There is a growing interest in the medical use of cannabis, especially CBD, considering the reported benefits, such as pain reduction, anti-inflammatory properties, antioxidant, antidiabetic, anticancer, anxiolytic, anticonvulsive, antiepileptic, and the absence of psychotropic effects induced by THC^3,14. The lack of psychotropic effects is one of the most attractive differences from its close derivate THC. However, CBD's regulatory role on inflammasome and HIV infection has not been studied. Previously published studies were focused on the neuroprotective attributes of CBD as well as anti-inflammatory features. However, Little is known with respect to the effect and mechanism of CBD and THC and a combination of both cannabinoids on HIV infected microglia cells.

A study published by Lowe et al.¹⁵ proved a possible antiviral activity of CBD against Hepatitis C Virus, which was comparable to the inhibitory effect of the interferon-alpha. Similarly, 2022 Nguyen et al. suggested the inhibition of SARS-CoV-2 virus in lung cells¹⁶. However, a study found that CBD led to downregulating interferon-stimulated genes and decreased anti-HIV activity in recently infected macrophage cells. Interestingly, the same study reported significant reductions in HIV RNA and gag HIV protein in long-term infected cells treated with CBD¹⁷. In addition, DeMarino et al., reported the induction of a protective effect by reducing the release of extracellular vesicles from HIV-1 Infected monocytes and viral RNA¹⁸.

Agudelo et al.¹⁹, demonstrated that Δ(9)-THC enhanced the expression of the HIV Long Terminal Repeat gene in monocyte-derived dendritic cells isolated from human leukopacks and infected with HIV. In contrast with our results, Molina et al.²⁰, reported that THC administration attenuated the progression of Simian Immunodeficiency Virus (SIV). Chronic Δ(9)-THC did not increase the viral load or exacerbate the morbidity of SIV. Moreover, Δ(9)-THC ameliorated the progression of SIV disease and attenuated the inflammation caused by the virus.

Our study did not detect an increment in GFP-positive cells, suggesting that CBD did not enhance the HIV infection; on the contrary, a reduction of GFP-positive cells and LTR gene expression was detected. However, more investigation is in need to elucidate the mechanisms behind this reduction. Specifically, our study shows that CBD significantly reduced the expression of HIV positive cells, while THC did not have this effect.

The expression of the Cannabinoid receptors (CNR) in glia cells is conditioned to the phenotype and activation of these cells. For example, CNR 1 is highly expressed in neuronal cells and lower in glial cells. On the contrary, glial cells express more CNR2. It has been reported the upregulation of the expression of CNR2 under neuroinflammation and neurodegeneration conditions in microglia cells, specifically after the stimulation with GM-CSF and IFNγ²¹ Moreover, the activation of cannabinoid receptor type 2 mitigated the neuroinflammation inducing the reduction of NLRP3²². In addition, HIV studies conducted in 293T cells showed the inhibition of HIV-1 LTR by synthetic, selective agonists of cannabinoid receptor 2²³. Our results demonstrated an upregulation of CNR2 when HIV is activated and decreased the infection with cannabinoid treatments. However, there were no significant differences between the THC and CBD used alone or in combination after the induction of HIV. A different scenery was obtained when we did not treat with poly IC, and the HIV infection was not upregulated. In this case, the addition of CBD or THC did not reduce the cannabinoid receptors with respect to HIV control. However, CNR2 showed a significant dysregulation when CBD was compared against THC.

The inflammasome complex, a tool of the innate immune system, is composed of proteins responsible for activating the inflammatory process. The inflammasome activation is initiated when cytosolic pattern recognition receptors identify microbe-derived pathogen-associated or danger-associated molecular patterns generated by the host cell.

The inflammasome complexes activate effector molecules such as caspase-1 or Interleukin-1 converting enzyme, which cleaves and activates immature pro-inflammatory cytokines, interleukin IL-1β and IL-18 and Gasdermin-D²⁴. The cytokines are secreted and propagate the inflammatory responses to the neighboring cells. Hence, we studied the impact of CBD compared to Δ9-THC the inflammasome activation. We specifically studied the expression of the "effector" Caspase 1, IL-1ß, and other inflammatory cytokines^14,25–27.

Activated microglia, astrocytes, neurons, and T-cells release inflammatory mediators contributing to neuroinflammation and neurodegeneration. Furthermore, immune cells and mediators from the periphery can cross the defective BBB and augment neuroinflammation^8,9,11,28,29. A study on HIV patients who consume cannabis, demonstrated that cannabis and its compounds decrease the number of inflammatory CD16+ monocytes, which are implicated in neuroinflammation and the secretion of pro-inflammatory cytokines and T cell activation^30–34. Moreover, after exploring diverse functional pathways in the brains from HIV infected patients with NeuroHIV diseases, the viral loads correlated with the increase of cytokine expression³⁵. Studies that reverted the latency in HIV-infected microglia cells by proinflammatory cytokines, demonstrated the activation, translocation, and binding of NF-κB p65/p50 or IRF3 to the HIV-1 promoter regions in the long terminal repeat (LTR)²⁹. This may explain the reason why CBD can prevent HIV reactivation and propagation.

Neurodegeneration and neuroinflammation are caused by neurotoxic mediators and proinflammatory cytokines such as: IL-1β, IL-6, IL-8, IL-33, TNF-α, CCL2, CCL5, matrix metalloproteinase (MMPs), GM-CSF, glia maturation factor (GMF), substance P.^{9,13,19,28,36,37}. Our study showed that CBD and THC led to differential effects on the inflammatory pattern at similar concentrations in HIV-infected microglial cells, with CBD producing a stronger anti-inflammatory response.

CBD significantly decreased the expression of cytokines and chemokines such as MIF, SERPIN E1, IL-6, IL-8, GM-CSF, MCP-1, CXCL1, and CXCL10 compared to THC under simillar experimental conditions. MIF, a pro-inflammatory cytokine, stimulates the production of immune mediators in the brain by microglia, astrocytes, and neurons and contributes to neuroinflammation and neurodegeneration^38–40.

In addition, SERPIN E1, also known as Plasminogen Activator Inhibitor-1, is upregulated during inflammation, and injury. The knockdown of SERPIN E1 suppressed inflammation in a neuronal cell line from mice and TNF-α, IL-6, IL-1β, and TGF-β1 levels. Furthermore, reduced levels of SERPIN E1 were associated with inhibition of hemin-induced neuronal apoptosis^41–43.

IL-6 is a potent proinflammatory cytokine. The upregulation of this cytokine plays a pathological impact on chronic inflammation and autoimmunity. IL-6 is recognized as a neuroinflammatory marker widely expressed in HIV infected patients^43–45. Our study results aligned with the outcomes reported by Kozela et al. They demonstrated the decreased production of pro-inflammatory cytokines such as IL-1ß, IL-6, and IFN ß in mouse microglial cells after LPS, Δ9-THC, or CBD treatments. Furthermore, they stated that the inflammatory suppression mechanism did not involve cannabinoid receptors. Remarkably, CBD significantly reduced the activity of the NF-kB, a major regulator of the inflammation process, and the upregulated STAT3, a transcription factor implicated in inducing anti-inflammatory events. Moreover, both Δ9-THC and CBD diminished the expression of STAT1, a key regulator of IFNß -dependent proinflammatory progression³². Likewise, Δ9-THC reduced the expression of MCP-1 and IL-6 in the co-culture of human astrocytes and monocyte TLR-7 stimulated. These authors reported that THC acts through the CB2 receptor and suppresses IL-1ß mRNA and caspase-1 activity In the brain⁴⁶.

MCP-1 (Monocyte chemoattractant protein-1), a beta chemokine, has been associated with various neuroinflammatory disorders, such as multiple sclerosis (MS), Alzheimer's disease (AD), and Parkinson's disease. Notably, high Serum levels of MCP-1 levels are increased in mild cognitive impairment and mild AD^47–49. Similarly, IL-8 cytokine is associated with neuroinflammatory disorders, and CBD significantly mitigated the LPS-induced NF-κB activity, IL-8, and MCP-1 in human macrophages⁵⁰. Our study showed similar results, Muthumalage et al. used a higher concentration of CBD. We evaluated the effect of 1uM of CBD after the activation of HIV infection with poly IC, an agonist of TLR3, in human microglia cells.

Granulocyte macrophage colony-stimulating factor (GM-CSF) generates significant activation of microglia, and causes neuronal network dysfunction, which contributes to cognitive impairments, BBB leakage, and/or cell infiltration^28,51. GM-CSF is considered a serum inflammatory cytokine^52–55. Moreover, in airway basal cells isolated from non-smoker HIV patients receiving antiretroviral drugs, GM-CSF is being released continuously, leading to lung inflammation⁵². Our study shows that CBD did not induce this important cytokine, unlike THC.

Additionally, we observed an upregulation in the expression of RANTES after CBD treatment (Fig. 4). CCL5/RANTES is a pro-inflammatory chemokine highly expressed in monocytes and lymphocytes, which acts as a chemotactic agent for immune cells. Previous studies concluded that RANTES has an HIV-suppressive function in a dose-dependent manner, and its antiviral function was reversed when neutralized antibodies against RANTES were used in inhibition assays^56,57. Our study shows that CBD increases the expression of RANTES, which may attract immune cells to the infection site. This result places CBD in a preferential place for potential medical use^3,6,14,58. On the contrary, elevated peripheral levels of CCL5 have been found in patients with major depressive disorder⁵⁹. More studies are needed to confirm and understand the mechanisms of potential antiviral activity of CBD.

Granulocyte Colony-Stimulating Factor (G-SCF) was associated with decreased bacteremia and increased survival in neutropenic HIV-Infected patients⁶⁰. Furthermore, it was upregulated to promote neuronal survival, proliferation, and differentiation after neuronal damage, serving as a neuroprotective mechanism⁶¹. Our study shows the upregulation of G-SCF.

The inflammasome process is primordial to host defense. However, it must be strictly regulated to prevent damage to the host. Therefore, it has an intrinsic regulation where a variety of pattern recognition receptors (the acidic transactivation domain, pyrin domain, caspase recruitment domain (CARD), and baculoviral inhibitory repeat (BIR)-like domains acid) can be assembled into the inflammasome complex. This regulates the production of inflammation, resulting in caspase activation as a final common pathway of inflammasome regulation. The continued activation of inflammasome has been associated with chronic inflammatory disorders such as HIV, neurocognitive disorders, etc. Therefore, reducing the triggering of the inflammasome pathway and inflammatory cytokines by CBD in the absence of psychotropic effects could be beneficial for HIV patients^8,9,11,28. CBD has been used as an anti-inflammatory agent. However, little is known about its effect on HIV-infected glial cells. In this paper, we evidenced that CBD prevented HIV reactivation in microglia cells and inclusively reduced inflammatory cytokines such as SERPIN E1, IL-6, IL-8, and IL-1 ß under reactivation conditions. In addition, CBD deactivated the activity and gene expression of Caspase 1 at a low level of HIV infection at different time points and after 24 h of activation and treatment. On the other hand, THC showed no significant reduction in the gene expression of caspase 1 with respect to HC69.5 in the presence or absence of polyIC at 6 h, and only was significant after 24 h of added polyIC cultures. However, we could not find differences between CBD and THC in gene expression, but we did for caspase1 activity. Interestingly, the combination of both cannabinoids did not show a reduction. Moreover, the expression of NLRP3 was differentially dysregulated by these cannabinoids, whereas in infected cultures treated with CBD, a decrease of NLRP3 was detected. This reduction was not achieved in THC treated HIV infected cultures. The Caspase 1 Glo kit elicits a stable signal proportional to caspase activity, and inhibits nonspecific proteasome-mediated cleavage of the substrate. Our results demonstrated that when HIV was not reactivated, CBD and Δ(9)-THC did not enhance the caspase activity. Further, when the cells were treated with poly IC, the caspase 1 was activated, and CBD reversed that activation, not in the case of Δ(9)-THC. Thus CBD provided greater protection against caspase 1 activation, a molecule that initiates downstream cleavage to induce an inflammatory response (Fig. 5B). The regulation of inflammasome results in caspase 1 activation as a final common pathway. Rizzo and collaborators⁴⁶ reported the downregulation of the caspase 1, IL-1 β, and NF-κB reduction in monocytes when stimulated with LPS or treated with Δ9-THC. Previously, a study reported that Δ9-THC inhibited caspase-1 activity at a concentration as low as 0.5 μM in human astrocyte-monocyte co-culture in vitro^46,62,63. Moreover, CBD Inhibited LPS-Induced IL-1β secretion and NLRP3 Inflammasome in THP-1 Monocytes⁶⁴. Our results support previous findings, as CBD and Δ(9)-THC did not enhance the caspase-1 activation in non-activated HIV cells, while activation of caspase 1 was detected only in HIV activated cells. Furthermore, CBD offers greater protection from the activation of the inflammasome pathway. Thus, our study corroborated with the previous reports concerning the inhibition of the IL-1β, both in latent and active HIV infected microglia cells.

There are several mechanisms proposed behind the CBD effects. One of them is the inhibition of the fatty acid amide hydrolase (FAAH), which raises the anandamide due to the absence of its breaking enzyme, FAAH. Consequently, the endogenous cannabinoid could help to reduce inflammation, pain, etc. Further, it has been shown that CBD exerts its effects by the activation of transient receptor potential cation channel subfamily V member 1 (TrpV1), which suppresses the inflammatory cytokines. Moreover, the inhibition of the NF-kB was reported due to the upregulation of its inhibitory protein IκBα. Consequently, CBD suppressed the mRNA and protein expression of NLRP3,and cytokines such as TNF-α, IFN-γ, IL-6, IL-1β, IL-2, IL-17A, and chemokines CCL-2 in T cells^14,27,58. Corpetti et al. reported Cannabidiol as an inhibitor of the inflammasome pathway, which was activated by the stimulation of Caco-2 cells with the spike protein from SARS-CoV-2 virus. Specifically, Corpetti et al. reported that CBD reduced the expression of NLRP3 and Caspase-1⁶⁵.

Additionally, in HIV-1 infection, the NLRP3 inflammasome pathway is activated and produces chronic inflammation^25–27. A schematic representation of the mechanisms of the CBD is shown in Fig. 8.

Mboumba et al. conducted a pilot study with an oral combination of Δ9-tetrahydrocannabinol (THC): cannabidiol (CBD) (THC 2.5 mg/CBD 2.5 mg) or CBD-only (CBD 200 mg) in HIV patients under retroviral regimen. This study found that patients did not show safety concerns, and it was well-tolerated. However, there was no report about inflammatory levels, CD4 cell count, viral load, co-morbidities, etc⁶⁶. Incorporating CBD or THC as therapeutic agents for HIV patients still requires more investigation to collect evidence on the HIV disease progression, viral latency, interaction with ART regimens, neurocognition, safety, and other co-morbidities. To date, there are no randomized controlled trials to demonstrate the potential benefits of cannabis in people with HIV.

When both cannabinoids were simultaneously used, a significant reduction of CNR2 with respect to HIV only was observed, conserving the effect of the individual drugs. Moreover, the impact on the production of caspase 1 was different. Upregulation of the expression of the gene caspase 1 and NLRP3 was observed, similar to the one found in HIV-only cultures. However, we did not detect the IL-1 β in the assayed supernatants for CBD-THC treatments after 24 h of activation and drug treatment. Al-Ghezi et al. reported an enhancement of antiinflammatory responses mediated by raised levels of IL-10 and TGF ß when a combination of CBD + THC was used in a murine model of autoimmune encephalomyelitis, multiple sclerosis. These effects were attributed to altering the gut microbiome metabolome, and anti-inflammatory cytokines were found in the serum and splenocyte supernatants⁶⁷. Likewise, the same group of authors found that the combination of THC + CBD also showed the suppression of neuroinflammation by the dysregulation of 9 microRNAs (miR-21a-5p, miR-31-5p, miR-122-5p, miR-146a-5p, miR-150-5p, miR-155-5p, miR-27b-5p, miR-706-5p, and miR-7116) involved in anti-inflammatory response⁶⁸. Moreover, the combination of these cannabinoids decreased the expression of TNF-α and increased Brain-derived neurotrophic factor in multiple sclerosis in vivo model⁶⁹.

In our study, the activation of the HIV infection was induced by the adding of poly IC, leading to the activation of caspase 1 and NLRP3. Interestingly, this activation was significantly reversed by CBD, which inactivated the expression of caspase 1, a final common molecule involved in regulating the inflammasome pathway, and NLRP3. This inhibition could lead to a decrease in inflammatory cytokines such as IL-1β, IL-6, IL-8, MCP-1, SERPIN E1, etc.

In summary, our results demonstrate that CBD had a suppressive effect on the expression of LTR-HIV, CNR2, caspase 1, NLRP3, and IL-1β, after HIV activation by Poly IC. These suppressions were not observed with Δ(9)-THC, indicating a specific anti-inflammatory effect of CBD in this context. Inhibition of caspase 1, NLRP3 expression and decrease of IL-1β levels suggest a potential role for CBD in controlling the inflammasome pathway, which is involved in the regulation of the immune response. Further research is needed to fully understand the mechanisms and assess the therapeutic potential of CBD and combined cannabinoids (CBD-THC) in HIV-associated inflammation and immunological disorders.