Neurode generation and inflammation were functional annotations identified in BIOCARTA. Given the large degree of overlap between these networks , we applied a merge network function in Cytoscape, which is shown in Fig. 7. The visualization of this gene network indicates that both HIV and cannabis increase genes with functions in neurode generation and inflammation , but cannabis decreased key contributors to the inflammatory process such as IL1b, TLR2, MyD88 and PARK7, as well as RASGRP1 . HIV infection in the context of cannabis indicated patterns that were similar to cannabis alone, with decreased expression in the same genes. Moreover, cannabis in the context of HIV elevated TLR2, TLR4 and MyD88, but had no or mild effects, or decreased a number of genes in this network . Another gene network related to neurode generation and inflammation in Fig. 6 but low overlap and smaller but significant enrichment , was functionally annotated to chemokines and cytokines . This network was highly sensitive to all the group conditions, with higher expression of most genes in cells from HIVþ/CAN- compared with HIV-/CAN- , and an effect of cannabis that was characterized by decreased expression of several genes, regardless of HIV . Yet, the genes decreased by cannabis differed in a context-dependent manner. For instance, the effects of cannabis alone as well as of HIV in the context of cannabis showed a lower expression of TLR2, GNAI3, AKT3,PRKACA, ITGAM, and GNG2, and a mild decrease of A1BG, Ly96, PIK3CD, BRAF, NAT1, NAT2, PRKAR2A and AADAC. Decrease in PLCB2 and RAC1 was a characteristic of cannabis alone , while decrease in RAF1 and NFKBIA was characteristic of HIV in the context of cannabis . The effects of cannabis in the context of HIV was also characterized by lower expression of MAPK3, PLCB3, CXCR6 and NFKB1 . Functional annotations associated with leukocyte-vascular adhesion and transmigration capacity were also sorted from pathway interactions. These functions were affected by HIV and cannabis . A large number of genes in this network were differentially increased by HIV and by cannabis . Yet cannabis lowered the expression of a large number of genes with cytoskeleton and signaling properties, including RHOA, AKT3, RAC1, BRAF and BCL2 .

HIV in the context of cannabis had also lower MAPK1 and CTNNB1 compared to uninfected cannabis users . HIVþ cannabis users had a high number of genes that were lower or mildly changed compared to HIV non-cannabis users . Inflammation is highly regulated by a kinases. The effects were differential and context-dependent. All the conditions showed decrease in CAMK4,vertical farming in comparison to respective controls . HIV alone decreased mTOR, CSF1R, EPHA4, PDPK1 and DGKE . Cannabis alone, as well as HIV in the context of cannabis , decreased ATK3 and MAPKPK2. Cannabis alone decreased CALM1 . HIV in the context of cannabis decreased the expression of PGK1 and RAF1 . Cannabis in the context of HIV decreased several genes in this network that were either not modified or increased by the other conditions . These included MAP2K1, MAPK9, MAPK3, PRKCA and PDPK1 .Networks analyzed above have shown distinct effects of cannabis, which differed between cannabis alone and in the context of HIV. We used iRegulon to make predictions on transcription factors usage associated with these context-dependent patterns, in order to identify regulatory and co-regulatory elements. Fig. 11 shows the same gene network assembled based on pathway interactions in Fig. 3, but now reorganized based on the expression of transcription factor motifs in these genes’ promoters. The table legend in Fig. 11 shows the transcription factors mostly associated with the genes in the network. 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 metallo proteinase 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.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 poly substance 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 immuno suppressive 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,flood tray 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 , 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 poly substance 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 polysubstance use. Other factors not examined here may include cannabis use frequency and dose.