Tuberculosis (TB) remains a longstanding global health challenge, claiming 1.5 million lives in 2020. Two-thirds of new cases come from the eight highest-burdened nations, with six of them in South and Southeast Asia. (1)population is latently infected with TB. The risk of reactivation of latent Mycobacterium tuberculosis (MTB) is rising due to international travel, migration, immunosuppressive co-morbidities, and medication use, affecting both developed and developing countries. (2) In order to achieve the World Health Organization goal of reducing 90% of new TB cases by 2035, it is vital to better understand the key causes of reactivation of latent TB. Multiple risk factors for TB are clustered in different subpopulations. (3–5) Epidemiological data have shown that long-term opioid users are more susceptible to TB than the rest. (6) As per a comprehensive community-based case-control study, higher TB risk was independently associated with tobacco smoking, drug use (especially injectable drugs OR = 5.67; 95%CI: 2.68, 11.98), homelessness and area-level deprivation. The strongest risk factor among the intermediate social determinants was misuse of class A injectable drugs (e.g., Ecstasy, Cocaine, Crack Cocaine, Heroin), with five times higher TB odds (OR = 5.67; 95%CI = 2·68, 11.98) compared to those who never misused class A drugs (adjusted for age, sex, BCG vaccination status and long stays in high TB area). (7) A multivariate analysis has shown that drug use was associated with smear-positive TB (OR 2.2, CI 311 – 401, p<0.001). (8) Even accounting for genetic, environmental, socio-economic, and culture-related risk factors, raises the possibility of an underlying immunopathological basis leading to immune suppression. (6, 9–11) TB is often the most common opportunistic infection in endemic areas. (11) This highlights the importance of exploring independent pathophysiological mechanisms causing immune impairment in chronic drug use separately in different infections despite the multiple confounding factors causing generalised immune suppression.

The World Drug Report of 2021 has reported that around 275 million people used illicit substances in 2020 globally, highlighting another growing health challenge. Opioids are the most heavily used substance. Over 75% of substance users live in developing countries, where the prevalence of TB is also highest. (12) Long-term use takes different forms, including misuse of prescription opioids, habitual use without dependence, and increased use as a long-term analgesic. (13) A recent review published in the Lancet has highlighted the TB burden among vulnerable groups worldwide with variable prevalence. (14) Table 1 further summarises studies assessing a link between drug use and TB. Except for one study, others point towards a strong link between drug use and TB risk. As multiple confounders co-exist in these vulnerable populations, causality cannot be independently assessed. Epidemiological data assessing TB infection among drug users taking it as an independent variable is a future need. More specific details of the nature of drug use and the immune status will further help fill the missing data gap.

Immunomodulatory properties of opioids vary in the presence of different microbial antigens. Detailed characterisation of immune pathways affected by opioids in the presence of specific microbial pathogens is essential to characterise mechanisms of immunopathogenesis further. (20, 21) Due to the widespread testing of morphine in multiple previous pre-clinical studies (22), this review collates the immunomodulatory properties of morphine, and it suggests a potential role for opioids during chronic use in the immune pathogenesis of TB.

Opioids act on their receptor (OR), a G protein-coupled receptor (GPCR). They are categorised into two distinct groups: classical OR, which includes morphine (mu) MOR, ketocyclazocine (kappa) KOR, and vas deferens (delta) DOR, and non-classical OR, which includes nociceptin Orphanin FQ peptide receptor (NOR). (23, 24) ORs are located in multiple immune cells, including macrophages and lymphocytes, and are widely distributed in the human body. Both endogenous opioid peptides and exogenous synthetic opioids with different molecular properties act on the same receptors, giving rise to variable downstream effects. (21, 25)

Chronic morphine administration is known to cause impairment of both innate and adaptive arms of the immune response. (26) The concept of direct and indirect morphine action was on the immune system first introduced through work in preclinical studies, which indicated that the MOR mediates morphine-induced immunosuppression and that although some functions are amplified in the presence of cortisol or sympathetic activation. (27) The activation of KOR has also been shown to reduce antibody production, inhibit phagocytic cell activity, inhibit T cell development and alter the production of various pro-inflammatory cytokines, chemokines, and the receptors for these mediators. (28) In vitro experiments have shown that the DOR agonist KNT-127 causes immune suppression in rat models with colitis. (29) It has further shown the functional differentiation of OR subtypes located on the immune cells responding differently to endogenous and exogenous opioids. The novel M3OR subtype has been characterised as an opioid peptide-insensitive and opiate alkaloid-selective GPCR that is functionally linked to constitutive nitric oxide synthase activation. Opioid peptides stimulate granulocyte and immunocyte activation and chemotaxis via the activation of a novel leukocyte D2OR subtype. However, opiate alkaloid M3OR agonists inhibit these same cellular activities. (30)

Chemokine receptors (CCR), which mediate chemokine response, belong to the same class of GPCR as OR and possibly share a common evolutionary origin. The evidence suggests that these receptors cross-desensitise each other, whereas morphine that binds to OR can block CCR signalling and vice versa. (31, 32) The mechanisms underlying heterogenous desensitisation could be the formation of receptor heterodimers and protein kinase C-mediated phosphorylation of Serine, Threonine and Tyrosine moieties. Heterogenous desensitisation may be one mechanism of immune suppression by opioids with high doses and long-term exposure. (33, 34) Bivalent molecules can be tested to modify the complex and its cellular effects. Different immune effects mediated by OR in the presence of mycobacterial antigens and morphine are discussed in the subsequent sections. Bivalent molecules that selectively modulate the OR-CCR complex have therapeutic potential, such as VZMC013, which targets the MOR-CCR5 heterodimer to inhibit opioid-exacerbated HIV1 entry into the immune cells. Similar molecules may have applications in managing opioid-induced immune suppression, including the potential TB risk (35).

A focused literature search in Google Scholar, PubMed and Medline was carried out to extract the studies conducted to find the effects of ‘morphine’ on the ‘immune system’. Tables 2 , 3 comprehensively summarise the immune cells and mediators influenced by morphine administration. Morphine suppresses multiple immune cells, including macrophages, which play a crucial role in the immunopathogenesis of TB. The consequences of morphine exposure extend to inhibiting chemotaxis and multiple cellular functions in macrophages, including respiratory burst activity, phagocytosis, and colony formation ( Table 2 ).

Suppression of the critical immune mediators such as tumour necrosis factor (TNF α), interferon (IFN γ), and nitrous oxide (NO) produced by macrophages was also reported in multiple studies ( Table 3 ). Figure 1 illustrates the immune pathways affected during the co-stimulation of macrophages by mycobacterial antigens and morphine. Suppression of the neutral killer (NK) cell by morphine is modulated via direct and centralised mechanisms. Lymphocytes are inhibited by multiple means, including reduced cytotoxicity and altered CD4/CD8 cell ratios.

TLRs are found in various immune cells and play a critical role in recognising molecular patterns of pathogens to trigger the immune system. Multiple TLRs, including TLR2, TLR4, TLR8, and TLR9, interact with various mycobacterial antigens. Morphine interacts with TLR2, TLR4, and TLR9, and co-stimulation by morphine and mycobacterial antigens may lead to complex immune effects downstream (49, 101–105).

Multiple mycobacterial antigens interact with immune cells via TLR-dependent and independent mechanisms. Figure 1 illustrates the co-binding of mycobacterial antigen and opioids with the TLR4 and its adaptor proteins. Rapidly growing, non-pathogenic mycobacteria containing AraLAM in their cell walls activate CD14 cells expressing TLR2 and macrophages. In contrast, slow-growing pathogenic MTB containing ManLAM has shown a relative inability to activate macrophages independent manner, potentially contributing to their virulence ( Figure 2 ). However, other soluble and cell wall-associated mycobacterial antigens distinct from LAM can mediate immune cell activation via TLR. For example, a soluble heat-stable and protease-resistant factor mediates TLR2-dependent activation of immune cells, whereas a heat-sensitive cell-associated mycobacterial factor mediates TLR4-dependent activation of them. (106–112) Interestingly, induction of adaptive T cell response in TB does not require TLR2/4/9. In TLR2/4/9-deficient mice, mycobacterial replication is controlled by TLR-independent mechanisms to induce an adaptive T-cell response (113).

Morphine interacts with TLR2, TLR4, and CD14 cells, causing inhibitory effects. ( Figures 2 , 3 ) These effects of morphine are exerted on different immune cell types interrupting their functions, which are vital for the immune defence against TB. Further, endomorphin-1, the endogenous form of opioids, has been shown to down-regulate TLR expression as a part of the negative feedback control. Consequently, external opioids, when strongly influencing the same pathway, may contribute to impaired and delayed antigen processing. (114) Consequently, the cells’ capacity to interact with mycobacterial antigens and trigger a protective immune response is ultimately reduced.

Figure 3 summarises the cross-talk between OR, TLR, and nucleotide-binding and oligomerisation domain-like receptors (NLR) 2 via multiple cytosolic second messengers during the interaction with morphine and mycobacterial antigens. In the central nervous system, cross-taking between OR and TLR share common cytosolic molecules such as MAPK, β-arrestin-2/TRAF6 complex, and the DNA-binding protein HMGB1. (115) NLR2 on the immune cells interacts directly with mycobacterial antigens and cross-talks with TLR 2/9 to modulate the immune response. (61) Cross-talk between MOR and TLR in cancer models has shown decreased NK cytotoxicity, decreased leucocyte migration, suppression of mast cell recruitment, and the induction of M2 cell polarisation, which may contribute to the immune impairment in TB (116). Further exploration of the role of these compounds within the immune system in modulating cellular function is required.

Co-stimulation of TLRs with antigenic material of a pathogen, together with morphine, has shown entirely different effects than the binding of either alone. Extensive in vitro and in vivo studies have consistently shown that morphine binding to TLR4 triggers a proinflammatory cytokine response downstream. In contrast, its binding to OR (opioid receptors) elicits an anti-inflammatory response. (104, 117) We hypothesise that opioid compounds interact with TLR as other natural compounds, modulating the host immune response, and it needs direct testing of this in pre-clinical models in the presence of tuberculosis antigens. ( Figure 3 ).

Morphine causes an antiinflammatory response in dendritic cells (DC) cells via TLR2 and NLR2 when co-stimulated with S. pneumoniae, in contrast to the proinflammatory response induced by S. pneumoniae alone ( Figure 3 ). (49) Morphine has also been shown to inhibit the TLR9 pathway when co-binding with HIV, promoting its replication in macrophages. (105) Similar variations of the immune effects have been observed with morphine and mycobacterial antigens in preclinical studies. Plasmacytoid DC expresses TLR9 in both humans and mice. (118–120) M. tuberculosis and morphine cotreatment have significantly upregulated TLR9 expression in mice. Its role is more proinflammatory, enhancing the levels of critical cytokines including TNF α, (interleukin) IL1β, and IL6, which contrasts with the antiinflammatory response exerted by TLR2/4 when co-stimulated by the same. (20, 121) This contracting proinflammatory action of TLR9 compared to other TLRs indicates the downstream receptor action heterogeneity, possibly explained by the unique binding of TB antigens with TLR9.

A TB granuloma is a unique pathological entity comprising activated macrophages, monocytes, DC, neutrophils, and T lymphocytes ( Figure 4 ). An established granuloma contains infected activated macrophages and epithelioid cells forming a central necrotic core and activated macrophages and layers of CD4+ and CD8+ T cells defining a dense cellular wall encircling the necrotic core. (122, 123) It is a dynamic structure that controls two processes: the induction of apoptosis of infected macrophages and the recruitment of uninfected macrophages by creating a chemotactic gradient (123).

Granuloma formation is triggered by the mycobacterial virulence factor ESX-1 (124) It triggers matrix metallopeptidase (MMP)9 secretion by the epithelial cell matrix surrounding a granuloma ( Figure 4 ). (125) It potentially induces the chemotaxis of macrophages via (chemokine ligand (CCL)7, a substrate for MMP9 produced by macrophages. (126–129) Both well-coordinated processes of new macrophage recruitment and infected macrophage apoptosis are essential to maintain the immune integrity of TB granulomas. Morphine decreases the levels of MMP9 and increases the tissue inhibitor of MMP1, dysregulating this process ( Figure 4 ) (108).

M. tuberculosis–induced TNF α production appears to be controlled via TLR2. (122) Both TNF α deficiency and excess can lead to granuloma necrosis. (147, 148) The TNF α signalling deficiency in mice produced disorganised tuberculous granulomas. (149, 150) Deficient TNF α signalling increases intra-macrophage mycobacterial load and accelerates the formation of disorganised granulomas, ultimately leading to granuloma necrosis (130, 151). Morphine has been shown to suppress TNFα levels in many studies, which may enhance the progression locally ( Tables 2 , 3 ). It may further affect disorganised secondary granuloma formation in distal organs, leading to disseminated disease.

Morphine upregulates CCR expression while downregulating CCL levels, causing a net deficiency of CCL. ( Figure 2 ) (21) IL6 stimulates macrophage and cytotoxic T-cell differentiation. At the same time, IL10 inhibits proinflammatory cytokines, blocks the generation of ROI and NOI, blocks antigen processing and presentation in different APCs, and diminishes T-cell responses. IL12 is a crucial cytokine in developing and maintaining type 1 cellular response in MTB infection. IL12 binds to its receptor IL12R-β2 and activates the JAK-STAT pathway, inducing IFN γ to differentiate CD4+ T cells into Th1 effectors. Preclinical evidence has shown that IL-12 p40−/− deficient mice could not control bacterial growth, which appeared to be linked to the absence of both innate and acquired sources of IFN-γ. (152) This shows the central role played by IL12 in the defence against TB infection. IL23 induces IL17 production by memory T cells, creating an inflammatory response by Th17 cells, and it generates protective cellular responses. Morphine blocks the synthesis of all these vital mediators and damages the chemical coordination in the immune defence against TB. Moreover, IL12 induces inflammation by suppressing TGF ß and stimulating NK cells, contributing to increased CCL2 and CCL3 levels. (153) However, this effect may be counteracted by overall anti-inflammatory actions caused by morphine and the virulence mechanisms of mycobacterial antigens. ( Table 3 , Figure 4 ).

Chronic morphine administration causes suppression of multiple protective immune pathways vital in the defence against MTB. Multiple cellular receptors in immune cells, including OR, TLR2, and NLR 2, play critical roles in immunosuppression via complex intracellular cross-talk. Various cell types and their mediators involved in granuloma formation are inhibited by morphine via multiple mechanisms. This leads to a state of immunodeficiency that likely contributes to the reactivation, progression, and dissemination of MTB. Further studies are required to characterise potential therapeutic immunomodulatory targets in chronic opioid users at risk of infection with/reactivation of MTB.

In vitro and in vivo preclinical studies assessing the immunomodulatory properties of opioids have been mostly limited to the testing of morphine. Considering the wide structural diversity and functional variation of opioids, direct testing of other categories of opioids and their antagonists is needed to delineate further the postulated mechanisms of the immunopathogenesis of TB potentiated by chronic opioid use. The duration and dosage of morphine use in patients may vary widely, with the added effect of the landscape of genetic heterogeneity across cultures, the impact of this review may be biased towards findings reported from the majority of studies originating from the Western, further studies are desperately needed from third-world nations investigating this phenomenon associating opioid use and the predisposition to TB. Chronic opioid use is associated with confounding factors, socioeconomic deprivation, malnutrition and infections associated with IVDU which may contribute to immune suppression.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

VB: Conceptualization, Data curation, Methodology, Software, Visualization, Writing – original draft. HS: Data curation, Formal Analysis, Writing – review & editing. NR: Supervision, Writing – review & editing. LD: Supervision, Writing – review & editing.