULTS
Qualitative health assessment of CB1−/−/CB2−/− and wild-type mice challenged with PR8
CB1−/−/CB2−/− and wild-type mice treated with corn oil or Δ9-THC and intranasally instilled with saline were normal in appearance and activity level. Infection with PR8 in the presence or absence of Δ9-THC treatment led to marked differences in the gross appearance of CB1−/−/CB2−/− and wild-type mice. Specifically, CB1−/−/CB2−/− mice were notably more gaunt in comparison with wild-type mice, suggesting that the mice were dehydrated. In addition, the CB1−/−/CB2−/− mice were lethargic and displayed unkempt fur. In contrast, wild-type mice infected with PR8 in the presence or absence of Δ9-THC treatment maintained normal grooming habits and exhibited similar activity levels as the saline-instilled controls. Upon gross examination of the lungs, CB1−/−/CB2−/− mice had extensive hemorrhage throughout all lung lobes. Wild-type mice also displayed evidence of hemorrhage, albeit to a much lesser extent than CB1−/−/CB2−/− mice.
The viral load of PR8 in the pulmonary airways of CB1−/−/CB2−/− and wild-type mice on Day 7 after challenge
To estimate the viral load of PR8 infection, the levels of mRNA for the viral surface protein, H1, were assessed by quantitative real-time PCR. For CB1−/−/CB2−/− and wild-type mice, viral H1 mRNA was elevated markedly above detection levels set by saline controls at 7 dpi in the lungs of all mice challenged with PR8 (Fig. 1 ). However, vehicle-treated wild-type mice exhibited markedly greater viral H1 mRNA levels than vehicle-treated CB1−/−/CB2−/− mice. Infection of Δ9-THC-treated CB1−/−/CB2−/− mice with PR8 resulted in an increase (P=0.056) in H1 mRNA levels when compared with mice instilled with PR8 alone. In Δ9-THC-treated wild-type mice infected with PR8, there was a significant increase in viral H1 mRNA levels when compared with wild-type mice instilled with PR8 alone. The overall level of expression of H1 mRNA was markedly less in CB1−/−/CB2−/− mice when compared with wild-type mice in the absence or presence of Δ9-THC treatment.
Δ9-THC enhanced vascular permeability induced by PR8 infection of the airways in CB1−/−/CB2−/− and wild-type mice
The detection of increased levels of total BALF protein is indicative of increased vascular permeability at the alveolar/capillary interface during the inflammatory response to PR8. The total BALF protein levels in PR8-infected CB1−/−/CB2−/− and wild-type mice, in the absence of Δ9-THC treatment, were two-fold greater than saline controls (Fig. 2 ). Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 exhibited a 33% increase in total BALF protein when compared with CB1−/−/CB2−/− mice challenged with PR8 alone. Conversely, Δ9-THC-treated wild-type mice infected with PR8 had a 50% decrease in total BALF protein when compared with wild-type mice challenged with PR8 alone. When comparing CB1−/−/CB2−/− and wild-type mice, the amount of total protein detected in mice treated with Δ9-THC and infected with PR8 was two-fold greater in CB1−/−/CB2−/− mice than amounts of total protein observed in wild-type mice receiving the same treatment.
CB1−/−/CB2−/− mice treated with Δ9-THC exhibit a distinctly different composition of leukocytes recruited to the airways in response to PR8 infection when compared with wild-type mice
Primary influenza infection elicits an inflammatory response consisting of a mixed population of leukocytes that infiltrates the pulmonary airways. In particular, there is a marked influx of neutrophils and lymphocytes into the airways with monocytes and eosinophils, representing a smaller portion of the total population of BALF leukocytes. In the current study, the total number of leukocytes in BALF was three-fold greater in PR8-infected wild-type mice and four-fold greater in CB1−/−/CB2−/− mice than in their respective noninfected controls (Fig. 3A ). Δ9-THC-treated mice infected with PR8 exhibited a two-fold increase above saline-instilled controls in CB1−/−/CB2−/− and wild-type mice. However, the difference in total leukocytes retrieved in the Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 was significantly less than CB1−/−/CB2−/− mice challenged with PR8 alone. To further assess differences in individual leukocyte populations retrieved by lavage, differential cell counts were performed. Neutrophils (Fig. 3B) were increased markedly in PR8-infected CB1−/−/CB2−/− and wild-type mice. Specifically, Δ9-THC-treated wild-type mice infected with PR8 exhibited two-fold increases in neutrophils when compared with wild-type mice challenged with PR8 alone. Conversely, there were marked decreases in the number of BALF neutrophils for PR8-infected CB1−/−/CB2−/− mice treated with Δ9-THC when compared with CB1−/−/CB2−/− mice challenged with PR8 alone. BALF lymphocytes were increased markedly (Fig. 3C) with PR8 infection in CB1−/−/CB2−/− and wild-type mice. Δ9-THC-treated CB1−/−/CB2−/− and wild-type mice infected with PR8 exhibited attenuation in the number of lymphocytes retrieved. There was no change in the number of monocytes retrieved in BALF from PR8-challenged, wild-type mice as compared with saline controls. A two-fold increase was observed, however, in the number of BALF monocytes from PR8-infected CB1−/−/CB2−/− mice that was attenuated in Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 (Fig. 3D) . Lastly, PR8 infection of CB1−/−/CB2−/− and wild-type mice resulted in an increase in the number of eosinophils retrieved in BALF (Fig. 3E) . Δ9-THC treatment did not affect the number of eosinophils in BALF induced by PR8.
BALF CD4+ and CD8+ T cell levels following PR8 challenge in CB1−/−/CB2−/− and wild-type mice
To evaluate the contribution of CD4+ and CD8+ T cells within the pool of BALF lymphocytes responding to PR8 challenge, the number of CD4+ T cells (Fig. 4A ) and CD8+ T cells (Fig. 4B) was enumerated by flow cytometry. Background levels of CD4+ T cells were greater in corn oil and Δ9-THC-treated CB1−/−/CB2−/− mice than in wild-type mice with the same treatments. There were no observed differences in the number of CD4+ T cells retrieved in BALF between saline-instilled controls and CB1−/−/CB2−/− mice infected with PR8 alone. Alternatively, Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 had significantly increased numbers of CD4+ T cells above background. The increase in BALF-associated CD4+ T cells in CB1−/−/CB2−/− mice treated with Δ9-THC and infected with PR8 was comparatively greater than the respective treatment group in wild-type mice. CD8+ T cells were not detected in BALF from saline-instilled controls in CB1−/−/CB2−/− and wild-type mice. However, there were marked increases in the number of BALF-associated CD8+ T cells for CB1−/−/CB2−/− and wild-type mice challenged with PR8. In addition, there was a trend toward decreased numbers of BALF-associated CD8+ T cells in Δ9-THC-treated wild-type mice infected with PR8.
CB receptor-deficient mice exhibit unique differences in epithelial and leukocytic chemokine and cytokine secretion in the pulmonary airways
One mode of cellular communication within a mixed population of leukocytes and between infected epithelium and leukocytes is through secretion of cytokines and chemokines. PR8-infected wild-type and CB1−/−/CB2−/− mice had significant increases in levels of the chemokine MCP-1 (Fig. 5A ) and proinflammatory cytokines TNF-α (Fig. 5B) , IL-6 (Fig. 5C) , and IFN-γ (Fig. 5D) measured in BALF. In addition, there was a modest enhancement of IL-10 concentrations (Fig. 5E) following PR8 challenge in CB1−/−/CB2−/− and wild-type mice. There were no changes in IL-12p70 in BALF in the presence or absence of Δ9-THC treatment or PR8 challenge (Fig. 5F) in CB1−/−/CB2−/− or wild-type mice. Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 had an attenuation of IFN-γ and IL-10 concentrations in BALF as compared with corn oil-treated mice infected with PR8. Conversely, the concentrations of chemokines and cytokines detected in BALF in PR8-infected wild-type mice were unaffected by Δ9-THC treatment. There were, however, comparative differences between detectable levels of TNF-α and IFN-γ (P=0.061) in BALF of PR8-infected wild-type and CB1−/−/CB2−/− mice.
The TH2 cytokines IL-2 (Fig. 6A ), IL-4 (Fig. 6B) , and IL-5 (Fig. 6C) were also quantified in BALF by CBA. Of particular interest, PR8- infected CB1−/−/CB2−/− mice had marked increases in concentrations of IL-5, which were comparatively two-fold less than concentrations observed in the respective PR8-infected wild-type mice. The concentrations of IL-2 and IL-4 in BALF were unaffected by PR8 challenge in CB1−/−/CB2−/− mice. Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 had modestly attenuated concentrations of IL-2 in BALF but had no effect on the concentrations of IL-4 or IL-5. PR8-infected wild-type mice had marked increases in IL-5 concentrations in BALF, mild increases in the concentrations of IL-2, and no detectable differences for IL-4 when compared with saline-instilled mice on 7 dpi. Δ9-THC-treated wild-type mice infected with PR8 had a modest attenuation of IL-2 concentrations in BALF with no observed effect on IL-4 or IL-5 concentrations.
The absence of CB receptors CB1 and CB2 enhances the observed pulmonary histopathology
As reported previously by our laboratory, infection of mice with PR8 induced significant pulmonary inflammation 7 dpi in wild-type mice (Fig. 7A 7B 7C 7D ) [5]. Infection of the CB1−/−/CB2−/− mice with PR8 resulted in a similar inflammatory reaction (Fig. 7E) . As we have reported, the inflammation consisted of primarily lymphocytes and neutrophils with fewer monocytes and plasma cells centered upon the bronchoalveolar junction, which extended out into the surrounding parenchyma. Treatment of PR8-infected wild-type mice with Δ9-THC resulted in a significant decrease in inflammation 7 dpi compared with corn oil-treated mice infected with PR8 (Fig. 7D) . These mice often had no to few inflammatory cells present; the remaining inflammation was comprised of primarily lymphocytes and alveolar monocytes. In contrast, treatment of PR8-infected CB1−/−/CB2−/− mice with Δ9-THC resulted in a vigorous, inflammatory and cellular reaction in the mice (Fig. 7F) , where there were large numbers of mature lymphocytes around the conducting airways and extended into the surrounding alveolar parenchyma, where they were admixed with fewer monocytes and neutrophils. The bronchiolar epithelium was moderately hypertrophied. Treatment of wild-type and CB1−/−/CB2−/− mice with corn oil vehicle or Δ9-THC alone did not result in significant histologic changes in any of the lung sections examined.
Δ9-THC affects the magnitude of the inflammatory response to PR8 in wild-type and CB1- and CB2-deficient mice
The magnitude and severity of inflammation observed in tissue sections isolated from the left lung lobe were scored independently (0—3, where 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 large, inflammatory foci found in the alveolar parenchyma) and compared between treatment groups and wild-type and CB1−/−/CB2−/− mice at 7 dpi (Fig. 8 ). There was no inflammation observed in the lungs of wild-type mice intranasally instilled with saline. However, two out of eight CB1−/−/CB2−/− mice instilled with saline had a modest inflammation present. Corn oil-treated mice infected with PR8 had marked inflammation scores in wild-type and CB1−/−/CB2−/− mice as compared with uninfected control mice. Δ9-THC-treated wild-type mice challenged with PR8 had a marked suppression of the inflammatory response in the conducting airways. In contrast, Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 had a trend toward increased (P=0.079) inflammation of the pulmonary airways.
Effects of CB1 and CB2 deficiency on the numeric cell densities of apoptotic cells and metaplastic goblet cells
Bronchiolar epithelial cell apoptosis in influenza-infected mice is directed by cell-mediated immune responses to virally infected cells [26]. The numeric cell density for the apoptotic cell marker CAS-3 (Fig. 9 ) was measured in the epithelium lining generation 5 of the main axial airway. CAS-3 numeric cell densities were not distinguishably different between treatment groups in wild-type mice. In PR8-infected CB1−/−/CB2−/− mice, CAS-3-labeled cells were modestly increased above saline-instilled controls but were not different than cell densities enumerated for CB1−/−/CB2−/− mice infected with PR8 and treated with Δ9-THC. During the recovery from viral infection, a metaplastic change occurs in the epithelium lining the bronchi that includes increased numbers of mucus-producing goblet cells [27]. Consistent with our previous finding [5], there was no difference in the numeric cell density of AB/PAS-positive mucous cells (Fig. 10 ) quantified at 7 dpi in wild-type mice with any treatment. Interestingly, Δ9-THC-treated CB1−/−/CB2−/− mice, in the presence or absence of PR8 infection, had significant increases in the numeric cell density for AB/PAS-positive epithelial cells as compared with wild-type mice.
DISCUSSION
Δ9-THC administration has been reported to decrease host resistance to bacterial and viral pathogens in mouse models [2 3 4 5]. However, the role of the CB receptors CB1 and CB2 in altered immune competence to pathogen challenge remains poorly understood. The CB receptor CB1 antagonist SR141716A and CB2 receptor antagonist SR144528 have been used to study CB1/CB2-specific effects elicited by CB treatment, including efforts to uncover the CB1/CB2 receptors involvement in Δ9-THC suppression of cytokine production elicited by pathogen challenge [28], but the specificity of these pharmacologic agents and their putative off-target effects are always of concern. The objective of the present study was to use CB1/CB2 receptor null mice to rigorously evaluate previously characterized immunologic and pathologic responses to PR8 infection in the presence and absence of Δ9-THC treatment [5]. Results from this study indicated that Δ9-THC treatment of wild-type and CB1−/−/CB2−/− mice affected epithelial and immune cell responses to PR8 that were dependent and independent of the CB1/CB2 receptors as summarized in Table 1 . Perhaps even more important was the observation that CB1/CB2 null mice exhibited a significantly enhanced response to PR8 infection regardless of CB treatment, strongly suggesting an endogenous role for CB1 and/or CB2 in immune regulation. This finding provides a novel extension to our current understanding of host immunity and the relationship between a normal, "homeostatic" immune response regulated by CB receptor-linked signaling pathways and hyper-responsive, "unchecked" immunity compromised by the absence of the CB1/CB2 receptors.
In the current study, Δ9-THC was administered by oral gavage. As we have reported previously, the oral route of administration was selected as a result of the fact that Δ9-THC is a highly lipophilic molecule requiring a nonaqueous diluent for drug delivery, which has the potential for inducing irritation and damage to the airways. Conversely, oral administration of Δ9-THC in corn oil is well tolerated but poorly absorbed from the gastrointestinal tract. Oral administration of 75 mg/kg Δ9-THC for 5 consecutive days resulted in a serum concentration of 66.2 ng/ml of the parent compounds, 446.5 ng/ml of the 9-carboxyl metabolite, and 10.5 ng/ml of the 11-hydroxyl metabolite, 4 h after the last Δ9-THC dose [5]. The levels of Δ9-THC observed systemically correlate well with a previous report by Azorlosa and co-workers [29], in which peak human plasma levels ranged from 57 to 268 ng/ml. The rationale for the dosing paradigm of PR8 and Δ9-THC was to investigate the putative effects of Δ9-THC treatment on the immune response to PR8 during the early stages of the primary infection.
To assess whether the CB receptors CB1 and CB2 are critical to the cell-mediated immune response that clears influenza from the lungs, we compared the levels of H1 mRNA in lungs from wild-type and CB1−/−/CB2−/− mice. In the current study, we observed H1 mRNA levels in the lungs of Δ9-THC-treated CB1−/−/CB2−/− and wild-type mice challenged with PR8 that were greater than H1 mRNA levels in CB1−/−/CB2−/− and wild-type mice challenged with PR8 alone. Interestingly, H1 mRNA levels were reduced in the lungs of CB1−/−/CB2−/− mice by two orders of magnitude when compared with H1 mRNA levels observed for wild-type mice. The measurement of H1 mRNA serves as a marker of transcriptional activation of the influenza virus genome that could result from increases in viral copy number or increases in transcriptional activation. Whether Δ9-THC is exerting an effect on copy number or transcriptional activation or both is not presently known. It is tempting to speculate, however, that H1 mRNA levels are related to copy number, as there are marked differences in the magnitude of the inflammatory response to PR8 observed by histopathology. The profound reduction in H1 mRNA levels also suggests that the kinetics of the cell-mediated immune response to PR8 is markedly enhanced in CB1−/−/CB2−/− mice. The suggested hyper-responsive immunity toward PR8 infection brought about by the absence of functional CB receptors CB1 and CB2 is supported by our findings, in the current study, of increased background levels of CD4+ T cells in CB1−/−/CB2−/− mice in combination with a more vigorous and extensive inflammation of the alveolar parenchyma. Recently, Karsak and co-workers [24] reported a more vigorous, allergic response to the contact allergen 2,4-dinitroflourobenzene in CB1−/−/CB2−/− mice when compared with wild-type mice. The current study and that reported by Karsak and co-workers [24] suggest that the CB receptors CB1 and CB2 may play a crucial role in maintaining immune homeostasis and controlling the magnitude of the immune response through negative regulation. It is tempting to speculate that endoCBs may naturally provide a mechanism to temper the immune response. The absence of CB1 and CB2 receptors appears to potentially result in an enhanced immune response under certain conditions.
We have previously observed decreased recruitment of monocytes and lymphocytes to the airways in Δ9-THC-treated mice challenged with PR8 [5]. This observation was consistent with the suppressive effects of CBs on leukocyte chemotaxis reported by others [30 31 32 33]. To clarify whether mice lacking functional CB1/CB2 receptors exhibit patterns of leukocyte recruitment to the airways in response to viral challenge that are modulated by Δ9-THC in a CB1/CB2 receptor-independent manner, total and differential cell counts from BALF were assessed. In the current study, the magnitude of total leukocyte recruitment to the pulmonary airways and the cellular subsets present were similar between CB1−/−/CB2−/− and wild-type mice challenged with PR8, suggesting that mice lacking functional CB1/CB2 receptors remained capable of mounting an aggressive immune response toward PR8. Differential cell counts provided evidence that the immune response to PR8 by CB1−/−/CB2−/− mice was more robust than the immune response mounted against PR8 in wild-type mice. More specifically, in the BALF, monocytes and neutrophils were decreased in Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8. The decreased recruitment of monocytes and neutrophils to the airways might point to a CB receptor-dependent effect on the chemotaxis of these leukocytes. Contrary to findings with monocytes and neutrophils, there were remarkable similarities in the total number of lymphocytes in BALF among all treatment groups for CB1−/−/CB2−/− and wild-type mice. Upon further examination of the T cell subsets retrieved in BALF, there was evidence that the T cell response to PR8 challenge with Δ9-THC treatment involved predominantly CD4+ T cells in CB1−/−/CB2−/− mice. As mentioned previously, increased numbers of CD4+ T cells might suggest hyper-responsive immunity in CB1−/−/CB2−/− mice. However, the balance of the CD4+ TH subset (TH1 cell-mediated immunity vs. TH2 humoral immunity) remains unclear.
We have previously shown that PR8 challenge of the pulmonary airways leads to marked increases in total protein and chemokine/cytokines MCP-1, IL-6, TNF-α, and IFN-γ in BALF [5]. Δ9-THC and other CBs have been reported to modulate the expression and/or secretion of these chemokines and cytokines [20 , 33 34 35 36]. In the current study, we observed increased total protein in the BALF of wild-type and CB1−/−/CB2−/− mice challenged with PR8. Comparatively, a marked difference existed between Δ9-THC-treated CB1−/−/CB2−/− and wild-type mice infected with PR8, suggesting an increased severity in vascular leakiness or inflammatory cell secretions in CB1−/−/CB2−/− mice. Consistent with increased amounts of total protein in BALF, inflammation scores assessed by histopathology accurately reflected changes observed in vascular leakiness. In addition to total protein, we evaluated chemokines/cytokines in BALF. Although there were trends toward increases in BALF chemokines and cytokines (e.g., MCP-1, TNF-α, and IFN-γ) from CB1−/−/CB2−/− mice infected with PR8, in the absence of Δ9-THC, the collective findings did not provide evidence for CB1 and/or CB2 receptor-dependent or -independent regulation of the secretion of these cytokines. Chemokine and cytokine levels should be interpreted carefully, as they represent a concentration in the airways at a given moment in time with respect to an ever-changing, dynamic inflammatory process. It is difficult to speculate in the current study as to whether differences observed between treatment and control groups represent trends toward increased or decreased expression or secretion of chemokines or cytokines. Moreover, the cytokines measured in BALF are pleiotropic and redundant, as they may be derived from multiple immune cell types. Not surprisingly, cytokines and chemokines are regulated in their own unique manner with their own distinct kinetics, and the molecular mechanism by which certain cytokines and chemokines are further modulated by CBs is only partially understood. It has been suggested that Δ9-THC treatment modulates cytokine production, resulting in decreased TH1 cell-mediated immunity and increased TH2 humoral immunity [37 , 38]. As CB1−/−/CB2−/− mice had a marked, lymphocytic response consisting of greater numbers of CD4+ T cells, and Δ9-THC treatment of CB1−/−/CB2−/− mice challenged with PR8 resulted in decreased concentrations of IFN-γ, the concentrations of the TH2 cytokines IL-2, IL-4, and IL-5 were also evaluated in BALF. Only the cytokine IL-5 was influenced by PR8 infection in wild-type mice in the presence or absence of Δ9-THC. IL-5 concentrations were reduced in PR8-infected CB1−/−/CB2−/− mice in the presence or absence of Δ9-THC, and Δ9-THC treatment of CB1−/−/CB2−/− or wild-type mice infected with PR8 exhibited no effect on the levels of IL-5 when compared with CB1−/−/CB2−/− or wild-type mice infected with PR8 alone. Our results suggest that the increased numbers of CD4+ T cells observed in CB1−/−/CB2−/− mice were not actively secreting more TH2-type cytokines than wild-type mice and that there did not appear to be an imbalance between TH1-type and TH2-type cytokines secreted as a result of Δ9-THC treatment in response to PR8.
To evaluate the extent of the inflammatory response to PR8 in CB1−/−/CB2−/− mice as compared with wild-type controls, lung sections were taken at the levels of the 5th (proximal) and 11th (distal) airway generations, bifurcating away from the main axial airway of the left lung lobe and scored for the magnitude and severity of inflammation. Within saline-instilled wild-type mice, there was no inflammation observed. Conversely, in two out of eight CB1−/−/CB2−/− mice instilled with saline, in the presence or absence of Δ9-THC treatment, there was modest inflammation. The CB1−/−/CB2−/− mice were subjected to an extensive battery of serological screens prior to their use and were found to be negative for all pathogens tested. In a separate, unpublished study, two untreated CB1−/−/CB2−/− mice were necropsied and examined for microscopic evidence of lesions in the brain, eye, conjunctiva, myocardium, lung, stomach, small intestine, large intestine, liver, spleen, testicle, and nasal cross sections. Within the lungs, there was a mild interstitial, inflammatory infiltrate composed of lymphocytes and plasma cells. In addition, there were multifocal regions of atelectasis, which is the incomplete expansion of the lungs or the collapse of lung parenchyma, producing an area of relatively airless pulmonary parenchyma. Atelectatic parenchyma is prone to developing superimposed infections, which may partially account for the more vigorous, inflammatory responses observed within CB1−/−/CB2−/− mice. Regardless, inflammation scores were similar for lung sections from CB1−/−/CB2−/− and wild-type mice challenged with PR8, in the absence of Δ9-THC treatment, and the histopathology for PR8-infected CB1−/−/CB2−/− and wild-type mice had a similar pattern of inflammatory cells infiltrating the lung. In Δ9-THC-treated wild-type mice challenged with PR8, there was a marked reduction in the inflammation score, suggesting that Δ9-THC altered the kinetics of leukocyte chemotaxis to the airways in response to PR8. This is supported by the marked reduction or absence of inflammatory cells in the submucosa and alveolar parenchyma. In contrast, inflammation scores for lung sections from Δ9-THC-treated CB1−/−/CB2−/− mice challenged with PR8 were modestly enhanced with respect to scores assessed for lung sections from CB1−/−/CB2−/− mice challenged with PR8 alone. Indeed, CB1−/−/CB2−/− mice treated with Δ9-THC and infected with PR8 represented some of the most severely affected mice in the study and exhibited more vigorous, inflammatory responses than even observed in CB1−/−/CB2−/− mice challenged with PR8 alone.
We have previously demonstrated that epithelial changes in the pulmonary airways of mice challenged with PR8 included increased evidence for apoptosis and MCM following PR8 challenge, which was suppressed by Δ9-THC [5]. Cell death pathways initiated by cytotoxic T cells or NK cells or by the virus itself influence CAS-3 activation [39 , 40]. In the comparison of wild-type and CB1−/−/CB2−/− mice, PR8 challenge led to increased CAS-3 staining; however, there were no effects observed with Δ9-THC administration. In a similar manner, MCM was quantified by counting AB/PAS-positive epithelial cells lining the main axial airway at G5 of the left lung lobe. We have previously shown that goblet cells producing mucosubstances in the airway epithelium increase substantially by 10 dpi in wild-type mice. In the current study, we found that CB1−/−/CB2−/− mice had significantly greater numbers of goblet cells lining the airways at G5 when treated with Δ9-THC alone at 7 dpi. This finding suggests that Δ9-THC is interacting directly with a signaling pathway tied with increased mucin production in CB1−/−/CB2−/− mice or that exposure of CB1−/−/CB2−/− mice to Δ9-THC is handled by the host as an allergen leading to an alternative, hyper-responsive CD4+ TH2 T cell response that is capable of eliciting MCM.
In conclusion, Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 exhibited more robust immune responses than wild-type mice receiving the respective treatment. Moreover, CB1−/−/CB2−/− mice infected with PR8 had markedly decreased levels of viral mRNA as compared with wild-type mice. Furthermore, Δ9-THC treatment increased viral load when compared with mice infected with PR8 alone, irrespective of the presence or absence of CB1 and CB2 receptors. Therefore, the current study demonstrated that Δ9-THC influenced the immune response to influenza infection in a manner that was dependent and independent of the CB1/CB2 receptors. Future studies will examine the effect of Δ9-THC on viral infection of lung parenchymal cells and establish the kinetics of lymphocyte, monocyte, and neutrophil chemotaxis to the lungs of PR8-infected CB1−/−/CB2−/− mice.
Source, Graphs and Figures: Targeted deletion of cannabinoid receptors CB1 and CB2 produced enhanced inflammatory responses to influenza A/PR/8/34 in the absence and presence of
We have previously reported that Δ-9-tetrahydrocannabinol (Δ9-THC)-treated mice challenged with influenza virus A/PR/8/34 (PR8) developed increased viral hemagglutinin 1 (H1) mRNA levels and decreased monocyte and lymphocyte recruitment to the pulmonary airways when compared with mice challenged with PR8 alone. The objective of the present study was to examine the role of cannabinoid (CB1/CB2) receptors in mediating the effects of Δ9-THC on immune and epithelial cell responses to PR8. In the current study, Δ9-THC-treated CB1/CB2 receptor null (CB1−/−/CB2−/−) and wild-type mice infected with PR8 had marked increases in viral H1 mRNA when compared with CB1−/−/CB2−/− and wild-type mice challenged with PR8 alone. However, the magnitude of the H1 mRNA levels was greatly reduced in CB1−/−/CB2−/− mice as compared with wild-type mice. In addition, Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 had increased CD4+ T cells and IFN-γ in bronchoalveolar lavage fluid with greater pulmonary inflammation when compared with Δ9-THC-treated wild-type mice infected with PR8. Δ9-THC treatment of CB1−/−/CB2−/− mice in the presence or absence of PR8 challenge also developed greater amounts of mucous cell metaplasia in the affected bronchiolar epithelium. Collectively, the immune and airway epithelial cell responses to PR8 challenge in Δ9-THC-treated CB1−/−/CB2−/− and wild-type mice indicated the involvement of CB1/CB2 receptor-dependent and -independent mechanisms.
INTRODUCTION
In humans, the primary routes of exposure to Δ-9-tetrahydrocannabinol (Δ9-THC) occur through inhalation as a complex mixture of chemicals, including cannabinoid (CB) compounds and pyrolysis products of smoked marijuana or via oral consumption, which can include a synthetically derived therapeutic for the treatment of AIDS-induced wasting or chemotherapy-induced emesis (e.g., dronabinol). Despite its highly debated therapeutic use, it is well established that the plant-derived CB Δ9-THC has immunosuppressive properties. CBs, such as Δ9-THC, have been found to increase host susceptibility to pathogens in a number of in vivo experimental models [1], including HSV [2 , 3], Listeria monocytogenes [2], Legionella pneumophila [4], and more recently, influenza virus A/PR/8/34 (PR8) [5]. The mechanism for increased susceptibility to pathogens appears to be correlated with the suppression of cell-mediated immune responses by Δ9-THC, which suppresses immune cell function by decreasing cytokine secretion and expression of costimulatory molecules by APC [6], suppressing the migration of monocytes and T cell subsets (CD4+ and CD8+ T cells) into the infected tissue [5], altering the cytoplasmic polarization of cytolytic T cells toward pathogen-infected cells [3], and suppressing the release of cytokines, such as IL-12 and IFN-γ, which are essential for competent, cell-mediated immunity regulated by TH1 cells [7]. Additionally, in a mouse model of allergic airway disease, Δ9-THC attenuated the TH2 cytokine response to an inhaled allergen, decreasing mRNA expression of the cytokines IL-2, IL-4, IL-5, and IL-13 elicited by OVA challenge [8]. Production of IL-2 is especially crucial to T cell-dependent responses via its role in T cell clonal expansion. Accordingly, Δ9-THC and other CB compounds have been shown to suppress lymphocyte proliferation in vitro [9] as well as markedly suppress IL-2 expression, in part, through inhibition of the NFAT [10 , 11]. The precise mechanism by which Δ9-THC modulates APC and T cell function as well as the role of CB1 and CB2 have remained elusive.
We have recently reported [5] that Δ9-THC increased host susceptibility to influenza virus PR8, a hemagluttinin 1, neuraminadase 1 (H1N1) influenza strain, in the C57BL/6 mouse. Influenza virus is a common respiratory pathogen that infects epithelial cells lining the respiratory tract, as well as mononuclear cells [12]. During the course of infection, inflammatory cells comprised primarily of lymphocytes and neutrophils infiltrate the pulmonary airways [13]. Viral clearance is achieved through immunocompetent, virus-specific T cell responses. We have recently reported that treatment of mice with increasing doses of Δ9-THC led to a dose-dependent decrease in recruitment of monocytes and lymphocytes (CD4+ and CD8+ T cells) with a concomitant increase in viral load at 7 days postinfection (dpi) [5]. These results suggest that Δ9-THC disrupted cell-mediated immune responses that are critically involved in viral clearance and recovery from influenza infection.
Two subtypes of CB receptors have been characterized: CB1 (brain receptor) and CB2 (peripheral receptor). The CB2 receptor is predominantly expressed in cells of the immune system, including neutrophils, monocytes, NK cells, T cells, and B cells [14 , 15], and has been thought to mediate many of the immunomodulatory effects produced by CBs. In particular, the CB-induced inhibition of antigen processing and presentation in macrophage [16 , 17] and antitumor activity in a murine model of lung cancer [18] has been shown to be mediated by the CB2 receptor. There is, however, emerging evidence for CB-mediated effects on cytokine production pertinent to T-dependent humoral and cell-mediated immune responses that occur independently of CB1/CB2 receptors. Specifically, the suppression of IL-2 production by a variety of immunosuppressive CBs is not attenuated by the CB receptor antagonists SR141716A and SR144528 [19]. Likewise, the endogenous CB (endoCB), 2-arachidonyl glycerol, suppressed PMA/ionomycin-elicited IFN-γ expression similarly in splenocytes obtained from CB receptor null mice (CB1−/−/CB2−/−) as compared with splenocytes from wild-type mice [20]. Additional evidence for CB receptor-independent effects of CBs is illustrated by the plant-derived CB, cannabidiol, which possesses weak affinity for the CB receptors CB1 and CB2, yet exhibits potent anti-inflammatory activity including the reduced production of PGE2, NO and other oxygen-derived free radicals in response to carrageenan-induced inflammation [21], inhibition of the release of IL-1, TNF-α, and IFN-γ by PBMC [22], and suppressed MIP-1α, MIP-1β, and IL-8 in the human T cell lymphotropic virus 1 B cell line [23]. Collectively, these studies illustrate different aspects of host immunity that are CB receptor (CB1 and CB2)-dependent and -independent.
The objective of the present study was to evaluate the role of CB1 and CB2 receptors in the suppression of cell-mediated immunity in mice infected with PR8 by Δ9-THC. Accordingly, CB1−/−/CB2−/− mice and background strain C57BL/6 mice were compared by using a mouse model of host resistance to infection with murine-adapted PR8. Measurements of viral load and immune cell recruitment and secretion of cytokines in the airways were determined. In addition, previously characterized, morphologic changes in airway epithelium, including apoptosis and mucous cell metaplasia (MCM), were quantified as a measure of viral and immune cell-mediated, pathologic outcomes [5]. Results from this study suggest that Δ9-THC is modulating immunological and pulmonary airway responses to influenza virus through CB receptor (CB1 and CB2)-dependent and -independent mechanisms.
MATERIALS AND METHODS
Animals
Two independent experiments were conducted as a part of this study. In the first experiment, 32 female C57BL/6 mice (8—10 weeks old, from Charles River, Portage, MI, USA) and 32 female CB1−/−/CB2−/− mice (8—16 weeks old), bred at Michigan State University (MSU; East Lansing, MI, USA) from breeding pairs provided by Dr. Andreas Zimmer [24], were randomly assigned to one of four experimental groups (n=8 per group) by genotype. The mice were transferred to plastic cages containing sawdust bedding (four mice per cage) and quarantined for 1 week in an animal holding room that was maintained at 21—24°C and 40—60% relative humidity with a 12-h light/dark cycle. The mice were provided food (Purina Certified Laboratory Chow) and water ad libitum and not used for experimentation until their body weight was 17—20 g. In the second experiment, 30 female C57BL/6 mice (8—10 weeks old) and 34 female CB1−/−/CB2−/− mice (8—16 weeks old) were randomly assigned to one of four experimental groups (n=5—14 per group) by genotype. CB1−/−/CB2−/− mice were subjected to an extensive battery of serological screens prior to breeding, as well as designated offspring sentinels after breeding. Tests for all pathogens assayed were negative. All mice used in this study were kept free of pathogens and respiratory disease and used in accordance with guidelines set forth by the Institutional Animal Care and Use Committee at MSU.
Δ9-THC
Δ9-THC was provided by the National Institute on Drug Abuse (Bethesda, MD, USA) as a resin, which was solubilized in corn oil at concentrations that yielded a dose of 75 mg/kg for administration to mice by oral gavage in a volume of 100 μl.
Influenza PR8 instillation
The laboratory of Dr. Alan Harmsen (Montana State University, Bozeman, MT, USA) generously supplied influenza PR8. Mice were anesthetized with 4% isoflurane in oxygen, and 50 μl PR8 in pyrogen-free saline was instilled as 25 μl per nare at a total dose of 50 pfu.
Experimental design
In both experiments, mice were orally administered Δ9-THC for 5 consecutive days at a dose of 75 mg/kg. On the 3rd day of dosing, mice were instilled intranasally with influenza virus (50 pfu) 4 h prior to Δ9-THC administration. All mice were killed at 7 dpi. In the first experiment, the right lung lobes were ligated and removed for RNA analysis, and the left lung lobe was lavaged with saline for cellular and biochemical measurements and then fixed in 10% neutral-buffered formalin for immunohistochemical analysis as described below. In the second experiment, the entire lung was immersed in TRI reagent (Sigma Chemical Co, St. Louis, MO, USA) for RNA isolation and analysis.
These experiments required the breeding of a sufficient number of female mice to support a large n value per treatment group. As breeding was a limiting factor in the design of these experiments, it was deemed necessary in the initial experiment to divide the right and left lung lobes to perform mRNA, bronchoalveolar lavage fluid (BALF), and immunohistochemical measurements. The assumption was made that the virus and associated immune response would be equally distributed between the right and left lung lobes and that there would be uniform and most importantly, discernible differences between treatment with Δ9-THC or vehicle. The trends in the results were similar to those observed previously [5]; however, a second experiment was performed to provide clarity in our measurement of viral hemagglutinin 1 (H1) mRNA for whole lung homogenates.
Necropsy, lavage collection, and tissue preparation
Mice were first anesthetized by an i.p. injection of 0.1 ml 12% pentobarbital solution, and then a midline laparotomy was performed, and mice were exsanguinated by cutting the abdominal aorta. Immediately after death, the trachea was exposed and cannulated, and the heart and lung were excised en bloc. The right lung lobes were ligated, removed, and immersed in TRI reagent and stored at −80°C until RNA was isolated. The left lung lobe was lavaged with 1 ml sterile saline, instilled through the tracheal cannula, and withdrawn to recover BALF. The left lung lobe was then inflated under constant pressure (30 cm H2O) with 10% neutral-buffered formalin (Sigma Chemical Co.) 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 the second study, the entire lung was immersed in TRI reagent and stored at −80°C until RNA was isolated.
RNA isolation
Total RNA was isolated from the lung using TRI reagent (Sigma Chemical Co.). The evaluation of the relative levels of H1 mRNA were determined using the TaqMan real-time multiplex RT-PCR with custom-designed TaqMan primers and probe to the target gene and the manufacturer's predeveloped primers and probe to 18S (Applied Biosystems, Foster City, CA, USA). The primers and probe to the target gene and endogenous reference gene were designed specifically to exclude detection of genomic DNA. Aliquots of isolated tissue RNA (1 μg total RNA) were converted to cDNA using random hexamers (Invitrogen, Carlsbad, CA, USA). 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 rRNA), and Taqman Universal Master Mix to a final volume of 30 μl. Following PCR, amplification plots (change in dye fluorescence vs. 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 comparative threshold cycle (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.
BALF cellularity
The total number of leukocytes in BALF was counted with a hemacytometer. In brief, 10 μl BALF supernatant was added to a hemacytometer, and the number of leukocytes in the four-etched corner quadrants was counted and multiplied by 2500 to yield the number of leukocytes per ml of sample. The percent of total leukocytes consisting of eosinophils, lymphocytes, monocytes, and neutrophils was determined from counts of 200 cells in a cytospin sample stained with Diff-Quick (Dade Behring, Newark, DE, USA). The percentage of eosinophils, lymphocytes, monocytes, and neutrophils was multiplied by the total number of leukocytes determined from the hemacytometer to yield the respective number of each cell type per ml of sample.
Total protein
Total protein was quantified in BALF using the bicinchoninic acid (BCA) method as provided by the manufacturer (Pierce, Rockford, IL, USA). In brief, BALF samples were centrifuged at 300 g for 5 min to pellet cellular debris. Supernatants were removed and stored at —20°C until testing was performed. BALF supernatant (25 μl) or BSA 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, BCA, and sodium tartrate in 0.1 M sodium hydroxide) and B (4% cupric sulfate) for 30 min at 37ºC. Samples were read on a Biotek microplate reader at 562 nm.
Inflammatory and TH1/TH2 cytokines
BALF samples were centrifuged at 300 g for 5 min to pellet cellular debris. BALF supernatants were 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 (CBA) mouse inflammation kit (BD PharMingen, San Diego, CA, USA), and the TH1/TH2 cytokines IL-2, IL-4, IL-5, IFN-γ, and TNF-α were detected simultaneously by using the mouse TH1/TH2 kit. In brief, 50 μl BALF supernatant from each sample was incubated individually with a mixture of capture beads and 50 μl PE detection reagent consisting of PE-conjugated anti-mouse IL-6, IL-10, MCP-1, IFN-γ, TNF-α, and IL-12p70 or 50 μl PE detection reagent consisting of PE-conjugated anti-mouse IL-2, IL-4, IL-5, IFN-γ, and TNF-α. The samples were incubated at room temperature for 2 h in the dark. After incubation, samples were washed once and resuspended in 300 μl wash buffer before analysis on a BD FACSCalibur flow cytometer. Data were analyzed using CBA software (BD PharMingen). Standard curves were generated for each cytokine using the mixed cytokine standard provided with the respective kits. The concentration of each cytokine was determined by interpolation from the corresponding standard curve that spanned concentrations ranging from 20 to 5000 pg/ml for each cytokine measured.
T-lymphocyte flow cytometry
After counting the number of cells retrieved in BALF, cells were pelleted by centrifugation at 300 g for 5 min, supernatants were removed, and cells were reconstituted in 150 μl flow cytometer (FCM) buffer [PBS supplemented with 2% (w/v) BSA and 0.09% (w/v) sodium azide] with purified anti-mouse CD16/CD32 (FcγRIII/II) antibody (BD Biosciences, San Jose, CA, USA) to block for 30 min. The samples were then divided into two groups of equal volume, washed, and reconstituted in FCM buffer. One group received antibodies directed against CD3 (APC anti-mouse CD3ε), CD4 [PE anti-mouse CD4 (L3T4)], and CD8 [FITC anti-mouse CD8a (Ly-2)], and the other group received the isotype control antibodies. Samples were allowed to incubate 1 h at 4°C, washed twice, and then fixed for 10 min with Cytofix (BD Biosciences). 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 3000.
Microdissection
The intrapulmonary airways of the fixed left lung lobe from each mouse were microdissected according to a modified version of the technique of Plopper et al. [25]. Beginning at the lobar bronchus, airways were 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. Tissue blocks were embedded in paraffin, sectioned at a thickness of 5 μm, and then stained with H&E for light microscopic examination. Other paraffin sections were stained with alcian blue (pH=2.5)/periodic acid Schiff (AB/PAS) to detect neutral and acidic mucosubstances in the respiratory epithelium lining the intrapulmonary-conducting airways (large and small diameter bronchioles).
Immunocytochemistry
Hydrated paraffin sections (5—6 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 peroxidase and then incubated with a polyclonal antibody to caspase-3 (CAS-3; Abcam, Cambridge, MA, USA) at 20 μg/ml for 1 h. Immunoreactive CAS-3 was visualized with the Vectastain Elite ABC kit (Vectastain Laboratories Inc., Burlingame, CA, USA) using 3′,3′-diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) as a chromagen.
Histopathology scores for inflammation
A histopathology score was established based on a previously established scoring system [5]. In brief, the scores assigned were: 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 large inflammatory foci found in the alveolar parenchyma. A board-certified pathologist scored the two left lung sections (generations 5 and 11) independently and without prior knowledge of treatment groups. A mean score with the SEM was calculated for each treatment group.
CAS-3 and mucosubstance numeric cell densities
Slides of lung sections stained immunohistochemically for CAS-3 or stained for mucosubstances (AB/PAS) 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 CAS-3-positive epithelial cells per mm basal lamina. In a similar manner, numeric cell densities were determined for epithelial cells staining with AB/PAS. The numeric cell density of epithelial cells staining for mucosubstances was expressed as the number of AB/PAS-positive epithelial cells per mm basal lamina.
Statistical analysis
Data are expressed as mean ± SEM. Outliers were identified and excluded from sample sets by using Grubb's test. The differences in outcomes between treatment groups within each genotype were analyzed by two-way ANOVA with multiple comparisons made by the Student Newman Keuls post hoc test using SigmaStat software, Version 2.03, from Jandel Scientific (San Rafael, CA, USA). Differences between treatment groups and genotypes were made using a Student's t-test. The criterion for significance was taken to be P < 0.05.
RESULTS
Qualitative health assessment of CB1−/−/CB2−/− and wild-type mice challenged with PR8
CB1−/−/CB2−/− and wild-type mice treated with corn oil or Δ9-THC and intranasally instilled with saline were normal in appearance and activity level. Infection with PR8 in the presence or absence of Δ9-THC treatment led to marked differences in the gross appearance of CB1−/−/CB2−/− and wild-type mice. Specifically, CB1−/−/CB2−/− mice were notably more gaunt in comparison with wild-type mice, suggesting that the mice were dehydrated. In addition, the CB1−/−/CB2−/− mice were lethargic and displayed unkempt fur. In contrast, wild-type mice infected with PR8 in the presence or absence of Δ9-THC treatment maintained normal grooming habits and exhibited similar activity levels as the saline-instilled controls. Upon gross examination of the lungs, CB1−/−/CB2−/− mice had extensive hemorrhage throughout all lung lobes. Wild-type mice also displayed evidence of hemorrhage, albeit to a much lesser extent than CB1−/−/CB2−/− mice.
The viral load of PR8 in the pulmonary airways of CB1−/−/CB2−/− and wild-type mice on Day 7 after challenge
To estimate the viral load of PR8 infection, the levels of mRNA for the viral surface protein, H1, were assessed by quantitative real-time PCR. For CB1−/−/CB2−/− and wild-type mice, viral H1 mRNA was elevated markedly above detection levels set by saline controls at 7 dpi in the lungs of all mice challenged with PR8 (Fig. 1 ). However, vehicle-treated wild-type mice exhibited markedly greater viral H1 mRNA levels than vehicle-treated CB1−/−/CB2−/− mice. Infection of Δ9-THC-treated CB1−/−/CB2−/− mice with PR8 resulted in an increase (P=0.056) in H1 mRNA levels when compared with mice instilled with PR8 alone. In Δ9-THC-treated wild-type mice infected with PR8, there was a significant increase in viral H1 mRNA levels when compared with wild-type mice instilled with PR8 alone. The overall level of expression of H1 mRNA was markedly less in CB1−/−/CB2−/− mice when compared with wild-type mice in the absence or presence of Δ9-THC treatment.
Δ9-THC enhanced vascular permeability induced by PR8 infection of the airways in CB1−/−/CB2−/− and wild-type mice
The detection of increased levels of total BALF protein is indicative of increased vascular permeability at the alveolar/capillary interface during the inflammatory response to PR8. The total BALF protein levels in PR8-infected CB1−/−/CB2−/− and wild-type mice, in the absence of Δ9-THC treatment, were two-fold greater than saline controls (Fig. 2 ). Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 exhibited a 33% increase in total BALF protein when compared with CB1−/−/CB2−/− mice challenged with PR8 alone. Conversely, Δ9-THC-treated wild-type mice infected with PR8 had a 50% decrease in total BALF protein when compared with wild-type mice challenged with PR8 alone. When comparing CB1−/−/CB2−/− and wild-type mice, the amount of total protein detected in mice treated with Δ9-THC and infected with PR8 was two-fold greater in CB1−/−/CB2−/− mice than amounts of total protein observed in wild-type mice receiving the same treatment.
CB1−/−/CB2−/− mice treated with Δ9-THC exhibit a distinctly different composition of leukocytes recruited to the airways in response to PR8 infection when compared with wild-type mice
Primary influenza infection elicits an inflammatory response consisting of a mixed population of leukocytes that infiltrates the pulmonary airways. In particular, there is a marked influx of neutrophils and lymphocytes into the airways with monocytes and eosinophils, representing a smaller portion of the total population of BALF leukocytes. In the current study, the total number of leukocytes in BALF was three-fold greater in PR8-infected wild-type mice and four-fold greater in CB1−/−/CB2−/− mice than in their respective noninfected controls (Fig. 3A ). Δ9-THC-treated mice infected with PR8 exhibited a two-fold increase above saline-instilled controls in CB1−/−/CB2−/− and wild-type mice. However, the difference in total leukocytes retrieved in the Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 was significantly less than CB1−/−/CB2−/− mice challenged with PR8 alone. To further assess differences in individual leukocyte populations retrieved by lavage, differential cell counts were performed. Neutrophils (Fig. 3B) were increased markedly in PR8-infected CB1−/−/CB2−/− and wild-type mice. Specifically, Δ9-THC-treated wild-type mice infected with PR8 exhibited two-fold increases in neutrophils when compared with wild-type mice challenged with PR8 alone. Conversely, there were marked decreases in the number of BALF neutrophils for PR8-infected CB1−/−/CB2−/− mice treated with Δ9-THC when compared with CB1−/−/CB2−/− mice challenged with PR8 alone. BALF lymphocytes were increased markedly (Fig. 3C) with PR8 infection in CB1−/−/CB2−/− and wild-type mice. Δ9-THC-treated CB1−/−/CB2−/− and wild-type mice infected with PR8 exhibited attenuation in the number of lymphocytes retrieved. There was no change in the number of monocytes retrieved in BALF from PR8-challenged, wild-type mice as compared with saline controls. A two-fold increase was observed, however, in the number of BALF monocytes from PR8-infected CB1−/−/CB2−/− mice that was attenuated in Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 (Fig. 3D) . Lastly, PR8 infection of CB1−/−/CB2−/− and wild-type mice resulted in an increase in the number of eosinophils retrieved in BALF (Fig. 3E) . Δ9-THC treatment did not affect the number of eosinophils in BALF induced by PR8.
BALF CD4+ and CD8+ T cell levels following PR8 challenge in CB1−/−/CB2−/− and wild-type mice
To evaluate the contribution of CD4+ and CD8+ T cells within the pool of BALF lymphocytes responding to PR8 challenge, the number of CD4+ T cells (Fig. 4A ) and CD8+ T cells (Fig. 4B) was enumerated by flow cytometry. Background levels of CD4+ T cells were greater in corn oil and Δ9-THC-treated CB1−/−/CB2−/− mice than in wild-type mice with the same treatments. There were no observed differences in the number of CD4+ T cells retrieved in BALF between saline-instilled controls and CB1−/−/CB2−/− mice infected with PR8 alone. Alternatively, Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 had significantly increased numbers of CD4+ T cells above background. The increase in BALF-associated CD4+ T cells in CB1−/−/CB2−/− mice treated with Δ9-THC and infected with PR8 was comparatively greater than the respective treatment group in wild-type mice. CD8+ T cells were not detected in BALF from saline-instilled controls in CB1−/−/CB2−/− and wild-type mice. However, there were marked increases in the number of BALF-associated CD8+ T cells for CB1−/−/CB2−/− and wild-type mice challenged with PR8. In addition, there was a trend toward decreased numbers of BALF-associated CD8+ T cells in Δ9-THC-treated wild-type mice infected with PR8.
CB receptor-deficient mice exhibit unique differences in epithelial and leukocytic chemokine and cytokine secretion in the pulmonary airways
One mode of cellular communication within a mixed population of leukocytes and between infected epithelium and leukocytes is through secretion of cytokines and chemokines. PR8-infected wild-type and CB1−/−/CB2−/− mice had significant increases in levels of the chemokine MCP-1 (Fig. 5A ) and proinflammatory cytokines TNF-α (Fig. 5B) , IL-6 (Fig. 5C) , and IFN-γ (Fig. 5D) measured in BALF. In addition, there was a modest enhancement of IL-10 concentrations (Fig. 5E) following PR8 challenge in CB1−/−/CB2−/− and wild-type mice. There were no changes in IL-12p70 in BALF in the presence or absence of Δ9-THC treatment or PR8 challenge (Fig. 5F) in CB1−/−/CB2−/− or wild-type mice. Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 had an attenuation of IFN-γ and IL-10 concentrations in BALF as compared with corn oil-treated mice infected with PR8. Conversely, the concentrations of chemokines and cytokines detected in BALF in PR8-infected wild-type mice were unaffected by Δ9-THC treatment. There were, however, comparative differences between detectable levels of TNF-α and IFN-γ (P=0.061) in BALF of PR8-infected wild-type and CB1−/−/CB2−/− mice.
The TH2 cytokines IL-2 (Fig. 6A ), IL-4 (Fig. 6B) , and IL-5 (Fig. 6C) were also quantified in BALF by CBA. Of particular interest, PR8- infected CB1−/−/CB2−/− mice had marked increases in concentrations of IL-5, which were comparatively two-fold less than concentrations observed in the respective PR8-infected wild-type mice. The concentrations of IL-2 and IL-4 in BALF were unaffected by PR8 challenge in CB1−/−/CB2−/− mice. Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 had modestly attenuated concentrations of IL-2 in BALF but had no effect on the concentrations of IL-4 or IL-5. PR8-infected wild-type mice had marked increases in IL-5 concentrations in BALF, mild increases in the concentrations of IL-2, and no detectable differences for IL-4 when compared with saline-instilled mice on 7 dpi. Δ9-THC-treated wild-type mice infected with PR8 had a modest attenuation of IL-2 concentrations in BALF with no observed effect on IL-4 or IL-5 concentrations.
The absence of CB receptors CB1 and CB2 enhances the observed pulmonary histopathology
As reported previously by our laboratory, infection of mice with PR8 induced significant pulmonary inflammation 7 dpi in wild-type mice (Fig. 7A 7B 7C 7D ) [5]. Infection of the CB1−/−/CB2−/− mice with PR8 resulted in a similar inflammatory reaction (Fig. 7E) . As we have reported, the inflammation consisted of primarily lymphocytes and neutrophils with fewer monocytes and plasma cells centered upon the bronchoalveolar junction, which extended out into the surrounding parenchyma. Treatment of PR8-infected wild-type mice with Δ9-THC resulted in a significant decrease in inflammation 7 dpi compared with corn oil-treated mice infected with PR8 (Fig. 7D) . These mice often had no to few inflammatory cells present; the remaining inflammation was comprised of primarily lymphocytes and alveolar monocytes. In contrast, treatment of PR8-infected CB1−/−/CB2−/− mice with Δ9-THC resulted in a vigorous, inflammatory and cellular reaction in the mice (Fig. 7F) , where there were large numbers of mature lymphocytes around the conducting airways and extended into the surrounding alveolar parenchyma, where they were admixed with fewer monocytes and neutrophils. The bronchiolar epithelium was moderately hypertrophied. Treatment of wild-type and CB1−/−/CB2−/− mice with corn oil vehicle or Δ9-THC alone did not result in significant histologic changes in any of the lung sections examined.
Δ9-THC affects the magnitude of the inflammatory response to PR8 in wild-type and CB1- and CB2-deficient mice
The magnitude and severity of inflammation observed in tissue sections isolated from the left lung lobe were scored independently (0—3, where 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 large, inflammatory foci found in the alveolar parenchyma) and compared between treatment groups and wild-type and CB1−/−/CB2−/− mice at 7 dpi (Fig. 8 ). There was no inflammation observed in the lungs of wild-type mice intranasally instilled with saline. However, two out of eight CB1−/−/CB2−/− mice instilled with saline had a modest inflammation present. Corn oil-treated mice infected with PR8 had marked inflammation scores in wild-type and CB1−/−/CB2−/− mice as compared with uninfected control mice. Δ9-THC-treated wild-type mice challenged with PR8 had a marked suppression of the inflammatory response in the conducting airways. In contrast, Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 had a trend toward increased (P=0.079) inflammation of the pulmonary airways.
Effects of CB1 and CB2 deficiency on the numeric cell densities of apoptotic cells and metaplastic goblet cells
Bronchiolar epithelial cell apoptosis in influenza-infected mice is directed by cell-mediated immune responses to virally infected cells [26]. The numeric cell density for the apoptotic cell marker CAS-3 (Fig. 9 ) was measured in the epithelium lining generation 5 of the main axial airway. CAS-3 numeric cell densities were not distinguishably different between treatment groups in wild-type mice. In PR8-infected CB1−/−/CB2−/− mice, CAS-3-labeled cells were modestly increased above saline-instilled controls but were not different than cell densities enumerated for CB1−/−/CB2−/− mice infected with PR8 and treated with Δ9-THC. During the recovery from viral infection, a metaplastic change occurs in the epithelium lining the bronchi that includes increased numbers of mucus-producing goblet cells [27]. Consistent with our previous finding [5], there was no difference in the numeric cell density of AB/PAS-positive mucous cells (Fig. 10 ) quantified at 7 dpi in wild-type mice with any treatment. Interestingly, Δ9-THC-treated CB1−/−/CB2−/− mice, in the presence or absence of PR8 infection, had significant increases in the numeric cell density for AB/PAS-positive epithelial cells as compared with wild-type mice.
DISCUSSION
Δ9-THC administration has been reported to decrease host resistance to bacterial and viral pathogens in mouse models [2 3 4 5]. However, the role of the CB receptors CB1 and CB2 in altered immune competence to pathogen challenge remains poorly understood. The CB receptor CB1 antagonist SR141716A and CB2 receptor antagonist SR144528 have been used to study CB1/CB2-specific effects elicited by CB treatment, including efforts to uncover the CB1/CB2 receptors involvement in Δ9-THC suppression of cytokine production elicited by pathogen challenge [28], but the specificity of these pharmacologic agents and their putative off-target effects are always of concern. The objective of the present study was to use CB1/CB2 receptor null mice to rigorously evaluate previously characterized immunologic and pathologic responses to PR8 infection in the presence and absence of Δ9-THC treatment [5]. Results from this study indicated that Δ9-THC treatment of wild-type and CB1−/−/CB2−/− mice affected epithelial and immune cell responses to PR8 that were dependent and independent of the CB1/CB2 receptors as summarized in Table 1 . Perhaps even more important was the observation that CB1/CB2 null mice exhibited a significantly enhanced response to PR8 infection regardless of CB treatment, strongly suggesting an endogenous role for CB1 and/or CB2 in immune regulation. This finding provides a novel extension to our current understanding of host immunity and the relationship between a normal, "homeostatic" immune response regulated by CB receptor-linked signaling pathways and hyper-responsive, "unchecked" immunity compromised by the absence of the CB1/CB2 receptors.
In the current study, Δ9-THC was administered by oral gavage. As we have reported previously, the oral route of administration was selected as a result of the fact that Δ9-THC is a highly lipophilic molecule requiring a nonaqueous diluent for drug delivery, which has the potential for inducing irritation and damage to the airways. Conversely, oral administration of Δ9-THC in corn oil is well tolerated but poorly absorbed from the gastrointestinal tract. Oral administration of 75 mg/kg Δ9-THC for 5 consecutive days resulted in a serum concentration of 66.2 ng/ml of the parent compounds, 446.5 ng/ml of the 9-carboxyl metabolite, and 10.5 ng/ml of the 11-hydroxyl metabolite, 4 h after the last Δ9-THC dose [5]. The levels of Δ9-THC observed systemically correlate well with a previous report by Azorlosa and co-workers [29], in which peak human plasma levels ranged from 57 to 268 ng/ml. The rationale for the dosing paradigm of PR8 and Δ9-THC was to investigate the putative effects of Δ9-THC treatment on the immune response to PR8 during the early stages of the primary infection.
To assess whether the CB receptors CB1 and CB2 are critical to the cell-mediated immune response that clears influenza from the lungs, we compared the levels of H1 mRNA in lungs from wild-type and CB1−/−/CB2−/− mice. In the current study, we observed H1 mRNA levels in the lungs of Δ9-THC-treated CB1−/−/CB2−/− and wild-type mice challenged with PR8 that were greater than H1 mRNA levels in CB1−/−/CB2−/− and wild-type mice challenged with PR8 alone. Interestingly, H1 mRNA levels were reduced in the lungs of CB1−/−/CB2−/− mice by two orders of magnitude when compared with H1 mRNA levels observed for wild-type mice. The measurement of H1 mRNA serves as a marker of transcriptional activation of the influenza virus genome that could result from increases in viral copy number or increases in transcriptional activation. Whether Δ9-THC is exerting an effect on copy number or transcriptional activation or both is not presently known. It is tempting to speculate, however, that H1 mRNA levels are related to copy number, as there are marked differences in the magnitude of the inflammatory response to PR8 observed by histopathology. The profound reduction in H1 mRNA levels also suggests that the kinetics of the cell-mediated immune response to PR8 is markedly enhanced in CB1−/−/CB2−/− mice. The suggested hyper-responsive immunity toward PR8 infection brought about by the absence of functional CB receptors CB1 and CB2 is supported by our findings, in the current study, of increased background levels of CD4+ T cells in CB1−/−/CB2−/− mice in combination with a more vigorous and extensive inflammation of the alveolar parenchyma. Recently, Karsak and co-workers [24] reported a more vigorous, allergic response to the contact allergen 2,4-dinitroflourobenzene in CB1−/−/CB2−/− mice when compared with wild-type mice. The current study and that reported by Karsak and co-workers [24] suggest that the CB receptors CB1 and CB2 may play a crucial role in maintaining immune homeostasis and controlling the magnitude of the immune response through negative regulation. It is tempting to speculate that endoCBs may naturally provide a mechanism to temper the immune response. The absence of CB1 and CB2 receptors appears to potentially result in an enhanced immune response under certain conditions.
We have previously observed decreased recruitment of monocytes and lymphocytes to the airways in Δ9-THC-treated mice challenged with PR8 [5]. This observation was consistent with the suppressive effects of CBs on leukocyte chemotaxis reported by others [30 31 32 33]. To clarify whether mice lacking functional CB1/CB2 receptors exhibit patterns of leukocyte recruitment to the airways in response to viral challenge that are modulated by Δ9-THC in a CB1/CB2 receptor-independent manner, total and differential cell counts from BALF were assessed. In the current study, the magnitude of total leukocyte recruitment to the pulmonary airways and the cellular subsets present were similar between CB1−/−/CB2−/− and wild-type mice challenged with PR8, suggesting that mice lacking functional CB1/CB2 receptors remained capable of mounting an aggressive immune response toward PR8. Differential cell counts provided evidence that the immune response to PR8 by CB1−/−/CB2−/− mice was more robust than the immune response mounted against PR8 in wild-type mice. More specifically, in the BALF, monocytes and neutrophils were decreased in Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8. The decreased recruitment of monocytes and neutrophils to the airways might point to a CB receptor-dependent effect on the chemotaxis of these leukocytes. Contrary to findings with monocytes and neutrophils, there were remarkable similarities in the total number of lymphocytes in BALF among all treatment groups for CB1−/−/CB2−/− and wild-type mice. Upon further examination of the T cell subsets retrieved in BALF, there was evidence that the T cell response to PR8 challenge with Δ9-THC treatment involved predominantly CD4+ T cells in CB1−/−/CB2−/− mice. As mentioned previously, increased numbers of CD4+ T cells might suggest hyper-responsive immunity in CB1−/−/CB2−/− mice. However, the balance of the CD4+ TH subset (TH1 cell-mediated immunity vs. TH2 humoral immunity) remains unclear.
We have previously shown that PR8 challenge of the pulmonary airways leads to marked increases in total protein and chemokine/cytokines MCP-1, IL-6, TNF-α, and IFN-γ in BALF [5]. Δ9-THC and other CBs have been reported to modulate the expression and/or secretion of these chemokines and cytokines [20 , 33 34 35 36]. In the current study, we observed increased total protein in the BALF of wild-type and CB1−/−/CB2−/− mice challenged with PR8. Comparatively, a marked difference existed between Δ9-THC-treated CB1−/−/CB2−/− and wild-type mice infected with PR8, suggesting an increased severity in vascular leakiness or inflammatory cell secretions in CB1−/−/CB2−/− mice. Consistent with increased amounts of total protein in BALF, inflammation scores assessed by histopathology accurately reflected changes observed in vascular leakiness. In addition to total protein, we evaluated chemokines/cytokines in BALF. Although there were trends toward increases in BALF chemokines and cytokines (e.g., MCP-1, TNF-α, and IFN-γ) from CB1−/−/CB2−/− mice infected with PR8, in the absence of Δ9-THC, the collective findings did not provide evidence for CB1 and/or CB2 receptor-dependent or -independent regulation of the secretion of these cytokines. Chemokine and cytokine levels should be interpreted carefully, as they represent a concentration in the airways at a given moment in time with respect to an ever-changing, dynamic inflammatory process. It is difficult to speculate in the current study as to whether differences observed between treatment and control groups represent trends toward increased or decreased expression or secretion of chemokines or cytokines. Moreover, the cytokines measured in BALF are pleiotropic and redundant, as they may be derived from multiple immune cell types. Not surprisingly, cytokines and chemokines are regulated in their own unique manner with their own distinct kinetics, and the molecular mechanism by which certain cytokines and chemokines are further modulated by CBs is only partially understood. It has been suggested that Δ9-THC treatment modulates cytokine production, resulting in decreased TH1 cell-mediated immunity and increased TH2 humoral immunity [37 , 38]. As CB1−/−/CB2−/− mice had a marked, lymphocytic response consisting of greater numbers of CD4+ T cells, and Δ9-THC treatment of CB1−/−/CB2−/− mice challenged with PR8 resulted in decreased concentrations of IFN-γ, the concentrations of the TH2 cytokines IL-2, IL-4, and IL-5 were also evaluated in BALF. Only the cytokine IL-5 was influenced by PR8 infection in wild-type mice in the presence or absence of Δ9-THC. IL-5 concentrations were reduced in PR8-infected CB1−/−/CB2−/− mice in the presence or absence of Δ9-THC, and Δ9-THC treatment of CB1−/−/CB2−/− or wild-type mice infected with PR8 exhibited no effect on the levels of IL-5 when compared with CB1−/−/CB2−/− or wild-type mice infected with PR8 alone. Our results suggest that the increased numbers of CD4+ T cells observed in CB1−/−/CB2−/− mice were not actively secreting more TH2-type cytokines than wild-type mice and that there did not appear to be an imbalance between TH1-type and TH2-type cytokines secreted as a result of Δ9-THC treatment in response to PR8.
To evaluate the extent of the inflammatory response to PR8 in CB1−/−/CB2−/− mice as compared with wild-type controls, lung sections were taken at the levels of the 5th (proximal) and 11th (distal) airway generations, bifurcating away from the main axial airway of the left lung lobe and scored for the magnitude and severity of inflammation. Within saline-instilled wild-type mice, there was no inflammation observed. Conversely, in two out of eight CB1−/−/CB2−/− mice instilled with saline, in the presence or absence of Δ9-THC treatment, there was modest inflammation. The CB1−/−/CB2−/− mice were subjected to an extensive battery of serological screens prior to their use and were found to be negative for all pathogens tested. In a separate, unpublished study, two untreated CB1−/−/CB2−/− mice were necropsied and examined for microscopic evidence of lesions in the brain, eye, conjunctiva, myocardium, lung, stomach, small intestine, large intestine, liver, spleen, testicle, and nasal cross sections. Within the lungs, there was a mild interstitial, inflammatory infiltrate composed of lymphocytes and plasma cells. In addition, there were multifocal regions of atelectasis, which is the incomplete expansion of the lungs or the collapse of lung parenchyma, producing an area of relatively airless pulmonary parenchyma. Atelectatic parenchyma is prone to developing superimposed infections, which may partially account for the more vigorous, inflammatory responses observed within CB1−/−/CB2−/− mice. Regardless, inflammation scores were similar for lung sections from CB1−/−/CB2−/− and wild-type mice challenged with PR8, in the absence of Δ9-THC treatment, and the histopathology for PR8-infected CB1−/−/CB2−/− and wild-type mice had a similar pattern of inflammatory cells infiltrating the lung. In Δ9-THC-treated wild-type mice challenged with PR8, there was a marked reduction in the inflammation score, suggesting that Δ9-THC altered the kinetics of leukocyte chemotaxis to the airways in response to PR8. This is supported by the marked reduction or absence of inflammatory cells in the submucosa and alveolar parenchyma. In contrast, inflammation scores for lung sections from Δ9-THC-treated CB1−/−/CB2−/− mice challenged with PR8 were modestly enhanced with respect to scores assessed for lung sections from CB1−/−/CB2−/− mice challenged with PR8 alone. Indeed, CB1−/−/CB2−/− mice treated with Δ9-THC and infected with PR8 represented some of the most severely affected mice in the study and exhibited more vigorous, inflammatory responses than even observed in CB1−/−/CB2−/− mice challenged with PR8 alone.
We have previously demonstrated that epithelial changes in the pulmonary airways of mice challenged with PR8 included increased evidence for apoptosis and MCM following PR8 challenge, which was suppressed by Δ9-THC [5]. Cell death pathways initiated by cytotoxic T cells or NK cells or by the virus itself influence CAS-3 activation [39 , 40]. In the comparison of wild-type and CB1−/−/CB2−/− mice, PR8 challenge led to increased CAS-3 staining; however, there were no effects observed with Δ9-THC administration. In a similar manner, MCM was quantified by counting AB/PAS-positive epithelial cells lining the main axial airway at G5 of the left lung lobe. We have previously shown that goblet cells producing mucosubstances in the airway epithelium increase substantially by 10 dpi in wild-type mice. In the current study, we found that CB1−/−/CB2−/− mice had significantly greater numbers of goblet cells lining the airways at G5 when treated with Δ9-THC alone at 7 dpi. This finding suggests that Δ9-THC is interacting directly with a signaling pathway tied with increased mucin production in CB1−/−/CB2−/− mice or that exposure of CB1−/−/CB2−/− mice to Δ9-THC is handled by the host as an allergen leading to an alternative, hyper-responsive CD4+ TH2 T cell response that is capable of eliciting MCM.
In conclusion, Δ9-THC-treated CB1−/−/CB2−/− mice infected with PR8 exhibited more robust immune responses than wild-type mice receiving the respective treatment. Moreover, CB1−/−/CB2−/− mice infected with PR8 had markedly decreased levels of viral mRNA as compared with wild-type mice. Furthermore, Δ9-THC treatment increased viral load when compared with mice infected with PR8 alone, irrespective of the presence or absence of CB1 and CB2 receptors. Therefore, the current study demonstrated that Δ9-THC influenced the immune response to influenza infection in a manner that was dependent and independent of the CB1/CB2 receptors. Future studies will examine the effect of Δ9-THC on viral infection of lung parenchymal cells and establish the kinetics of lymphocyte, monocyte, and neutrophil chemotaxis to the lungs of PR8-infected CB1−/−/CB2−/− mice.
Source, Graphs and Figures: Targeted deletion of cannabinoid receptors CB1 and CB2 produced enhanced inflammatory responses to influenza A/PR/8/34 in the absence and presence of
