Increased plasmacytoid dendritic cell maturation and natural killer cell activation in HIV-1 exposed, uninfected intravenous drug users
Tomescu, Costina; Duh, Fuh-Meib; Lanier, Michael Ac; Kapalko, Angelad; Mounzer, Karam Cd; Martin, Maureen Pb; Carrington, Maryb; Metzger, David Sc; Montaner, Luis Ja
aThe Wistar Institute, HIV Immunopathogenesis Laboratory, Philadelphia, Pennsylvania
bCancer and Inflammation Program, Laboratory of Experimental Immunology, SAIC-Frederick, Inc., NCI Frederick, Frederick, MD 21702 and Ragon Institute of MGH, MIT and Harvard, Boston, Massachusetts, USA
cThe University of Pennsylvania, Department of Psychiatry, HIV Prevention Division, USA
dPhiladelphia FIGHT, The Jonathan Lax Treatment Center, Philadelphia, Pennsylvania, USA.
Received 10 March, 2010
Revised 30 June, 2010
Accepted 7 July, 2010
Correspondence to Dr Luis J. Montaner, The Wistar Institute, 3601 Spruce Street, Room 480, Philadelphia, PA 19104, USA. Tel: +1 215 898 3934; fax: +1 215 573 9272; e-mail: Montaner@wistar.org
Background: Increased natural killer (NK) activation has been associated with resistance to HIV-1 infection in several cohorts of HIV-1 exposed, uninfected individuals. Inheritance of protective NK receptor alleles (KIR3DS1 and KIR3DL1high) has also been observed in a subset of HIV-1 exposed, uninfected individuals. However, the exact mechanism contributing to NK activation in HIV-1 exposed, uninfected intravenous drug users (EU-IDU) remains to be elucidated.
Objective: We investigated the role of both host genotype and pathogen-induced dendritic cell modulation of NK activation during high-risk activity in a cohort of 15 EU-IDU individuals and 15 control, uninfected donors from Philadelphia.
Design: We assessed the activation status of NK cells and dendritic cells by flow cytometry and utilized functional assays of NK-DC cross-talk to characterize the innate immune compartment in EU-IDU individuals.
Results: As previously reported, NK cell activation (CD69) and/or degranulation (CD107a) was significantly increased in EU-IDU individuals compared with control uninfected donors (P = 0.0056, n = 13). Genotypic analysis indicated that the frequency of protective KIR (KIR3DS1) and HLA-Bw4*80I ligands was not enriched in our cohort of EU-IDU individuals. Rather, plasmacytoid dendritic cells (PDC) from EU-IDU exhibited heightened maturation (CD83) compared with control uninfected donors (P = 0.0011, n = 12). When stimulated in vitro, both PDCs and NK cells from EU-IDU individuals maintained strong effector cell function and did not exhibit signs of exhaustion.
Conclusion: Increased maturation of PDCs is associated with heightened NK activation in EU-IDU individuals suggesting that both members of the innate compartment may contribute to resistance from HIV-1 infection in EU-IDU.
Along with CD8 ‘killer’ T cells, natural killer (NK) cells function as cytolytic lymphocytes capable of seeking out and destroying virally infected target cells in the body. Unlike antigen specific T cells, NK cells use the coordinated interaction of both inhibitory and activating receptors to regulate their cytotoxic activity against target cells. NK activity is also modulated by accessory cells that can augment NK activation and killing potential. In particular, plasmacytoid dendritic cells (PDC), through the secretion of interferon-alpha (IFN-α), have been shown to be essential during the host response to viral infection [1–3]. PDCs recognize ssRNA and dsDNA pathogens through the use of their intracellular toll-like receptors, TLR7 and TLR9, and comprise the main IFN-α secreting cell type in the blood. In vitro, PDC secretion of IFN-α has been shown to be necessary for NK-mediated lysis against several virally infected target cell types including herpesvirus-infected fibroblasts [4–8] and HIV-infected autologous CD4+ primary T cells .
Overall, several main types of NK inhibitory receptors exist that can be distinguished based on their expression pattern and ligand specificity. Of these, the killer inhibitory receptors (KIRs) exhibit a restricted pattern of expression and interact with only a limited subset of MHC class I ligands [10,11]. Nevertheless, inheritance of specific KIR alleles has profound implications for individual susceptibility to infectious diseases [12,13]. In HIV-1 infected individuals, the KIR family of inhibitory and activating receptors has been directly implicated in control of viral replication. Inheritance of KIR3DL1 inhibitory receptor alleles that exhibit high expression (KIR3DL1high) have been shown to be associated with delayed progression to AIDS when coinherited with their corresponding HLA-Bw4*80I ligands . Similarly, KIR3DS1, an activating allele of the same locus has likewise been associated with delayed progression to AIDS . Recently, the KIR3DL1/S1 locus has also been associated with resistance to HIV-1 infection. Inheritance of protective KIR3DL1high and KIR3DS1 receptor alleles were observed to be over-represented in a high-risk cohort of HIV-1 exposed, uninfected intravenous drug users (EU-IDU) and their sexual partners [16,17].
Among intravenous drug users, HIV-1 exposed, uninfected individuals have been described that remain uninfected with HIV-1 in spite of repeated high-risk behavior, as defined by a history of long-term needle exchange with persons of known/unknown HIV-1 infection . Previously, increased NK activation has been associated with resistance to HIV-1 infection in a cohort of intravenous drug users from Vietnam . Increased NK activation has also been associated with resistance to infection in HIV-discordant couples from Columbia and perinatally exposed children born to HIV-1 infected mothers [20,21]. Together with genotypic data showing an enrichment of protective NK receptor alleles in EU-IDU individuals, these results suggest that increased NK activity may be associated with protection from HIV-1 during high-risk activity. However, it remains unknown if the heightened NK activation observed in EU-IDU individuals is related to the inheritance of protective NK receptor alleles or is due to other factors. Here, we investigated the role of both host genotype and pathogen-induced dendritic cell modulation of NK activation during high-risk behavior in a cohort of EU-IDU from Philadelphia, USA.
Materials and methods
Subject criteria and peripheral blood mononuclear cell purification
HIV-1 exposed, uninfected injection drug users (EU-IDU) were enrolled over a 3-year period from the city of Philadelphia via community-based street outreach by staff experienced in recruiting high-risk injectors. Currently, the city of Philadelphia has a rate of new infections that is estimated to be more than five times the national average [22,23], and over 30% of these are attributable to injection drug use . Within Philadelphia, injection drug use is clustered in specific neighborhoods previously defined as ‘risk pockets’ , where drugs are sold, drugs are used, and sex is exchanged for drugs in close proximity. In agreement with a previous report , we estimate the HIV-1 sero-prevalence within these ‘risk pockets’ in Philadelphia to be approximately 20% among high-risk needle-sharing IDU individuals.
We utilized the ‘Prognostic Model for Seroconversion Among Injection Drug Users’  to identify high-risk HIV exposed, uninfected individuals for our study based upon their frequency of injection drug use and needle sharing behavior in areas identified as ‘risk pockets’ containing a high prevalence of new HIV-1 infections. Individuals from known ‘risk pockets’ were identified as EU-IDU, if they reported a history of greater than 2 years of daily injection and frequent (monthly or greater) needle sharing with partners of unknown HIV status. In addition to HIV testing, all individuals were offered counseling and referral for additional services. HIV-1 sero-positive individuals were excluded from the study but received counseling and referral to care settings. Blood was then drawn from 15 HIV-1 exposed, uninfected individuals at the Jonathan Lax Treatment Center at Philadelphia FIGHT according to Institutional Review Board approval and informed consent. As a control, blood was drawn from a panel of 15 healthy, HIV-1-seronegative donors from the greater Philadelphia area at The Wistar Institute Blood Donor Program. All blood was processed within 3 h of draw and peripheral blood mononuclear cells (PBMC) were collected by standard ficoll density gradient centrifugation as previously described .
Although all individuals were injecting heroin, six (40%) reported also injecting stimulants (cocaine and/or methamphetamine). EU-IDU individuals were confirmed to be negative for HIV-1 at the time of blood draw using an anti-HIV antibody serum ELISA test (BioChain Institutue, Hayward, California, USA) and later screened for the absence of CCR5 delta 32 homozygocity as described further. All individuals were analyzed for the presence of anti-HCV antibodies in serum with HCV antigen-coated ELISA plates (Core, E2, NS3, NS4, and NS5) according to the manufacturer instructions (BioChain Institutue, Hayward, California, USA). In confirmation of their high-risk needle sharing behavior, greater than 70% of the EU-IDU documented within our study were HCV infected.
Number of peripheral blood mononuclear cell 2 × 106 PBMC from HIV-1 exposed, uninfected injection drug users and control uninfected individuals were frozen in DNAzol (Molecular Research Center, Cincinnati, Ohio, USA) and genotyped at HLA Immunogenetics Laboratory in The National Cancer Institute. Presence or absence of each KIR gene was determined as previously described . The HLA class I loci were typed by the sequence based typing method as recommended by the 13th International Histocompatibility Workshop (http://www.ihwg.org/tmanual/TMcontents.htm).
All cell surface antibodies and isotype controls were preconjugated and used at the recommended dilution of 0.25 μg antibody per million cells in PBSA (PBS with 0.09% sodium azide). PBMCs were stained with antibodies to phenotypic and functional markers for 15 min at RT°C in the dark, washed twice and fixed with Cytofix Buffer (BD Cytometry Systems, San Jose, California, USA). The following antibodies and their appropriate isotype controls were used in this study CD56 v450 (BD), CD69 FITC (BD), CD107a PE (BD), CD3 PERCP (BD), Lineage FITC (BD), CD83 PE (BD), HLA-DR PERCP (BD), BDCA-4 or BDCA-1 APC (Miletyni Biotech, Auburn, California, USA). For intracellular cytokine staining, cells were permeabilized with the Cytofix/Cytoperm kit (BD) as described by the manufacturer and stained for 15 min at RT°C in the dark with 0.25 μg of anti-IFN-gamma FITC antibody (BD) per million cells. A minimum of one hundred thousand events were collected on a BD LSR-II Flow Cytometer and samples were subsequently analyzed with FlowJo software (Tree Star Incorporated, Ashland OR). Prior to analysis, all samples were gated by forward and side scatter to exclude dead cells.
CD107a degranulation assay
Number of peripheral blood mononuclear cell 1 × 106 were co-cultured alone (no target control) or with K562 cells at a 10: 1 effector/target ratio in the presence of 20 μl anti-CD107a monoclonal antibody for 3 h in a 200 μl volume. PBMC were then washed and stained with antibodies to NK phenotypic markers (CD56/CD3) for 15 min at RT°C. NK cells were gated by CD56+/CD3− staining and CD107a expression was determined based on background levels of staining exhibited by no target control cells.
Plasmacytoid dendritic cell-stimulated natural killer activation
Number of peripheral blood mononuclear cell 2.5 × 106 were stimulated for 18 h with 10 μg/ml CpG-ODN 2216 in a 1 ml volume. PBMCs were then washed and stained with antibodies to the NK phenotypic markers CD56, CD3, and the activation marker, CD69, for 15 min at RT°C. NK cells were gated by CD56+/CD3− staining and CD69 expression was determined based on isotype control.
Supernatants from overnight stimulation of PBMC with CpG-ODN 2216 were collected and frozen at −80°C. Supernatants were thawed and tested in duplicate for IFN-α secretion using the Verikine multisubtype interferon alpha ELISA kit (PBL Biomedical Laboratories, Piscataway, New Jersey, USA) according to the manufacture's instructions. The Verikine multisubtype interferon alpha ELISA kit detects 13 of 15 known IFN-α subtypes but does not exhibit cross reactivity with human IFN-β, human IFN-γ or human IFN-ω.
Paired statistical analyses were performed with Prism software (GraphPad Software, La Jolla, California, USA) using Wilcoxon matched pair, nonparametric t-tests. In all cases, P values were two-sided with significance < 0.05.
NK cells from EU-IDU exhibit increased activation and/or constitutive degranulation compared with controls.
Utilizing the definition of a high-risk exposed, uninfected intravenous drug-user (EU-IDU) detailed in Materials and Methods and Table 1, we tested the phenotype and function of NK cells in a panel of 15 EU-IDU and 15 control donors from the greater Philadelphia area. As previously described [28,29], we observed that NK cells from EU-IDU individuals constitutively exhibited an increased expression of the CD69 activation marker and/or heightened CD107a degranulation compared with control donors (Fig. 1a and b). Although there was no change in the overall frequency of CD56dim/CD3− NK cells in EU-IDU individuals (Fig. 1c), NK cells from EU-IDU individuals exhibited a statistically significant increase (P = 0.0056, n = 13) in CD69 and/or CD107a expression compared with control donors (Fig. 1d). Together, these results suggested a qualitative difference in the activation state of NK cells from EU-IDU.
We have previously shown that NK cells from healthy uninfected donors possess the capacity to re-degranulate and lyze tumor targets repeatedly without a loss in NK function or viability . Likewise, we tested if NK cells from EU-IDU individuals also maintained strong effector function despite evidence of heightened activation and recent degranulation. We incubated PBMC with K562 tumor cells to induce target cell lysis and measured CD107a degranulation on NK cells. We also stimulated PBMC with IL-12 and IL-15 to induce the production of interferon-gamma (IFN-γ) by NK cells. As shown in Fig. 1e and f, we observed that NK cells from EU-IDU possessed a normal capacity to degranulate in response to target cell stimulation and there was no statistical difference in K562 cell-induced degranulation between EU-IDU and controls. Similarly, we observed that the production of IFN-γ by NK cells following IL-12/IL-15 stimulation was similar between EU-IDU and controls (data not shown). Together, these results suggested that NK cells from EU-IDU exhibit a constitutive activation profile ex vivo, but maintain the capacity for normal function when restimulated in vitro.
PDC cells from EU-IDU exhibit increased maturation but maintain the functional capacity to mediate NK/DC cross-talk.
Owing to the critical role that plasmacytoid dendritic cells (PDC) play in augmenting NK responses, we next measured the phenotype and function of PDC in EU-IDU individuals. Although there was no change in the overall frequency of PDC cells in EU-IDU individuals, we observed that PDCs from EU-IDU individuals exhibited increased constitutive expression of the maturation-specific marker CD83 [31,32] compared with controls (Fig. 2a and b). The heightened CD83 expression observed on PDC from EU-IDU was statistically significant compared with controls (P = 0.0011, n = 12) and was specific for the PDC subset (Fig. 2d). Myeloid dendritic cells (MDCs), the other main circulating dendritic cell subset, did not exhibit heightened activation compared with controls (data not shown).
We next tested if PDCs from EU-IDU exhibited signs of functional exhaustion owing to the state of persistent innate immune activation. We utilized the PDC-specific [33–35] toll-like receptor 9 agonist, CpG-ODN 2216, and measured the ability of PDC to secrete interferon-alpha (IFN-α) and induce NK activation as previously described . Owing to the documented dysfunction in PDC function observed in HCV-infected individuals [36–39], we stratified EU-IDU individuals based on their HCV serostatus. As shown in Fig. 3a, we observed no difference in the secretion of IFN-α between HCV-negative EU-IDU individuals and control donors following CpG-ODN 2216 stimulation. However, there was a statistically significant decrease in CpG-induced IFN-α secretion between HCV sero-positive EU-IDU individuals and control donors (P = 0.0221, n = 7). Nevertheless, we observed that the ability of PDC to mediate NK activation following CpG-ODN 2216 stimulation was similar between HCV sero-positive EU-IDU individuals and control donors (Fig. 3b). Together, these results suggest that HCV infection negatively impacts IFN-α secretion, but PDC activation of NK cells is maintained among EU-IDU individuals allowing for sustained NK/PDC cross-talk.
Finally, we observed that the heightened maturation status of PDC from EU-IDU was independent of HCV status. As shown in Fig. 3c, PDC from both HCV seropositive and HCV seronegative EU-IDU individuals exhibited high levels of CD83 expression compared with control donors.
No enrichment of protective NK KIR3DS1 receptor or HLA-Bw4*80I ligands in EU-IDU individuals.
The increased inheritance of the protective NK KIR3DL1high and KIR3DS1 receptor alleles has been previously observed in high-risk HIV-1 exposed, uninfected intravenous drug users as a correlate of protection [16,17]. We tested if inheritance of protective KIR receptor alleles and their corresponding HLA-Bw4*80I ligands were also correlated with the heightened innate immune activation phenotype we described in our cohort of EU-IDU individuals from Philadelphia. As shown in Table 2, we observed a similar frequency of the activating receptor KIR3DS1 among EU-IDU (4/14, 29%) and control uninfected donors (4/12, 33%) reflecting the fact that both groups are composed from the same geographic area. Although our primer/probe design did not allow us to be able to distinguish NK KIR3DL1high protective alleles from KIR3DL1low nonprotective alleles, we tested instead for the presence of MHC-Class-I ligands of the HLA-Bw4*80I family whose inheritance is also required for protection. We observed that only five of 14 EU-IDU individuals from our cohort possessed the corresponding HLA-Bw4*80I ligands for interaction with protective KIR3DL1high alleles (Table 2). Of the five EU-IDVU individuals that possessed the corresponding HLA-Bw4*80I ligands, only one exhibited heightened NK activation (data not shown). Together, these results suggested that inheritance of protective NK receptor alleles and their corresponding MHC Class-I ligands likely did not account for the increased NK activation in the majority of EU-IDU individuals from our cohort.
Here we confirm previous reports of increased NK activation in exposed uninfected individuals, and show for the first time that increased PDC maturation is also a marker of the heightened innate immune activation state observed in EU-IDU individuals. Despite a state of persistent activation, we show that both PDCs and NK cells from EU-IDU maintained strong effector cell function and did not exhibit signs of exhaustion. Overall, our results highlight the role of PDCs in augmenting NK function and show how both members of the innate immune response are comodulated during high-risk activity in EU-IDU individuals.
Upon the basis of previous findings indicating an enrichment of protective NK KIR3DL1high and KIR3DS1 receptor alleles in high-risk HIV-1 exposed, uninfected individuals [16,17], we correlated NK phenotype and function with genotypic analysis of KIR and their HLA-Bw4*80I ligand alleles. Our findings indicate that inheritance of protective KIR alleles or their corresponding HLA ligands was not enriched in our cohort of EU-IDU individuals compared with control uninfected donors from the same geographic area. Rather, our results indicate that increased maturation of PDCs is associated with heightened NK activation in EU-IDU individuals. However, a greater number of individuals will be required to draw firm conclusions regarding KIR allele frequency and resistance to infection at the population level in high-risk individuals.
Our observations are of interest in light of the controversial role of opioids on NK cell function where contrasting data suggests that opioids can inhibit NK activity or can lead to a ‘tolerant’ state that is dependent on frequency of drug use and timing of NK analysis relative to drug exposure [40–42]. Upon the basis of the frequency and duration of injection drug use among the EU-IDU individuals in our cohort (see Table 1), we speculate that the innate immune response of EU-IDU individuals reaches an equilibrium state after years of prolonged injection-drug use. Indeed, work from several groups supports a loss of innate function during acute exposure to opioids or upon opioid withdrawal, rather than chronic opioid usage [43,44]. As has been described previously for other cohorts of EU-IDU individuals [19,28], we observed that NK cells from EU-IDU maintained strong effector cell function when stimulated in vitro (Fig. 1e and f). We now extend these observations to include PDC cells from EU-IDU individuals, which remained functional in vitro (Fig. 3) despite evidence of heightened PDC maturation ex vivo.
It is also important to note that whereas high-risk drug-use is a hallmark of the EU-IDU individuals, approximately half of the individuals from our cohort also reported a history of recent high-risk sexual activity. This included a high frequency of sexual events without the use of a condom as well as a high number of sexual partners over the previous 3-month period (Table 1). Interestingly, we did not observe any correlation between the frequency of high-risk sexual practices or the number of sexual partners and the extent of innate immune activation in EU-IDU from our cohort. Future work will be needed to determine to what extent sexual exposure (if any) can enhance the observed innate immune activation phenotype among IV drug users as our data does not allow us to specifically address this point.
In addition to the influence of opioid usage on innate immune function, the covariable of HCV exposure in injection drug users proved informative in interpreting the functional data from our cohort. As described, we observed impairment in the ability of the HCV seropositive individuals to secrete IFN-α following CpG-ODN 2216 stimulation (Fig. 3a). However, PDC-dependent NK activation was similar between HCV seropositive and HCV seronegative EU-IDU individuals suggesting that PDC/NK crosstalk is sustained in EU-IDU despite evidence of HCV infection (Fig. 3b). Interestingly, the increase in PDC maturation we observed among EU-IDU individuals was independent of HCV status, as both HCV seropositive and HCV seronegative EU-IDU individuals exhibited increased CD83 expression compared with controls (Fig. 3c). This finding suggests that HCV seropositivity is not a requirement for the heightened PDC maturation observed in many EU-IDU individuals. Nevertheless, the high incidence of HCV seropositivity in our cohort of EU-IDU (10/14, 71%) suggests that persistent exposure to HCV virus is likely encountered during prolonged IV-drug use and may contribute to PDC maturation.
Overall, our results highlight NK cells and PDCs, as candidate cell types whose retained function and heightened activation status may contribute to resistance upon HIV-1 exposure.
We thank Jonathan Davis at the Jonathan Lax Treatment center at Philadelphia FIGHT for his contribution as phlebotomist specializing in drawing blood from IDU individuals. We thank Deborah Davis at The Wistar Institute for the recruitment of control donors and her contribution as phlebotomist for this study. We also thank David E. Ambrose, Daniel Hussey, and Jeffrey S. Faust at The Wistar Institute Flow Cytometry Core facility. This study was supported by grants from the National Institutes of Health (NIDA R01 DA028775, R01 AI073219, RO1 AI065279, Core grant P30 CA10815), the Philadelphia Foundation, and funds from the Pennsylvania Commonwealth Universal Research Enhancement Program.
This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. There are no conflicts of interest.
Author contributions: C.T. (completed all of the in-vitro assays, co-wrote the manuscript, completed statistical analysis), F.-M.D. (completed all of the HLA and CCR5 genotyping), M.L. (coordinated subject recruitment, analyzed subject behavioral data), A.K. (coordinated subject blood draws), K.C.M. (coordinated blood draws, edited manuscript), M.P.M. (completed all of the KIR genotyping, edited the manuscript), M.C. (coordinated KIR, HLA and CCR5 genotyping, edited the manuscript), D.S.M. (coordinated EU-IVDU subject cohort, analyzed subject behavioral data, co-wrote the manuscript), L.J.M. (coordinated the study, analyzed all of the functional data, co-wrote the manuscript).
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