Higher expression of the chemokine receptors C-C chemokine receptor type 5 (CCR5) and C-X-C chemokine receptor type 4 (CXCR4), which act as co-receptors for HIV, increases susceptibility of cells to HIV infection  and viral replication [2,3], whereas chemokines as natural ligands for these receptors can antagonise these effects [4–6]. Injecting drug use (IDU) is a risk factor for HIV transmission, but it may also change the natural course of HIV infection [7–9]. We previously showed that HIV-infected individuals with a history of IDU have a more rapid CD4+ cell decline in the absence of antiretroviral therapy (ART) . One possible explanation is the use of heroin and other opioids, as in-vitro studies have shown that opioids can up-regulate the expression of HIV co-receptors [10–12] and affect the expression of CCR5-binding chemokines RANTES [13–16], MIP-1α  and MIP-1β . It is unknown if this also takes place in vivo. We therefore studied the relationship of opioid use with CCR5 and CXCR4 expression, and ex-vivo chemokine production in HIV-infected individuals in Indonesia, which has a concentrated HIV epidemic strongly driven by IDU .
We included 84 ART-naïve HIV-infected adults with no signs of opportunistic infections and CD4+ cell counts above 100 cells/μl in West Java, Indonesia. Four groups of study participants were included: current heroin injectors; current users of methadone maintenance therapy (MMT); previous users of heroin or methadone (>1 year previously); or non-users. After consent was obtained, study participants underwent interviews and blood taking. This study was approved by the Health Research Ethics Committee at the Faculty of Medicine of Padjadjaran University/Dr Hasan Sadikin General Hospital in Bandung, Indonesia.
Using three-colour flow cytometry (BD FACSCalibur, CellQuest Pro Software), CCR5 and CXCR4 expression was measured as a proportion (%) of positive cells and mean fluorescence intensity (MFI). For whole blood stimulation, blood was diluted 1 : 5 with culture medium only or in combination with either live Candida albicans (106 cells/ml), Mycobacterium tuberculosis lysate (10 μg/ml) or Escherichia coli lipopolysaccharide (LPS) (10 ng/ml). After 24 or 48 h incubation, the expression of chemokines stromal cell-derived factor 1 (SDF-1) (which binds CXCR4), and monocyte chemotactic protein (MPC)-2, macrophage inflammatory protein (MIP)-1α, MIP-1β and regulated upon activation, normal T cell expressed and secreted (RANTES) (which bind CCR5) was measured using a multiplex beads assay (Merck Millipore, Billerica, Massachusetts, USA), and corrected for CD4+ cell counts (picogram per 105 CD4+ cells).
We compared differences between groups using chi-square for categorical variables, analysis of variance for normally distributed variables, and Kruskal–Wallis and Mann–Whitney analyses for non-normally distributed variables. If characteristics were different between groups, we also examined the association between these variables and our outcomes. Results were found statistically significant (two-sided) at a level of 0.05, resulting in 0.016 after Bonferroni adjustment for multiple tests. All statistical analyses were performed using the SPSS, version 18.0 (SPSS Inc., Chicago, Illinois, USA) and graphs were created using GraphPad Prism, version 5.0 (GraphPad Software Inc., San Diego, California, USA).
In total, 84 ART-naïve HIV-infected individuals were included: 17 active heroin users, 18 individuals receiving MMT, 19 individuals who had stopped using opioids and 30 individuals who never used heroin. Participants were mostly men (85.7%, n = 72), with a median CD4+ cell count of 336 cells/μl [interquartile range 214–505 cells/μl]. The median CD4+ cell count was 387 cells/μl for heroin users, 237 cells/μl for MMT clients, 385 cells/μl for former users and 290 cells/μl for controls (P = 0.518). Compared to the other groups, individuals who had never used any opioid were slightly younger (P = 0.001), but no differences were found between groups in sex (P = 0.479) or total lymphocyte count (P = 0.592).
Fewer CD4+ lymphocytes expressed CXCR4 in active heroin users (P = 0.071), methadone clients (P = 0.092) and former users (P = 0.005) compared to non-users, whereas the MFI of CXCR4-producing cells was not statistically different. Similar proportions of CD4+ lymphocytes expressed CCR5, but the MFI of CCR5-expressing cells was significantly higher in active heroin users (P = 0.007), MMT clients (P = 0.024) and former heroin users (P = 0.003), compared to non-users (Fig. 1a). The average MFI was 9.2 for heroin users (SEM = 0.92), 7.8 for MMT (SEM = 0.93), 7.8 for former heroin users (SEM = 1.2) and 6.5 for controls (SEM = 0.32). Age was not associated with the MFI CCR5-expressing CD4+ cells (P = 0.613) and did not affect the association between drug use and the MFI CCR5-expressing CD4+ cells (P = 0.534 in multivariable analyses).
Production of chemokines was not related to sex (lowest P value was 0.171) or age (lowest P value was 0.110). SDF-1 exclusively binds to the CXCR4 receptor and we found higher production of SDF-1 in controls after exposure to C. albicans (P = 0.011) and M. tuberculosis (P = 0.047), but not after E. coli LPS (P = 0.450). MCP-2, MIP-1α, MIP-1β and RANTES are ligands for the CCR5 receptor. After stimulation with LPS, the production of MCP-2, MIP-1α and MIP-1β were lower in heroin users (Fig. 1b). C. albicans stimulation resulted in lower MCP-2 production in current and former opioid users compared to controls (P = 0.025). After exposure to M. tuberculosis current and former heroin users produced less MIP-1α (Fig. 1c) and MIP-1β (Fig. 1d). We observed no differences in the production of RANTES.
In this study, we show that active heroin users have an increased expression of CCR5 on CD4+ lymphocytes, and a lower ex-vivo production of CCR5-binding chemokines. These results suggest that opioids could affect the HIV susceptibility by modulating the expression of the CCR5 HIV co-receptors or its natural ligands.
Previous in-vitro studies have shown activation of the μ-opioid receptor induces up-regulation of HIV co-receptors CCR5 and CXCR4 [12,17]. However, little is known about immunomodulating effects of chronic opioid use in vivo, especially in humans. In rhesus macaques, chronic morphine administration increased CCR5 and decreased CXCR4 expression on CD4+ and CD8+ lymphocytes , which is in line with our results. Chemokines, as the natural ligands of CCR5 and CXCR4, may also affect HIV infection. Previous studies have shown an inverse relationship between circulating chemokine concentrations and susceptibility to HIV . We found that cells from HIV-infected opioid users showed lower chemokine production after ex-vivo stimulation with various pathogens. Interestingly, we also observed increased CCR5 expression and reduced chemokine production in individuals who had stopped using opioids. Possibly, opioids induce long-term immunomodulating effects through epigenetic changes .
No HIV-RNA measurements were conducted for this study, but in a previous study in the same setting, plasma HIV-RNA concentrations were similar between patients with and without a history of IDU [9,18]. In addition, we do not have any information on the prevalence of CCR5Δ32 polymorphism in our study population. Exposure to other infections could possibly affect the expression of CCR5 and its ligands. In Indonesia, drug use is often associated with higher education and socio-demographic background, and therefore exposure to infections, such as tuberculosis, is unlikely to be very different from people who do not use drugs [9,18].
Methadone is used as an opioid substitution to reduce drug injecting and HIV transmission. However, our data suggest that methadone could also affect susceptibility to, and progression of, HIV infection. If true, this would have important implications for methadone use in HIV-infected individuals. Our results could have larger implications than just among HIV-infected individuals, as opioids are also used as painkillers in healthcare settings, and modulation of chemokines may be important for numerous diseases [21–23].
We previously showed that that a history of IDU on average was associated with a 33% CD4+ cell decline after 1 year without ART, versus 22% among non-users . The results of the current study fit those findings and provide a possible mechanism.
We would like to thank Dr Bayu Wahyudi, Director of Hasan Sadikin General Hospital, and Professor Tri Hanggono Achmad, Dean of the Medical Faculty, Universitas Padjadjaran, for encouraging and accommodating research at their institutions. Everyone working at the HIV and methadone clinic is thanked for providing HIV care and collecting the data used for this study. In particular, we would like to thank Yusandi Sastra Atmaja, Nuni Haeruni, Dwi Febni Ratnaningsih and Diah Wulandari for their support during patient inclusion. Great appreciation to all Rumah Cemara staff, especially Dehan Mulyana and Hendra Ferdian, for helping us with this project, but also for showing us the work they do and all the care they provide. Also, we would like to thank Arif Rusman for taking us to all over Bandung to include participants.
Author contributions: All authors have significantly contributed to this study. Rv.C., Cd.J., Avd.V., R.W., A.I. and H.M. were responsible for the conception and design of this study. H.M., A.I., S.S., F.U., R.W., and B.A. collected and assembled the data in Indonesia. H.M. undertook the primary data analysis in collaboration with A.I., and under supervision of R.W. and Avd.V. All authors had an opportunity to contribute to the interpretation of the results. H.M., A.I. and Avd.V. wrote the first draft of the manuscript; all other authors contributed to further drafts, and all authors approved the final manuscript.
This work was supported by the European Commission (SANTE/2005/105–033).
Conflicts of interest
The authors have no conflicting interests to declare. Hinta Meijerink and Rudi Wisaksana had fellowships from Radboud University Medical Centre in Nijmegen; Bachti Alisjahbana had a post-doctoral fellowship from the Royal Dutch Academy of Arts and Sciences (KNAW); Reinout van Crevel has a VIDI fellowship from the Netherlands Organisation for Scientific Research (NWO).
1. Platt EJ, Wehrly K, Kuhmann SE, Chesebro B, Kabat D. Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1
. J Virol
2. Reynes J, Portales P, Segondy M, Baillat V, Andre P, Reant B, et al. CD4+ T cell surface CCR5 density as a determining factor of virus load in persons infected with human immunodeficiency virus type 1
. J Infect Dis
3. de Roda Husman AM, Blaak H, Brouwer M, Schuitemaker H. CC chemokine receptor 5 cell-surface expression in relation to CC chemokine receptor 5 genotype and the clinical course of HIV-1 infection
. J Immunol
4. Blaak H, Ran LJ, Rientsma R, Schuitemaker H. Susceptibility of in vitro stimulated PBMC to infection with NSI HIV-1 is associated with levels of CCR5 expression and beta-chemokine production
5. Cocchi F, DeVico AL, Garzino-Demo A, Arya SK, Gallo RC, Lusso P. Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells
6. Arenzana-Seisdedos F, Parmentier M. Genetics of resistance to HIV infection: role of co-receptors and co-receptor ligands
. Semin Immunol
7. Nath A, Hauser KF, Wojna V, Booze RM, Maragos W, Prendergast M, et al. Molecular basis for interactions of HIV and drugs of abuse
. J Acquir Immune Defic Syndr
2002; 31 (Suppl 2):S62–S69.
8. Roy S, Ninkovic J, Banerjee S, Charboneau RG, Das S, Dutta R, et al. Opioid drug abuse and modulation of immune function: consequences in the susceptibility to opportunistic infections
. J Neuroimmune Pharmacol
9. Meijerink H, Wisaksana R, Iskandar S, den Heijer M, van der Ven AJ, Alisjahbana B, et al. Injecting drug use is associated with a more rapid CD4 cell decline among treatment naive HIV-positive patients in Indonesia
. J Int AIDS Soc
10. Finley MJ, Happel CM, Kaminsky DE, Rogers TJ. Opioid and nociceptin receptors regulate cytokine and cytokine receptor expression
. Cell Immunol
11. Kapadia F, Vlahov D, Donahoe RM, Friedland G. The role of substance abuse in HIV disease progression: reconciling differences from laboratory and epidemiologic investigations
. Clin Infect Dis
12. Steele AD, Henderson EE, Rogers TJ. Mu-opioid modulation of HIV-1 coreceptor expression and HIV-1 replication
13. Campbell LA, Avdoshina V, Rozzi S, Mocchetti I. CCL5 and cytokine expression in the rat brain: differential modulation by chronic morphine and morphine withdrawal
. Brain Behav Immun
14. Avdoshina V, Biggio F, Palchik G, Campbell LA, Mocchetti I. Morphine induces the release of CCL5 from astrocytes: potential neuroprotective mechanism against the HIV protein gp120
15. Wetzel MA, Steele AD, Eisenstein TK, Adler MW, Henderson EE, Rogers TJ. Mu-opioid induction of monocyte chemoattractant protein-1, RANTES, and IFN-gamma-inducible protein-10 expression in human peripheral blood mononuclear cells
. J Immunol
16. Happel C, Steele AD, Finley MJ, Kutzler MA, Rogers TJ. DAMGO-induced expression of chemokines and chemokine receptors: the role of TGF-beta1
. J Leukoc Biol
17. Mahajan SD, Schwartz SA, Shanahan TC, Chawda RP, Nair MP. Morphine regulates gene expression of alpha- and beta-chemokines and their receptors on astroglial cells via the opioid mu receptor
. J Immunol
18. Wisaksana R, Indrati AK, Fibriani A, Rogayah E, Sudjana P, Djajakusumah TS, et al. Response to first-line antiretroviral treatment among human immunodeficiency virus-infected patients with and without a history of injecting drug use in Indonesia
19. Cornwell WD, Lewis MG, Fan X, Rappaport J, Rogers TJ. Effect of chronic morphine administration on circulating T cell population dynamics in rhesus macaques
. J Neuroimmunol
20. Wang Z, Yan P, Hui T, Zhang J. Epigenetic upregulation of PSD-95 contributes to the rewarding behavior by morphine conditioning
. Eur J Pharmacol
21. Yao L, Herlea-Pana O, Heuser-Baker J, Chen Y, Barlic-Dicen J. Roles of the chemokine system in development of obesity, insulin resistance, and cardiovascular disease
. J Immunol Res
22. Cardona SM, Garcia JA, Cardona AE. The fine balance of chemokines during disease: trafficking, inflammation, and homeostasis
. Methods Mol Biol
23. Stuart MJ, Baune BT. Chemokines and chemokine receptors in mood disorders, schizophrenia, and cognitive impairment: A systematic review of biomarker studies
. Neurosci Biobehav Rev