*5.2. In Vivo Animal Studies*

The observational studies citing the toxic effects of marijuana smoke on the pulmonary system have prompted in vivo studies on animals to further investigate the extent of the effects. As expected, animal studies have evidenced a large variety of lung lesions, including neutrophil, lymphocyte, and macrophage infiltration, goblet cell hyperplasia, endothelial proliferation, emphysema and airway hyperresponsiveness (AHR) doubled by increased cytokines and inflammatory pathway activation [114]. Interestingly, when the cannabinoids were removed from the inhaled smoke, the alveolar inflammation and wall thickening, pneumonitis, tracheobronchial fibrosis, inflammation and sputum excess were still observed [115]. These findings prompted the need for research using *per se* cannabinoids in controlled studies.

A study investigating the effects of the synthetic cannabinoid CP55,940, a full CB1 and CB2 agonist, in C57BL6/J mice showed that the oropharyngeal instillation of the compound caused CB1 activation and the subsequent increase of TNF-α, IL-1β, IL-6, C-C Motif Chemokine Ligand 2 (CCL) 2 and 3, C-X-C motif chemokine ligand 10 (CXCL10), and various pro-inflammatory transcription factors [116].

However, the in vivo effects of cannabinoids were most commonly investigated in animal-induced inflammatory conditions, most commonly using LPS to induce ALI. The intraperitoneal administration of CBD is able to reduce TNF-α, IL-6, MCP-1 and MIP-2 in the bronchoalveolar lavage fluid (BALF), as well as being able to decrease the lung myeloperoxidase activity and the pulmonary infiltration of leukocytes [117,118]. Other cannabinoids exhibited similar effects. WIN 55,212-2, THC and AEA caused a dose-dependent decrease of TNF-α and neutrophil recruitment in BALF after intranasal administration, while PEA only decreased the TNF-α levels [119]. Conversely, pro-inflammatory effects were cited for

CBD in this animal model of ALI. The oral administration of CBD amplified the production of TNF-α, IL-5, IL-23 and G-CSF in C57BL/6 mice, while also increasing the infiltration of neutrophils and monocytes in the BALF [120].

Another animal model of lung inflammation was used by Arruza et al. to test the effects of CBD on newborn piglets with hypoxic–ischemic brain damage [121]. CBD decreased IL-1, the protein content and leukocyte infiltration in the BALF and the extravascular lung compartment; interestingly, the authors identified that the serotonin 1A (5-HT1A) receptor participated in the mediation of these effects.

Some pulmonary anti-inflammatory effects of cannabinoids appear to be carried out through non-cannabinoid receptors. Tauber et al. showed that WIN55,212-2 causes a decrease in MMP-9 production in mice with lung inflammation induced by cigarette smoke via the extracellular signal-regulated kinase (ERK) signaling pathway consecutive to TRPV1 activation [122].

Asthma is a serious pulmonary disease with an important associated inflammatory component. Mice with ovalbumin (OVA)-induced asthma were treated with 5 mg/kg intraperitoneal CBD, and a reduction of TNF-α and ILs 4, 5, 6 and 13 was observed [123]. In the same animal model, CBD also decreased AHR and the collagen fiber content alongside a decrease of the inflammatory markers in the BALF [124]. Conversely, JWH-133 caused an increase in eosinophil migration, chemotaxis and ROS generation, aggravating the AHR [125]. Furthermore, a study using a CB2 knockout mouse model showed that CB2 activation triggers pro-inflammatory effects with increased IFN-γ production by pulmonary NK cells in the BALF [126].

Cannabinoids are also able to modulate the inflammation related to various infectious diseases. In a series of articles, Tahamtan et al. showed that the activation of CB1 and CB2 receptors by JZL184 and JWH-133, respectively, decreased the production of cytokines and the influx of cells, alleviating lung pathology in Balb/c mice infected with the respiratory syncytial virus (RSV) [127,128]. THC also exhibits anti-inflammatory properties in mice with influenza infection via CB1 and/or CB2 activation by decreasing the levels of IL-17 and IFN-γ, as well as the macrophage infiltration in the BALF; however, THC also caused immunosuppression by decreasing the recruitment of CD4+ and CD8+ T-cells and macrophages with a subsequent increase of the viral load [129,130]. THC is capable of reducing the cell proliferation and levels of IFN-γ, inhibiting the phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) pathway and decreasing the *Staphylococcal* enterotoxin B-induced lung toxicity in C3H/HeJ mice [131]. PI3K/Akt pathway activation was also achieved in C57BL/6 mice treated with JWH-133 for paraquat-induced ALI, causing a CB2 mediated decrease of TNF-α, IL-6 and MPO activity while improving lung function [132].

JWH-133 reduces the serum and tissue levels of TNF-α, IL-6 and IL-1β in a polymicrobial sepsis model in rats while increasing the IL-10 levels [133]. Additionally, neutrophil recruitment, bacteremia and lung injury are decreased when CB2 synthetic agonist GP1a is used in septic C57BL/6J wild-type mice [134]. Other CB2 agonists, such as melilotus, exhibit similar effects in this animal model, while also decreasing neutrophils and lymphocyte infiltration and blocking NF-κβ activity [135].

Various other pulmonary disease models have been used to evaluate the efficacy of cannabinoids. A dose of 1 mg/kg of JWH-133 improves neurogenic pulmonary edema at 24 h after subarachnoid hemorrhage in rats, decreasing the MPO activity and leukocyte infiltration while improving the lung permeability and tight junction protein levels [136]. Furthermore, AEA demonstrated anti-inflammatory effects by increasing the expression of heat shock proteins (HSP) 25 and 70 in the lungs of rats injected with 1 mg/kg AEA [137]. Moreover, the oral or intraperitoneal administration of β-Caryophyllene prevents neutrophil infiltration and the decreased production of IL-12, NO, leukotriene B4 and CXCL1/keratinocytes-derived chemokine (KC) in C57Bl/6 mice with *Mycobacterium* bovis-induced pulmonary inflammation [138].
