THC and CBD, two major cannabinoids found in Cannabis sativa, are well recognized for their immunosuppressive properties. Specifically, THC and CBD have been evidenced to attenuate inflammatory cytokine production from CD16⁺ monocytes, a monocyte subset implicated in crossing the BBB and contributing to neuroinflammation in HAND (Fischer-Smith et al. 2001; Rizzo et al. 2018, 2019b; Sermet et al. 2025). Furthermore, up to 50% of people with HIV still experience some level of HAND, despite effective viral suppression with cART (Cysique et al. 2004; Heaton et al. 2010). Clinical studies report that cannabis use is associated with reduced CSF levels of neuroinflammatory chemokine MCP-1, supporting the need for further investigation of cannabis and major cannabinoids as a potential therapeutic strategy to limit monocyte contributions in neuroinflammation (Watson et al. 2021). To explore the therapeutic potential of cannabinoids in managing monocyte induced inflammation, the present study examined how THC and CBD influence CD16⁺ monocyte-initiated astrocyte inflammatory responses and monocyte migration, using a primary human coculture model of CD16⁺ monocytes and astrocytes in combination with assessments of chemotaxis. We have demonstrated that both THC and CBD suppressed CD16⁺ monocyte-induced production of inflammatory mediators IL-6, IL-8, and MCP-1 from astrocytes. Additional studies established that monocytes isolated from HIV+ individuals displayed enhanced chemotaxis towards conditioned media from astrocytes treated with recombinant IL-1ß relative to their HIV- counterparts. Pre-treatment of monocytes with THC resulted in reduced chemotaxis in both donor groups, while CBD had no measurable effect. Moreover, short-term exposure to THC also decreased F-actin levels and reduced surface expression of the active conformations of LFA-1 and MAC-1. While both cannabinoids dampened CD16⁺ monocyte-initiated inflammatory cytokine production from astrocytes in coculture, only THC additionally impaired monocyte chemotactic responses.

Under normal conditions, astrocyte produced inflammatory mediators, such as IL-6, IL-8, and MCP-1, exert neuroprotective effects by maintaining CNS homeostasis (Capogna et al. 2023; Choi et al. 2014; Kim et al. 2014; Kummer et al. 2021). However, if persistently elevated, these same mediators can become neuroinflammatory and disrupt neuronal function (Conductier et al. 2010; Kummer et al. 2021; Sokolova et al. 2009). Furthermore, IL-6 functions primarily as a pro-inflammatory cytokine that directly promotes inflammatory signaling. In contrast, MCP-1 and IL-8 are chemokines, which recruit monocytes and neutrophils, respectively, to the site of infection (Bickel 1993; Deshmane et al. 2009; Mukaida et al. 1998; Tanaka et al. 2014). Notably, peripheral blood CD16⁺ monocytes have been implicated in neuroinflammatory processes across various neurodegenerative conditions, such as HAND, multiple sclerosis, and Alzheimer’s disease (Clay et al. 2007; Fischer-Smith et al. 2001; Muñoz-Castro et al. 2023; Tian et al. 2025). Therefore, elucidating how THC and CBD regulate CD16⁺ monocyte-induced astrocyte inflammatory responses can have broad implications.

Results from this current study indicated that TLR7 and TLR8 activation of cocultures significantly induced the secretion of IL-6, IL-8, and MCP-1. Because these measurements were taken from coculture supernatants, the cellular source of the mediators was initially unclear. However, the incorporation of parallel monoculture samples demonstrated that astrocytes produced substantially more IL-6, IL-8, and MCP-1 compared to CD16⁺ monocytes, suggesting that the astrocytes are the primary contributors to mediator production under coculture conditions (Figs. 2 and 4). This finding is consistent with the design of the coculture, wherein the astrocytes are seeded in a 20:1 ratio relative to the monocytes. Furthermore, both THC and CBD significantly suppressed TLR7-induced release of IL-6, IL-8, and MCP-1 from cocultures (Fig. 1). In TLR8-activated cocultures, THC and CBD also suppressed supernatant release of inflammatory mediators, although CBD did not significantly affect IL-8 secretion.

Given that previous neutralization studies have identified monocyte-derived IL-1ß as an inducing factor of astrocyte inflammatory mediators, CD16⁺ monocyte monoculture supernatant IL-1ß levels were quantified (Rizzo et al. 2019a). Consistent with previously published work from our laboratory, TLR7 and TLR8 activation significantly induced CD16⁺ monocyte IL-1ß secretion, which was markedly suppressed by THC and CBD (Sermet et al. 2025). IL-1ß levels in astrocyte monoculture supernatants fell below the detection limits, indicating that astrocytes do not produce IL-1ß in this culture (data not shown). Interestingly, the concentrations of THC and CBD that suppressed monocyte IL-1ß secretion correlated with cannabinoid concentrations that inhibited IL-6, IL-8, and MCP-1 in coculture supernatants, suggesting that cannabinoid-mediated suppression of monocyte-derived IL-1ß may have influenced astrocyte production of inflammatory factors. This is further supported by the findings from the astrocyte monoculture results wherein recombinant IL-1ß enhanced astrocyte secretion of IL-6, IL-8, and MCP-1, and was not directly suppressed by THC and CBD. These monoculture supernatant data suggest that THC and CBD reduce inflammatory cytokine production in cocultures by inhibiting monocyte IL-1ß secretion, indirectly suppressing astrocyte secretion of inflammatory factors.

Under normal conditions, the BBB restricts the entry of peripheral immune cells into the CNS. However, under neurodegenerative conditions, such as HAND, BBB integrity can be disrupted, facilitating immune cell infiltration and exacerbating neuroinflammation (Bush et al. 1999; Kim et al. 2014). Moreover, post-mortem brain samples obtained from HIV+ subjects diagnosed with HAND demonstrate peripheral CD16⁺ monocyte infiltration into the CNS (Clay et al. 2007; Fischer-Smith et al. 2001). Having demonstrated that CD16⁺ monocytes induced MCP-1 secretion from astrocytes in coculture, we next investigated whether conditioned media from IL-1ß-stimulated astrocytes would trigger monocyte chemotaxis, and whether migratory capacity would differ based on HIV status. Monocytes from both HIV- and HIV+ subjects exhibited enhanced chemotaxis towards astrocyte-conditioned media regardless of whether astrocytes were stimulated with recombinant IL-1ß, compared to the fresh media control. In contrast, recombinant MCP-1 alone was insufficient to trigger migration, which indicates that astrocyte-conditioned media likely contain a combination of soluble factors that together generate a more potent migratory signal. Interestingly, monocytes from HIV+ donors exhibited enhanced chemotaxis towards IL-1ß-treated astrocyte-conditioned media relative to their HIV- counterparts. These findings differ from earlier findings by Eugenin and colleagues, who observed robust chemotaxis toward MCP-1 in monocytes acutely infected with HIV ex vivo, which was attributed to elevated CCR2 expression (Eugenin et al. 2006). Taken together, our data instead suggest that in the context of chronic HIV infection, monocytes appear primed to respond to multifactorial signals released by activated astrocytes rather than to a single chemokine, such as MCP-1.

Given that THC and CBD have been demonstrated to inhibit the inflammatory contribution of monocytes on astrocyte responses, we considered whether they could additionally regulate monocyte migration. THC pre-treatment significantly suppressed the capacity of monocytes to migrate in response to astrocyte-conditioned media in a comparable magnitude in both HIV + and HIV- monocytes, whereas CBD had no effect. While this chemotaxis assay did not directly model BBB-mediated transmigration, it provides a controlled system to assess cannabinoid effects on chemotactic behavior. Accordingly, we examined cytoskeletal and integrin-dependent mechanisms that mediate both chemotaxis and transmigration across the BBB. Consistent with the chemotaxis assay findings, assessments of cytoskeletal polymerized actin and high-affinity conformational states of LFA-1 and MAC-1 revealed that pre-treatment with THC impaired monocyte expression of key molecular processes required for chemotaxis. The findings from this study differ, in part, from those reported by Raborn et al., who used microscopy-based approaches to demonstrate that THC inhibited Tat-induced U937 monocyte adhesion to extracellular matrix proteins by reducing ß1 integrin-expression and altering F-actin distribution (Raborn et al. 2014). Notably, in the absence of Tat, THC treatment alone increased overall ß1 integrin expression while suppressing F-actin levels in U937 monocytes (Raborn et al. 2014). An important distinction between the studies by Raborn et al. and our study lies in the integrin populations examined and the methods used to assess them. While Raborn et al. evaluated total ß1 integrin expression, the present study focused on high-affinity conformational states of LFA-1 and MAC-1, ß2 integrins that play critical roles in monocyte adhesion and migration (Li et al. 2018; Raborn et al. 2014; Sumagin et al. 2010). Given that integrin-mediated adhesion is controlled both by surface expression as well as activation state, selective assessment of high-affinity LFA-1 and MAC-1 may account for the apparent differences between our findings and those reported by Raborn et al. (Li et al. 2017; Takagi et al. 2002). Interestingly, despite differences in experimental context and integrin readouts, both studies indicate that THC alters integrin-associated adhesion mechanisms and F-actin dynamics. Based on the findings from this current study, we hypothesize that THC pre-treatment may suppress monocyte transmigration across the BBB.

Actin polymerization and integrin activation states are interconnected through a process termed “inside-out integrin signaling”, wherein intracellular signaling pathways induce conformational changes of integrins (Springer and Dustin 2012; Zhu et al. 2008). Actin polymerization is critical for stabilizing integrins in their high-affinity state and can promote inside-out integrin signaling (Zhu et al. 2008). Based on the results from this current study, it remains unclear whether THC suppresses LFA-1 and MAC-1 high-affinity states indirectly by inhibiting actin polymerization or whether it affects both processes independently. Nevertheless, these findings suggest that even a brief, 1-hour pre-treatment with THC was sufficient to diminish monocyte responsiveness to migratory cues, indicating that short-term exposure to THC can rapidly alter monocyte cytoskeletal dynamics and integrin activation. In contrast, CBD treatment did not affect monocyte chemotaxis, actin polymerization, or integrin expression. These results highlight clear immunopharmacological differences between these cannabinoids and point to a selective role for THC in suppressing monocyte chemotactic function.

THC primarily binds CBR2 to elicit its immunomodulatory effects however, can also engage with additional targets, including proliferator-activated receptor gamma (PPAR-γ), GRP55, and transient receptor potential V2 (TRPV2) (Adams et al. 1940; Devane et al. 1988; Lauckner et al. 2008; Munro et al. 1993; O’Sullivan et al. 2005; Vara et al. 2013; Zhang et al. 2022). In contrast, CBD has low binding affinity for the CBRs, and its molecular targets remain widely debated (de Almeida and Devi 2020; Kaplan et al. 2008). Nonetheless, studies have implicated fatty acid amide hydrolase, PPAR-γ, TRPV1, and adenosine A_2A receptors as targets by which CBD mediates its effects on the immune system (Bisogno et al. 2001; De Petrocellis et al. 2008; Mecha et al. 2013; Rodrigues et al. 2024; Watanabe et al. 1996). While THC and CBD share some overlapping targets, they may act through distinct mechanisms, which explains why their immune-modulating effects throughout this current study are similar in some respects yet divergent in others. Furthermore, previously published work from our laboratory has also identified differences by which THC and CBD suppress CD16⁺ and CD16⁻ monocyte secretion of IL-1ß, TNF-α, and IL-6 (Sermet et al. 2025). Regardless, this current study demonstrated that THC and CBD treatments may have therapeutic value in attenuating monocyte-initiated or enhanced astrocyte inflammatory responses. While both cannabinoids reduced monocyte-initiated astrocyte IL-6, IL-8, and MCP-1 secretion, only THC additionally inhibited monocyte chemotaxis, suggesting that THC may exert broader immune-regulating effects compared to CBD in the context of monocyte-mediated neuroinflammation. Additionally, through our previous findings, we identified that THC-mediated effects on monocyte inflammatory mediator secretion, including IL-1ß, are CBR2-independent (Sermet et al. 2025). In this context, we speculate the indirect effects of THC and CBD observed in the present investigation on astrocyte inflammatory cytokine secretion through inhibition of monocyte-derived IL-1ß may similarly reflect CBR2-independent mechanisms, although should be directly assessed in future investigations. It would also be valuable for future studies to assess whether any cannabinoid-mediated effects involve non-CBR mechanisms, such as PPAR-γ and TRP channels, especially given their role in regulating IL-1ß maturation and secretion (Hong et al. 2015; Scheraga et al. 2016; Wu et al. 2023; Yang et al. 2021).

Importantly, the concentrations of THC and CBD utilized throughout these studies are physiologically relevant to human exposure. A controlled human inhalation study following a strict smoking protocol and rigorous sampling revealed that after smoking, peak plasma concentrations of THC can reach an average of 152 µg/L (0.5µM), which falls within our tested concentrations (Grotenhermen 2003; Huestis et al. 1992; Perez-Reyes et al. 1982). Notably, this peak concentration of THC was observed after smoking research-grade cannabis, which is estimated to have THC content of less than 3.55% THC per dry weight. Furthermore, the THC content of research-grade cannabis is substantially lower than the average THC content found on the market today, which can reach levels as high as 30% THC, thus indicating the relevance of the higher concentrations utilized throughout this currently study. While peak plasma concentrations of CBD following cannabis use are not well studied, one study reported concentrations reaching 191 µg/L (0.6µM) after participants smoked a cannabis cigarette containing 20 mg CBD (Ohlsson et al. 1986). However, a large proportion of commercial CBD-containing products are intended for oral administration, which results in significant first-pass metabolism. Pharmacokinetic studies conducted for the FDA-approved CBD therapeutic, Epidiolex^® reported an average peak plasma concentration of 330 µg/L (1µM) CBD following a 20 mg/kg/day oral dosing regimen (FDA 2018). It is possible that individuals using low-dose THC- and CBD-containing products may not achieve plasma concentrations assessed within this current study. Nevertheless, due to the lipophilicity of THC and CBD, repeated or chronic exposure can result in tissue accumulation, wherein these concentrations may be reached over time.

We acknowledge that the outlined studies had several limitations. First, the coculture system utilized represents a simplified model that does not capture the full cellular complexity of the brain, which also includes microglia, pericytes, neurons, and brain microvascular endothelial cells. This specific coculture was used to examine how THC and CBD influence CD16⁺ monocyte contributions to astrocyte inflammatory responses. Future studies will need to incorporate a more complex multicellular culture that better mimics the brain environment to define CD16⁺ monocyte contributions to neuroinflammation as a whole. Additionally, the chemotaxis assay employed was not intended to model BBB transmigration. While THC clearly altered monocyte chemotactic responses, these results do not directly address its effects on monocyte recruitment to the CNS from the periphery. Additionally, we acknowledge the lack of access to donor demographic information for the HIV- control population, wherein potential contributors to observed inter-donor variability in cytokine responses, including differences in age, sex, or other biological factors could not be evaluated in the present study. We also note that HIV- and HIV+ participants in this study were recruited from different geographic locations. Finally, we recognize that the overall sample sizes within these studies were modest. However, the effects observed with cannabinoid treatments were relatively consistent across donors despite this limitation.

Overall, this study used a primary human in vitro model to expand the current body of knowledge on CD16⁺ monocyte-mediated astrocyte inflammatory responses and the potential immunomodulatory roles of THC and CBD. CD16⁺ monocytes induced astrocyte secretion of inflammatory IL-6, MCP-1, and IL-8 in TLR7- and TLR8-activated primary human cocultures. Furthermore, treatment of activated cocultures with THC and CBD inhibited the astrocyte inflammatory response indirectly by attenuating monocyte IL-1ß secretion. Additionally, we observed that monocytes from HIV+ donors exhibited enhanced chemotaxis toward astrocyte-conditioned media compared to their HIV- counterparts. Chemotaxis was diminished by short-term pre-treatment of monocytes with THC and was associated with reduced expression of F-actin and active conformations of integrins, LFA-1 and MAC-1. Taken together, this study provides evidence to support that THC, and to a lesser extent CBD, exert anti-inflammatory effects on CD16⁺ monocyte-mediated inflammatory and migratory responses that may be associated with HAND.