Modulatory Potential of Cannabidiol on the Opioid-Induced Inflammatory Response

Abstract

Opioids are effective analgesics; however, there are many negative consequences of chronic use. One important side effect of chronic opioid use is the continuous engagement of the immune response that can exacerbate chronic pain. The opioid, morphine, initiates a Toll-like receptor 4 (TLR4) signaling cascade that drives the activation of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome proteins, resulting in cytokine production and effectively creating a positive feedback loop for continuous TLR4 activation. In addition to driving cytokine production, morphine drives changes in proinflammatory lipid signaling. The alteration of both cytokine and lipid signaling systems by morphine suggests that its chronic use leads to a pathological immune response that would benefit from targeted therapy. Engaging the endogenous cannabinoid system has shown therapeutic benefit, particularly regarding its anti-inflammatory and immunosuppressive effects. Promising preclinical and clinical investigations suggest that cannabidiol (CBD) is an effective adjuvant for treatment of symptoms of opioid use disorders; however, the mechanism through which CBD drives this outcome is unclear. One potential source of insight into this mechanism is in how CBD regulates immune regulators such as cytokines and lipid signaling systems, including endocannabinoids and related immune-responsive lipids. In this review, we outline the immune response to chronic opioid use as well as CBD in the context of a lipopolysaccharide-induced immune response and speculate on the mechanism of CBD as a modulator of chronic opioid-induced immune system dysregulation.

Introduction

Immune responses are vital to preparing the body to fight against foreign invaders as well as internal danger signals. During a typical immune response, an alarm signal is recognized and downstream signaling results in the production of proteins and lipids and both can amplify the alert and protect against its potentially harmful effects. One well-studied sensor of foreign and endogenous danger signals is Toll-like receptor 4 (TLR4). While TLR4 signaling is beneficial to maintaining homeostasis as part of a typical immune response, chronic activation can have deleterious effects. As opioids continue to be used as effective analgesics and prescribed over extended periods of time, it is important to understand the maladaptive immune response caused by morphine, such as chronic activation of TLR4.

In this study, we discuss how the immune response to chronic morphine can be evaluated in cellular and animal model systems and how it parallels with well-studied immune reactions to agents such as lipopolysaccharide (LPS). A third system being discussed are endogenous cannabinoids (eCBs), which are a growing system of receptors, endogenous ligands (e.g., anandamide [AEA]), and enzymes that are modulated by the active compounds in Cannabis (e.g., cannabinoids) such as Cannabidiol (CBD). We use these comparisons to illustrate how CBD is effective in modulating the LPS response and propose that similar cellular mechanisms are likely at play with CBD as a potential adjuvant therapeutic to modulate the maladaptive immune response that occurs with chronic opioid use.

TLR4, Inflammasomes, and the eCB System

There are many components of the immune response that are potential targets for therapeutics. In this study, we focus on pathways that are regulated in specific types of innate activity, as well as those exacerbated by chronic opioid use. Figure 1 provides a graphical representation of cellular signaling with LPS activation in immune cells, such as mast cells and microglia, which is explained in more detail below. These pathways are not meant to be the exhaustive summary of the LPS-induced immune response; however, they are those that most closely correspond to the morphine-induced immune response that will be described in this review.

FIG. 1.

Selected LPS-induced immune pathways. More comprehensive details of these pathways are provided in the text and use the following references.^{4,8,19,20,34,35,45,46,49,51} For clarification, short summary findings are provided on the figure highlighting each of the references listed. LPS, lipopolysaccharide.

TLR4 is a pattern recognition receptor that is activated by exogenous pathogen-associated molecular patterns, such as LPS, and endogenous danger-associated molecular patterns (DAMPs), such as high-mobility box group 1 (HMGB1) and heat shock proteins.¹ HMGB1 is a DAMP whose disulfide form binds the TLR4 accessory protein myeloid differentiation protein 2 (MD-2), activating TLR4 and resulting in increases in cytokines.^2,3 To connect to downstream kinases and signaling molecules, TLRs require adaptor proteins such as MyD88, which initiates a unique, MyD88-dependent signaling cascade.^4–6

The inflammasome is a multiprotein complex that activates caspase-1, which cleaves pro-IL-1β into mature IL-1β.^5,7 Expression of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes in the macrophage cell line NR8383 is modulated by MD-2 after LPS treatment, linking the NLRP3 inflammasome system with TLR4 activation.⁸ In a feedback dynamic, TLR4 activation leads to NLRP3 priming, then the subsequent activation of NLRP3 (and its downstream effects) leads to further TLR4 activation.^9,10 TLR4 is also coupled with protein kinase C ɛ (PKCɛ) following LPS treatment, an effect dependent on MyD88.¹¹

A key result of TLR4 activity is the generation of transcription factors AP-1,¹² IRF3,¹³ and NFκB,¹⁴ resulting in synthesis of a variety of proteins that include precursors to proinflammatory cytokines, such as pro-IL-1β,¹⁵ and cytokines themselves, such as TNF.¹⁶ TLR4 has also been implicated in the “two-hit hypothesis,” in which an initial immune activation leads to a heightened response following a second activation (e.g., chronic morphine treatment) (see review in Zhang et al.⁹). An additional pathway for LPS-derived NLRP3 activity is through the activation of purinergic P2X7 receptors coupled to pannexin-1 activity driving increases in ATP.¹⁷ Pannexin-1 has been linked to the eCB system because the blockade of Panx1 in hippocampal slices produces elevated levels of the eCB ligand, AEA,¹⁸ which provides an important link between these systems.

eCBs and related lipids are also modulated with LPS activity. In mast cells, TLR4 activation by LPS leads to an increase in the eCB ligand, 2-arachidonlyl glycerol (2-AG), which then acts through cannabinoid receptor type 2 (CB₂) to upregulate negative modulators of TLR4 and prevent TNF secretion as part of endotoxin tolerance.¹⁹ Systemic administration of the fatty acid amide hydrolase (FAAH) inhibitor PF3845 before LPS treatment results in increases in AEA, N-palmitoyl ethanolamine, and N-oleoyl ethanolamine (OEA) and an attenuation of LPS-induced increases in IL-1β, IL-6, IL-10, TNFα, COX2, and iNOS mRNA, but not IL-1ra or mPGES in the frontal cortex 2 h after LPS treatment.²⁰ Twenty-four hours after LPS treatment, animals coadministered PF3485+LPS have even higher levels of IL-1β and TNFα in the frontal cortex than animals treated with LPS alone. Taken together, this suggests that FAAH inhibition does not eliminate the effects of LPS, but rather delays them. The effects of PF3485 are due, in part, to transient receptor potential vanilloid 1 (TRPV1) channel activation because the TRPV1 antagonist IRTX prevents the decrease in IL-6.²⁰

Increasing 2-AG levels in the brain through monoacylglycerol lipase inhibition protects against increases in iNOS—but not TNFα or IL-1β—after chronic exposure to the environmental pollutant sulfur dioxide, an effect that occurs in a cannabinoid receptor type 1 (CB₁)/CB₂-dependent manner.²¹ AEA exerts a protective effect against hyperhomocysteinemia-induced increases in Caspase-1 and IL-1β in murine podocytes and prevent inflammasome assembly in glomeruli in vivo. This effect is not seen in NLRP3^−/− mice and is prevented by coadministration of the COX-2 inhibitor celecoxib, suggesting that the AEA metabolite PGE₂-EA is a key player. Confirming the inhibitory effects of PGE₂-EA on the NLRP3 inflammasome, PGE₂-EA can prevent inflammasome assembly and activation at a low concentration, in which AEA exerted no effect (10 μM).²²

Prostaglandins (PGs) are integrally involved in immune responses and TLR4 signaling. In a study examining the effects of Staphylococcus aureus infection on primary peritoneal macrophages, compared to cells derived from wild-type animals, cells derived from NLRP3^−/−, TLR4^−/−, and TLR2^−/− animals exhibit significantly lower levels of mPGES-1 and COX-2 mRNA and protein after infection. In addition, NLRP3^−/−, TLR2^−/−, and TLR4^−/− cells produce less IL-1β, TNFα, PGE₂, and RANTES (chemokine) in response to S. aureus lipoproteins, while both TLR4^−/− and NLRP3^−/− secrete less IL-1β. COX-2 inhibition with CAY10404 or NS398 decreases protein levels of NLRP3 and TLR2 (but not TLR4 surface protein), and decreases secretion of IL-1β and RANTES secretion (but not TNFα). This suggests a role for PGE₂ in the regulation of TLR2 and NLRP3 signaling in response to bacterial infection. Application of exogenous PGE₂ was also capable of inducing increases of TLR4 surface expression, but did not influence NLRP3 expression. Treating cells with PGE₂ before S. aureus infection resulted in increased activation of JNK, p38, p65, and caspase-1, as well as enhanced expression of IL-1β and pro-IL-1β.²³

TLR4 activation in bone marrow-derived macrophages has been linked to broad changes in the lipidome. When lipids were extracted 24 and 48 h after treating cells with the TLR4 agonist LPS, the lipidome of treated cells was markedly distinct from the lipidome of cells stimulated with other TLR agonists. In the same study, 48 h after LPS treatment, an additional examination of how lipids changed when MyD88 and TRIF were blocked showed that the two adaptor proteins resulted in different lipidomic profiles that act in opposition.²⁴ These data suggest that an important target in modulating each of these immune system signaling pathways may be the enzymes that modulate larger classes of lipids and not simply specific species.

Implications of Chronic Opioid Activity on TLR4, Inflammasome, and eCB/Lipid Signaling

There are four different opioid receptors: (μ-opioid receptor [MOR]), Δ-, κ-, and nociceptin, all of which are G protein-coupled (reviewed in Refs.^25,26). The receptors are located both centrally and peripherally in a variety of cell types such as neurons, astrocytes, and immune cells, including microglia (see review in Machelska and Celik²⁷). Despite the common use of morphine as an analgesic, chronic administration can result in negative side effects, including hyperalgesia^28,29 and tolerance.²⁸ Figure 2 provides a graphical representation of the modulatory actions of morphine highlighted in red text superimposed on the immune responses outlined in Figure 1.

FIG. 2.

Selected morphine-induced immune pathways. This figure uses the pathways outlined in Figure 1 with changes induced by chronic morphine highlighted in red. More comprehensive details of these effects are provided in the text and use the following references.^{10,22,30,32,33,35,36,38,39} For clarification, short summary findings are provided on the figure highlighting each of the references listed in red.

Microglial MORs are involved in the development of analgesic tolerance as evidenced by the delayed tolerance shown in animals with conditional deletion of MOR from microglia.²⁸ In the spinal cord, the microglial pannexin-1 channel is identified as acting in morphine withdrawal,³⁰ but not tolerance or hyperalgesia.³¹ While analgesia due to high-dose morphine (3 mg/kg) has been hypothesized to occur through an alternative pathway that involves MORs in a non-nociceptor neuronal population, hyperalgesia and hyperalgesia priming due to low-dose morphine (0.03 mg/kg) treatment have been shown to require TLR4 and PKCɛ in nociceptors.³² Because TLR4 receptors are expressed in microglial cells,^29,33 the relationship between morphine and activation of TLR4 is of interest when studying the effects of morphine on the central nervous system (CNS).

Treating microglial cells with LPS along with a low dose of morphine (100 nM) leads to increased NFκB mRNA through μ-opioid receptor and PKCɛ activation, whereas the effects of administering a high dose of morphine (10 μM) on NFκB require TLR4 and PKCɛ activity.³⁴ More specifically, evidence exists that morphine contributes to TLR4 activation by binding to the TLR4 accessory protein MD-2, which then induces oligomerization of TLR4.³⁵ In BV2 (immortalized murine microglia) cells, morphine initiates a proinflammatory response that is similar to the effects of LPS stimulation via TLR4.³⁵ The involvement of TLR4 in the effects of opioids might have practical implications, in that coadministration of the TLR4 inhibitor TAK242 and morphine prevented opioid-induced hyperalgesia after tibial fracture in mice.²⁹ In the case of morphine-induced persistent pain sensitization after chronic constriction injury (CCI), the effect depends on microglia, TLR4, P2X7, and NLRP3 inflammasomes in the dorsal spinal cord.³⁶ Inflammasomes have been connected to both TLR4 and the downstream effects of morphine and are of particular interest because they provide a possible mechanism for positive feedback of TLR4 signaling as outlined above.

In a model of morphine-induced hyperalgesia after CCI, HMGB1 protein levels are increased and pharmacological blockade of TLR4, P2X7, and caspase-1 attenuates this increase. Inhibiting HMGB1, biglycan, and HSP90 prevented persistent sensitization. Thus, TLR4 and NLRP3 have been identified as part of a positive feedback loop, in which morphine leads to the synthesis of DAMPs by NLRP3 inflammasome, which then activate TLR4.¹⁰

Potassium channels also play an important role in the relationship between morphine, TLR4, and NLRP3. Chronic morphine treatment has been shown release neuronal HSP70, a process which depends on the K_ATP channel and subsequently activates TLR4 on microglia, leading to increases in phosphorylated p38 MAPK, NFκB p65, mRNA for IL-1β, and TNFα, and levels of NLRP3 protein, as well as activating caspase-1 and IL-1β when ATP is present.³⁷

Large conductance calcium-activated potassium (BK) channels located in spinal microglia have also been identified in morphine-induced hyperalgesia and anti-nociceptive tolerance, as illustrated by morphine-induced increases in membrane and protein levels of P2X4Rs, which alter outward BK currents. This is hypothesized to occur through a pathway involving arachidonic acid (AA) or its metabolites (e.g., PGs) because morphine-elicited BK currents in MG6 cells are attenuated by PLA₂ inhibition. Application of extracellular AA leads to slightly delayed generation of BK currents, whereas intracellular application leads to immediate BK currents. In addition, intracellular PGE₂ administration leads to BK currents, and indomethacin (COX1/2 inhibitor), NS398 (COX2 inhibitor), and baicalein (12/15 lipoxygenase inhibitor) inhibit BK currents after morphine treatment.³⁸

Chronic morphine treatment in rats results in a significant decrease in levels of 2-AG across the brain, independent of AEA levels.³⁹ Such a decrease in 2-AG may be indicative of an impaired microglial response. In contrast, acute morphine does not result in significant changes in spinal levels of endogenous lipids PGE₂ or AEA on its own, nor does acute morphine treatment before carrageenan inflammation significantly alter these compounds or palmitic, stearic, and AA.⁴⁰ Taken together, the modulatory effects of AEA, 2-AG, and their metabolites across multiple models of immune activation suggest that they may also exert a protective effect against immune assaults from chronic morphine.

CBD, TLR4, the NLRP3 Inflammasome, and eCB/Lipid Signaling

While data suggest that CBD acts as a negative allosteric modulator of CB₁,⁴¹ CB₂⁴² and opioid receptors,⁴³ as well as an antagonist at GPR55,⁴⁴ it has effects on the immune response discussed here that appear to be largely independent of this activity. Figure 3 graphically highlights how CBD effects the TLR4-inflammasome pathways in the context of LPS stimulation shown in Figure 1 and these changes are highlighted in green superimposed on the figure to show where the distinctions are known and are further outlined in the text here.

FIG. 3.

Effects of CBD on selected LPS-induced immune pathways. This figure uses the pathways outlined in Figure 1 with changes induced by CBD treatment highlighted in green. More comprehensive details of these effects are provided in the text and use the following references.^{45,46,49–51,54} For clarification, short summary findings are provided on the figure highlighting each of the references listed in green. CBD, cannabidiol.

When microglial cells are activated by the TLR4 agonist LPS (24 h), CBD (1, 10 μM) reduces microglial activation, p NFκB p65, reactive oxygen species (ROS), TNFα, IL-1β, glucose uptake, and NADPH synthesis. This effect is suggested to be the result of an inhibition of ROS and NFκB signaling.⁴⁵ In the BV2 microglial cell line, pretreatment with CBD attenuates LPS-induced increases in proinflammatory cytokines IL-1β and IL-6 and attenuates LPS-induced increases in IL-1β and IFNB mRNA. This effect results, in part, from inhibition of the signaling pathway that leads to NFκB activation.⁴⁶ Transcriptional changes after CBD treatment are not limited to IL-1β and IFNB.

BV2 microglia cells treated with 10 μM CBD for 6 hours exhibit increases in mRNA levels for 680 genes as well as decreases for genes involved in inflammation, including CX3CR1, CCL2, CCL7, CXCL14, CCL6, and CCL9.⁴⁷ Acute CBD administration also leads to transcriptional changes, including an increase in heat shock proteins in T98G and U87MG human glioblastoma cell lines.⁴⁸ In the THP-1 human macrophage cell line, CBD (45 min) pretreatment before LPS (30 min) affects the TLR4 MyD88-independent pathway and prevents nuclear accumulation of IRF3 and increases in CXCL20, and IFN-β, but does not influence IκB degradation or increases in TNFα or CXCL8 protein seen after LPS. This effect is CB₁, CB₂, and PPARɣ independent. Importantly, the effects of CBD pretreatment on IL-1β were not investigated in this study.⁴⁹ Treating THP-1 cells with CBD (1 h) before LPS (24 h) also attenuates LPS-induced increases in IL-1β, but not TNFα.⁵⁰ Consequently, these data show that CBD can drive long-term signaling and transcriptional changes, some of which are involved in inflammatory pathways.

In line with its activity in modulating molecules involved in immune responses, CBD protects against inflammasome activation. In the RAW264.7 murine macrophage cell line, LPS treatment combined with ATP results in increased NFκB activation, as well as increased levels of inflammasome proteins (NLRP3 and ASC) and mRNA levels of IL-1β, TNFα, and MCP-1. Pretreating cells with CBD attenuated these effects, possibly through regulation of NFκB signaling; treating cells with the NFκB inhibitor BAY11-7082 resulted in significant attenuation of the effects, while CBD+BAY11-7082 resulted in further attenuation.⁵¹ The previously mentioned anti-inflammatory effect of CBD on THP-1 monocytes was also indicative of NLRP3 inflammasome inhibition: pretreating cells with CBD before activating the inflammasome (by LPS and nigericin) reduced IL-1β in a way that was comparable to known inhibitors (oridonin, MCC950). Pretreatment with CBD also prevented increased K⁺ release from the cells that occurs after nigericin or LPS+nigericin treatment, potentially by modulating the activity of P2X7.⁵⁰

A prevalent hypothesis suggested that CBD drives increases in AEA through FAAH inhibition⁵²; however, this was primarily because CBD caused robust increases in AEA, and FAAH is the primary metabolic enzyme for AEA. Work from Dale Deutsch's laboratory provides evidence that CBD is not acting as a FAAH inhibitor; but that it is being transferred into the cell through fatty acid-binding proteins, which facilitate its ability to increase AEA.⁵³ Recently, we demonstrated that the CBD-induced increases in AEA and related lipids occur through a NAPE-PLD-dependent pathway.⁵⁴ NAPE-PLD is the primary biosynthetic enzyme for AEA⁵⁵; therefore, the current working hypothesis is that CBD is causing an upregulation in NAPE-PLD activity. We also showed that CBD causes decreases in PGs in a NAPE-PLD-independent pathway manner.⁵⁴

In two models of hypertension (spontaneous and DOCA-salt), chronic CBD (10 mg/kg for 14 days via i.p. injection) modulated lipid signaling levels in the heart. In the DOCA model, CBD decreased 2-AG, OEA, N-docosahexaenoyl ethanolamine (DEA), and AA in cardiac tissue, as well as DEA, N-linoleoyl ethanolamine, AA, and docosahexaenoic acid in the plasma; in the spontaneous model, CBD only decreased N-stearoyl ethanolamine, and the only change in receptor expression after CBD treatment was a decrease in GPR55. Surprisingly, chronic CBD treatment in animals without hypertension resulted in a decrease in GPR18 (spontaneous control), plasma AA (both controls), and linoleic acid (LA) (DOCA control), as well as increases in LA and AA in both controls.⁵⁶ Taken together, these studies suggest that chronic CBD influences lipid signaling differently, depending on treatment schedule (acute vs. chronic) and the pathology of the animal (i.e., normal, spontaneously hypertensive, DOCA-salt hypertensive) and the tissue examined. These results support further investigation of chronic CBD treatment in morphine-dependent animals to better understand its potential effects on lipid signaling in this context.

Conclusions

In addition to its effects on inflammation, investigations of CBD as a potential treatment for opioid use disorders (OUD) have shown promising results in both human and animal studies. Acute CBD administered to rats prevents the typical reactivation of an extinguished morphine-induced conditioned place preference following exposure to triggering stimuli (an acute injection of morphine or stressful event).⁵⁷ In humans recovering from heroin use disorder, 3 days of CBD treatment attenuated drug cravings and anxiety in response to drug-related cues.⁵⁸ While these studies are important in demonstrating a link between CBD and favorable behavioral outcomes for OUD treatment, it remains unclear what specific activity CBD may have at the molecular level.

In this review, we have outlined how morphine mimics many of the LPS-derived cellular responses and drives the activation of multiple immune pathways that cause chronic increases in proinflammatory responses. We also highlight how CBD drives a significant downregulation on these LPS-derived proinflammatory pathways. Therefore, we hypothesize that at least part of the mechanism for the therapeutic benefits of CBD in OUD treatments is the reduction of these chronic immune responses. It will be an important avenue to investigate how CBD effects the morphine-derived immune responses as there are currently no published data focusing on this interaction.

Abbreviations Used

2-AG

2-arachidonlyl glycerol

AA

arachidonic acid

AEA

anandamide

BK

calcium-activated potassium

CB₁

cannabinoid receptor type 1

CB₂

cannabinoid receptor type 2

CBD

cannabidiol

CCI

chronic constriction injury

DAMPs

danger-associated molecular patterns

DEA

N-docosahexaenoyl ethanolamine

eCB

endogenous cannabinoids

FAAH

fatty acid amide hydrolase

HMGB1

high-mobility box group 1

LA

linoleic acid

LPS

lipopolysaccharide

MD-2

myeloid differentiation protein 2

MOR

μ-opioid receptor

NLRP3

NOD-, LRR- and pyrin domain-containing protein 3

OEA

N-oleoyl ethanolamine

OUD

opioid use disorders

PGs

prostaglandins

PKCɛ

protein kinase c ɛ

ROS

reactive oxygen species

TLR4

Toll-like receptor 4

TRPV1

transient receptor potential vanilloid 1

Author Disclosure Statement

No competing financial interests exist.

Funding Information

CTJ received funding from DA024628.

Cite this article as: Johnson CT, Bradshaw HB (2021) Modulatory potential of cannabidiol on the opioid-induced inflammatory response, Cannabis and Cannabinoid Research 6:3, 211–220, DOI: 10.1089/can.2020.0181.