We have identified a significant number of binding motifs to BHLHE40, BACH1, SPl1, NFKB1 , JUND, CEBPE, SRF, PRDM14, ATF4 and USF2. Mapping of genes regulated by these factors have revealed sub-clusters characterized by co-regulation. Interestingly, the visualization of effects of cannabis alone and of cannabis in the context of HIV suggests that the genes most affected by cannabis in the HIVþ subjects are more likely to be coregulated by at least two transcription factors. A closer visualization of these genes is shown in Fig. 12, mapping the majority of the blue genes . Of these, four genes were predictors of uninfected cannabis users: a disintegrin and metalloproteinase domain-containing protein 10 , the Calcium/Calmodulin Dependent Protein Kinase II Delta , the cAMP responsive element binding protein 1 , and the CREB binding protein . Four genes were predictors of HIV and were also affected by HIV and cannabis interactions: Beclin 1 , Plexin C1 , survival of motor neuron 1 , and the class II major histocompatibility complex molecule HLA-DRA . Four genes marked HIV- cannabis users and also significantly distinguished between HIV- and HIVþ cannabis users: the Protein Phosphatase 3 Catalytic Subunit Beta and subunit gamma,the K-Ras gene and Cullin 2.Overall, there was a trend to regulation of gene transcription by cannabis in the context of HIV, but not in the uninfected group, further highlighting interaction effects.Table 3 shows the domain scores by METH group indicating that the pattern we observed in terms of global cognition, that is, potential benefit of cannabis use on cognition in HIVþ, is reduced in the context of other drug use .
When the four HIV/CAN groups are stratified by METH use , we did not observe any significant differences across the HIV/CAN groups in the METHþ group, but in the METH- group, the pattern described above is observed globally, and across domains, with the most prominent differences observed in the domains of memory and working memory, and the least differences for the speeded information processing domain, trimming trays as shown by the calculation of effect sizes . Similarly, genes showing distinct patterns in mixed models with METH as a covariate highlight the challenges of biomarker research in the context of substance use disorders. The findings suggest that polysubstance use in HIV may explain variability and limit discovery power. The analysis of the effects of interaction between HIV and other drugs such as METH, alcohol and cocaine using mixed models has shown that these drugs further increase the expression of a number of biomarkers in the HIVþ group, but cannabis decreases these effects . For instance, METH dependence in HIVþCAN- subjects, but not in HIVþCANþ, was associated with increased NPC2 , FYN and TLR2 . METH use increased the expression of CAST in all groups, but not in HIVþCANþ subjects . A similar effect was observed in the expression of Perforin 1 when alcohol dependence was tested as a co-variate , and in the expression of CD44 in cocaine users . Overall this indicates that cannabis is a strong confounder in biomarker associated with HIV, drug abuse and polysubstance use interactions. On the other hand, although cannabis reverted the expression of markers that were elevated by HIV and METH, effects of cannabis on cognition were beneficial in non-METH users. Fig. 15 shows Global T scores, where higher values indicate better overall cognitive performance. In the context of METH-/CAN-, HIVþ participants performed worse; in the context of HIV-CAN-, METHþ participants perform worse overall. However, in the context of HIVþ, cannabis users had better neurocognitive performance if they did not have a history of lifetime METH dependence. This suggests confounding effects of polysubstance use and that the potentially beneficial effect of cannabis on HIV biomarkers may be reduced when other drugs are used.
The screening of a large number of transcripts associated with neurological disorders has shown that the effects of cannabis differed drastically between HIV- and HIVþ groups, particularly in gene networks playing a role in inflammation, neurodegeneration, apoptosis and leukocyte adhesion and transmigration. The results indicate that cannabis in the context of HIV may have beneficial effects. However, in individual genes, we identified detrimental effects that were associated with polysubstance use as a covariate, particularly methamphetamine. Effects of cannabis, one of the most widely used drugs, on HIV and particularly on biomarkers of inflammation and cognition, are largely unknown, diverse or anecdotal. By examining a large number of transcripts associated with neurological disorders and pathways of inflammation in peripheral leukocytes, we fill a gap on the understanding of how drugs of abuse impact cellular phenotypes, with the goals of identifying biomarkers of HIV neurocognitive disorders that are sensitive to interactions with substance use. In this study, we examined cells from 102 subjects evenly distributed as HIVþ/-and CANþ/. In order to increase the power, the cohort was homogeneous in sex, age and education. The use of other substances was limited but not excluded, due to characteristics of the population. The sample size was a limitation for the identification of strong predictors. However, systems biology strategies helped us identify genes that exhibited interactive properties based on their co-involvement in highly overlapping molecular pathways. Visualization strategies helped identify gene networks with a concerted behavior in different groups, highlighting important trends in effects of cannabis use dependence. Our results show that cannabis has strong effects on the expression of a number of genes in peripheral leukocytes, which serve as reporters of biological processes that are relevant both to HIV infection as well as to neurological disorders.
For instance, the pathway identified as viral host interactions included class II HLA-DRA, CCR5 and CCR2. While HIV in the context of cannabis developed to lower expression of HLA-DRA,cannabis did not lower the transcription of CCR5, suggesting a limited impact on viral entry . On the other hand, HIV in the context of cannabis and cannabis alone showed detectable decrease in the transcription of SIRT1, a histone deacetylase with epigenetic silencing properties . We have previously reported that the transcriptional decrease of SIRT1 may be one factor in the dysregulation of the inflammatory environment and others have suggested that SIRT1 regulates viral transcription . Whether the effects of cannabis in this pathway have real implications to the infection remains to be addressed. In this cohort, we did not find correlations between the activation of these pathways in leukocytes and plasma or CSF viral load . Yet, the attenuating effects of cannabis observed in the context of HIV links and expands to pathways in inflammation and neurodegeneration, as well as to apoptosis, due to overlap in genes and transcriptional co-regulators . The actions of cannabis on the expression of genes involved in vascular adhesion and leukocyte transmigration have indicated that in HIVþ cannabis users, peripheral leukocytes may be less likely to focally adhere to endothelial cells and migrate into tissues. This may be beneficial at preventing inflammation in end-organs including the brain, while potentially impairing surveillance, but also viral spread . The implications of this findings must be addressed using experimental models. Overall, the findings were consistent across pathways, suggesting that, like HIV alone, cannabis alone may increase the expression of a number of inflammation-associated genes, but this may differ in the context of HIV, where cannabis use was associated with attenuated or decreased expression of pathway components. In end organs, the actions of cannabis may differ due to effects via distinct receptors.
Cannabinoid receptor 1 is largely expressed in CNS but also in several tissues with links to psychoactive and physiological effects, while CB2R is expressed mainly by immune cells with described anti-inflammatory and immunosuppressive properties.Given the differences in distribution and signaling between the receptors, effects of cannabis or cannabinoids may differ in the presence or absence of inflammatory cells, or in the context of infection, where pro-inflammatory signals are occurring. Other less studied cannabinoid receptors and endocannabinoids may also play a role. Our data supports this idea that cannabis effects on molecular markers and biological processes is context-dependent, potentially driven by infection and inflammation, and the resulting differences in numbers and activation status of CB2R-expressing innate and adaptive immune cells. The examination of changes in expression patterns in kinase networks can inform mechanisms of action by cannabis in the context of HIV. Aberrant kinase activity is linked to a wide range of diseases including neoplastic diseases, central nervous system disorders, vascular disorders, and chronic inflammatory diseases. The analysis of a gene networks assigned to kinases indicated that cannabis in the context of HIV decreased transcription of components of the p38 MAPK pathway, which is involved in a diversity of biological functions . The blockage of p38 MAPK by cannabinoids has been previously reported in other models, with both suppressor and stimulating effects . Suppression of this pathway may be associated with blockage of oxidative stress . The anti-oxidant activity of cannabis and cannabinoid compounds has been previously acknowledged , although healthy cannabis users do not differ from non-users regarding oxidative stress markers . HIV infection promotes changes in the number of immune cells, quality and activation status of cell subsets. The infection and the broken homeostasis are likely critical in the determination of the effects of cannabis as a therapy or a complication. It has been suggested that the effects of cannabinoids on macrophages are critical to resulting T-cell mediated responses and may differ according to those cells activation status and to stimuli . Moreover, here we have shown by transcription factor usage predictions, that the effects of cannabis are associated with transcriptional co-regulation at the individual gene promoters, by multiple factors that may vary by context. Co-regulation by different transcription factors is a critical factor in determination of transcriptional levels and kinetics ,trim tray for weed and is highly influenced by covariates and comorbidities.
Cannabis use may be considered as a confounder in biomarker investigations as it tended to mask the expression of molecules upregulated by HIV, particularly if cognitive function was not improved in parallel with markers, for instance when other drugs were present. Cannabis users had better neurocognitive performance, overall and in learning and memory subdomains, particularly if they did not have a history of lifetime METH dependence. Such effect was stronger in METH users, but also observed in markers sensitive to HIV/alcohol and HIV/cocaine. This suggests differential effects of cannabis in the context of polysubstance use and how the potentially beneficial effect of cannabis on HIV biomarkers may be relative when other drugs are also used. Overall, our work has screened effects of cannabis on an extensive number of transcript biomarkers of inflammation and neurological outcomes, which were peripherally expressed by uninfected and HIV infected subjects. Systems biology strategies have aided the identification of gene networks assigned to processes relevant to neuroHIV, which exhibited orchestrated behaviors in response to HIV or cannabis alone, or their interactions. Cannabis effects were largely dependent on context, with infection as the most significant interacting factor followed by poly substance use. Other factors not examined here may include cannabis use frequency and dose. The results suggest that cannabis may be beneficial in the context of HIV when other substances are not concomitantly used.In 2018, the global 12-month-prevalence of cannabis use was estimated at 3¢9%. Based on surveys from 2015 to 2020, it is estimated that 15¢4% of EU inhabitants aged 15 to 35 used cannabis in the previous year. Occasional and more so heavy use of cannabis is associated with a number of adverse outcomes, including cannabis use disorder and an increased risk of psychosis, acute cognitive impairment as well as other outcomes such as motor vehicle accidents, respiratory problems, testicular cancer and lower birthweight of offspring . In 2019, the Global Burden of Disease study estimated that 0¢6% of the European population met criteria for CUD, resulting in 158,000,000 disability adjusted life years. In addition, there are indications that cannabinoids are genotoxic and epigenotoxic with yet to determined multi-generational implications on population health. At least 23 European countries permit some form of cannabis use for medical purposes, albeit regulatory frameworks differ largely.