Morphine exerts a potent analgesic effect primarily by interacting with mu opioid receptors (MOR). Its widespread utilization in pain management for conditions such as cancer is attributed to its rapid onset and prompt relief (Badshah et al., 2023). However, the chronic administration of morphine inevitably leads to various undesirable effects such as morphine tolerance (MT) and dependence, respiratory depression, nausea, vomiting, and sedation, which pose challenges to its clinical application (Ouyang et al., 2022). The development of morphine tolerance is a complex and multifaceted process, encompassing various factors such as the desensitization and internalization of opioid receptors (Narita et al., 2006), the formation of heterodimers between G protein-coupled receptors (Szentirmay et al., 2013), activation of glial cells, modifications in glutamate receptor function (Wang et al., 2021), stimulation of the cyclic adenosine monophosphate (cAMP) pathway (Coffey et al., 2022) and mitogen-activated protein kinase (MAPK) pathway (Fu and Zhang, 2023), among others. Considering the inadequate therapeutic outcomes and significant burden associated with morphine tolerance, it is imperative to identify potential underlying risk factors that contribute to its occurrence.

The human gut, which contains approximately 13 trillion bacterial cells, plays a crucial role in regulating various bodily functions such as vitamin production, colonization resistance, and immunological balance (Su et al., 2023). Recent research has suggested that the composition of gut microbiome may also be linked to conditions involving pain such as neuropathic pain, visceral pain and headaches (Minerbi and Shen, 2022). Studies have shown that morphine can disrupt the natural balance of bacteria in the gut leading to dysbiosis while also increasing intestinal permeability thereby facilitating bacterial translocation (Muchhala et al., 2023). The presence of microbial dysbiosis, specifically a reduction in Bifidobacteria and Lactobacillaeae populations, was observed in individuals undergoing MT. The administration of probiotics containing these bacterial communities demonstrated the ability to mitigate the development of analgesic tolerance in mice treated with morphine (Zhang et al., 2019). Additionally, recent studies have highlighted the significance of inflammatory factors released by immune cells in relation to MT (Eidson and Murphy, 2019). There is a growing body of research indicating that morphine has the potential to induce neuroinflammation (Wan et al., 2022). This process involves the activation of microglia (Mélik Parsadaniantz et al., 2015) and an increase in the production of proinflammatory cytokines, including IL-1β, TNF-α, CXCL12, IL-33, and IL-6 (Eidson et al., 2017; Bai et al., 2022), leading to heightened synaptic transmission and central sensitization (He et al., 2019). However, further investigation is needed to establish a definitive link between gut microbiota, immune cells, and the risk of MT due to possible biases.

Mendelian randomization (MR) is a highly effective approach to infer causality, employing genetic variants as instrumental variables (IVs) to investigate the causal impact of exposure on outcome (Wang et al., 2023). In this study, gut microbiota and immune cells were chosen as the exposure variables, while MT was considered as the outcome variable for MR analysis. The aim was to explore potential causal relationships and establish a theoretical foundation for further investigations into the intricate mechanisms and risk factors associated with MT.

To our understanding, this MR analysis report appears to be the initial study establishing a potential causal link between gut microbiota/immune cells and MT. Through a two-sample MR investigation, we have discovered that there is evidence of causal correlation between 6 specific gut microbial taxa and 16 immune cells with regards to MT (Figure 4). Nevertheless, when BMI was included in the MVMR analyses, the impact of these factors was diminished.

Previous studies have extensively discussed the potential mechanisms by which the gut microbiota contributes to the development of MT. Disruption of the gut barrier and translocation of bacteria are observed as a result of morphine-induced dysbiosis, leading to local inflammation in the gut and subsequent activation of proinflammatory cytokines, ultimately driving MT. Both animal experiments and human studies have provided evidence for morphine’s detrimental effects on intestinal microbial composition, which further contribute to gastrointestinal disorders experienced by patients (Wang et al., 2018). The introduction of subcutaneous morphine granules or intraperitoneal morphine injections in mice resulted in an increase harmful bacteria, such as Flavobacterium, Enterococcus, Fusobacterium, Sutterella, Clostridium, Rikenellaceae and Ruminococcus, observed in fecal samples (Lee et al., 2018). Similarly, prolonged administration of morphine to mice led to alterations in the bacterial composition found in fecal samples. This included a decrease in Bacteroidetes and Firmicutes populations alongside an increase in Proteobacteria (Kang et al., 2017). Furthermore, there were reported elevations specifically within the phylum Firmicutes (Banerjee et al., 2016). Moreover, mice that developed tolerance to morphine exhibited a significant reduction in beneficial bacteria from the Lactobacillus and Bifidobacterium. However, this tolerance was mitigated following treatment with probiotics (Zhang et al., 2019). These studies indicate that chronic administration of morphine can lead to imbalances in the composition of gut bacteria. The close association between reductions in bacteria such as Lactobacillus or Bifidobacterium at the intestinal level and the development of dysbiosis emphasizes their crucial role in maintaining the integrity of the intestinal epithelium (Guo et al., 2017). Limited clinical research has been conducted to explore the connection between dysbiosis and the opioid system. A study involving cirrhotic patients who received long-term opioid treatment revealed a reduction in Bacteroidaceae relative abundance among those diagnosed with hepatic encephalopathy (HE). This decrease coincided with lower levels of aromatic amino acids and metabolites from specific bacterial strains while also showing elevated endotoxin and IL-6 levels (Acharya et al., 2017). Our findings align with prior research on the beneficial effects exerted by Bacteroidaceae. Furthermore, our investigation suggests that an increased presence of Flavonifractor, Prevotella9, RuminococcaceaeUCG005, Ruminococcus1 is linked to an augmented risk for MT.

The expression levels of opioid receptors have been found to be associated with the composition of the gut microbiota. For instance, there is a positive correlation between the abundance of Bacteroides spp. in fecal matter and MOR mRNA in colonic tissue among rats (Aguilera et al., 2013). The chronic administration of morphine induces changes in intestinal epithelial function, resulting in increased permeability and bacterial translocation mediated by Toll-like receptor (TLR) (Meng et al., 2013). However, these effects were mitigated when mice were treated with a combination of antibiotics. In addition, the impact of opioids on the intestinal microbiota could worsen the effects of opioids themselves by causing inflammation. In a mouse model of sepsis, it was observed that blocking IL-17A after morphine treatment, which increases levels of Gram-positive bacteria, led to improved inflammation and function of the intestinal barrier (Meng et al., 2015). Administering a probiotic like Lactobacillus acidophilus in rats with irritable bowel syndrome (IBS) increased their pain threshold and alleviated hypersensitivity. This was achieved by activating the inflammatory NF-кB pathway to enhance MOR expression (Rousseaux et al., 2007).

Fecal microbiota transplantation (FMT) is currently being investigated as a potential approach to alleviate morphine tolerance in rodents with opioid-induced dysbiosis (Muchhala et al., 2023). Prolonged exposure to morphine reduces the presence of Regenerating islet-derived 3 gamma (Reg3γ), an antimicrobial peptide, in the ileum. This leads to decreased intestinal antimicrobial activity against Gram-positive bacteria like L. reuteri. However, when FMT was performed using fecal samples from mice not exposed to morphine, it restored the antimicrobial activity and expression of Reg3γ in the intestine. Consequently, this prevented an increase in intestinal permeability and the development of antinociceptive tolerance in mice dependent on morphine. In vitro strains, such as Staphyloccocus aureus, S. epidermidis, Streptoccocus pneumoniae, S. pyogenes, S. faecalis, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa showed no growth inhibition upon exposure to a 2 mg/mL concentration of morphine (Rosenberg and Renkonen, 1985). In contrast, the growth of pathogenic bacteria in culture, such as E. coli and S. epidermidis, could be completely inhibited by tramadol at a concentration of 25 mg/mL (Farzam et al., 2018). In accordance with the aforementioned research, our study indicated a causal link between a decreased risk of MT and the heightened prevalence of Lachnospiraceae and RuminococcaceaeUCG011.

On the contrary, MT is also influenced by immune cells in its occurrence and development (Zhang et al., 2022). Extensive research has been dedicated to understanding the molecular mechanism behind morphine tolerance in recent years. A growing body of evidence supports the notion that morphine can initiate neuroinflammation, characterized by microglia activation and increased production of pro-inflammatory cytokines like tumor necrosis factor-α (TNF-α), Interleukin (IL)-6, and IL-1β. This subsequently exacerbates neuroinflammation, ultimately leading to MT (Guan et al., 2023).

Our research discovered the risk of morphine tolerance decreased with an increase in the proportion of BAFF-R on B cell, BAFF-R on CD20⁻ CD38⁻, BAFF-R on IgD⁺ CD24⁻, BAFF-R on IgD⁺ CD38dim, CD8 on CD39⁺ CD8br, CD14 on CD33br HLA DR⁺ CD14dim, CD14 on Mo MDSC, CD24⁺ CD27⁺ %lymphocyte, CX3CR1 on monocyte. B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor superfamily and plays a critical role in the proliferation, maturation, and differentiation of B lymphocytes. BAFF-R has the ability to impede the interaction between soluble BLyS and receptors on cell membranes, thereby inhibiting NF-κB activation and suppressing immunoglobulin production by B cells in peripheral blood (Xu and Chen, 2017). The increased levels of B-cell activating factor (BAFF) are a consequence of both acute and chronic inflammation (Matharu et al., 2013). Research findings indicate that the inhibition of the TLR4/NF-κB signaling pathway can effectively suppress microglia activation and neuroinflammatory responses, leading to an improved analgesic effect of morphine (Yang et al., 2023). HLA-DR, a cell surface receptor belonging to the MHC class II family, is encoded by the human leukocyte antigen complex located in chromosome 6 region 6P21. The reduced expression of HLA-DR in monocytes during chronic inflammation highlights its anti-inflammatory properties (Söderlund et al., 2009).

Furthermore, it is worth mentioning that the occurrence of MT was observed to be linked with a rise in the percentage of B cell % CD3⁻ lymphocyte, CD14⁻ CD16⁻ AC, CD14 on CD14⁺ CD16⁺ monocyte, CD24 on IgD⁺ CD38br, CD24 on unsw mem, CD25 on unsw mem, CD25hi CD45RA-CD4 not Treg %CD4⁺. Based on the differential expression of surface markers CD14 and CD16, monocytes can be categorized into two subgroups: CD14⁺ CD16⁻ and CD14⁺ CD16⁺. Notably, elevated levels of CD14⁺ CD16⁺ have been observed in conditions such as inflammatory pain and ANCA-associated vasculitis, leading to their identification as inflammatory monocytes. Research suggests that this subgroup’s mechanism of action may involve increased expression of relevant inflammatory receptors along with pro-inflammatory microRNAs (Xia et al., 2021). Based on the differential expression of CD25 on CD4⁺ T cells, they can be categorized into two groups: effector T cells (Teff) with low CD25 expression and regulatory T cells (Treg) with high CD25 expression. Both types can be activated through antigen stimulation via the signaling pathway of the T cell receptor (TCR). However, while Teff promotes immune inflammatory reactions, Treg maintains tolerance in the body, exhibiting distinct functions and roles. Morphine has been found to enhance the expression of several crucial pro-inflammatory chemokines as well as C-C Motif Chemokine Receptor 5 (CCR5) on T cells, monocytes, and macrophages. Moreover, morphine has the ability to elevate IL-10 levels while simultaneously reducing IL-17 secretion from cultured CD4⁺ T cells (Shao et al., 2022). Previous studies have reported a significant increase in the numbers of circulating Treg cells and the functional activity of Th17 cells following chronic exposure to morphine. Additionally, there is an observed increase in T cell populations expressing surface markers associated with gut-homing (CD161 and CCR6) as well as susceptibility to HIV-1 infection (CCR5 and β7 integrin) (Cornwell et al., 2013). However, limited research has been conducted on the role of monocytes and T cells in MT.

The advantages of this study are as follows: MR utilizes genetic variants as proxies for environmental exposure in order to establish a causal association between an exposure and the occurrence of a disease. Given that genetic variations are presumed to be randomly determined prior to birth, they exhibit a strong independence from environmental factors and become firmly established long before the onset of illness. This characteristic helps circumvent issues related to residual confounding and reverse causation commonly encountered in conventional observational studies. This research utilizes openly accessible datasets to obtain more accurate estimates and increased statistical potency as a result of the extensive sample sizes in GWAS. The findings were not influenced by horizontal pleiotropy or other variables. In summary, this study had sufficient statistical power to identify a significant correlation between gut microbiota/immune cells and MT. However, there were certain limitations that need to be acknowledged. Firstly, in order to minimize the impact of population stratification bias, the majority of participants included in our analysis were of European descent, which could potentially introduce some bias into our findings. Secondly, due to the unavailability of demographic information such as gender and ethnicity in the original dataset, we were unable to conduct subgroup analyses. Thirdly, since the number of SNPs meeting the genome-wide significance threshold (p < 5 × 10⁻⁸) was insufficient for further investigation, we only focused on SNPs reaching locus-wide significance level (p < 5 × 10⁻⁵). These constraints may limit the generalizability of our results and could have potentially affected the accuracy of this study.

In summary, we have extensively verified the link between gut microbiota/immune cells and MT. Specifically, four bacterial characteristics and seven immune cells exhibited a positive causal relationship with MT, while two other bacterial features and nine immune cells displayed a negative causal association with MT. These microbial strains could serve as innovative biomarkers and offer valuable insights for preventing and treating MT.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

SH: Conceptualization, Data curation, Formal analysis, Writing – original draft, Writing – review & editing. JiG: Writing – original draft. ZW: Writing – original draft. YX: Writing – review & editing. YG: Writing – review & editing. YL: Funding acquisition, Writing – review & editing. JuG: Conceptualization, Funding acquisition, Writing – review & editing.