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Official websites use. Share sensitive information only on official, secure websites. Antiretroviral therapy has been effective in suppressing HIV viral load and enabling people living with HIV to experience longer, more conventional lives. However, as people living with HIV are living longer, they are developing aging-related diseases prematurely and are more susceptible to comorbidities that have been linked to chronic inflammation. Coincident with HIV infection and aging, drug abuse has also been independently associated with gut dysbiosis, microbial translocation, and inflammation. Here, we hypothesized that injection drug use would exacerbate HIV-induced immune activation and inflammation, thereby intensifying immune dysfunction. Plasma immune profiles were characterized by immunoproteomics, and cellular immunophenotypes were assessed using mass cytometry. In conclusion, a comprehensive analysis of inflammatory mediators and cell immunophenotypes revealed remarkably similar patterns of immune dysfunction in HIV-infected individuals and in people who inject drugs with and without HIV-1 infection. Injection drug use IDU of opioids and synthetic opioids i. Moreover, chronic diseases such as diabetes, cardiovascular diseases, premature aging, and neurologic diseases seen in PLWH could further be enhanced due to IDU, coinfection, dysbiosis, and chronic inflammation. In this cross-sectional study, we investigated the potential synergistic impacts of chronic HIV infection and IDU on immune functions by comprehensively analyzing the inflammatory cytokines and mediators, as well as the immune cell population profiles between PLWH and PWID. Written informed consent was obtained from all study participants, and all study data were deidentified before analysis and publication. After completion, each participant is given recruitment coupons that they can pass out to people they know who qualify for the study. Those who are recruited through coupons are then given more coupons and recruitment spreads through the local community. Participants were eligible for an incentive for completing the survey and for each recruitment coupon that resulted in a new participant. Overall, most eligible participants who responded to solicitation i. We were unable to estimate the proportion of coupons that were not passed on to potential new participants i. At the study site after obtaining written informed consent and conducting the survey, we performed HIV rapid antibody tests cat. A portion of whole blood was reserved for mass cytometry staining. The Cytodelics whole-blood preservation kit was used to stabilize the stained whole blood for storage and shipping cat. Importantly, our study performed the staining of fresh whole blood prior to stabilization, freezing, and shipping in order to avoid any negative staining effects. Graphical representation of study design and methodology. Whole-blood samples were collected and partitioned into plasma while reserving some whole blood. The plasma was subjected to the Olink proximity extension assay with the Olink Target 96 inflammation panel. The reserved whole blood was heparin blocked and isotope labeled before stabilization, fixation, washing, and quantification on the mass cytometer at OMRF. Created with BioRender. MDIPA contains a standard panel of 30 antibodies that can identify 37 immune cell populations. Adding the T-cell expansion pack 1 allows further characterization of T-cell activation and exhaustion. Cytodelics Stabilizer was added to 2-mL cryovials ratio with whole blood and equilibrated to room temperature for 5 to 10 min. Finally, the stained whole blood was transferred from the MDIPA tube to the cryovials containing the Cytodelics Stabilizer, mixed by inverting 10 to 15 times, and incubated at room temperature for 10 min. The cells were fixed with fresh 1. The normalized FCS files were used for downstream analyses. The percentage of lineage parent was calculated for each cell population and used for downstream analysis i. GraphPad Prism v9. The well plate was sealed, frozen, and shipped to Olink on dry ice. HIV-1 plasma viral load was quantified as described previously. Statistical analysis was performed in GraphPad Prism v9. These analyses were performed in R, and results are provided in tabular format in the supplemental materials Supplementary Table 3. Finally, multivariable linear regression with main effects for HIV status, IDU status, and their interaction was performed with adjustment for the potential confounding variables discussed above. Due to the use of all observations in the study for these comparisons, the assumption of residual normality is more likely to hold due to the central limit theorem, whereas this was not the case in unadjusted pairwise comparisons. We did not correct the P values here for studying multiple outcomes since this analysis was exploratory in nature, which may limit the power to detect observed differences in outcomes as was done in previous studies. Continuous variables are shown as median with interquartile range and compared using Mann-Whitney tests. The P values are italicized and significant P values are bolded. The self-reported non-injection drug use NIDU characteristics, treatment program history, and the results of urine toxicology tests performed at the time of blood sample collection were also compared between the groups Table 2. Injectors were more likely to have engaged in NIDU in the past 12 mo and to have participated in drug treatment programs than non-injectors Table 2. Finally, the self-reported use of multiple non-injection and injection drugs was analyzed for common combinations of drugs and visualized using heatmaps of the row percentages Supplementary Fig. For example, 1 of 38 speedball injectors 2. The most frequently reported non-injection drug was alcohol Supplementary Fig. Buprenorphine and alcohol were the most frequently co-occurring substances; of the 22 individuals who reported using buprenorphine through non-injection routes, Drug abuse has been shown to affect the levels of isolated cytokines in various studies, 16 — 18 , 21 , 22 but no study in humans has comprehensively investigated the extent to which IDU affects peripheral immune soluble inflammatory mediators. To evaluate differentials in plasma inflammatory profiles between injectors and non-injectors, we employed an established Olink proximity extension assay Target 96 Inflammation Panel; Olink Bioscience AB Fig. Finally, outlier samples were not detected. Exploratory PCA of inflammatory mediators and cellular immunophenotypes. In each case, the input data were standardized and centered. Studies to define the impact of IDU on the immune cell populations have focused on limited cell lineages. To develop a more comprehensive appreciation of how IDU alters peripheral immune cell phenotypes, we employed a mass cytometry panel of 36 isotopically defined mass markers to detect 47 immune cell populations, including granulocytes, in a single assay MDIPA; Fluidigm Fig. Surprisingly, this group included the exhausted T-cell subsets. This HIV association was not observed in the remaining excluded populations. We calculated the percentage of lineage parent i. Then, we applied PCA, and outliers were not detected Fig. Data sets for both inflammatory mediators and cellular immunophenotypes were merged for 59 participants, and PCA was applied Fig. Since the merged data set provided the best group segregation, we projected the PCA results in 3 dimensions Supplementary Fig. High variation between samples within groups and lack of tight clustering highlight the heterogeneity intrinsic to hard-to-reach human populations in a non-clinical setting. To ensure that potentially confounding demographic differences between groups Table 1 were not immunologically confounding factors, adjustment for age, homelessness, gender, marital status, employment status, and education was performed as described in the Materials and Methods and applied to all downstream analyses. Nevertheless, pairwise comparisons are needed to elucidate finer resolution differences between the groups and identify any potential additivity or synergy between HIV infection and IDU. In this confounder-adjusted analysis, 34 of 74 detectable inflammatory mediators were significant in at least 1 comparison. C GO enrichment analysis was performed for the inflammatory mediators with significant interaction effects in B, and the most specific subclass of the hierarchical ontology cluster is shown. To investigate the functional roles of these cytokines, we performed GO enrichment analysis Fig. While these GO enrichments and interaction effects are significant, they have small coefficient estimates i. We would therefore caution against overinterpretation. Because it was anticipated that different types of drugs abused and the frequency of use could differentially affect inflammatory profiles, we also investigated the associations of inflammatory mediators with drug use parameters collected during the study surveys Table 2 , Table 3. Nine inflammatory mediators correlated with the extent of polysubstance abuse evident in the urine test Supplementary Fig. These results indicate that the number of drugs abused can additionally influence plasma inflammatory mediator profiles. EM, effector memory; TE, terminal effector. Surprisingly, significant differences in the neutrophil and monocyte populations were not detected, contrary to what was expected since injectors would likely have increased exposure to pathogens. Thus, we employed an unbiased clustering algorithm of our live, singlet population. No global cell population differences were detected between our cohorts using this strategy Supplementary Fig. Further, no cell populations had a statistically significant correlation with the number of positive drugs in urine tests Supplementary Fig. Like the soluble mediators, there were significant interaction effects, but the coefficient estimates i. Thus, the biological significance of these subtle synergies is unclear. Overall, these data suggest that HIV has a larger effect on the peripheral immune cell populations than IDU but only a marginal synergistic effect in the context of IDU. Although previous studies have analyzed aspects of the effects of HIV and IDU on the immune system, this study, to our knowledge, provides the most comprehensive peripheral immune profiling data set available on PWID. Furthermore, this study focused on a unique population of Puerto Rican PWID and PLWH for whom the potential impacts of both insults on immune dysfunction have not been previously analyzed. Despite their significance, these interaction effects were accompanied by weak coefficient estimates, reinforcing the subtlety of any potential synergy. Moreover, the considerable differences in inflammatory mediators between PLWH and PWID vs controls did not translate into notable changes in cell phenotypes, and only weak to moderate correlations were noted between cell phenotypes and their associated inflammatory mediators Supplementary Fig. This is further reinforced by the lack of concordance between inflammatory mediators and cell populations with significant HIV, IDU, or interaction effects Fig. The inflammatory mediators may be originating from tissue-resident cells i. The effects of HIV infection and injection drug use on the peripheral immune repertoire. The upregulated cytokines are shown in the cloud Venn diagram for HIV infection, injection drug use, and those that are in common. The upregulated cytokines and dysregulated cell populations with significant interaction effects synergy between HIV infection and injection drug use are shown. The smaller text size indicates a less significant effect, while the larger text size indicates a more significant effect. Albeit through potentially different mechanisms, both drug abuse and HIV infection have been associated with gut dysbiosis and microbial translocation. Such translocation could explain the similarity in peripheral immune profiles. Microbial translocation can result from gut dysbiosis, weakening of the mucosal barrier, or an immune deficiency 35 and has been implicated in systemic inflammation. To our knowledge, this study is the first to apply Olink technology and mass cytometry to the study of immune dysfunction in PWID. The results are concordant with published studies using other assays in the context of drug abuse. Importantly, this did not translate into any functional innate or adaptive differences, 32 suggesting that although we detect differential cell population numbers, these are unlikely to translate into functional changes in the immune response. Further, in the study of abstinent cocaine users seeking treatment, Araos et al. Given the anticipation of high HCV prevalence and the aforementioned rates of self-reported HCV infection, we were unable to control for HCV infection status in our study and therefore did not seek to quantify HCV viral load. This supports the notion that our PWID have already been infected by HCV, which was likely not a confounding factor in the cytokine dysregulation detected in our study. This cross-sectional study has described the most comprehensive and contemporary data set on comparative immune profiling of PWID with and without HIV infection. While cause and effect cannot be established, this study has generated preliminary results using novel contemporary approaches upon which future studies can build. Finally, other recent studies have reported minimal and complex synergistic effects between HIV infection and heroin use. However, the disproportionality is representative of the natural populations. Additionally, it is important to note that the demographics of San Juan are not necessarily representative of the rest of Puerto Rico or the continental United States. It would be important for future studies to obtain a large sample from diverse regions and poverty levels, allowing the study to be more generalizable and evaluate the subtle differences between groups that may prove biologically or therapeutically relevant. There was also a higher proportion of males in our injectors group than non-injectors. Again, this is representative of the natural populations 6 and was accounted for in the confounder-adjusted analyses. However, when we removed the non-injectors with evidence of NIDU, our results remained largely the same. While many inflammatory mediators were significantly different between the mono- and dual-affected individuals and controls, there were very few differences between the mono- and dual-affected individuals themselves. We also identified subtle synergistic effects between HIV infection and IDU, but these effects may not be biologically relevant and did not translate into differences in peripheral immune cell phenotypes. Future studies with larger sample sizes and a focus on single drug-using populations would better elucidate how each drug of abuse affects the peripheral immune profile, and analysis of the gut microbiome, microbial translocation, and functional analysis of immune cells would paint a complete picture of how IDU and HIV infection, in combination, affect the immune system. Supplementary materials are available at Journal of Leukocyte Biology online. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. J Leukoc Biol. Published in final edited form as: J Leukoc Biol. Find articles by Sydney J Bennett. Find articles by Carmen Ana Davila. Find articles by Zahiraliz Reyes. Find articles by Kim Gocchi Carrasco. Find articles by Roberto Abadie. Find articles by M Caleb Marlin. Find articles by Marci Beel. Find articles by Andrew G Chapple. Find articles by Samodha Fernando. Find articles by Joel M Guthridge. Find articles by Kathy S Chiou. Find articles by Kirk Dombrowski. Find articles by John T West. Find articles by Charles Wood. Author contributions J. PMC Copyright notice. The publisher's version of this article is available at J Leukoc Biol. Open in a new tab. Conflict of interest statement. None declared. Supplementary material Supplementary materials are available at Journal of Leukocyte Biology online. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. 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Immune profiling in Puerto Rican injection drug users with and without HIV-1 infection

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The Illegal drug trade in Puerto Rico is a problem from a criminal, social, and medical perspective. Located in the Caribbean , Puerto Rico has become a major transshipment point for drugs into the United States. Crimes related to drugs are not the only crimes plaguing the island. Along with gang violence the island has also been victim to Police and political corruption. The Government of Puerto Rico has struggled to combat illegal drug use and the resulting crime since the mids. In the s the increase in drug use, particularly among those under the age of 25, became a major concern in Puerto Rico. Many Puerto Ricans have attributed the increase in crime to the drug trade. This led to a major focus on crime and drugs in Puerto Rican politics. Strategies used by the government of Puerto Rico include long sentences for criminals, increased funding for law enforcement equipment, and the construction of new prisons. In the early s, law enforcement began targeting white collar drug users. The start of the 'Drug Wars' in Puerto Rico was in in a conflict between Police and drug dealers which police wounded and killed two men. This occurred in Naranjito, Puerto Rico, the place where the drug dealers distributed their drugs to many places in Puerto Rico. In the house they found AK, M9 pistols and the drugs. Also they found that they were working with corrupt politicians to approve marijuana legalization and the export of exotic animals. In police captured 16 drug dealer 'clans' that had the same information and they worked together to defeat opposing dealers. They also started to 'clean house' in , when they killed about 44 men attached to some drug dealers or those that were compromising the dealer. In the death toll was 1, at one point, due to many big drug arrests different gangs went into war with each other for the control of drugs in the island. In it was discovered that many of the killings were to cover up some corrupt politicians. In , around 1, murders were reported. Also in Bayamon, a major city of Puerto Rico, many drug cartels started to defend their dealers by getting snipers and attackers on rooftops. Police captured a sniper in late , the sniper had a Dragunov Caliber Sniper Rifle and was arrested with 5 other drug dealers. In early , two assassinations occurred in Humacao involving the Carteli. As early as , Puerto Rico had already become a transshipment point for illegal drugs that are smuggled from source countries like Colombia , Venezuela , and Peru into the U. Most of it is transported to and through the island from drug trafficking organizations in the Dominican Republic and Colombia, and criminal organizations in Puerto Rico. Go-fast boats are the most favorable, as they are fast and stealthy, and have been used to intercept drug shipments that have been dropped off into the open water from other larger ships or airdropped from aircraft. In June , 36 people were arrested in Puerto Rico and the U. The smuggling had been taking place since In , a group of researchers from Puerto Rico's Mental Health and Anti Addiction Services Administration, published the results of a study involving out-of-treatment drug users in the San Juan area. Their study found that In , U. Customs and Border Protection in Puerto Rico seized 56 kilograms of heroin from commercial aircraft at airports and 28 kilograms of heroin from commercial maritime vessels in Puerto Rico. That same year, government officials arrested seven individuals in Puerto Rico, for swallowing between 36 and 98 condoms full of heroin, when they arrived from Aruba on a cruise ship. In June , drug detecting dogs detected and CBP seized 24 kilograms of heroin in a cargo storage area on a pier in San Juan. That same year federal law enforcement officials in San Juan seized 1. Addicts 'shooting up' with dirty needles has created a public health emergency in Puerto Rico with an HIV positive rate that is 4 times higher than the U. In , a resident of San Juan was arrested at the airport, when government officials found In , four police officers in Puerto Rico were arrested by the Federal Bureau of Investigation FBI , including a Lieutenant with 33 years on the force, for extortion and distribution of cocaine and heroin. In , one of the biggest police corruption busts in U. The yearlong undercover operation was initiated by the FBI, after authorities got a tip about the police possibly being involved in drug dealing, and protecting cocaine dealers and shipments and movement throughout the island. On hand were a range of Bureau personnel—crisis negotiators, evidence response team members, canines and their handlers, and 80 medical personnel from first responders and nurses to a trauma surgeon and a veterinarian. The central thread of the corruption was law enforcement officers providing protection and other services to drug traffickers. Over 1, agents of the FBI conducted the raids. Many of them were flown in secretly. The agency characterized the action as, 'likely the largest police corruption case in the FBI's history. It is cheap and easy to buy and deal to the public in housing projects in Puerto Rico, leading to the second highest homicide rates in the United States or its territories. They are in the illegal drug trade. The Puerto Rican government has implemented a series of law enforcement operations in relation to the federal ' war on drugs ' in order to minimize drug-related crimes and trafficking on the island. The operation uncovered money laundering schemes from within financial institutions and from the sale of illegal lottery tickets. Federal agents raided 10 banks and arrested 17 people on money laundering charges. They tried to stop the circulation of the illegal money and mobilized to arrest the individuals connected to the money. Contents move to sidebar hide. Article Talk. Read Edit View history. Tools Tools. Download as PDF Printable version. In other projects. Puerto Rico is a transshipment point for drugs. This article needs to be updated. Please help update this article to reflect recent events or newly available information. September Chronology \[ edit \]. Drug trafficking \[ edit \]. Cocaine \[ edit \]. Heroin \[ edit \]. Marijuana \[ edit \]. Police corruption \[ edit \]. Gangs \[ edit \]. Crime reduction \[ edit \]. See also \[ edit \]. References \[ edit \]. Archived from the original on 26 December Retrieved 4 November Virgin Islands Drug Threat Assessment. Department of Justice. Archived PDF from the original on 2 May Retrieved 26 August ISBN The New York Times. Archived from the original on 26 May Retrieved 20 October Internet Archive. Retrieved 18 September Archived from the original on 31 August Retrieved 31 August Associated Press. Archived from the original on 18 September El Nuevo Dia. Archived from the original on 27 August Primera Hora. Archived from the original on 25 August Archived from the original on 11 August Department of Homeland Security in Spanish. Archived from the original on 18 October Federal Bureau of Investigation. Archived from the original on 20 June The Times — News. Retrieved 18 October Retrieved 26 October P R Health Sci J. PMID Retrieved 9 April Primera Hora in Spanish. General OneFile. Archived from the original on 7 October Retrieved 28 July Retrieved 20 January Archived from the original on 11 July Retrieved on 20 March InSight Crime 13 March External links \[ edit \]. Puerto Rico articles. Endemic flora. Amphibians Birds Vieques Mammals Reptiles. Outline Index. Crime in the United States by state. Washington, D. Virgin Islands. Illegal drug trade in the Americas. 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