Plasmacytoid dendritic cells (pDCs) compose a minor population (0.2%–0.5%) of circulating peripheral blood mononuclear cells (PBMCs) that play a crucial role in bridging the innate and adaptive antiviral immune response.1–4 On activation, pDC secrete 1000-fold more type I interferon (IFN) than any other population of PBMCs to stimulate other leukocytes including NK cells,5,6 B cells,4 and T cells.6,7
Somewhat paradoxically, pDC number and function is suppressed in association with certain types of viral infections including hepatitis C virus and HIV.8,9 In a rhesus macaque model of HIV infection, using simian immunodeficiency virus (SIV), the number of circulating pDC is reduced during the acute stage of SIV infection as pDC migrate to the gut.10 In both HIV and SIV infection, gut lymphoid tissue is a key site of viral replication and, therefore, a target for pDC recruitment. However, pDC may be susceptible to productive HIV infection because of their expression of CD4. Specifically, infection by HIV may perturb pDC function resulting in reduced secretion of interferon alpha (IFNα).11 This reduced capacity for IFNα secretion during infection would hinder an appropriate host response, as evidenced by protection against HIV-mediated CD4+ T cell depletion in a humanized mouse model on administration of IFNα,12 and lead to an inability to appropriately control the infection.13 HIV-infected pDC may also directly facilitate the infection of CD4+ T cells during the acute phase of HIV infection.14 Furthermore, the loss of pDC in circulation correlated with an increase in HIV viral serum titer such that fewer circulating pDC translated into a deficiency in antiviral response.15 Collectively, these results have broader implications for the health of HIV+ patients as loss of pDC function could exacerbate susceptibility to opportunistic viral infection.
In 2015, the Centers for Disease Control and Prevention estimated that 1.2 million people were infected with HIV in the United States and 36.9 million globally. Antiretroviral therapy (ART) is the primary therapy for patients with HIV in the United States and has been since the mid 1990s.16 Although effective, ART therapy can also induce nausea and reduce appetite.17 Furthermore, HIV infection, even when properly controlled by ART, is associated with physical wasting18,19 and anxiety,20,21 both of which can have deleterious effects on the host immune response. The effects of both HIV infection and ART have led to a significant number of patients with HIV using cannabinoid-based therapies such as medical marijuana (Cannabis sativa) and dronabinol (Marinol).22–24
Δ9-Tetrahydrocannabinol (THC, also known as dronabinol or Marinol) is the primary psychoactive cannabinoid in marijuana and is a well-characterized immune modulator.25–27 In mouse models of herpes simplex virus type II,28,29Listeria monocytogenes,29 and influenza virus type A,30,31 THC administration exacerbated disease progression. Although THC has been shown to have suppressive effects on the function of many different immune cell populations, THC-mediated suppression of IFN secretion was demonstrated in all the aforementioned models of disease.32 Suppression of IFN (type I and II) secretion by THC is likely a key mechanism by which viral infections are potentiated.
Currently, the utilization of cannabinoid-based therapies in HIV infection is controversial. Utilization of cannabinoids has been found to reduce the concentration of circulating antiretroviral drugs, and these studies indicated little effect of cannabinoids on retroviral therapy efficacy or immune cell function.23,33 However, in these cases, it is difficult to distinguish between the direct effects of the cannabinoids on leukocyte function and possible confounders. Furthermore, suppression of peripheral IFNα secretion through utilization of medicinal cannabinoids may reduce certain HIV-associated comorbidities, thereby lending potential support for cannabinoid-based therapies. The objective of this study was to determine the effects of THC on IFNα production by pDCs using leukocytes from HIV+ patients on ART and healthy donors as controls.
MATERIALS AND METHODS
PBMC Isolation and Cell Identification
Leukocyte packs were purchased from the Gulf Coast Regional Blood Center (Houston, TX). Blood was diluted 1:1 with Hanks balanced salt solution from Gibco (Grand Island, NY) and layered on 15 mL Ficoll Paque Plus (GE Healthcare Life Sciences, Pittsburgh, PA) in SepMate 50-mL conical tubes by StemCell Technologies (Vancouver, BC, Canada). Leukocytes were centrifuged at ×1300g for 25 minutes at 4°C. The leukocyte layer was resuspended in RPMI Media from Gibco containing 5% Human AB Serum (Sigma-Aldrich, St. Louis, MO), 1% Penicillin-Streptomycin (Gibco), and 0.035% β-mercaptoethanol. pDCs were identified using mouse anti-human antibodies by Miltenyi Biotec GmbH (Bergisch Gladbach, Germany) as CD303+ CD123+ cells.
pDC Purification by Magnetic-Activated Cell Sorting
pDCs were isolated by negative selection using magnetic-activated cell sorting (MACs) isolation kits from Miltenyi Biotec per the manufacturer's instructions. Briefly, PBMC cell concentrations were determined using a coulter cell counter, and the appropriate volume of non-pDC antibody cocktail was incubated with PBMC followed by washing and incubation with magnetic beads. Labeled PBMCs were then passed through a MACS depletion column affixed to a MACS magnet with unstimulated pDC being collected in the flow through. The number of PBMCs in a single leukocyte pack range from 3.0 to 11 × 108 total PBMC with an average of 6 × 108 total PBMC and 0.9–1 × 106 pDC per leukocyte pack containing 6 × 108 total PBMC when accounting for isolation efficiency.
Gene Expression Analysis
RNA was isolated using Qiagen RNeasy kits (Germantown, MD) per the manufacturer's instructions. Briefly, cells were lysed using lysing buffer containing β-mercaptoethanol and stored at −20°C. Lysates were then purified and treated with DNAse from Promega SV Total RNA Isolation Kit (Madison, WI). RNA concentrations were determined by Nanodrop (Thermo-Fisher Scientific, Waltham, MA). Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed using High-Capacity cDNA RT-PCR kit by Applied Biosystems (Foster City, CA). cDNA was frozen at −20°C. Gene analysis was determined by real-time quantitative PCR (Qt-PCR) using TaqMan probes for CNR1 (Hs00275634_m1) and CNR2 (Hs00275635_m1) by Life Technologies (Compendia Bioscience, Ann Arbor, MI) with 18sRNA as a loading control.
Treatment With Cannabinoids or Vehicle Control and Cell Stimulation
THC was supplied by the National Institute of Drug Abuse. Purified, unstimulated pDCs or PBMCs were treated with either Δ9-THC, cannabidiol (CBD), or vehicle control (–0.026% ethanol). The appropriate concentrations were prepared in complete RPMI. The prepared cell suspensions and appropriate treatments were added to flat bottom 96-well tissue culture plates. Cells were then incubated at 37°C and 5% CO2 for 30 minutes. After incubation, cells were stimulated with CpG-ODN type A 2216 (15 μg/mL) (InvivoGen; San Diego, CA).
IFNα Capture Assay
Secretion of IFNα was determined using the IFNα capture assay by Miltenyi Biotec per the manufacturer's directions. Treated cells were bound with IFNα capture reagent and placed into warm media and incubated under continuous motion for 30 minutes. Cells were then washed and incubated with IFNα detection antibody. Cells were fixed using CytoFix buffer by BD Biosciences (San Jose, CA), and IFNα secreting pDC were quantified by flow cytometry.
Treated PBMCs were washed, and pDCs were stained as described. pIRF7 levels were determined using Phosflow antibodies and the harsh detergent method by BD Biosciences. In brief, cells were fixed using BD Cytofix buffer for 10 minutes at 37°C then permeabilized using ×1 of Perm buffer IV, stained for 1 hour under continuous motion using Fluorescence Activated Cell Sorting (FACS) buffer and 5% Human AB Serum, washed 3X with ×0.5 perm buffer, and analyzed by flow cytometry.
IFNΑ2 Gene Expression by PrimeFlow
PrimeFlow RNA assay (eBiosciences; San Diego, CA) was performed per manufacturer's directions. Treated PBMCs were fixed, permeabilized, and bound with IFNΑ2 probe. The mRNA signal was then amplified and detected using Alexa Fluor 647 detection probes (Thermo-Fisher Scientific, Waltham, MA). Relative gene expression was determined using flow cytometry.
Measuring Secreted IFNα
IFNα secretion was determined using the LEGENDplex cytometric bead array by BioLegend per the manufacturer's directions. Detection beads were sonicated and incubated with media from purified pDC. The BD Canto II was used for data acquisition and accompanying LEGENDplex software was used for analysis.
GraphPad Prism 5.0 was used for statistical analysis. Where appropriate, samples were normalized to 0 μM THC + CpG, which was considered 100% maximum response for each individual donor, and the appropriate statistical test was performed (Figs. 2–5).
HIV+ Donor Recruitment and Data Management
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.
Profile of CNR1 and CNR2 Expression in pDC and PBMC From HIV+ Donors Versus Healthy Donors
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.
pDCs From HIV+ Donors Are More Sensitive to THC-Mediated Suppression of IFNα Secretion Compared With Healthy Donors
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.
pDC were identified as CD303+ CD123+ cells (Fig. 2A), and secretion of IFNα was then quantified by flow cytometry (Fig. 2B). The induction of IFNα+ pDC after CpG-ODN treatment from HIV+ donors was comparable with pDC from healthy donors (Fig. 2C). Treatment of PBMCs with THC decreased the number of IFNα-secreting pDC from both healthy and HIV+ donors (Figs. 2D, E). Conversely, the closely related cannabinoid congener CBD, which possesses low affinity for both CB1 and CB2, produced no effect on the percentage of IFNα-secreting cells in response to CpG-ODN activation (Figs. 2D, E). Neither THC nor CBD exhibited cytotoxic effects on pDC at any of the concentrations used in these determinations.
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.
Δ9-THC Directly Suppressed Secretion of IFNα in Healthy Donors
Given that pDCs are a minor population within the PBMC (Fig. 2A), studies were conducted to determine whether THC acts directly on pDC to suppress IFNα production or indirectly through bystander cell effects. The aforementioned studies were repeated using highly purified pDC (Fig. 3A) which showed that treatment with THC decreased the percent of IFNα-secreting pDCs in a manner comparable with that observed in the PBMC preparation (Fig. 3B), indicating THC acts directly on pDC.
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).
THC Directly Suppressed IFNα mRNA Levels by Impairment of Interferon Regulatory Factor 7 Phosphorylation
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).
Honda et al35 demonstrated that phosphorylation of IFN regulatory factor 7 (IRF-7) is a master regulatory event of type I IFN responses. In this study, THC treatment suppressed the phosphorylation of IRF-7 in pDC from healthy and HIV+ donors in a concentration-dependent manner. Treatment with CBD had no effect on healthy pDC, but suppressed pIRF7 in pDC from HIV+ donors (Figs. 4D, E). IFNα mRNA expression is dependent on nuclear translocation of pIRF-7, which is in turn controlled, at least in part, through osteopontin.36 Treatment with both THC and CBD had no significant effect on osteopontin levels in pDC from healthy donors (Fig. 4C).
THC-Suppressed TLR-9–Mediated Induction of Costimulatory Molecule CD83 on pDC From Healthy and HIV+ Donors
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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