Mu-opioid receptor (MOR), encoded by the OPRM1 gene, is a receptor for morphine and morphine derivatives and plays an important role in mediating morphine's analgesic properties and the development of tolerance. Considering the crucial role of μ-opioid receptor signaling, a large number of studies have focused on studying opioid-induced epigenetic modifications that affect the OPRM1 gene promoter and its expression in relation to opioid-induced behavioral changes. Opioid use is associated with aberrant DNA methylation at the OPRM1 promoter region, and ethnicity has been identified as an important player in determining the DNA methylation changes. For example, the DNA methylation of the OPRM1 promoter was higher in the African American population than in the Hispanic and Caucasian populations (Nielsen et al., 2009). Hypermethylation of the OPRM1 promoter region was identified in the lymphocytes (−18 and +84 CpG sites) of Caucasian heroin addicts on methadone maintenance (Nielsen et al., 2009), the blood and sperm (5, 9, 10, 11, 18, 23, and 24 CpG sites) of male opioid addicts (Chorbov et al., 2011), the blood (126 CpG) of heroin users, and the blood (−60, −32, +27, and +126+140 CpG) of opioid users in the Chinese Han population (Xu et al., 2018). Even though hypermethylation at the OPRM1 promoter region was observed in opioid users of various ethnicities, the location and extent of hypermethylation seem to depend on the ethnic background.

Hypermethylation of the OPRM1 promoter was observed in cancer patients taking opioids for pain management, and the extent of methylation was dose-dependent and associated with the lack of inadequate pain relief (Viet et al., 2017). Hypermethylation of the OPRM1 promoter region was also observed in the saliva of postoperative patients with short-term clinical opioid use, and the extent of methylation correlated with opioid dosage (Sandoval-Sierra et al., 2020), suggesting that even short-term clinical opioid use results in an epigenetic change. Heroin use has also been associated with differential DNA methylation patterns in neurons isolated from the prefrontal cortex region of the postmortem brains of users who died of a heroin overdose (Kozlenkov et al., 2017). Ethnicity appears to play an important part in determining the extent and location of DNA methylation, and it is important to note that different dietary habits and the concomitant effect of diet on the microbiome among people of different ethnicities may be contributing factors to the changes in DNA methylation.

A single nucleotide polymorphism (SNP) at position +188 of the OPRM gene promoter resulted in the introduction of a new CpG site at this location, thereby leading to hypermethylation and downregulation of the OPRM1 gene in the postmortem brain tissue of chronic opioid addicts compared to opioid addicts who did not harbor the SNP in the OPRM1 gene (Oertel et al., 2012). The A118G SNP in OPRM1 was significantly associated with the onset and treatment of opioid addiction (Taqi et al., 2019). In a study by Doehring et al., DNA methylation of the OPRM1 promoter (+126, +159 CpG) was higher in the blood leukocytes of opiate addicts. In a separate cohort of postmortem brain tissue (somatosensory cortex and lateral thalamus), no significant changes in the OPRM1 gene expression were observed in heroin addicts compared to controls (Doehring et al., 2013). In an opioid cohort of postmortem brain tissue from heroin addicts, DNA methylation of the OPRM1 promoter region did not significantly correlate with OPRM1 expression levels (Knothe et al., 2016). These findings imply that the OPRM1 levels observed in peripheral blood may not be indicative of brain OPRM1 methylation levels, and may not represent brain OPRM1 transcript levels.

The role of the gut microbiome in modulating these changes is not known and needs to be addressed. However, supplementation with probiotic Lactobacillus acidophilus was shown to attenuate abdominal pain by mediating the expression of OPRM1 and cannabinoid receptors (CB2) in the intestinal epithelial cells in an Nf-kb-dependent mechanism (Rousseaux et al., 2007), implying that gut microbiota plays a role in pain perception by regulating OPRM1 gene expression. Notably, morphine use has been associated with a decrease in the species Lactobacillus in the gut (Meng et al., 2013, 2020; Banerjee et al., 2016; Sharma et al., 2019; Zhang et al., 2019). These results collectively imply that the microbiome plays an important role in modulating morphine-induced hyperalgesia. Experimental evidence employing μ-opioid receptor knock out mice also indicated that the endogenous opioid system sets the tone for the basal composition of the microbiome (Banerjee et al., 2016), indicating bidirectional regulation between the μ-opioid receptor and the microbiome.

These studies collectively indicate that morphine use leads to complex changes in global DNA methylation levels that are in part mediated by the microbiome. Evidence from separate studies shows that the microbiome is an important player in the regulation of DNA methylation; however, direct evidence linking the microbiome to opioid-induced changes in DNA methylation is lacking.

Histone acetylation is one of the best-characterized acyl modifications. The acetylation of histones neutralizes the positive charge of the histone tails, thereby weakening the interaction between the negatively charged DNA and positively charged histones and opening the chromatin to the transcriptional transcription. Histone acetylation is therefore often linked to transcriptional activation. Histone acetylation is maintained by the addition of the acetyl group from acetyl-CoA by a group of enzymes termed histone acetyltransferases (HATs), and the acetyl group from the lysine present on histone N-terminal tails is removed by the action of enzymes termed histone deacetylases (HDACs). Class I (HDAC 1, 2, 3, and 8) and class II HDACs (HDAC 5, 7, 6, 9, and 10) are zinc-dependent, and class III HDACs belong to the family of NAD+ dependent sirtuins (Sirt1–7) (Lennartsson and Ekwall, 2009; Bannister and Kouzarides, 2011; MolinaSerrano et al., 2019).

In recent years, it has been shown that microbial fermentation of complex dietary fibers results in the production of SCFAs: butyrate, acetate, and propionate. These SCFAs are known to inhibit the activity of HDACs, thus contributing to the changes in the epigenome (Stilling et al., 2014; Sook Lee et al., 2017; Silva et al., 2020). Microbially derived butyrate is a potent inhibitor of HDACs belonging to classes I and II, and it leads to hyperacetylation of the histones and results in increased gene expression. Approximately 2% of the mammalian genome has butyrate-responsive elements, and gene activation and repression by HDAC are mediated through the binding of transcription factors SP1 and SP2 to the butyrate-responsive genes (Davie, 2003). In a recent study by Yuille et al., cell-free culture supernatants from 79 common commensal bacteria were used to screen for SCFA production capacity and HDAC inhibition potential. The cell-free culture supernatants from three strains: Megasphaera massiliensis MRx0029, Roseburia intestinalis MRx0071, and Bariatricus massiliensis MRx1342, had the strongest HDAC inhibitory capacity, and metabolite analysis revealed that all three strains are potent butyrate producers. The strain M. massiliensis MRx0029 had the maximum inhibitory effect and is a producer of butyrate and valeric acid, identifying valeric acid as one of the potential HDAC inhibitors of microbial origin (Yuille et al., 2018). Furthermore, M. massiliensis MRx0029 was shown to inhibit the secretion of IL-6 in vitro via the production of SCFAs (Ahmed et al., 2019).

The epigenome, particularly the acetylation and methylation status of histones H3 and H4 in the colon, liver, and adipose tissue, is altered by the microbiome through the production of SCFAs and other bacterial metabolites in a diet-dependent manner. Conventionally raised mice were found to have higher levels of histone acetylation and methylation than germ-free mice, and microbiota transfer increased the acetylation and methylation levels of histones in germ-free mice (Krautkramer et al., 2016). Additionally, the microbiome was shown to regulate microglial properties, functions, and colonization by creating an open chromatin state through the production of SCFAs (Thion et al., 2018). These results clearly indicate that the gut microbiome can alter the epigenome of the host through the production of SCFAs and other metabolites necessary for methylation reactions. A recent study conducted by us showed that opioid use in an HIV model of humanized mice leads to exacerbated microbial dysbiosis, gut barrier damage, and impaired intestinal renewal through HDAC1-mediated suppression of NOTCH signaling. We also showed that morphine treatment leads to decreased HDAC inhibition capacity of luminal content, and pharmaceutical inhibition of HDAC by suberoylanilide hydroxamic acid blocked morphine's suppression of NOTCH signaling and stem cell proliferation (Meng et al., 2020). In another previous study, we showed that morphine treatment inhibits IL-2 production in T-cells by inhibiting acetylation trimethylation of histones and decreasing DNA demethylation at IL-2 promoter regions, thus affecting the T-cell response and increasing susceptibility to infections (Wang et al., 2007). The role of the gut microbiome was not investigated in the latter studies; however, since Interleukin-2 (IL-2) has multiple, sometimes opposing, functions during an inflammatory response, it is plausible that opioid-induced dysbiosis that results in sustained inflammation may drive IL-2 expression, thus contributing to both the induction and the termination of inflammatory immune responses.

Opioid use is associated with increased histone H3 acetylation in the spinal cord and the development of OIH. HAT inhibition by curcumin attenuated the development of OIH, tolerance, and dependence, whereas HDAC inhibition had the opposite effect and increased side effects, implicating increased histone acetylation in the development of OIH, tolerance, and dependence (Liang et al., 2014). Increased acetylation of lysine at position 9 in histone H3 tail (H3K9 acetylation) at the promoters of BDNF and pdyn (prodynorphin) was observed with morphine treatment in the dorsal horn neurons of the spinal cord, contributing to the development of OIH; treatment with the HDAC inhibitor SAHA led to prolonged OIH (Liang et al., 2014). Additionally, early-stage inflammatory pain was associated with decreased acetylation of histone H3 in dorsal root ganglia neurons, and the use of morphine in the mouse model of inflammatory pain resulted in the recovery of histone H3 acetylation in glial cells and neurons, along with increased H3 acetylation in astrocytes (Li et al., 2012), indicating the role of acetylation in mediating inflammatory pain. Preoperative chronic morphine use was associated with increased OIH and reduced analgesic tolerance mediated through increased H3K9 acetylation at BDNF and pdyn and concomitant increased expression of these genes in the spinal cord, thus limiting the clinical use of morphine for surgical pain (Sahbaie et al., 2016).

CaMKII is a critical mediator of opioid-induced hyperalgesia (OIH), neuropathic pain (Chen et al., 2009, 2010), and inflammatory pain (Luo et al., 2008). Increased CaMKII expression has been observed in the spinal cord during OIH and is required for both its initiation and maintenance (Chen et al., 2009, 2010). HAT inhibition by curcumin was shown to delay morphine tolerance and dependence by attenuating the activity of CaMKIIα in the prefrontal cortex (Hu et al., 2015) and by attenuating OIH by inhibiting the activity of CaMKIIα in the spinal cord (Hu et al., 2016). In addition to inhibiting CaMKII activity, curcumin treatment was also shown to block morphine-induced upregulation of BDNF and the development of tolerance (Matsushita and Ueda, 2009). In a rat model of bone cancer pain, tumor cell inoculation resulted in the downregulation of OPRM1, which correlated with increased HDAC expression. Furthermore, HDAC inhibition was shown to attenuate morphine tolerance and restore MOR expression, highlighting the role of histone acetylation in the development of morphine tolerance during the progression of bone cancer pain (He et al., 2018).

These results highlight the role of histone acetylation in the development of morphine analgesic tolerance, dependence, and OIH. Pharmacological HDAC inhibition or modulation of the microbiome aimed at the production of bioactive compounds responsible for altering histone acetylation may help overcome the development of tolerance, dependence, and OIH and improve the clinical use of opioids for pain management.

Opioid use is associated with alterations in histone acetylation in the brain reward systems, contributing to reward-related learning, memory, drug-seeking behaviors, and addiction. Accumulating evidence suggests that histone acetylation plays an important role in mediating morphine's rewarding behavior in mouse models of morphine-induced conditioned place preference (CPP). For example, pretreatment with HDAC inhibitor trichostatin A (TSA) was shown to facilitate memory formation during the acquisition and extinction of morphine-induced CPP in the basolateral amygdala through increased H3K14 acetylation and a concomitant increase in BDNF, Delta FOSB, and CREB activation (Wang Z. et al., 2014). A similar result of induced CPP was observed with HDAC inhibition by sodium butyrate in the striatum (Sanchis-Segura et al., 2009). Increased histone acetylation of the Dlg4 gene (disk large homolog 4) encoding for postsynaptic density protein (PSD-95) mediated by pCREB in the ventral tegmental area (VTA) contributed to morphine-induced CPP and rewarding behavior (Wang Y. et al., 2014). Additionally, increased histone acetylation was observed at the promoter of BDNF in the ventral medial prefrontal cortex following conditioned place aversion (CPA), and HDAC treatment facilitated the extinction of aversive memory associated with morphine withdrawal (Wang et al., 2012).

Increased acetylation has also been implicated in morphine-induced neuroinflammation, as increased acetylation and upregulation of cxcl12 were observed in hippocampal CA1 neurons, driving morphine-seeking behavior (Liu et al., 2020). Morphine use was also associated with synaptic abnormalities mediated through increased HDAC2 expression. HDAC2-mediated decrease in H3K9 acetylation was observed in ventral tegmental area (VTA) dopamine neurons and is reversible by HDAC2 inhibition with the drug CI-994 (Authement et al., 2016).

Depletion of the microbiome by prolonged antibiotic treatment resulted in decreased SCFAs and increased cocaine-mediated CPP, while supplementation with SCFAs resulted in a behavioral phenotype similar to that of regular mice, implying that microbial SCFAs are responsible for cocaine-seeking behavior (Kiraly et al., 2016). However, the results observed with cocaine cannot be directly extrapolated to other substances of abuse. The vast wealth of data obtained through studies aimed at pharmaceutical manipulation of HAT and HDAC implies that histone acetylation plays an important role in mediating morphine's rewarding behavior. While these studies established that aberrant epigenetic modifications at candidate genes, such as OPRM1, BDNF, PDYN, CaMKII, and CXCL12, and pharmaceutical manipulation of HAT and HDAC modulate tolerance development, OIH, the perception of pain, and addiction, it must be noted that treatment with inhibitors of HAT and HDAC can have global effects and result in epigenetic changes in the promoters and enhancers of many other genes, in addition to the investigated candidate genes. Therefore, there is a need to comprehensively study the histone acetylation changes induced by opioid treatment and the alterations induced by the use of pharmaceutical inhibitors of HATs and HDACs.

Histone lysine crotonylation (HKcr) is a dynamic, evolutionarily conserved acyl modification that is regulated by the action of acylases, which add the modification, and deacylases, which remove the modification (Tan et al., 2011). The well-established HAT, p300/CREB binding protein, performs crotonyl transferase activity, and histone crotonylation levels are dependent on the intracellular levels of crotonyl-CoA (Sabari et al., 2015). Class I histone deacetylases, HDAC1–3, and Sirtuin 3, were identified to remove histone crotonylation (Bao et al., 2014; Wei et al., 2017). HKcr, which is primarily observed in active gene promotors and enhancers, is linked to gene activation, and changes in HKcr were observed in various disease states (Sabari et al., 2015; Wang et al., 2021). Changes in histone lysine crotonylation were implicated in acute kidney injury (Ruiz-Andres et al., 2016), DNA damage response, and stress-induced depressive-like behaviors (Liu et al., 2019). The induction of crotonylation at the LTA of HIV is known to reactivate latent HIV (Jiang et al., 2018). Histone crotonylation was identified to be involved in neuropathic pain, the induction of neuroinflammation, and hyperalgesia (Zou et al., 2022). Histone lysine 18 crotonylation (HK18cr) is an abundant mark in the intestine, and the gut microbiota was shown to regulate histone crotonylation levels in the gut via SCFAs. Depletion of the microbiome and the consequent reduction in SCFA, especially butyrate, was shown to lead to a decrease in crotonylation (Fellows et al., 2018). Since morphine treatment was shown to modulate SCFA production (Cruz-Lebrón et al., 2021), microbiome-mediated changes in HKCr are to be expected with opioid use. However, to date, no studies have explored the changes in histone lysine crotonylation mediated by opioids.

Histone methylation involves the addition of mono-, di-, and tri-methylation to lysine and arginine residues at the N-terminals of histones. These modifications serve as binding sites for the proteins. The reader proteins, which specifically recognize the different histone-lysine modifications, are responsible for the downstream events. Histone lysine methylation is catalyzed by SET domain-containing histone lysine methyltransferases, while enzymes containing the Jumonji domain catalyze the demethylation of histone lysine residues. Methylation of H3K9 and H3K27 is linked to heterochromatin formation and transcriptional silencing, resulting in gene repression. H3K4 methylation and H3K36 methylation are generally associated with active gene expression (Lennartsson and Ekwall, 2009; Bannister and Kouzarides, 2011; MolinaSerrano et al., 2019).

Few studies have explored the role of lysine methylation in modulating morphine-induced behavioral changes. The repressive histone mark H3K9me2 was studied for its role in modulating behavioral outcomes in response to chronic morphine treatment. Repeated morphine treatment decreases G9a, a core component of the histone lysine methyltransferase that primarily catalyzes the addition of H3K9me2 in the NAc. Reduced G9a is involved in the morphine reward pathway by affecting histone methylation at fosB, glutamate receptors, and repetitive elements. Interestingly, overexpression of G9a reduced the rewarding response to the drug and withdrawal symptoms (Sun et al., 2012). Similar changes were also observed with cocaine self-administration and stress (Maze et al., 2010). In addition, the expression of the methyl binding protein (MeCP2) in the central nucleus of the amygdala is altered by morphine and inflammatory pain. Morphine-induced MeCp2 binds and represses the activity of G9a, contributing to morphine's rewarding behavior (Zhang et al., 2014).

While the role of the microbiome was not assessed in the abovementioned studies, the microbiome and microbial metabolism are known to affect histone methylation by modulating substrates/methyl donors (choline, betaine, methionine, SAM, and SAH) and cofactors (folate, cobalamin, riboflavin, and niacin) of the one-carbon metabolism and folate cycle (Friso et al., 2017), as detailed in the earlier DNA methylation section. In support, the low availability of SAM was shown to affect histone lysine methylation (H3K4me3) at the promoters of genes involved in innate immunity and affect the survival of Pseudomonas infection in Caenorhabditis elegans (Ding et al., 2015). Methionine restriction was shown to decrease the methylation at histone H3K4me3 in the liver by altering the one-carbon metabolism, thus affecting the SAM and SAH levels in the liver (Mentch et al., 2015). Further research is needed to identify the role of the microbiome in regulating histone methylation and its influence on behavioral responses to opioids.