Cannabidiol differentially regulates basal and LPS-induced inflammatory responses in macrophages, lung epithelial cells, and fibroblasts

https://doi.org/10.1016/j.taap.2019.114713 Get rights and content

Highlights

  • Cannabidiol (CBD) containing products are available in a plethora of flavors and forms.

  • CBD showed differential pro- and anti-inflammatory effects by ROS levels.

  • CBD significantly attenuated LPS-induced NF-κB activity and IL-8 and MCP-1.

  • CBD and dexamethasone reduced the IL-8 level induced by LPS via MCPIP.

  • CBD has a differential inflammatory response and acts as an antagonist with steroids.

Abstract

Introduction

Cannabidiol (CBD) containing products are available in a plethora of flavors in oral, sublingual, and inhalable forms. Immunotoxicological effects of CBD containing liquids were assessed by hypothesizing that CBD regulates oxidative stress and lipopolysaccharide (LPS) induced inflammatory responses in macrophages, epithelial cells, and fibroblasts.

Methods

Epithelial cells (BEAS-2B and NHBE), macrophages (U937), and lung fibroblast cells (HFL-1) were treated with varying CBD concentrations or exposed to CBD aerosols. Generated reactive oxygen species (ROS) and inflammatory mediators were measured. Furthermore, monocytes and epithelial cells were stimulated with LPS in combination with CBD or dexamethasone to understand the anti-inflammatory effects of CBD.

Results

CBD showed differential effects on IL-8 and MCP-1, and acellular and cellular ROS levels. CBD significantly attenuated LPS-induced NF-κB activity, IL-8, and MCP-1 release from macrophages. Cytokine array data depicted a differential cytokine response due to CBD. Inflammatory mediators, IL-8, serpin E1, CXCL1, IL-6, MIF, IFN-γ, MCP-1, RANTES, and TNF-α were induced, whereas MCP-1/CCL2, CCL5, eotaxin, and IL-2 were reduced. CBD and dexamethasone treatments reduced the IL-8 level induced by LPS when the cells were treated individually, but showed antagonistic effects when used in combination via MCPIP (monocytic chemotactic protein-induced protein).

Conclusion

CBD differentially regulated basal pro-inflammatory response and attenuated both LPS-induced cytokine release and NF-κB activity in monocytes, similar to dexamethasone. Thus, CBD has a differential inflammatory response and acts as an anti-inflammatory agent in pro-inflammatory conditions but acts as an antagonist with steroids, overriding the anti-inflammatory potential of steroids when used in combination.

Introduction

Cannabidiol (CBD) is the non-psychoactive derivative of the Cannabis sativa (marijuana) plant. It has been widely used for medicinal benefits by all age groups, including children. CBD is self-administered for its anxiolytic, antiemetic, anti-inflammatory, and anti-cancer properties (Fraguas-Sanchez and Torres-Suarez, 2018). Numerous consumer products are available in the market containing CBD. These products include edibles, personal care products, health supplements, and e-liquids used in ENDS (electronic nicotine delivery systems). Routes of administration of CBD oil are oral, sublingual, topical, and inhalation via ENDS. The majority of CBD liquid/oil users purchase it online or at local retailers and do not require a prescription. Currently, there are no FDA regulations on these commercially available CBD containing products. In 2018, for severe forms of epilepsy, such as Dravet syndrome, the FDA approved Epidiolex, a purified form of CBD oil, as a prescribed medicine for children above 2 years old. It is important to assess the effects of exposure to these CBD containing products, such as vaporizable liquids used in e-cigarettes, before being available in the market.

Inhaled toxicants can generate reactive oxygen species (ROS), giving rise to an inflammatory response (Wong et al., 2016). As a result, inflammatory mediators, such as interleukin-8 (IL-8) and monocytic chemoattractant protein-1 (MCP-1), are secreted enhancing the recruitment of inflammatory cells (Turner et al., 2014). These inflammatory cells, such as alveolar macrophages and neutrophils, release more ROS, such as superoxide and hydrogen peroxides during respiratory burst (Kawabata et al., 2002; Glennon-Alty et al., 2018). These secondary reactive species act as a positive feedback loop dysregulating the oxidative homeostasis leading to inflammation as well as tissue injury. Gene expression of these inflammatory cytokines depends upon Nuclear Factor kappa beta (NF-κB) transcription factor (Ueda et al., 1994; Hildebrand et al., 2013). Therefore, we assessed the activity of NF-κB upon the treatment of CBD to understand the signaling mechanism. To further understand the mechanism of cytokine regulation, monocyte chemotactic protein-induced protein-1 (MCPIP-1) protein expression was determined in cells. MCPIP-1 has been known to dampen the inflammatory cytokine response by inhibiting NF-κB activation and toll-like receptor signaling (Li et al., 2017).

As sublingual and inhalation are the primary routes of CBD administration, in this study, we tested normal primary human bronchial cells (NHBE), bronchial epithelial cells (BEAS-2B), monocytes (U937), and human lung fibroblasts (HFL-1). It is vital to assess cellular changes and the inflammatory response upon exposure to CBD containing liquids as xenobiotics are known to cause inflammatory disorders due to chronic ROS exposure (Upham and Wagner, 2001; Pagano, 2002). We further studied the effect of CBD on LPS-induced inflammatory responses and compared the anti-inflammatory properties between dexamethasone and CBD containing liquids.

We have previously shown that e-liquids and their flavoring chemicals disrupt oxidative homeostasis in cells, thus inducing inflammation (Gerloff et al., 2017; Muthumalage et al., 2017). Many vaping products are emerging with CBD as a beneficial constituent in the e-liquid, but the validity of these claims has not been scientifically proven. Thus, we attempted to assess the CBD containing liquids and their toxicological effects on cells. We hypothesized that exposure to CBD containing e-liquids regulates inflammation and oxidative stress in monocytes/macrophages and various lung cells.

Section snippets

Ethics statements

We used a rigorous and unbiased approach throughout the experimental plans mentioned in this manuscript. Reproducibility has been ensured by repeating the experiments. All the key biological and chemical resources that are used in this study were validated except for the tested CBD oil/liquid containing products. Our results adhere to NIH standards of reproducibility and scientific rigor.

Procurement of CBD containing liquids

CBD oil-containing liquids, Green Roads (100 mg, 300 mg, and 550 mg), and Hemplucid (1000 mg) were purchased

Cellular ROS generation by CBD

To assess the cellular ROS generation by CBD containing liquids RAW264.7cells were treated with 10.6 μM and 21.2 μM of the Green Roads CBD oil (100 mg) and stained with cellROX green stain. The intensity of the whole image increased with the CBD treatments compared to the untreated counterpart. CBD 21.2 μM treatment showed significantly greater image intensity (ROS intensity) compared to the 10.6 μM CBD treatment. CBD (10.6 μM) slightly increased the intensity but was not significant compared

Discussion

Pharmacological benefits of pure CBD have been shown in ameliorating ailments (Nagarkatti et al., 2009). We tested commonly available CBD liquids which are primarily administered sublingually and orally. Composition analysis of these liquids showed that most of these e-liquids also contain other ingredients, such as metals, trace amounts of THC, and flavoring chemicals. Elements that were present in these liquids included chromium, copper, and lead, all of which cause detrimental health

Acknowledgments

This research was supported by the Toxicology Training Program grant T32-ES007026, and National Institute of Health NIH 2R01HL085613, HL137738, HL135613, and the WNY Center for Research on Flavored Tobacco Products (CRoFT) under cooperative agreement U54CA228110. Melanie Prinz and Thomas Lamb helped with the acellular ROS assays. Alan Friedman, Ph.D. at the University at Buffalo, performed GC–MS analysis of the CBD containing liquids. Thanks to Tom Scrimale and Matthew Rand of the Element

Authors' contributions

TM conducted most of the experiments and data analysis.

TM and IR conceived/designed the experiments and prepared the manuscript.

Declaration competing of interests

The authors declare no conflict of interests.

Disclaimer

The authors have nothing to claim or disclaim about any products used here to test their toxicological and biological effects. The authors have no personal interests or gains from the outcome of this study. The products tested are available commercially to consumers/users.

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