Neuroimmune mechanisms of opioid use disorder and recovery: Translatability to human studies, and future research directions

Abstract

Opioid use disorder (OUD) is a major current cause of morbidity and mortality. Long-term exposure to short-acting opioids (MOP-r agonists such as heroin or fentanyl) results in complex pathophysiological changes to neuroimmune and neuroinflammatory functions, affected in part by peripheral mechanisms (e.g., cytokines in blood), and by neuroendocrine systems such as the hypothalamic-pituitary-adrenal (HPA) stress axis. There are important findings from preclinical models, but their role in the trajectory and outcomes of OUD in humans is not well understood. The goal of this narrative review is to examine available data on immune and inflammatory functions in persons with OUD, and to identify major areas for future research. Peripheral blood biomarker studies revealed a pro-inflammatory state in persons with OUD in withdrawal or early abstinence, consistent with available postmortem brain studies (which show glial activation) and diffusion tensor imaging studies (indicating white matter disruptions), with gradual abstinence-associated recovery. The mechanistic roles of these neuroimmune and neuroinflammatory changes in the trajectory of OUD (including recovery and medication management) cannot be examined practically with postmortem data. Collection of longitudinal data in larger-scale human cohorts would allow examination of these mechanisms associated with OUD stage and progression. Given the heterogeneity in presentation of OUD, a precision medicine approach integrating multi-omic peripheral biomarkers and comprehensive phenotyping, including neuroimaging, can be beneficial in risk stratification, and individually optimized selection of interventions for individuals who will benefit, and assessments under refractory therapy.

Introduction

Causing high rates of morbidity and mortality, opioid use disorders (OUD) continue to cause escalating public health concerns (Brown et al., 2022, Garcia et al., 2022, Kulkarni et al., 2019, Mattson et al., 2021, Wakeman et al., 2020). Acute mortality due to non-medical use of abused opioids (primarily agonists at μ-opioid receptors, MOP-r) is driven by respiratory depression mediated by brainstem mechanisms (Baldo and Rose, 2022). Preclinical studies and a relatively small number of studies in humans with OUD further implicate immune and inflammatory alterations after MOP-r agonist exposure, detected in the periphery and in the CNS. However, the roles of these immune and inflammatory mechanisms in the trajectory and morbidity of OUD in humans are not well understood. The goal of this review is to examine and conceptualize human data in this field, and to identify major areas for future research.

Organization of the Review:

Initial sections provide background and context for the trajectory and phenomenology of OUD and its interaction with immune and inflammatory functions (with effects in vitro and in vivo). We then consider core issues in translation of results between rodent models and human studies.

The literature supporting a role for the immune system in humans with OUD will be organized in five major sections with accompanying Tables:

1. Functional changes in peripheral blood leukocytes and in blood cytokines

2. mRNA and transcriptomics from peripheral blood leukocytes

3. Postmortem brain studies, including immune and inflammatory changes

4. Effects of pharmacological interventions on immune or inflammatory targets

5. Mechanisms that can interact with immune and inflammatory functions as part of the phenomenology of OUD.

We will conclude the review with a synthesis of the major future areas for human research.

Background and context

Trajectory and phenomenology of opioid use disorders in humans – potential effects on immune and inflammatory processes:

The trajectory of OUD includes initial exposure to mu-opioid receptor (MOP-r) agonists, followed by escalation and heavy use, and withdrawal that occur in cycles. Early exposure to MOP-r agonists can be iatrogenic (for example with prescription opioids such as oxycodone, for the treatment of pain), or happen in the context of illicit drug use. As the disease progresses, many persons with OUD use heroin or synthetic compounds such as fentanyl by the intravenous route (Comer and Cahill, 2019). The standard of care for OUD includes chronic treatment with MOP-r ligands, especially methadone (an orally available MOP-r agonist) or buprenorphine (a MOP-r partial agonist and KOP-r antagonist) (Kreek et al., 2019, Strang et al., 2020). Acute and chronic exposure to MOP-r agonists can have immune and inflammatory effects at different stages in the OUD trajectory.

Persons in the active stage of severe OUD usually self-administer short-acting compounds (e.g., heroin or fentanyl analogs) several times per day, whereas maintenance medications cause a stable level of activation (Kreek, et al., 2019, Strang et al., 2020). Opioid use and withdrawal cause robust hypothalamic-pituitary-adrenal (HPA)-axis activation, resulting in high levels of systemic glucocorticoids (Cami et al., 1992), which can potentially affect a large number of downstream immune and inflammatory processes (Cain and Cidlowski, 2017, McGregor et al., 2016). Therefore, through their chronic drug use in which persons with OUD can be exposed for years, there are long-term disruptions in their HPA-axis leading to downstream changes in immune and inflammatory processes. Different stages in OUD (e.g., active use, withdrawal, medication maintenance with methadone) are thought to be associated with different immune profiles. For example, persons who were actively using heroin exhibited decreased natural killer (NK) cell activity compared to healthy controls, but persons in stable methadone maintenance were indistinguishable from controls (Novick et al., 1989). Other studies in humans also showed that during the early post-withdrawal period (e.g., approximately 15 days), there were elevations in some cytokines (e.g., IL-6 and RANTES), that normalized over more extended post-withdrawal periods (e.g., 1 year) (Re et al., 2022). These studies therefore show that immune profiles change dynamically over the course of active use and recovery and recovery with medication maintenance.

Adding to the direct acute or chronic immune perturbations of abused MOP-r agonists is the broader phenomenology of OUD in humans. Unlike standard designs in laboratory rodents, humans with OUD are exposed to infections [e.g., human immunodeficiency virus (HIV) and hepatitis C virus (HCV)] as a result of risky behaviors including syringe sharing and unsafe sex. In addition, the often years-long course of OUD can include poly-drug and alcohol exposure (Butelman et al., 2021, Hassan and Le Foll, 2019), disruptions in sleep patterns (Mukherjee et al., 2021), poor nutrition , and increases in socioeconomic stressors (Chavez and Rigg, 2020, Forati et al., 2021). Stress exposure, sex differences, psychiatric and medical comorbidities, and sleep disruptions can each have immune and inflammatory consequences, based on in vitro and in vivo studies, as well as in rodent studies (Aubry et al., 2022, Costi et al., 2021, Deonaraine et al., 2020, Langstengel and Yaggi, 2022, Rodas et al., 2021, Wang et al., 2022). Therefore, these interacting mechanisms, as well as sex differences, can potentially contribute to individual differences in immune and inflammatory – mediated functions in humans with OUD, as also shown in some rodent models (Hofford et al., 2019, Lucerne et al., 2021, Parekh et al., 2023).

Effects of MOP-r agonists on immune or inflammatory functions in vitro:

MOP agonists act through their canonical target, MOP-r, to directly affect immune and inflammatory processes in leukocytes (either from humans or from rodent models) (Eisenstein, 2019, Machelska and Celik, 2020, McCarthy et al., 2001, Novick, et al., 1989, Wybran et al., 1979). Some studies suggest that exogenous opioids may also produce immunomodulatory effects by acting through toll-like receptor 4 (TLR4), enriched on myeloid cells (i.e., monocytes and neutrophils), but the impact of this alternative (non MOP-r) mechanism has not been examined extensively in persons with OUD (Grace et al., 2015).

In vitro studies often involve the extraction of a subset of leukocytes (peripheral blood mononuclear cells, PBMCs), followed by stimulation with exogenous compounds such as lipopolysaccharide (LPS, which act primarily as ligands of TLR4) or phytohemagglutinin (PHA). Early studies reported that beta-endorphin (one of the major endogenous MOP-r neuropeptide) affected responsiveness of human PBMC cultures in vitro, including an increase in natural killer cell activity (Kay et al., 1984, Mandler et al., 1986, Sharp et al., 1987). Later studies also showed effects of MOP-r agonists on release of several cytokines from PBMC cultures, including transforming growth factor-β, and TNF-α (Chao et al., 1992, Chao et al., 1993). Other in vitro studies also show that MOP-r receptors and specific cytokine receptors (e.g., CCR5) can form functional heterodimers in the same cells (Huang et al., 2021, Kong et al., 2022, Szabo et al., 2002). This demonstrates complex interactions between MOP-r and cytokine receptors, their cognate ligands and immune cell activity.

Long-term MOP-r agonist-induced changes to immune or inflammatory functions can be mediated by epigenetic changes (e.g., DNA methylation or histone modifications), resulting in changes in mRNA transcription and protein levels. For example, chronic exposure to MOP-r agonists in T-cells in vitro can cause long-term changes in IL-2 expression, mediated in part by histone modifications (Wang et al., 2007). This can be a bidirectional process, as exposure to a cytokine (IL-4) can upregulate expression of mRNA for OPRM1, the gene encoding MOP-r, also in part through histone modifications (Kraus et al., 2010).

Overall, the immune effects of MOP-r studies in vitro are highly dependent on the types of cells studied, and also on multiple methodological conditions, including incubation and challenge parameters (Peterson et al., 1998). This variability underscores the importance of in vivo studies (either in preclinical species or humans) in examining pathophysiological mechanisms that underlie OUD (see text below).

Effects of MOP-r agonists on immune or inflammatory functions in vivo:

Early studies showed that injection of MOP-r agonists to rodents can cause diverse immunosuppressive effects, including decreasing the release of the cytokine interferon following a chemical challenge (Bryant et al., 1987, Geber et al., 1977, Shavit et al., 1986). MOP-r receptors have been detected in human T lymphocytes (Wybran, et al., 1979), supporting potential mediation of these effects by opioid receptors on peripheral immune cells. Importantly, immune effects of MOP-r agonists in vivo can be mediated indirectly through the HPA axis (through corticosteroid release with its own immunosuppressant effects), as shown in rodents (Bryant et al., 1991). This mechanism can be especially relevant during advanced stages of OUD, when cycles of dependence and withdrawal can cause increased and dysregulated adrenal secretion of corticosteroids (see also below). Among the earlier studies, fresh blood collected from persons in methadone maintenance therapy showed decreased production of reactive oxygen species after a challenge with a chemical activator, phorbol ester (Peterson et al., 1989). Other studies in persons in the active stage of OUD or in withdrawal showed other immunosuppressive effects, such as decreases in peripheral blood T-lymphocyte proliferation after stimulation with PHA (Govitrapong et al., 1998).

persons with OUD had immune cell distributions different than controls’, especially showing increases in regulatory T cells (T_regs) with a “fragile” profile (i.e., they have decreased immunosuppressive function) (Zhu et al., 2023). This study also reported that opioid withdrawal was positively correlated with increased migration of regulatory T cells into the brains of opioid-exposed mice (Zhu, et al., 2023).

The immune effects of MOP-r agonists in vivo can be mediated by several interacting mechanisms, including through different types of leukocytes, and also by direct effects on glia, based on studies from human samples, or in rodent models (Cuitavi et al., 2023, Machelska and Celik, 2020). Furthermore, acute opioid withdrawal causes hyperactivation of the HPA-axis (Kreek and Koob, 1998, Ueno et al., 2011). Peripheral immune and inflammatory disruptions can result in increased migration of specific types of leukocytes (especially monocytes and macrophages) into the CNS, in part due to changes in functional properties of the blood-brain barrier, as detected in rodent models (Wohleb et al., 2014). Also, when these macrophages penetrate into the brain parenchyma, they can embody properties of residing microglia, as shown in mice (Wohleb et al., 2013). Such migration of immune cells can further exacerbate immune and inflammatory changes in the CNS.

Aside from direct action on immune cells, MOP-r activation can also affect the functional status of different glial cells, as examined in rodent models (Avey et al., 2018). Some glia including astrocytes have important structural and functional interactions with neurons, therefore MOP-r agonists have been shown to affect glial-neuronal signaling in rodent brain (Kruyer et al., 2022, Kruyer et al., 2020). Microglia, the resident immune cells in the brain, can express MOP-r, based on mouse studies (Maduna et al., 2018). Therefore, in vivo exposure to MOP-r agonists could result in CNS inflammation by acting directly on microglia (Maduna, et al., 2018, Reiss et al., 2020). Also, changes in the expression of several genes in oligodendrocytes (the glia responsible for myelination in the brain) were detected in the nucleus accumbens (NAc) after exposure to MOP-r agonists in rats (Reiner et al., 2022). It is unclear if this effect is caused by MOP-r expressed in oligodendrocytes, or indirectly by changes in cytokines, neuronal or other glial function. Nevertheless, at a morphological level, chronic exposure to the MOP-r agonist oxycodone in rats resulted in several white matter changes, including deformation of axonal tracts (Fan et al., 2018).

Of relevance to potential effects in oligodendrocytes and myelinaton, recent diffusion tensor imaging studies indicate that persons with OUD have white matter impairments, both whole brain and in specific tracts (Gaudreault et al., 2022, King et al., 2022, Zhu et al., 2022), and that recovery and abstinence are associated with normalization and potential remyelination (Wang et al., 2011, Zhu, et al., 2022).

Translational considerations:

Among the difficulties in translational studies of OUD are the greater variability in duration, amounts and patterns of opioid exposure in humans versus designs typically used in preclinical species (primarily rodents) (Ahmed et al., 2000, Butelman, et al., 2021, Zernig et al., 2007). For example, exposure drug patterns in humans with severe OUD can extend for several years and can involve extensive poly-drug exposure (e.g., to cocaine and alcohol) (Butelman et al., 2019, Butelman, et al., 2021). Such patterns, and their trajectory throughout the lifespan, are inherently difficult to model in preclinical species. Several regulatory functions for the effects of OUD may also differ between rodents and humans. For example, promoters of several genes (important for the regulation of transcription of mRNA), can differ functionally in rodents and humans (Dermitzakis and Clark, 2002, Hagai et al., 2018, Rockman et al., 2005). Other regulatory elements, including long noncoding RNA and gene enhancers (which affect gene transcription), can also differ between rodents and humans (Fullard et al., 2019, Gao and Qian, 2019, Sarropoulos et al., 2019). Other relevant mechanisms that can differ between humans and commonly used nocturnal species, such as rats or mice, include the sleep cycle and entrained circadian hypothalamic-pituitary-adrenal hormonal systems (e.g., ACTH and corticosteroids), which affect immune status (Baldo et al., 2013, McKillop and Vyazovskiy, 2020, Yasenkov and Deboer, 2012). Also, potential differences have been reported in microglial function in humans versus rodents (Smith and Dragunow, 2014).

Literature findings in persons with OUD:

Section 1: Peripheral mechanisms in blood, including leukocytes and cytokines

Table 1 summarizes major studies that have examined cytokines and peripheral cellular biomarkers in persons with OUD. Early studies examining immune cell responsivity in persons with active stage OUD showed primarily a decreased response, consistent with peripheral immunosuppression (Brown et al., 1974, Eisenstein, 2019, Govitrapong, et al., 1998, Novick, et al., 1989). More recently, several studies showed that the pro-inflammatory cytokines TNF-alpha (Shi and Pamer, 2011), TGF-alpha and IL-8 were increased in the blood of persons with OUD (Table 1), especially during early abstinence (e.g., within 1 week) (Piepenbrink et al., 2016, Purohit et al., 2022, Salarian et al., 2018). Some, but not all, studies found that such proinflammatory changes were decreased with more prolonged abstinence, and also during stable long-term MAT (Novick, et al., 1989, Re, et al., 2022, Wang et al., 2018, Zhang et al., 2021).

Section 1 (Table 1):

Peripheral mechanisms in blood, including leukocytes and cytokines

In-vivo Sample (Dx, N, age, sex) Stage in OUD	Target	Direction of effect, compared to healthy controls	Other patient characteristics and notes	Citation
Males with OUD (n=48), mean age=32 Within 24 h of last opioid exposure	Serum cytokine panel	↑TNF-α, ↑IL-10 ↓IL-6		(Purohit, et al., 2022)
Heroin users (n=81), mean age=35, males Abstinence periods ranged: 7 days – 12 mo	Plasma cytokine panel (27 targets)	↑IL-17A, ↑IL-6 ↑IL-10, ↑IL-1ra, ↑IL-4, ↑RANTES High at early abstinence, then decrease over time ↑IL-7, ↑IL-17A and other cytokines High at early abstinence and remained high		(Re, et al., 2022)
Males with OUD in acute (7–14 days; n=10) or protracted (1 year; n=21) abstinence, mean age=37	Plasma cytokine panel	↑IL-4, ↑G-CSF, ↑IL-7, ↑IL-10, ↑IL-17A, IL-2 In acute abstinence	Some poly-drug use (esp. methamphetamine) Several of the cytokine changes normalized by 1 year abstinence	(Zhang, et al., 2021)
Heroin IVDU (n=19), mean age=28, 68%male Injected heroin within 3 days of study	Plasma and cellular panel	↑TNF-α, ↑TGF-alpha, ↑IL-8	Poly-drug use reported (e.g., cocaine). In syringe exchange program	(Piepenbrink, et al., 2016)
Males with heroin addiction (n=90), age range 18–45, in buprenorphine treatment.	Neutrophil and platelet: lymphocyte ratios	Ratios increased, indicating greater systemic inflammation	Ratios correlated with duration of addiction	(Cicek et al., 2018)
Males with OUD (n=20), mean age 36 In methadone maintenance.	TNF-α levels before and in early methadone maintenance	↑TNF-α at early abstinence, decreases over 2 weeks of methadone.	All OUD patients were also tobacco smokers	(Salarian, et al., 2018)
OUD patients (n=47), mean age 37, 87%males In methadone maintenance.	Plasma cytokine panel and cognitive tests over 12 weeks of MMT	Negative correlation between TNF-α levels and visual index, and IL-6 and verbal memory index	Inflammatory markers and verbal memory tended to recover during MMT.	(Wang, et al., 2018)
Persons with OUD (n=33), mean age= 40, 90% male Blood samples obtained at the onset of methadone maintenance, and up to 12 weeks.	Plasma cytokine panel	↑TNF-α, ↑TIL-8	TNF-α levels remained elevated for at least 12 weeks of MMT.	(Chen et al., 2012)
Males with OUD (n=34), mean age=40. Samples obtained after at least 1 month of methadone maintenance.	Serum cytokine panel	↑IL-1β, ↑IL-6, ↑IL-8		(Chan et al., 2015)
Males with OUD (n=20), age range: 21–40	immune stimulation of blood samples.	↑IFN-γ, ↑IL-10 Production after stimulation	Phytohemagglutinin or lipopolysaccharide stimulation	(Azarang et al., 2007)
Males with OUD (n=19) and in heroin withdrawal (n=17). Mean ages 22–26.	immune stimulation of blood samples.	↓T-cell activation after stimulation in OUD. Decreases in CD4/CD8 ratios observed in early withdrawal.	Phytohemagglutinin stimulation	(Govitrapong, et al., 1998)
Males with OUD (n=11 heroin users, n=11 in methadone maintenance. Mean ages ranged 29–41	measurement of natural killer (NK) cell activity in vitro	↓NK activity in active heroin users	Recovery of NK activity in MMT	(Novick, et al., 1989)
Persons with OUD (n=38), age at least 21. Sex unspecified. Recruited during admission to methadone maintenance.	immune stimulation of blood samples in vitro.	↓T-cell activation after stimulation.		(Brown, et al., 1974)

Dx: diagnosis; MMT: methadone maintenance therapy

Comparison to preclinical studies:

A larger number of studies in rodents examined peripheral responses in leukocytes from sites other than blood (including spleen, thymus and peritoneum) (Pacifici et al., 1993, Peng et al., 2000), and detected an immunosuppressive effect caused by opioid withdrawal (Franchi et al., 2017, Rahim et al., 2004). Since leukocytes from these other sites are not commonly studied in humans with OUD due to ethical and practical reasons, it is unclear if all aspects of immune and inflammatory function are shared by humans with OUD and rodent models. Interestingly, in rodent models, chronic MOP-r agonist exposure increases blood to brain passage of some peripheral cytokines (e.g., IL-2) but not others (TNF-alpha) (Lynch and Banks, 2008). This result points to potentially highly specific patterns of blood-brain-barrier disruption following chronic MOP-r agonist exposure, but it is unknown whether these patterns are shared between humans and rodents.

Section 2: mRNA and transcriptomic changes in peripheral blood leukocytes:

Studies using transcriptomics (e.g., RNAseq) in peripheral leukocytes (Table 2) identified a complex array of genes in immune and cytokine pathways that were upregulated or downregulated in patients with OUD compared to controls (Dai et al., 2022). One study also examined men with OUD at acute (7–14 days) versus prolonged abstinence (approximately 1 year) (Zhang, et al., 2021) and found expression changes in mRNA of several cytokine and chemokine genes. This study also found changes in pathways including leukocyte trans-endothelial migration (possibly indicating blood-brain barrier dysfunction) and chemokine signaling (Zhang, et al., 2021). The recent development of droplet-based single cell RNA sequencing (scRNA-seq) approaches allows for single cell resolution profiling of gene expression in blood. A study (Karagiannis et al., 2020) explored the immune modulatory effect of opioids on PBMCs from 7 opioid-dependent individuals and 7 age/sex matched non-dependent controls. The study demonstrated that chronic opioid exposure either from opioid dependent individuals or after 24h in vitro morphine exposure caused widespread suppression of antiviral genes upon ex vivo endotoxin challenge, and also in interferon-stimulated genes. Mechanisms for this effect could include two major interferon signaling inhibitors (Karagiannis, et al., 2020) Overall, this study indicated that opioid exposure suppresses the antiviral program, predisposing the body’s defense response to potential infection.

Section 2 (Table 2):

mRNA and transcriptomic changes in peripheral blood leukocytes

In-vivo Sample (Dx, N, age, sex) Stage in OUD	Target(s)	Direction of effects, compared to healthy controls	Other patient characteristics and notes	Citation
Males with OUD (n=7), age 40–50 Stage in OUD not clearly delineated.	Total RNA from PBC, analyzed with RNAseq	Differentially expressed genes (DEG, including IL-22) in immune systems, and also several miRNA		(Dai, et al., 2022)
Males with OUD in early (7–14 days; n=10) or protracted (1 year; n=21) abstinence, mean age=37	RNAseq from PBC and also exosomes	Several immune-related pathways were affected in persons with OUD		(Zhang, et al., 2021)
Age/sex matched with/out OUD (n=14), mean age 24–45	Single cell RNA seq from PBMCs, exposed to LPS and control PBMCs exposed to morphine	Suppression of antiviral genes and interferon-stimulated genes (ISGs) in opioid-dependent PBMCs and of antiviral response under endotoxin stimulation except for CD8+ T cells and monocytes.		(Karagiannis, et al., 2020)

DEG: differentially expressed genes; LPS: lipopolysaccharide endotoxin; PBMC: peripheral blood mononuclear cells

Comparison to preclinical studies:

In rats examined after a 5-day morphine exposure protocol, mRNA microarrays in PBMC revealed an increase in expression of some immune genes (e.g., IL-1R) and a decrease in others (e.g., CCL2, also known as monocyte chemoattractant protein 1, MCP-1), during early withdrawal (Desjardins et al., 2008). Citing similar limitations in bulk sequencing from peripheral blood from humans, cell-type specific analyses are important to account for the heterogeneity of gene expression patterns, and remain to be performed in animal models. Altogether these results from both humans with OUD and animal models of OUD highlight the robust changes in gene expression in PBMCs following MOP-r agonist exposure. They also suggest a consequential effect in altering susceptibility to infection in humans. Future investigation is necessary in both humans and animal models to determine the extent and significance of cell specificity in transcriptional dysregulation in OUD.

Section 3: Postmortem brain studies in humans with OUD

Post-mortem studies are of great value because they allow for diverse molecular, tissue and neuroanatomical analyses of CNS samples. Table 3 summarizes brain post-mortem studies in persons with OUD that have focused on neuroimmune and inflammatory mechanisms. One study using semi-quantitative immunohistochemical methods (i.e., using antibodies directed at specific proteins) detected increased levels of TNF-alpha and IL-6 immunoreactivity in neurons and glia in OUD decedents (Neri et al., 2013). This result is consistent with an inflammatory state, as observed in peripheral biomarker studies mentioned above.

Section 3 (Table 3):

Postmortem brain studies in humans with OUD

In-vivo Sample (Dx, N, age, sex)	Target	Effects, compared to controls	Notes	Citation
Post-mortem brain of persons with OUD (n=20), mean age 47, 50% male	RNAseq of DLPFC and NAc	Several neuroinflammatory modules affected, including TNF-α and cytokine signaling.		(Seney, et al., 2021)
Post-mortem males with opioid abuse (n=30), mean age=52.	RNAseq of Ventral midbrain	Several immune-related modules (e.g., cytokine response) affected.		(Saad, et al., 2019)
Post-mortem males with OUD from microarray database (n=22), age range 15–35.	Expression microarray of NAc	Several immune-related gene modules affected in OUD, including IL4 as a hub gene.		(Zhang et al., 2020)
Post mortem brain from persons who died of heroin-induced causes (n=45), 93% male.	Measured expression of cytokine panel in slices of several brain areas.	↑expression in several cytokines, especially: TNF-α, IL6 and IL15, CD68 microglia marker.	Immunohistochemistry based on antibody labeling of cytokines.	(Neri, et al., 2013)
Post-mortem from opioid overdose, (n=45 cases, n=50 controls), 88% male	Single nucleus RNA seq on ventral midbrain	Widespread glial activation associated with expression of genes involved in inflammation, immune response, and downregulated synaptic function		(Wei, et al., 2023)
iPSCs stimulated forebrain organoids with individuals with OUD, (n=3), 100% male	Single nucleus RNA seq of organoids exposed to oxycodone or buprenorphine	Buprenorphine displayed significant influence on transcription regulation in glial cells; oxycodone induced type I interferon signaling via STAT1 in most cell types		(Ho, et al., 2022)

DLPFC: dorsolateral prefrontal cortex; NAc: Nucleus accumbens; iPSCs: induced pluripotent stem cells

Other recent studies using microarray and RNAseq technologies have examined a larger set of targets that may be affected in the post-mortem brains of persons with OUD (Saad et al., 2019, Seney et al., 2021). These studies detected activation of several immune pathways including those related to pro-inflammatory responses and TNF-α signaling, in areas including the prefrontal cortex and nucleus accumbens, involved primarily in executive functions and reward processing, respectively. Of note, some of these studies used bulk brain tissue and then predicted differential cell expression in neurons and glia using cell deconvolution analyses (Saad, et al., 2019, Seney, et al., 2021).

One study that used snRNAseq to examine gene expression from the ventral midbrain tissue samples from individuals who had died of opioid overdoses found evidence of widespread glial cell activation, including microglia and astrocytes (Wei et al., 2023). This glial activation was associated with changes in the expression of genes involved in inflammation, immune response, and synaptic function. In another study, scRNAseq was used to investigate the effects of oxycodone and buprenorphine exposure on brain organoids generated from induced pluripotent stem cells (iPSCs) from individuals with OUD (Ho et al., 2022). The results showed that oxycodone and buprenorphine elicited distinct transcriptional effects, in which oxycodone treatment evoked changes in gene expression involved in inflammation, oxidative stress, and mitochondrial function, whereas buprenorphine led to changes in synaptic function and neuroplasticity.

Comparison to preclinical studies and conclusions:

Mouse and rat models provide a controlled source of brain tissues for examination of immune and inflammatory changes after opioid exposure, including examination after specific times of exposure. Prior reviews of preclinical literature have noted differences in designs and methodology in many rodent studies, and concluded that complex pro- and anti-inflammatory changes may occur in the brain of opioid-exposed subjects (Hofford, et al., 2019). At a cell and protein level, repeated exposure to mu-opioid agonists in rodents can result in neuroinflammatory features, including microgliosis and astrogliosis and an increase in cytokine levels, including TNF-α (Zamani et al., 2023). Also, , mice who underwent 14 days of oxycodone self-administration showed changes in mRNA expression of numerous inflammatory and immune genes in bulk tissue of dorsal or ventral striatum, including CCR5, which is thought to be a co-receptor for MOP-r (Zhang et al., 2017). Another study in rats found that 17 days of i.v. fentanyl-self-administration resulted in changes in several immune and inflammatory genes in the hippocampus, NAc and to some extent in the prefrontal cortex (Ezeomah et al., 2020). Single nucleus RNAseq was was also used to investigate cell-type specific gene expression changes in the NAc of rats who self-administered morphine (Reiner, et al., 2022). Many of the differentially expressed genes are involved in synaptic transmission, ion transport and G-protein coupled receptor signaling and transcription factor activation, suggesting that morphine-induced changes in gene expression may be regulated by transcriptional mechanisms (Reiner, et al., 2022).

Overall, these results from human post-mortem studies and animal models highlight neuro-immune cell specificity in transcriptional responses following chronic MOP-r agonist exposure. However, future investigation is required to determine the extent and significance of neuroimmune cell specificity, interaction between glial immune cells and neurons, and possible brain and spinal cord regional differences in OUD.

Section 4: Effects of pharmacological interventions on immune or inflammatory targets:

Thus far, only a small number of studies have directly examined the effects of pharmacological interventions on neuroimmune and neuroinflammatory targets in persons with OUD (summary in Table 4). The antibiotic minocycline, known to decrease microglial activation, caused a decrease in oxycodone-induced subjective effects in non-dependent users (Mogali et al., 2021), but not in opioid withdrawal signs or craving in persons on medication maintenance therapy (Arout et al., 2019). Ibudilast, an anti-inflammatory agent that decreases microglial cell activation and may act through TLR4 receptors (Grace et al., 2014), caused a decrease in some subjective effects of oxycodone in opioid-detoxified volunteers, and in opioid withdrawal ratings, in opioid-dependent volunteers (Cooper et al., 2016, Metz et al., 2017). The effects of commonly used non-steroidal anti-inflammatory agents on the peripheral immune and cytokine markers in persons with OUD has not been investigated extensively (Li et al., 2021). Furthermore, the effects of major clinically available agents which modulate specific cytokine systems have not been examined extensively in persons with OUD.

Section 4 (Table 4):

Effects of pharmacological interventions on immune or inflammatory targets

In-vivo Sample Dx, N, age, sex	Intervention and target	Effect	Citation
Recreational opioid users (n=12), mean age=36, 83% males. non-dependent	Minocycline (immune modulator; 100 or 200 mg PO)	No effect on respiratory effects of oxycodone. Decreased some positive subjective effects of oxycodone.	(Mogali, et al., 2021)
Persons with OUD (n=20), ages 23–56, 70–80% males Dependent in MMT or buprenorphine treatment	Minocycline (200 mg PO, 15 days)	No effect on withdrawal severity or opioid craving. Improvement in cognitive performance (Go / No go task)	(Arout, et al., 2019)
Males with OUD (n=11), mean age=45 Post-detoxification, non-dependent	Ibudilast (50 mg PO, BID)	Ibudilast decreased liking and wanting of oxycodone, and decreased break-point for one dose of oxycodone (15 mg po).	(Metz, et al., 2017)
Morphine-maintained persons with OUD (n=10 group) dependent	Ibudilast (glial modulator; 20 or 40 mg PO, BID)	No consistent effect of ibudilast on oxycodone-induced subjective profile	(Cooper et al., 2017)
Morphine-maintained persons with OUD (n=31), mean age range 38–40, 80–90% male.	Ibudilast (20 or 40 mg PO, BID)	Overall SOWS and COWS withdrawal scores did not differ. Selected subjective rating of withdrawal decreased.	(Cooper, et al., 2016)
Non-dependent opioid users (n=17; 15 men); mean age=35	Pioglitazone (glial modulator PPAR-gamma agonist; 15 or 45 mg PO, daily for 14–16 days)	No effect on measures of oxycodone abuse liability.	(Jones et al., 2018)

SOWS: Subjective Opioid Withdrawal Scale; COWS: Clinical Opioid Withdrawal Scale

Comparison to preclinical studies and conclusions:

There is a larger number of preclinical studies on the effects of immune and inflammatory modulators on the rewarding effects of MOP-r agonists (see reviews) (Hofford, et al., 2019, Wu and Li, 2020)], showing some inconsistencies with the human literature described above. For example, minocycline decreased opioid withdrawal signs in rodents (Liu et al., 2021). Also, pioglitazone decreased withdrawal and relapse-like operant responding in mice (de Guglielmo et al., 2017). An antagonist for CCR5 (a potential MOP-r co-receptor) caused a decrease in oxycodone-induced place preference and relapse-like operant responding in rats (Iriah et al., 2021), but clinical studies of this target are not currently available.

Section 5: Mechanisms that can interact with immune and inflammatory functions as part of the phenomenology of OUD

Table 5 summarizes studies on major interacting mechanisms that could also affect immune and inflammatory functions in OUD. Firstly, there are known sex differences in immune and inflammatory function that could be due to hormonal or other factors, including genomic and epigenetic factors (Chlamydas et al., 2022, Pujantell and Altfeld, 2022). Of note, many genes in immune and inflammatory pathways are on the human X-chromosome. Therefore, the regulatory responses of these genes to opioid exposure could be, in part, sexually dimorphic. We have recently shown that overdose mortality due to synthetic opioids and heroin is robustly higher in men versus women, even taking into account different levels of misuse (Butelman et al., 2023). It is unknown if neuro-glial or cytokine changes over the course of OUD are involved in this sex difference.

Section 5 (Table 5):

Mechanisms that can interact with immune and inflammatory functions as part of the phenomenology of OUD

Mechanism	Molecular Target(s)	Effect	Source (central vs. peripheral)	Citation
Sex
Diverse immune-related genes and micro-RNA are X-linked in humans	Genes Include: CD40L, CD99, CRFL2, CXCR3, GMCSFR, IL2RG, IL3RA, IL13RA2, IL9R, IL13RA1, IRAK1, TLR7, TLR8	Sex as a complex mediating variable in different neuroimmune aspects of OUD.	Potentially both, depending on mechanism and contextual variables.	(Libert, et al., 2010)
Neurosteroid effects on innate immune function in macrophages	Likely TLR4	Allopreganolone (positive modulator of GABA-receptor) has sex-specific immunomodulatory effects after LPS in vitro exposure	In vitro study	(Balan et al., 2022)
Sex affects cytokine responses to LPS.	Likely TLR4 and other mechanisms (IL6, IL-10, TNF-α)	Female cells respond to LPS with higher IL-10 and lower TNF-α than males.	In vitro study	(Rodas, et al., 2021)
Differential transcriptional effects of X-liked in genes in leukocytes	TLR4 and other mechanisms	Leukocytes from males and females show sex-specific changes in transcription of 54 genes, after LPS.	In vitro LPS stimulation	(Stein et al., 2021)
i.v. LPS and hyperalgesia	TLR4 and other mechanisms	fMRI responses to inflammatory pain in rACC differ between sexes	Central responses	(Karshikoff et al., 2016, Karshikoff et al., 2015)
Poly-drug Use
Smoking status affects cytokine responses to LPS.	Likely TLR4 and other mechanisms (IL6, IL-10, TNF-α)	Smoking status Predicts differential cytokine responsiveness to LPS stimulation	In vitro study	(Rodas, et al., 2021)
Microglial imaging in persons with AUD	Effects of chronic alcohol on microglial function.	Brain TSPO binding lower in persons with AUD, but only if they were stratified by genotype	Brain PET study with TSPO radiotracer (11C-PBR-28)	(Kalk et al., 2017, Kim, et al., 2018)
Stress exposure
Acute social stress in males n=44 (age 21–65).	Acute psychological stress effects on HPA axis and neuroimmune function	Acute social stress causes ↓TNF-α and ↓IL-6 after LPS in vitro stimulation	Potentially central and hypothalamic, based on social stress exposure	(Wirtz, et al., 2007)
PTSD and cytokine levels (n=28, 50% females, mean age 41–42).	Chronic PTSD affects peripheral cytokines	↑levels of several pro-inflammatory cytokines in PTSD vs. controls: esp. TNF-α, IFN-γ, IL-6 and IL-1β	Unclear	(Hoge, et al., 2009)
Sleep and Circadian Rhythm
Simulated night shift work effects on cytokines (n=10, 90% male, mean age 27).	4 days of simulated night shift	Disruption in circadian cycle of cytokines in response to stimulation	Propose several central and peripheral mechanisms	(Cuesta, et al., 2016)
Chronic insomnia and cytokine function (n=11 young adults, 55% male).	4-day sleep study	Disruption of cytokine release in persons with insomnia (↑IL-6 and TNF-α, as well as cortisol in the daytime, compared to controls).	Diverse mechanisms possible (including HPA axis disruption)	(Vgontzas, et al., 2002)

AUD: Alcohol use disorder

HPA: hypothalamo-pituitary adrenal axis

LPS: Lipopolysaccharide (experimental TLR4 agonist stimulus)

rACC: rostral anterior cingulate cortex

TSPO: 18KDa-translocator protein; marker for activated microglia

Persons with OUD often have a history of using other substances (e.g., psychostimulants), alcohol and nicotine, each with its own potential effects on immune function (Ahearn et al., 2021, Alfonso-Loeches et al., 2010, Fox et al., 2012, Redwine et al., 2003, Rodas, et al., 2021). Common patterns of poly-drug use (e.g., opioids and cocaine) may also cause more severe neurotoxicity than opioid use alone (Cunha-Oliveira et al., 2010).

Sleep disturbances, common in persons with OUD (including insomnia during withdrawal)(Sharkey et al., 2011), also affect neuroinflammatory function (Cuesta et al., 2016, Furman et al., 2019, Redwine, et al., 2003, Vgontzas et al., 2002). Stress exposure and post-traumatic stress disorder, common in persons with OUD, can similarly have prominent effects on peripheral neuroimmune biomarkers (Hoge et al., 2009, Pfau et al., 2019, von Känel et al., 2007, Wirtz et al., 2007). Other co-morbid conditions that can interact with immune status in persons with OUD include nutritional deficiencies (Chavez and Rigg, 2020).

Comparison to preclinical studies and conclusions:

Numerous preclinical studies (primarily in mice or rats) also show sex differences in neuroimmune and inflammatory functions and mechanisms (Libert et al., 2010, Pujantell and Altfeld, 2022). However, mice and humans do not have identical sets of inflammatory and immune genes on the X chromosome (Libert, et al., 2010), potentially limiting cross-species examinations. Sleep disruption also results in changes in immune parameters and mRNA expression in rodents (Archer and Oster, 2015, Brager et al., 2013). Neuroimmune effects of poly-drug exposure (e.g., cocaine and other drugs) have also been detected in rodents (Hofford, et al., 2019, Wu and Li, 2020).

Overall conclusions and future research directions in persons with OUD

Most studies of acute abstinence or withdrawal in persons with OUD detect a peripheral pro-inflammatory state, and this is consistent with postmortem brain studies. Furthermore, neuroimaging studies in persons with OUD also show white matter changes in major tracts, which may recover with abstinence. As can be seen from Tables 1 and 2, the majority of subjects with OUD in these studies were men. Therefore comparative studies in men and women remain critically necessary, especially due to potential sex differences in inflammatory and immune regulation (Libert, et al., 2010).

The direct pharmacological manipulation of neuroimmune or neuroinflammatory function has only been examined with a very limited number of targets in persons with OUD (Table 4). Several other pharmacological targets could be selected (e.g., CCR5 and TNF-α-alpha) (Chen et al., 2007, Iriah, et al., 2021, Lee et al., 2021). Such interventional studies could more directly examine the causality of these processes at specific stages of OUD trajectory and recovery. Some immune/inflammatory pharmacological agents that modulated MOP-r agonist-induced effects in rodent models did not do so in humans (de Guglielmo, et al., 2017), suggesting further need for proof-of-mechanism studies in humans. Furthermore, pharmacological intervention studies can benefit from parallel neuroimaging readouts (especially with PET, fMRI and diffusion tensor imaging) to examine the functional status of these molecular targets, signal processing, and brain white matter at specific stages of OUD and recovery (Gaudreault, et al., 2022, Kim et al., 2018, Raval et al., 2022). Such combined pharmacological-neuroimaging studies could eventually guide novel mechanism-based prognostic or therapeutic approaches for OUD.

Major co-morbid conditions including poly-drug exposure, sleep disturbances and stress exposure (and post-traumatic stress disorders) can each have immune and inflammatory sequelae (Daskalakis et al., 2016, Marchese et al., 2022). Therefore, as differences in their status could underlie apparent discrepancies between studies (and inter-individual differences), these major mechanisms should be studied as covariates in persons with OUD. Such co-morbid and interacting factors are difficult to interrogate in preclinical models of OUD.

Peripheral blood biomarker studies will continue to be important in the study of persons with OUD. These studies could take a multi-level approach, including transcriptomics (e.g., RNA seq) and epigenetics of immune and inflammatory genes, neuroendocrine HPA status, as well as cytokine quantification. Longitudinal studies at specific stages in OUD trajectory may also provide critical advantages for the control of individual differences in diverse interacting mechanisms (e.g., stress exposure, co-infections, and sleep disruptions), and to examine the potential process of recovery.

Future studies should evaluate the functional status of the blood-brain-barrier at specific stages of OUD trajectory and recovery, with parallel biomarker and neuroimaging studies. These studies could interrogate the potential causality of blood-brain-barrier disruption on OUD, including the impact of leukocyte infiltration or altered cytokine permeability into the CNS.

Future studies could examine whether variability in OUD trajectory is associated with polymorphisms in immune or inflammatory genes, or genes regulating corticosteroid function (Levran et al., 2014). There is also growing evidence of shared genetic liabilities between OUD with other clinical conditions (Seney, et al., 2021). In addition to infectious diseases, chronic pain, other substance use disorders, and other neuropsychiatric illnesses also have shared genetic liabilities with OUD (Sanchez-Roige et al., 2021). Interestingly, polygenic risk score analyses for prescription opioid misuse have revealed widespread associations with cardiometabolic conditions that do have an inflammatory component (e.g., ischemic heart disease, stroke) (Sanchez-Roige, et al., 2021).

Bulk RNA transcriptomics employs a “whole genome” based approach to study immune mechanisms driving differential gene expression in OUD. This unbiased approach integrates genomics, computational biology, and systems biology to identify overlap in differentially expressed genes and transcription factors from peripheral blood and in post-mortem brains with OUD. However, a limitation of bulk RNA transcriptomic studies is the assumption that transcripts from the tissue are homogeneous (Munsky et al., 2012, Raj and van Oudenaarden, 2008, Stegle et al., 2015).

Overall, there is a major need for “omic” studies of peripheral blood biomarkers, in parallel with neuroimaging studies (including white matter investigations), to characterize neuroinflammatory and neuroimmune mechanisms systematically at different stages over the course of OUD and recovery. This will contribute toward a precision medicine approach to risk stratification for prevention and mechanistically-based therapeutic efforts, focusing on these potentially important molecular targets that have not been exploited to date in the treatment of opioid use disorders.

Figure 1:

Schematic of major postulated immune interactions with CNS in persons with opioid use disorders (OUD). The four main interactive compartments affected in OUD can be summarized as 1) Neuronal populations in different parts of the brain, 2) Central glia that can functionally interact with neurons, 3) Changes in blood-brain barrier structure and function resulting in pathological permeability to peripheral macrophages cells and cytokines, and 4) Changes in peripheral immune cells and their release of soluble mediators, including cytokines.

Figure 2:

Schematic of main vascular compartments, blood-brain barrier (BBB), and their potential changes in OUD. These changes can be caused directly by actions of self-administered mu-opioid receptor (MOP-r) agonists on immune cells and by neuroendocrine changes in the stress axis (e.g., affecting cortisol levels and its diverse downstream targets).

Highlights:

• Opioid use disorder (OUD) is a major current cause of morbidity and mortality

• Neuroimmune and neuroinflammatory mechanisms are changed in OUD

• These mechanisms can include peripheral components (cytokines and leukocytes).

• Glia, neurons and blood-brain barrier can also be affected by inflammation in OUD.

• Longitudinal multiomic biomarker / neuroimaging studies are necessary in OUD.

Immune and inflammatory processes involved in OUD.

Cytokines:

Diverse group of signaling proteins, which can be released by leukocytes into blood, or by glia in the CNS. Cytokines act on specific receptors and can have pleiotropic immune effects. Cytokines include several major families, including interleukins, chemokines, growth factors, interferons, and tumor-necrosis factor (TNF)-related proteins. See summary below.

Interleukins (IL):

A large group of signaling proteins that have pleiotropic effects on immune functions. Specific interleukins can have either pro- or anti-inflammatory effects.

Chemokines:

A large group of proteins involved primarily in the guidance of cell migration (chemotaxis).

Growth Factors:

Growth factors modulate the proliferation of diverse immune cell types (including cells that release other cytokines), as part of the immune response.

Interferons (IFN):

A group of proteins that are involved in intra-cellular and extracellular responses to infection or inflammation.

Tumor Necrosis Factor (TNF):

TNF-related proteins that are released at the site of inflammation of infection, can enhance several adaptive immune responses, and also result in tissue necrosis.

Glia:

Non-neuronal cells in the CNS (4 main types: astrocytes, microglia, oligo-progenitor cells and oligodendrocytes). Glia have complex and dynamic interactions with neurons.

Astrocytes:

glial cells with diverse roles in the brain, including regulation of neuronal maturation and function.

Microglia:

resident immune cells of the brain, also involved in synaptic maintenance and pruning.

Oligodendrocytes:

glial cells which regulate central neuronal myelination, thus affecting signal transmission.

Oligodendrocyte progenitor cells:

precursor cells to oligodendrocytes.

Leukocytes (white blood cells):

immune cells with differing functions. These can include the release of antibodies and also soluble signaling molecules, the cytokines. Some leukocytes can migrate into the CNS during inflammatory conditions.

Peripheral blood mononuclear cells (PBMC):

A subset of leukocytes found in blood, bone marrow and spleen that have a single nucleus. PBMCs include monocytes and macrophages, T cells, B cells and natural killer (NK) cells.

Abbreviations:

BBB

blood-brain barrier

CNS

Central nervous system

DTI

diffusion tensor imaging, measuring brain white matter anatomical connectivity

Dx

Diagnosis

HPA

hypothalamic – pituitary - adrenal axis, ultimately controlling the release of the main glucocorticoid cortisol in humans. Cortisol can affect the transcription of numerous immune and inflammatory genes

IVDU

intravenous drug user

KOP-r

kappa-opioid receptor

LPS

lipopolysaccharide; bacterial surface molecule often used as an experimental immune stimulator for blood leukocytes, acts through toll-like TLR4 receptors

MAT

medication-assisted therapy

MMT

methadone maintenance therapy

MOP-r

mu-opioid receptor

NK

natural killer cells

OPRM1

gene encoding MOP-r, the main target of abused opioids

OUD

Opioid use disorder

PBMC

Peripheral blood mononuclear cells

PHA

Phytohemagglutinin; plant-derived molecule often used as an in vitro experimental immune stimulator