HIV+ donors voluntarily enrolled in the Mid-Michigan HIV consortium (MMHC) under the institutional review board–approved protocol (IRB # 11-202) and into the MMHC Registry. Donors were recruited from clinics attended by Dr. Peter Gulick; HIV+ were males between the ages of 31 and 71 with an average age of 54.4 years. Donors received the standard of care and were not asked to change any lifestyle habits to participate. All subject questionnaires and their abstracted medical record data for the MMHC are managed using the Research Electronic Data Capture (REDCap) (Vanderbilt University), which supports 21 CFR Part 11 compliance for clinical research and trial data and HIPAA guidelines.
The profile of cannabinoid receptor (CNR1 and CNR2) expression has not previously been characterized in human pDC and was therefore investigated using purified pDC and compared with PBMC from healthy donors (Fig. 1A). Purified pDC were found to exhibit a very similar profile of CNR1 and CNR2 expression compared with other PBMC such that CNR2 mRNA levels were more highly expressed than CNR1 (Fig. 1B). These studies were extended to also quantify CNR1 and CNR2 levels in HIV+ donors. PBMC from HIV+ donors showed significantly augmented CB1 mRNA levels compared with healthy donors (Figs. 1C, D). By contrast, CB2 mRNA levels were similar in PBMC from healthy versus HIV+ donors (Figs. 1C, D). A sufficient amount of blood could not be collected from HIV+ donors to quantify CNR1 and CNR2 mRNA expression levels in purified pDC by RT/Qt-PCR.
HIV infection reduces both the number of circulating pDC and the ability for the remaining pDC to secrete IFNα.8,15,34 To extend the previous observations, PBMCs from HIV+ patients were treated with CpG-ODN, and the number of IFNα-secreting pDCs were quantified using the IFNα capture assay. THC is known to suppress IFN secretion in infection and inflammatory conditions.32 Here, the effects of THC on IFNα secretion were determined in CpG-ODN–induced human primary pDC.
HIV infection, and associated disease states, can cause prolonged stimulation of host immune cells and a chronic inflammatory state which can alter immune cell function. To determine possible differences in THC sensitivity of pDC between HIV+ and healthy donors, PBMCs from HIV+ donors were treated with THC and activated with CpG-ODN, as previously described. Treatment with THC significantly suppressed the number of IFNα-secreting pDCs from HIV+ donors (Fig. 2E), and the degree of suppression was greater than the suppression in pDC from healthy donors (Fig. 2F), indicating more pronounced sensitivity to cannabinoid-mediated suppression in pDC from HIV+ donors.
To determine whether THC also suppressed the quantity of total secreted IFNα, LEGENDplex cytometric bead array was used to quantify the amount of IFNα in the cell culture supernatants from purified healthy pDC preparations. THC treatment significantly suppressed the amount of IFNα secreted by the highly purified pDC (Fig. 3C).
To determine whether the suppression of IFNα by THC was tied to decreased IFNα mRNA levels, PrimeFlow, a flow cytometry–based method that allows quantification of gene-specific mRNA levels on a per cell basis, was employed (Fig. 4A). THC suppressed the transcription of IFNΑ2, a member of the IFNα gene cassette, in healthy pDC in a manner that paralleled the decrease of secreted IFNα (Fig. 4B).
CD83 is a surface protein on myeloid lineage cells, including pDCs, which serves as a costimulatory molecule to drive other immune cell activation.37–41 We found that CD83 is expressed early during pDC activation by CpG-ODN (within 6 hours) and that THC suppressed the number of pDC expressing surface CD83 in both healthy and HIV+ donors (Figs. 5A, B). Treatment with CBD did not alter CD83 expression by pDC from healthy donors (Fig. 5A) but did suppress CD83 expression in pDC from HIV+ donors (Fig. 5B).
Presented here is the first report of cannabinoid receptor expression and modulation by THC of pDC function. pDC expression of the canonical cannabinoid receptors (CNR1 and CNR2) was found to be comparable with other PBMC, with greater expression of CNR2 than CNR1. We also observed that treatment with THC, and not CBD, caused a concentration-dependent suppression of IFNα secretion by pDC in healthy donors but did have an effect at higher concentrations in pDC from HIV+ donors. Because CBD has much lower affinity for both CB1 and CB2 than THC, suppression of pDC secretion of IFNα by THC suggests the involvement of cannabinoid receptors rather than nonspecific mechanisms. Moreover, THC impaired IFNα secretion by purified pDC, ruling out the possibility for a bystander effect by other cell types. The direct suppression by THC of pDC-secreted IFNα is in agreement with previous findings showing pDC modulation by the endogenous cannabinoid, anandamide.42
The mechanism underlying the modulation of immune cell function by cannabinoids has been partially elucidated by our and other laboratory results.25,27,43 Here, we provide evidence that THC suppresses the phosphorylation of IRF-7, the master regulator of IFNα secretion, in pDC and that this suppression results in the loss of IFNα gene transcription. IRF-7 can be phosphorylated by Interleukin-1 Receptor Associated Kinase 1 & 4 (IRAK 1/4),44 phosphoinositide 3-kinase,45 and IκB kinase-α.46 PI3K signaling in particular has been identified in modulation of the innate immune cell response and is a putative target for the development of therapeutics.47 Activation of the cannabinoid receptors has been shown to directly modulate mTOR-AKT-PI3K signaling in neuronal cell differentiation and survival48,49 and disrupt T-cell stimulation by keratinocytes through suppression of the same pathway.50 Given the critical role of PI3K in IFNα secretion in pDC and the conservation of cannabinoid receptor-mediated suppression of mTOR-AKT-PI3K signaling across different cell types, the suppression of the mTOR-AKT-PI3K signaling axis is likely a means by which IFNα secretion is suppressed in pDC by THC. However, a comprehensive phosphoproteomic approach will be needed to elucidate the complexity surrounding the cannabinoid-mediated modulation of this signaling pathway.
pDC from HIV+ donors were found to be more sensitive to suppression by THC compared with pDC from healthy donors. This increased cannabinoid sensitivity may be linked to the significantly higher expression of CNR1 mRNA, and therefore a greater number of CB1 receptors, in PBMC from HIV+ donors compared with healthy donors. The higher expression of CNR1 mRNA might be linked to the chronic inflammatory state experienced by many HIV+ patients, as activation of T cells results in the upregulation of CNR1 and not CNR2.51 Patients with HIV, even those successfully treated by ART, experience a variety of inflammatory conditions (eg, “Leaky Gut Syndrome”) that can lead to systemic inflammation and higher levels of circulating inflammatory cytokines.52,53 It is tempting to speculate that higher levels of inflammatory cytokines lead to increased expression of CNR1, but proinflammatory cytokines can induce expression of both CNR1 and CNR2.54 Furthermore, it is noteworthy that in the current studies, CB1 and CB2 expression was quantified solely at the mRNA level (CNR1 and CNR2, respectively). Additional studies will be needed to confirm these findings at the protein level.
pDC can stimulate other immune cells by secretion of IFNα and through the expression of costimulatory molecules (CD83, CD86, CD80, and HLA-DR).55 Expression of CD83 by pDC has been associated with stimulation of both T and B cells.4 Here, we show that THC can impair CD83 surface expression by pDC within 6 hours after activation by CpG-ODN. Similarly, when CD83 signaling is ablated, dendritic cell induction of T-cell expansion was significantly reduced.38,39 Therefore, our results indicate that cannabinoid-based therapies may diminish pDC activation of the adaptive immune response by suppressing both the secretion of IFNα and the expression of a key costimulatory molecule, CD83. Future studies will reveal whether the suppression of CD83 by THC contributes to a functional deficit in pDC-mediated T-cell effector function.
The use of cannabis remains controversial in both healthy and HIV+ populations. The results presented here suggest that THC directly impairs pDC function, which may further compromise patients with HIV in responding to opportunistic viral infections. However, the actual implications of these results are mixed. HIV-Associated Neurocognitive Disorders (HAND) affect patients with HIV56,57 regardless of ART, and these neurocognitive deficits have been linked with a chronic neuroinflammatory state.52,58 pDCs have been implicated in neuroinflammatory disease,42,59–61 and elevated levels of IFNα in neuronal tissue have been associated with neuroinflammation and neurodegeneration.62,63 Although the direct role of pDC on IFNα levels in the CNS is unclear, the suppression of pDC activation may be protective against neuroinflammation associated with prolonged HIV infection. Furthermore, and consistent with the premise of medicinal marijuana use as potentially neuroprotective, cannabinoids have been shown to help maintain the integrity of the blood–brain barrier in patients with HIV,64 potentially reducing the migration of inflammatory cells from the periphery to the brain.
The data generated from HIV+ donors presented in this article were generated using PBMC provided by male donors exclusively, which comprise 80% of patients with HIV in the United States. However, over 240,000 women are infected with HIV in the United States, and modulation of pDC activity is of particular interest for these patients. Women progress more quickly than men from the establishment of HIV infection to the development of AIDS.65 Interestingly, pDC from women have an augmented IFN response compared with men when stimulated through Toll-like Receptor-7,66 and this difference may underlie the accelerated development of AIDS.65 Collectively, the presented data imply that the use of cannabinoids may be also beneficial for suppressing the activity of the cells, which play a role in the persistent activation of the immune system of patients with HIV who have been successfully treated by ART.
The authors thank Linda Dale for coordinating blood collection from HIV+ donors.
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