HIV-Associated Neurocognitive Disorders (HANDs) are becoming one of the largest problems in patients infected with HIV-1. The ability of infected cells such as monocytes and microglial cells to act as viral reservoirs causes extreme inflammation in the CNS and has led to several different types of neurocognitive problems. Specifically these HIV-1 infected monocytes are able to secrete inflammatory factors such as the regulatory protein Tat which acts as a chemoattractant for monocytes while also promoting the adhesion of leukocytes to the extracellular matrix (ECM). We have shown that one of the major features of the Tat protein is that it promotes cytoskeletal rearrangement resulting in increased adhesion. Specifically integrin and actin visualization was performed using confocal immunofluorescence while cytoskeletal morphology was shown with light and SEM. This microscopy work showed the Tat protein resulted in altered β1-integrin expression and distribution as well as changes in polymerized actin. These cytoskeletal changes resulted in increased adhesion to the ECM. Similarly we have also shown that these cytoskeletal changes of β1-integrin distribution and polymerized actin can be modulated through select cannabinoids THC and CP55940 that bind through the CB2 receptor which inhibits this adhesion as well as the morphological changes. The modulation of this reorganization is characteristic of a signal transduction pathway where a novel convergent point between extracellular Tat and the select cannabinoids THC and CP55940 exists. The aim of this project was to show the cytoskeletal reorganization using different microscopy techniques including light and scanning electron microscopy. This was followed by identifying and characterizing the convergent point along the signal transduction pathway linked to these changes. Different techniques were utilized in order to identify the putative convergent point in the signal transduction cascade including antibody arrays, RT-PCR, and western immunoblotting. The cytoskeletal rearrangements of β1-integrin and actin polymerization were shown successfully via light and scanning electron microscopy in the context of treatment with Tat in the presence and absence of select cannabinoids THC and CP55940. Several different pathways were identified as possibly linked to cannabinoid-mediated inhibition of signal transductional activation consequent of attachment to extracellular matrix proteins. However, the exact molecules implicated in specific signal transductional pathways as targets of cannabinoid-mediated action remain to be defined.

Cannabinoids have been shown to modulate the immune system in vitro and in animal models. A major area of interest is how cannabinoids impact the brain. A whole variety of neuropathies or brain disorders, such as AIDS dementia, Parkinson’s disease, Multiple Sclerosis and Alzheimer’s disease, are associated with a hyperinflammatory response within the brain. Microglia, the resident macrophages of the brain, are the major cell type responsible for the persistent elicitation of pro-inflammatory cytokines (IL-1a, IL-1b, IL-6, TNFa) and other mediators. In vitro experiments have demonstrated that the partial exogenous cannabinoid agonist delta-9-tetrahydrocannabinol (D9-THC) and the potent synthetic exogenous cannabinoid agonist CP55940 down-regulate the robust production of pro-inflammatory cytokines elicited in response to bacterial lipopolysaccharide (LPS) at the mRNA level. These observations suggest that cannabinoids, devoid of psychotropic properties, have the potential to betherapeutic agents. These highly lipophilic compounds can pass through the blood brain barrier and act through specific cannabinoid receptors, cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2). CB1 and CB2 are expressed in the brain and the periphery, respectively, and may serve as molecular targets for ablating chronic brain inflammation. Electrophoretic mobility shift assays (EMSA) were used to assess the effects of D9-THC and CP55940 on the LPS-induced binding interactions of the universal transcription factor NFkB to its cognate promoter binding site in BV-2 microglial-like cells. EMSA analyses demonstrated that the D9-THC and CP55940 down-regulated LPS-induced NFkB binding in BV-2 cells in a biphasic manner. Furthermore, reporter activity assays determined that D9-THC and CP55940 attenuated LPS-induced, NFkB transcriptional activity in the same biphasic manner. We then determined the specificity in which cannabinoids inhibit NFkB function. Real-Time RT-PCR analysis demonstrated that BV-2 cells did not express CB1 mRNA, but they do express CB2 mRNA when untreated and stimulated with IFN-g or LPS. We performed specificity studies using CB1 and CB2 selective agonists and antagonists with our reporter activity assays. The CB1-selective agonist ACEA did not affect NFkB transcriptional activity but the CB2-selective agonist O-2137 exerted a significant decrease in activity. Furthermore, the CB1 antagonist SR141716A could not reverse the inhibitory effects of CP55490 but those effects were blocked by the CB2 antagonist SR144528. Lastly, we determined the site of action in which cannabinoids inhibit NFkB function by assessing the effects of D9-THC and CP55940 on NFkB’s inhibitor protein IkBa. IkBa retains NFkB in the cytoplasm until stimulus-induced cell activation. Neither cannabinoid compound was able to inhibit the phosphorylation of IkBa, which initiates its degradation. However both cannabinoids inhibited the complete degradation of IkBa. Western immunoblot analysis also demonstrated that comparable levels of endogenous and phosphorylated p65, the transactivation subunit of the NFkB protein (p65/p50), were detected in the nucleus of LPS-stimulated BV-2 cells pre-treated with or without D9-THC. These results suggest that, in addition to inhibiting the proteolytic degradation of IkBa, there is also a mechanism of action in the nucleus that prevents the proper binding and subsequent transcriptional activity of NFkB. Collectively, these results suggest that cannabinoids suppress pro-inflammatory cytokine gene expression at the transcriptional level, but it is likely that there is more than one signal transduction pathway involved in the cannabinoid-mediated inhibition of NFkB function.

Cannabinoid treatment increases Th2 activity and previous reports showed B cells express the highest level of CB2 mRNA relative to other immune cells suggesting that cannabinoids play a critical role in B cell activation and maturation. To examine the direct effect of cannabinoids on B cell antibody class switching, mouse splenic B cells were purified by negative selection and cultured with IL4 and anti-CD40 in the presence or absence of the nonselective cannabinoid agonist, CP55940, or the CB1 selective agonist, methanandamide, or the CB2 selective agonist, JW015. The cultures were then analyzed at different times by flow cytometry for expression of B cell surface markers, such as CD19, CD138, CD40, MHCII, CD23, CD80, CD45R, immunoglobulins produced such as IgM, IgE, IgD, and IgG1, and Toll-like receptors such as TLR 2 and 4. Cells treated with CP55940 showed an increase in surface expression of IgE by day 5 in culture; methanandamide had no effect. CP55940 also induced an increase in secreted IgE in culture supernatants analyzed by ELISA. In addition, CB2 receptors were increased on B cells following stimulation with IL-4 and anti-CD40 and the class switching effect of CP55940 was attenuated by the CB2 antagonist, SR144528. We also observed that cannabinoid treatment of B cells modulates cell functions other than antibody class switching such as surface marker and TLR expression. CP55940 caused a significant increase in surface expression of TLR 4, but had no effect on other markers. Additional experiments with cannabinoid receptor selective agonists and antagonists suggested both CB1 and CB2 receptors were involved in the TLR effect. Receptor involvement and Gi coupling was supported by our findings that cannabinoids inhibit intracellular cAMP levels in forskolin stimulated B cells, and increasing intracellular cAMP with forskolin suppressed IgE antibody class switching in activated B cell cultures. These results suggest cannabinoids negatively regulate cAMP in B cells resulting in increased IgE. In conclusion, cannabinoids can directly affect the function of B cells by inducing antibody class switching to IgE and TLR4 expression through mechanisms involving CB1 and CB2 receptors suggesting the endocannabinoid system may be an important regulator of humoral immunity and the allergic response.