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. 2011 Nov;90(5):983-95.
doi: 10.1189/jlb.0511219. Epub 2011 Aug 26.

Deletion of cannabinoid receptors 1 and 2 exacerbates APC function to increase inflammation and cellular immunity during influenza infection

Peer W F Karmaus  1 , Weimin Chen, Robert B Crawford, Jack R Harkema, Barbara L F Kaplan, Norbert E Kaminski
Affiliations

Deletion of cannabinoid receptors 1 and 2 exacerbates APC function to increase inflammation and cellular immunity during influenza infection

Peer W F Karmaus et al. J Leukoc Biol. 2011 Nov.

Abstract

We and others have reported that simultaneous targeted deletion of CB(1) and CB(2) resulted in exacerbation of immune reactivity, suggesting a role of endocannabinoids in down-regulating immune function. In this study, we demonstrate that APC function is enhanced specifically in the absence of CB(1) and CB(2) signaling, resulting in an exacerbated immune response phenotype. After influenza infection, CB(1)(-/-)CB(2)(-/-) mice showed more pronounced pulmonary damage, increased inflammatory cell infiltrate, inflammation, and a greater cellular immune responses compared with WT mice, as evidenced by transcriptome analysis, more robust T cell activation, and effector cell cytokine production. After direct activation in vitro, there were no differences in the percentages of cytokine-producing CD4(+) T cells between CB(1)(-/-)CB(2)(-/-) and WT mice. However, untreated CB(1)(-/-)CB(2)(-/-) mice routinely had fewer naïve T cells compared with WT, suggesting dysregulation of APC immune homeostasis. Moreover, bmDCs and AM isolated from CB(1)(-/-)CB(2)(-/-) mice exhibited a more mature phenotype, with and without TLR stimulation, and bmDCs elicited T cells more robustly than WT mice. Collectively, these findings implicate a role for CB(1) and CB(2) on APCs in regulating immune responses and immune homeostasis.

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Figures

Figure 1.
Figure 1.. More severe virus-induced necrotizing bronchiolitis and loss of CCSP in CB1−/−CB2−/− compared with WT mice, 3 dpi.
Light photomicrographs of axial airways (AA; generation 5) and terminal bronchioles (TB; generation 5) from the left lung lobe of WT and CB1−/−CB2−/− mice intranasally instilled with SAL vehicle alone or PR8 influenza virus in SAL at 3 dpi. The insets are low magnification of tissue sections of the left lung lobe taken perpendicular to the axial airways at generation 5 (see Materials and Methods for microdissection details). All tissues were stained with H&E (A) or immunohistochemically stained for CCSP (red chromagen) located in bronchiolar epithelium (arrows) at 3 dpi (B). Virus-induced necrotizing bronchiolitis is evident in the axial airways of WT and CB1−/−CB2−/− mice with greater necrosis and exfoliation (arrows) of the surface epithelium (e) and interstitial cellular inflammation and edema (asterisks) in the CB1−/−CB2−/− mouse compared with the WT mouse. Similar necrotizing bronchiolitis is evident in the terminal bronchiole of the CB1−/−CB2−/− mouse but not in the virus-treated WT mouse. Compared with SAL-instilled control mice, there was an apparent loss of CCSP staining in the bronchiolar epithelium lining the large-diameter axial airways and smaller-diameter preterminal and terminal bronchioles of the virus-treated CB1−/−CB2−/− mouse. In virus-treated WT mice, there was a loss of CCSP only in large-diameter axial airways, but CCSP was still present in preterminal and terminal bronchioles. All CCSP-stained tissue sections were counterstained with hematoxylin. AD, Alveolar duct; ap, alveolar parenchyma; p, pleura; a, alveolus; bv, blood vessel; asterisk, necrotic cellular debris and inflammatory cells in airway lumen. Light microscopic examinations of histologic tissue sections were conducted using an Olympus BX41 clinical microscope with UPlan Apochromat 10×, 20×, and 40× and Achromat 60× brightfield objectives (www.olympusamerica.com). A 5.0-megapixel Qlympus Q-Color5 camera system was used for all light photomicroscopy. This included a high-resolution FireWire digital charged-coupled device color camera, designed for publication and documentation, with a two-third-inch Bayer color sensor and 2580 × 1944 pixel resolution. The camera was connected to a Dell Precision 380 computer and QCapture Suite software for Windows, allowing for real-time viewing and full computer control of the camera.
Figure 2.
Figure 2.. Relative transcript levels of genes associated with inflammation are increased in lungs of CB1−/−CB2−/− mice compared with WT mice basally and after influenza infection at 3 dpi.
RNA from lung samples (n=4) was converted to cDNA and analyzed using an Applied Biosystem's mouse immune panel. Genes were included based on 1.5-fold up- or down-regulation, and fold changes in gene expression levels were analyzed using factorial ANOVAs for significance. Genes with significantly different expression between CB1−/−CB2−/− and WT mice were visualized in a heat map with red, indicating an increase, and blue, a decrease, in expression in a log2 relative expression scale, as shown on the right side of the figure. The P value of the factorial ANOVA is shown on the left side of each gene. Arrangement of the heat map is as follows: (A) differentially regulated genes basally, without influenza infection between CB1−/− CB2−/− and WT mice; (B) gene expression differentially expressed in CB1−/−CB2−/− mice after influenza infection.
Figure 3.
Figure 3.. More rapid T cell activation after influenza infection in CB1−/−CB2−/− compared with WT mice.
At 1, 3, 5, and 7 dpi, cells were isolated from lung tissue (n=5), FcRs were blocked on the surface, and samples were stained for surface markers CD4, CD8, and CD69. FlowJo software was used to analyze samples and create histograms shown. Gating was performed on single cell populations, size, and expression of CD4 and CD8 (bottom left of figure). Within CD4+ (A) and CD8+ (B) populations, histograms depict the fluorescence intensity of CD69 staining. As a result of bimodal expression of CD69, percent positively gated cells, as shown in the first panel (topmost, A), were enumerated (CD4, C; CD8, D). Friedman's test for percentages was performed, and significance was indicated in figures as #P < 0.05 and ##P < 0.01 in comparison with WT and CB1−/−CB2−/−. FSC-H, Forward-scatter-height; FSC-A, forward-scatter-area; SSC-A, side-scatter-area.
Figure 4.
Figure 4.. Greater cytokine production by leukocytes from CB1−/−CB2−/− mice compared with WT mice.
Lungs from mice were mechanically disrupted and passed through a sieve (n=5). After removal of connective tissue, cells were counted and restimulated with PMA/Io for 5 h in the presence of Brefeldin A. (A) Cells were blocked with FcRs and stained with CD4, CD8, and NK1.1, in addition to cytokine staining with IFN-γ and IL-17 (B). Flow cytometry samples were gated as depicted in A. Cytokine secretors were identified as percent of positive cells within surface delineation, and unstimulated samples were used as controls. (C) Immune cell populations were enumerated for percent cytokine expression in FlowJo (type of cytokine indicated on the left) within effector cell populations (surface marker indicated on the right) in FlowJo, and bar graphs were generated using GraphPad Prism. Friedman's test for percentages was performed, and significance is indicated in figures as #P < 0.05 and ##P < 0.01 in comparison with WT mice and CB1−/−CB2−/− mice.
Figure 5.
Figure 5.. Lower number of naïve CD4+ T cells and no difference in T cell stimulation in vitro in CB1−/−CB2−/−mice compared with WT mice.
Splenocytes (n=3) were obtained from WT and CB1−/−CB2−/− mice and analyzed by flow cytometry with gating shown in A. Non-naïve T cells (CCR7–CD62L– or CD62L–) were gated for in B and analyzed statistically in C. Naïve (NA; CD62L+) and non-naïve (non NA) CD4+ cells were isolated from splenocytes and driven toward a Th17 phenotype in the presence of TGF-β and IL-6, as described in Materials and Methods. Cells were gated as shown in D and then analyzed for cytokine production in E. Statistical analyses of experimental data in Th17-polarizing conditions from E are shown in F and G. The experiment is representative of two repeat in vitro studies. *P = 0.05; **P = 0.01 CB1−/−CB2−/− compared with WT.
Figure 6.
Figure 6.. Increased expression of DC maturation markers in bmDCs from CB1−/−CB2−/− mice.
bmDCs were generated as described in Materials and Methods. In short, bm was isolated from femurs and tibias and cultured in the presence of GM-CSF (20 ng/mL) for 9 days, and then immature bmDCs were washed and stimulated with TLR agonists (n=3). (A) bmDCs gated for singlets, Live/Dead dye, and high CD11b and CD11c expression. (B) Surface expression of maturation markers MHCI, MHCII, CD80, and CD86 was analyzed after stimulation for 24 h with LPS (1 μg/mL) and ssRNA (5 μg/mL). (C) As a result of the Gaussian distribution of the fluorescent staining, mean fluorescence intensity (MFI) was used to compare samples using statistics. The results shown are representative of four identical experiments.
Figure 7.
Figure 7.. bmDCs from CB1−/−CB2−/− elicit OT-1 responses without requirement for maturation stimulus and more potently than WT.
OT-1 cells were elicited as described in Materials and Methods. In short, bmDCs were matured with different TLR agonists for 24 h, pulsed with SIINFEKL peptide for 2 h, washed, incubated with OT-1 cells for 4 days, and then restimulated for 5 h to induce cytokine secretion (n=3). (A) Gating scheme for OT-1 cells; singlet, size, and Live/Dead gating were applied before visualizing data. (B) Proliferation was assessed by Cell Trace dye dilution, and a gate depicting proliferated populations was drawn according to the WT-naive population shown in gray. The legend for the other experimental groups is below the figure, and the TLR agonist used is shown beside the proliferation graph. (C) Proliferation and concurrent IFN-γ secretion were visualized using dot plots. LPS (1 μg/mL) and ssRNA (5 μg/mL) were used to stimulate bmDC maturation as described previously (Fig. 6). Proliferation and IFN-γ secretion gates were drawn according to a WT-naive group without SIINFEKL peptide, which showed no proliferation or IFN-γ secretion. (D) Data from proliferation and cytokine plots are summarized using percentages obtained from shown gates. Statistical significance is indicated as #P ≤ 0.05 comparing WT with CB1−/−CB2−/− using Friedman's test for nonparametric data. The results shown are representative of three identical experiments.
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