The consumption of cannabis has a long history marked by controversial views about its legality and risks, both regarding its application for medical purposes and as psychoactive drug. Concerning the latter, it is the most frequently used illicit substance worldwide and, as such, a major public health issue due to its negative physical and psychological outcomes [1]. Chronic cannabis use (CCU) has been, among others, linked to respiratory and vascular issues [2] and an increased risk of developing psychotic disorders [3]. Given that the use of cannabis also affects brain development, adolescents are considered to be especially vulnerable to its negative effects: CCU in adolescence has been associated with a decline in cognitive function, comprising impaired learning, memory and thinking [4, 5]. Still, a trend toward increasing societal acceptance and a less harmful view of cannabis use is observed, particularly among adolescents [6]. The lifetime use of cannabis among German adolescents has been increasing from 6.7% in 2011 to 9.3% in 2021, with 3.5% of adolescent users reporting cannabis use during the previous month and 1.6% consuming cannabis on a regular basis (defined as more than 10 times within the last 12 months) [7].

In addition, more and more countries are legalizing cannabis and cannabis-based products for recreational and medical purposes. Of the numerous chemical compounds derived from the cannabis plant (Cannabis sativa), the two most extensively studied phytocannabinoids are cannabidiol (CBD) and trans-Δ9-tetrahydrocannabinol (THC). THC is known to be the major psychotropic component of cannabis, limiting its therapeutic use due to adverse effects, whereas the non-psychotropic CBD is considered to have a high potential for therapeutic use. The use of medicinal cannabis (containing both THC and CBD) in the treatment of, e.g., chronic pain, chemotherapy-induced nausea and vomiting, anxiety, seizure disorders and inflammatory bowel disease [8–10] illustrates that cannabis has diverse effects on a variety of physiological processes, including modulations of the immune system. Both THC and CBD have been shown to possess immuno-modulatory and anti-inflammatory properties in vitro and in animal models (reviewed by, e.g., [11, 12]). THC primarily exerts its effects through cannabinoid receptors of the endocannabinoid system (ECS): while cannabinoid receptor 1 (CB1) is mainly localized in the central nervous system [13] and mediates the psychotropic effects of THC, cannabinoid receptor 2 (CB2) is predominantly expressed by lymphoid organs and immune cells [14], with highest mRNA levels in B cells, followed by natural killer (NK) cells, monocytes, polymorphonuclear leukocytes, CD8 + T cells and CD4 + T cells [14]. Accordingly, the ECS is considered to be a key regulator of the immune system and to modulate both innate and adaptive immune responses [15]. In contrast to THC, CBD only displays weak affinity for cannabinoid receptors [16], in spite of its impact on immune functions. However, it has been shown that CBD (and THC) can act via several further receptors (e.g., PPARγ, GPR55) and ion channels (e.g., members of the TRPA/TRPV channel families) involved in ECS signaling [15, 17]. Notably, genetic variants of genes encoding such receptors and other proteins involved in the action, metabolism and transport of cannabinoids may explain the inter-individual differences observed in the therapeutic and adverse effects of phytocannabinoids [18]. With regard to immune function, for instance, a polymorphism in the CB2 receptor gene has been linked to altered endocannabinoid immune modulation [19].

The impact of cannabinoids on the immune system is regarded as biphasic, exhibiting both stimulatory and inhibitory effects contingent on the specific type and concentration of cannabinoids involved [20]. The immunosuppressive and anti-inflammatory effects of cannabinoids have been demonstrated in numerous experimental models, both in vitro and in vivo, and are exerted via multiple mechanisms. These comprise suppression of pro-inflammatory cytokines and chemokines [21], inhibition of immune cell proliferation / activation [11, 20], induction of immune cell apoptosis [22] as well as induction of regulatory T cells and of immunosuppressive myeloid-derived suppressor cells (MDSCs) [23]. Recent studies suggest that the molecular mechanisms mediating these effects involve, among others, regulation of gene expression via epigenetic modulations [23], such as DNA methylation (i.e., the attachment of a methyl group to a cytosine base, commonly in a CpG dinucleotide context). Several studies have shown that cannabis use can impact DNA methylation, both on a genome-wide scale and of candidate genes [24, 25]. For instance, induction of MDSC by THC has been associated with altered methylation profiles of key genes involved in the differentiation and function of these immune cells [26].

In medical conditions requiring modulation or suppression of immune responses, such as autoimmune diseases or organ transplantation, the proposed immune-modulatory and anti-inflammatory properties of cannabis-derived cannabinoids could be beneficial. On the other hand, animal studies have demonstrated an association between cannabinoids and a compromised resistance to different pathogens, e.g., Legionella pneumophila or certain viral infections [11, 27, 28]. Although these findings implicate that particularly CCU might have adverse immunological consequences, there is only a limited number of studies with inconclusive outcomes as to whether cannabis use impairs immune competence in human subjects [29, 30]. Several studies assessing immune-related effects of cannabis use in HIV patients have suggested that cannabis does not adversely affect their immune status [31–33]. In adult individuals without an infectious disease, there is limited evidence supporting a link between regular exposure to cannabis and a decrease in the production of several factors mediating inflammation [29]. However, such associations were not confirmed for CRP (C-reactive protein, a marker of inflammation) in cannabis-using adolescents and young adults [34, 35]. The few studies to date that have examined the numbers of circulating white blood cells (WBCs) in cannabis users also provide mixed results [25, 36–38]. In summary, more research is urgently needed to uncover the effects of CCU on immune function – especially in adolescence, since the vulnerability to the negative effects of cannabis is increased during this developmental period [5]. In the present exploratory study, we therefore aimed to investigate the association of CCU with leukocyte measures, including DNA methylation and expression levels of different immune cell markers, in an adolescent outpatient cohort. We hypothesize that CCU is linked to alterations in leukocyte proportions and the expression of associated immune cell markers in adolescent users.

A growing body of literature has shown that during adolescence, the developing body and brain are especially sensitive to the effects of cannabis use [5]. Experimental studies further provide evidence that cannabinoids have immune-modulatory and anti-inflammatory properties; however, little literature exists regarding the effect of cannabis use on circulating immune cells, particularly in adolescents. Our exploratory study examined the association between CCU and leukocyte characteristics in an adolescent outpatient cohort with self-reported CCU, defined as at least weekly use of cannabis during the past 12 months. Since our study was conducted with a clinical sample, it is important to note that adolescent patients who abuse cannabis often concurrently smoke tobacco [46]. Given this common co-occurrence, it becomes challenging to disentangle the distinct effects of cannabis and tobacco on immune markers in this cohort. To address this issue, we incorporated tobacco use as a covariate in our statistical models, thereby controlling for its potential confounding effect. Consequently, the originally observed higher proportion of granulocytes in CCU subjects was attenuated when controlling for past-year tobacco use. Furthermore, the observed differences in mean DNA methylation and gene expression of immune cell markers were not statistically significant. Importantly, the methylation-based quantification of immune cell type proportions revealed a significantly lower proportion of B cells in the CCU group. We also observed a trend to reduced gene expression of the B cell marker CD19. Although not statistically significant, a reduced expression would match the differences observed in the mean CpG site methylation levels of CD19, since increased methylation is commonly associated with reduced gene expression [47]. Previous research investigating WBC counts in cannabis users has yielded mixed results: one study observed a significant decrease only in the number NK cells, but not for T and B cells as compared to controls [37]. In the context of their methylome-wide association study (MWAS) investigating the effects of cannabis use on the methylome, Clark et al. [25] report slightly decreased B cell levels in adolescents with problematic cannabis use compared to non-problematic users, yet equal levels of T cells, monocytes and granulocytes. El-Gohary and Eid, who analyzed leukocyte profiles of young adults using bhang (an edible form of cannabis), found decreased numbers of NK, T and B cells [36]. In our study, we could not detect CCU-associated differences in the proportion of NK cells and CD4+ T cells. Still, our observation of a significant reduction in B cells, which are the central element of humoral immunity and a major component of the adaptive immune system, is in line with previous findings.

Although differential effects of dose (low vs. high) and origin of the acting cannabinoid (endo- vs. phyto- or synthetic) on B cell proliferation (stimulation vs. inhibition) are discussed [20], experimental studies investigating the effect of cannabinoids on B cells have provided evidence for enhanced apoptosis [23], decreased cell proliferation and reduced antibody production (reviewed by [20]) (Fig. 3). B cells express the highest amount of CB2 on their cell surface as compared to other leukocyte subtypes [14], implying that the underlying mechanism of action may involve a CB2-mediated reduction of B-cell proliferation or survival. The levels of CB2 mRNA in peripheral blood mononuclear cells (PBMCs) of chronic cannabis users have been found to be elevated [48], and CB2 mRNA levels to remain increased in PBMCs even after abstaining from cannabis for ≥ 6 months [49]. Yet there is still a paucity of studies identifying B cells as direct targets of the ECS [11]; cannabinoids could also act on B lymphocytes indirectly via other immune cells, such as MDSC or T cells [23], and through receptors other than CB2. A potential mechanistic link is suggested by Clark et al. [25]: based on their MWAS-results, they hypothesize that problematic cannabis use may alter methylation of genes relevant for DNA repair in B cells, with potential downstream effects on immune functioning. Such effects may involve impaired humoral immune responses to pathogens along with reduced vaccination-induced immunity [15], yet more research is needed to elucidate the mechanisms and clinical outcome of CCU-related effects on the adaptive immune system. Fig. 3

Schematic illustration* of hypothesized CCU effects on B cells. Studies investigating the effect of cannabinoids on B cells have provided evidence for enhanced apoptosis, decreased cell proliferation and reduced antibody production. B cells express the highest amount of CB2 on their cell surface as compared to other leukocyte subtypes, implying that the underlying mechanism of action may involve a CB2-mediated reduction of B-cell proliferation or survival. However, cannabinoids could also act on B lymphocytes indirectly via modulation of other immune cells, such as MDSC or T cells. MDSC: myeloid-derived suppressor cell; CB2: Cannabinoid receptor 2. *The Figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license, and illustrations provided by pixabay (https://pixabay.com/de/vectors/marihuana-blatt-gr%c3%bcn-topf-cannabis-34178/)

Other than B cells, granulocytes (or polymorphonuclear leukocytes) are effectors of the innate immune system. They comprise eosinophils, basophils and neutrophils, which are the most abundant subtype of circulating WBC (50–70%) and continuously generated in the bone marrow from myeloid precursors. Neutrophils are typically the first leukocytes to be recruited to injured or infected tissue, capable of eliminating pathogens by multiple mechanisms [50]. Our CCU group displayed an increased proportion of circulating granulocytes, along with lower mean CpG site methylation and increased expression of FUT4 (encoding the enzyme fucosyltransferase 4, which catalyzes the synthesis of the granulocyte marker CD15) and of MPO, encoding myeloperoxidase, which is released by activated neutrophils at the site of infection [50]. We suggest that the low and CCU-independent MPO expression levels measured in our whole blood samples may arise from the fact that the neutrophil granules containing MPO are formed during their maturation in the bone marrow [50].

Our finding of an increased fraction of granulocytes in adolescents with CCU is consistent with results obtained from an adult sample of the US National Health and Nutrition Examination Survey, where a modest association between heavy cannabis use and increased WBC count, primarily elevated neutrophils, was detected [38]. The author suggests that this increase may be related to the inflammatory effects of combustion by-products. This aligns with our sample, where the most common mode of cannabis use is smoking, akin to tobacco cigarette smokers who have shown elevated WBC [51, 52]. The adolescents in our CCU group did not differ from the NCU group in terms of their physical health status, so that acute inflammation or disease can be excluded as a reason for the differences in granulocyte proportions; however, the CCU group did use significantly more tobacco than the NCU group. Although in the study by Alshaarawy [38], the differences in neutrophil counts remained significant after adjustment for multiple variables, including BMI, alcohol drinking and tobacco smoking, accounting for past year tobacco use attenuated the differences in granulocyte proportions and FUT4 / MPO methylation levels in our sample. Nonetheless, CBD has been shown to exert direct effects on neutrophils by inhibiting their migration to sites of infection [53]. It should also be noted that chronic heavy smoking of cannabis has been associated with increased symptoms of chronic bronchitis, and CCU is suggested to have different effects on lung function than tobacco smoking [54, 55]. To determine the effects of cannabis vs. tobacco smoking on immune cells, future studies should try to adapt their sample accordingly, e.g. by including a further group of tobacco-only smoking participants, and investigating a larger sample size, which is one of the limitations of our exploratory study.

The results of our explorative study suggest that CCU in adolescents is associated with altered levels of circulating WBCs, especially a lower proportion of B cells, and possibly linked to epigenetic changes. Further studies with larger sample sizes are warranted to confirm our findings and to provide insights regarding functional and clinical consequences of CCU-associated changes in immune cell profiles.

Supplementary Material 1.