Human leukocytes differentially express endocannabinoid-glycerol lipases and hydrolyze 2-arachidonoyl-glycerol and its metabolites from the 15-lipoxygenase and cyclooxygenase pathways

2-Arachidonoyl-glycerol (2-AG) is an endocannabinoid with anti-inflammatory properties. Blocking 2-AG hydrolysis to enhance CB2 signaling has proven effective in mouse models of inflammation. However, the expression of 2-AG lipases has never been thoroughly investigated in human leukocytes. Herein, we investigated the expression of seven 2-AG hydrolases by human blood leukocytes and alveolar macrophages (AMs) and found the following protein expression pattern: monoacylglycerol (MAG lipase; eosinophils, AMs, monocytes), carboxylesterase (CES1; monocytes, AMs), palmitoyl-protein thioesterase (PPT1; AMs), α/β-hydrolase domain (ABHD6; mainly AMs), ABHD12 (all), ABHD16A (all), and LYPLA2 (lysophospholipase 2; monocytes, lymphocytes, AMs).We next found that all leukocytes could hydrolyze 2-AG and its metabolites derived from cyclooxygenase-2 (prostaglandin E2-glycerol [PGE2-G]) and the 15-lipoxygenase (15-hydroxy-eicosatetraenoyl-glycerol [15-HETE-G]). Neutrophils and eosinophils were consistently better at hydrolyzing 2-AG and its metabolites than monocytes and lymphocytes. Moreover, the efficacy of leukocytes to hydrolyze 2-AG and its metabolites was 2-AG ≥ 15-HETE-G >> PGE2-G for each leukocyte. Using the inhibitors methylarachidonoyl-fluorophosphonate (MAFP), 4-nitrophenyl-4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184), Palmostatin B, 4′-carbamoylbiphenyl-4-yl methyl(3-(pyridin-4-yl)benzyl)carbamate, Nmethyl-N-[[3-(4-pyridinyl)phenyl]methyl]-4′-(aminocarbonyl) [1,1′-biphenyl]-4-yl ester carbamic acid (WWL70), 4′-[[[methyl[[3-(4-pyridinyl)phenyl]methyl]amino]carbonyl]oxy]-[1,1′-biphenyl]-4-carboxylic acid, ethyl ester (WWL113), tetrahydrolipstatin, and ML349, we could not pinpoint a specific hydrolase responsible for the hydrolysis of 2-AG, PGE2-G, and 15-HETE-G by these leukocytes. Furthermore, JZL184, a selective MAG lipase inhibitor, blocked the hydrolysis of 2-AG, PGE2-G, and 15-HETE-G by neutrophils and the hydrolysis of PGE2-G and 15-HETE-G by lymphocytes, two cell types with limited/no MAG lipase. Using an activity-based protein profiling (ABPP) probe to label hydrolases in leukocytes, we found that they expressmanyMAFP-sensitive hydrolases and an unknown JZL184-sensitive hydrolase of ~52 kDa. Altogether, our results indicate that human leukocytes are experts at hydrolyzing 2-AG and its metabolites via multiple lipases and probably via a yet-to-be characterized 52 kDa hydrolase. Blocking 2-AG hydrolysis in humans will likely abrogate the ability of human leukocytes to degrade 2-AG and its metabolites and increase their anti-inflammatory effects in vivo

Loss of vascular CD34 results in increased sensitivity to lung injury

Survival during lung injury requires a coordinated program of damage limitation and rapid repair. CD34 is a cell surface sialomucin expressed by epithelial, vascular and stromal cells that promotes cell adhesion, coordinates inflammatory cell recruitment, and drives angiogenesis. To test whether CD34 also orchestrates pulmonary damage and repair, we induced acute lung injury in wild type (WT) and Cd34-/- mice by bleomycin (BLM) administration. We found that Cd34-/- mice displayed severe weight loss and early mortality compared to WT controls. Despite equivalent early airway inflammation to WT mice, CD34-deficient animals developed interstitial edema and endothelial delamination, suggesting impaired endothelial function. Chimeric Cd34-/- mice reconstituted with WT hematopoietic cells exhibited early mortality compared to WT mice reconstituted with Cd34-/- cells, supporting an endothelial defect. CD34-deficient mice were also more sensitive to lung damage caused by influenza infection, showing greater weight loss and more extensive pulmonary remodeling. Together our data suggest that CD34 plays an essential role in maintaining vascular integrity in the lung in response to chemical- and infection-induced, tissue damage

Treatment with the NR4A1 agonist cytosporone B controls influenza virus infection and improves pulmonary function in infected mice.

The transcription factor NR4A1 has emerged as a pivotal regulator of the inflammatory response and immune homeostasis. Although contribution of NR4A1 in the innate immune response has been demonstrated, its role in host defense against viral infection remains to be investigated. In the present study, we show that administration of cytosporone B (Csn-B), a specific agonist of NR4A1, to mice infected with influenza virus (IAV) reduces lung viral loads and improves pulmonary function. Our results demonstrate that administration of Csn-B to naive mice leads to a modest production of type 1 IFN. However, in IAV-infected mice, such production of IFNs is markedly increased following treatment with Csn-B. Our study also reveals that alveolar macrophages (AMs) appear to have a significant role in Csn-B effects, since selective depletion of AMs with clodronate liposome correlates with a marked reduction of IFN production, viral clearance and morbidity in IAV-infected mice. Furthermore, when reemergence of AMs is observed following clodronate liposome administration, an increased production of IFNs was detected in bronchoalveolar fluids of IAV-infected mice treated with Csn-B, supporting the contribution of AMs in Csn-B effects. While treatment of mice with Csn-B induces phosphorylation of transcriptional factors IRF3 and IRF7, the latter appears to be less indispensable since effects of Csn-B treatment on the synthesis of IFNs were slightly affected in IAV-infected mice lacking functional IRF7. Together, our results highlight the capacity of Csn-B and consequently of NR4A1 transcription factor in controlling IAV infection

Impact of cannabis, cannabinoids and endocannabinoids in the lungs

Since the identification of cannabinoid receptors in the 1990s, a research field has been dedicated to exploring the role of the cannabinoid system in immunity and the inflammatory response in human tissues and animal models. Although the cannabinoid system is present and crucial in many human tissues, studying the impact of cannabinoids on the lungs is particularly relevant because of their contact with exogenous cannabinoids is the context of marijuana consumption. In the past two decades, the scientific community has gathered a large body of evidence supporting that the activation of the cannabinoid system alleviates pain and reduces inflammation. In the context of lung inflammation, exogenous and endogenous cannabinoids have shown therapeutic potential because of their inhibitory effects on immune cell recruitment and functions. On the other hand, cannabinoids were shown to be deleterious to lung function and to impact respiratory pathogen clearance. In this review, we present the existing data on the regulation of lung immunity and inflammation by phytocannabinoids, synthetic cannabinoids and endocannabinoids

Treatment with cytosporone B reduces lung viral load and improves survival in mice infected with Influenza A virus.

<p><b>(A)</b> Mice (n = 5/group) were infected with IAV and daily treated with placebo or with increasing concentrations of Csn-B administered intraperitoneally (ip.). Lungs were harvested at day 5 post IAV-infection (p.i.) for viral load determination. For lung viral loads, differences were analysed using One-Way ANOVA followed by Tukey post-hoc test. ** <i>p</i> ≤ 0.01, *** ≤ 0.001 and ****<i>p</i> ≤ 0.0001 as compared to indicated groups. <b>(B)</b> Lung viral loads were assessed in IAV-infected WT and <i>Nr4a1</i>^<i>-/-</i> mice (50 PFU), daily treated with placebo or Csn-B (5 mg/kg ip.). Lungs were harvested at day 3, 5 and 7 post-infection. Results are presented as mean ± SEM of two independent experiments (total of 10 mice/group). For lung viral loads, differences were analysed using Two-Way ANOVA followed by Dunnett post-hoc test. *<i>p</i> ≤ 0.05 and ****<i>p</i> ≤ 0.0001 as compared to indicated groups. <b>(C)</b> WT and <i>Nr4a1</i>^<i>-/-</i> mice (n = 8/group) were infected with IAV (3000 PFU in.) and daily treated for 16 days with either placebo or Csn-B (5 mg/kg). Survival was monitored daily. Differences were analysed using a log rank test (*<i>p</i>≤0.05 as compared to WT mice treated with a placebo). <b>(D)</b> Amplification of IAV genes (M1, NS1 and PB2) was performed by RT-PCR at day 5 on lung homogenates of IAV-infected mice treated with Csn-B or placebo. bp: base pair. Positive control: MDCK cells infected with IAV (4000 PFU/ml). Fold increase of gene expression is expressed relative to the placebo group. Data are representative of two independent experiments (n = 5mice/group). Differences were analysed using One-Way ANOVA followed by Tukey post-hoc test. ** <i>p</i> ≤ 0.01 as compared to indicated groups.</p

Hypothetical scenario of the effects of Csn-B on the production of type 1 IFNs in bronchoalveolar lavages of mice infected with IAV.

<p>Influenza A virus infection of WT mice induces phosphorylation of IRF3 and IRF7 transcription factors. As primary mechanism, treatment with Csn-B, an agonist of NR4A1, potentiates phosphorylation of IRF3 (solid line) and then, to a lesser extent, phosphorylates IRF7 (dashed line). These effects consequently increase production of type 1 IFNs in alveolar macrophages.</p

Treatment with cytosporone B increases production of type I interferons in bronchoalveolar fluids of IAV-infected mice.

<p>Levels of IFN-β and IFN-α production in bronchoalveolar fluids (BALs) from IAV-infected (<b>A-B)</b> WT and <b>(C-D)</b> <i>Nr4a1</i>^{<b><i>-/-</i></b>} mice (n = 4 mice/group), daily treated with placebo or Csn-B (5 mg/kg). BALs were collected at day 3, 5 and 7 post-infection. Results are presented as mean ± SEM of two independent experiments. Differences were determined using Two-Way ANOVA followed by Tukey post-hoc test. ****<i>p</i> ≤ 0.0001 as compared to indicated groups.</p

IRF3 and IRF7 contribute to Cytosporone B-induced IFN secretion in response to IAV infection.

<p>WT, <i>Irf3</i>^<i>-/-</i> <i>and Irf7</i>^<i>-/-</i> mice were infected with IAV (50 PFU) and daily treated with placebo or Csn-B (5 mg/kg). Levels of <b>(A-C)</b> IFN-β and <b>(B-D)</b> IFN-α production in BALs from IAV-infected WT, <i>Irf3</i>^{<b><i>-/-</i></b>} and <i>Irf7</i>^{<b><i>-/-</i></b>} mice, daily treated with placebo or Csn-B (5 mg/kg). BALs were collected at day 5 post-infection. Levels of IFN-β and IFN-α production in BALs of <i>Irf3</i>^{<b><i>-/-</i></b>} and <i>Irf7</i>^{<b><i>-/-</i></b>} mice were negligible at day 3 and 7 post-infection (data not shown). Results are presented as mean ± SEM of two independent experiments (n = 4 mice/groups). Differences were determined using Two-Way ANOVA followed by Tukey post-hoc test. *<i>p</i> ≤ 0.05 **<i>p</i> ≤ 0.01 ***<i>p</i> ≤ 0.001 and ****<i>p</i> ≤ 0.0001 as compared to indicated groups.</p

Treatment with cytosporone B improves lung structure and function in mice infected with Influenza A virus.

<p><b>(A)</b> Haematoxylin and eosin stained of lung sections from not infected or IAV-infected (50 PFU) WT and <i>Nr4a1</i>^<i>-/-</i> mice, treated daily with placebo or Csn-B (5 mg/kg). Lungs were harvested at day 5 post-infection. Images are representative of two independent experiments (n = 3 mice/group). a: alveolar and b: bronchiolar structure (original magnification 100X). At day 5 pi., <b>(B)</b> airway resistance, <b>(C)</b> airway elastance, <b>(D)</b> tissue damping and <b>(E)</b> tissue elastance were measured (n = 6 mice/groups) upon methacholine challenge. Whilst baseline elastance was measured prior to methacholine administration using the FlexiVent apparatus. (<b>B, C</b>) Results are presented as mean ± SEM. **<i>p</i> ≤ 0.01 and ****<i>p</i> ≤ 0.0001 as compared to IAV + Csn-B. Differences in groups were determined using Two-Way ANOVA followed by Dunnett post-hoc test. (<b>D, E</b>) Results are presented as mean ± SEM. ****<i>p</i> ≤ 0.0001 as compared to IAV + Csn-B. Differences in groups were determined using One-Way ANOVA followed by Dunnett post-hoc test.</p

Selective depletion of alveolar macrophages reduces effects of Cytosporone B on IAV infection.

<p><b>(A)</b> Representative gating strategies showing alveolar macrophages (AMs) in naive mice after PBS or clodronate liposome administration. AMs were gated as CD45⁺, CD11b^-, CD11c^high, F4/80⁺, Siglec-F^high cells in lungs of naive and PBS or clodronate liposomes treated WT mice. Data are presented as the frequencies (%) of AMs on CD45⁺ cells and true count values of AMs at indicated time following PBS or clodronate liposome administration. <b>(B)</b> Lung viral loads were assessed in IAV-infected WT mice administered with PBS-lipo (control) or Clo-lipo and daily treated with placebo or Csn-B (5 mg/kg ip.). Lungs were harvested at day 4, 6, 8 and 10 post-clodronate administration. Results are presented as mean ± SEM of two independent experiments (n = 4 mice/group). Differences were determined using Two-Way ANOVA followed by Dunnett post-hoc test. *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01 and ***<i>p</i> ≤ 0.001 as compared to indicated groups. <b>(C)</b> WT mice injected with PBS-lipo or Clo-lipo (n = 9/group) were infected with IAV (lethal dose of 3000 PFU in.) and daily treated for 16 days with either placebo or Csn-B (5 mg/kg). Survival and body temperature were monitored daily. For survival data, differences were analysed using a log rank test. *<i>p</i> ≤ 0.05 as compared to Clo-lipo + IAV + Csn-B. ** <i>p</i> ≤0.01 as compared to Clo-lipo + IAV + placebo. For body temperature data, differences were analysed using Two-Way ANOVA followed by Dunnett post-hoc test. *<i>p</i> ≤ 0.05 and **<i>p</i> ≤ 0.01 and ***<i>p</i> ≤ 0.001 as compared to Clo-lipo + IAV + Csn-B. <b>(D)</b> Levels of IFN-β and IFN-α were assessed in BALs of IAV infected mice (50 PFU) treated with placebo or Csn-B at indicated time following Clo-lipo administration. Results are presented as mean ± SEM of two independent experiments (n = 4 mice/groups). Differences were determined using Two-Way ANOVA followed by Dunnett post-hoc test. **<i>p</i> ≤ 0.01 and ****<i>p</i> ≤ 0.0001 as compared to indicated groups.</p