Low back pain (LBP) is a global health concern that affects nearly 1 in 10 people worldwide and ranks highest in terms of disability (1). Over 85% of individuals that seek care for LBP have pain in the absence of a specific underlying condition (2, 3), making it difficult to treat. Although most individuals with LBP will experience a resolution of their pain within 4–6 weeks (acute low back pain; aLBP), around 20% of individuals will develop chronic low back pain (chronic low back pain; cLBP), which lasts beyond 12 weeks and negatively impacts normal activities and quality of life (4). Variables to predict if an individual will transition from aLBP to cLBP have yet to be established, however, it can likely be attributed to a combination of genetic, epigenetic, and environmental factors (5). Current therapies for chronic pain are not universally successful, likely due to complex interactions of these factors. Therefore, establishing the mechanisms underlying the transition from aLBP to cLBP, and developing novel and individualized therapies for cLBP are extremely important.

Our previous findings (6) as well as studies from other groups (7, 8), have demonstrated associations between genetic variation and the susceptibility to chronic pain. Most commonly, these variations have been in the form of single nucleotide polymorphisms (SNPs), but other types of variations in the linear DNA sequence, e.g., copy number variations, have also been implicated in individual differences in pain and analgesia (9–11). While polymorphisms change the genetic code, individual differences can also occur in the 3 dimensional structure of the genome through epigenetic modifications. Variations in the epigenome change the physical structure of the genome through processes including DNA methylation and histone modifications, both of which affect the ease with which the linear DNA sequence can be transcribed. DNA methylation occurs when a methyl group is added to the fifth carbon of a cytosine that is adjacent to a guanine, referred to as a CpG site (12). CpG islands, or DNA sequences with a high percentage of CpG sites, are usually found within the promotor region of a gene sequence (13), which make it possible for methylation to interfere with gene transcription, generally by decreasing gene expression (12). Chronic pain patients have been found to exhibit significant differences in DNA methylation patterns (14–19). In support of this, neuropathic pain symptoms were significantly correlated with higher methylation in the CpG island of the TRPA1 gene as well as lower TRPA1 mRNA expression (17, 20). It has even been suggested that alterations in DNA methylation serve as a “genomic memory of pain” and can influence long-term regulation of gene expression (21). Individuals with chronic pain also display differences in histone acetylation (13, 22), which occurs on the n-terminal of the histone tail and prevents chromatin from becoming compact. This makes it more accessible for transcription factors to bind (23) typically resulting in increased transcription (24, 25). A disruption in the appropriate balance of histone acetylation and deacetylation has been implicated in the etiology of neurological disorders, including pain (26). Finally, altered gene expression is also seen in preclinical models of neuropathic pain, where at least 10% of the transcriptome becomes dysregulated (22). Given that epigenetic modifications regulate gene expression, this link further points to a potential role of epigenetics in the development of chronic painful conditions.

In the present study, we measured global DNA methylation and H4 histone acetylation, as well as mRNA expression of 84 candidate genes with known roles in the transduction, maintenance, and/or modulation of pain in peripheral blood from individuals with new onset LBP at time of recruitment (<4 weeks duration). LBP status was tracked over a 6 month period, and only participants whose pain resolved within the 1st month (aLBP) or for whom pain was still present at 6 months (cLBP) were compared to healthy controls in the present analyses. Somatosensory functioning was assessed in all participants using multiple quantitative sensory testing (QST) measurements. We hypothesized that during the acute pain phase, individuals that eventually transition to cLBP would display differences in epigenetic markers, candidate gene expression levels, and somatosensory function (QST measures) compared to those whose pain would resolve (aLBP) and healthy controls. Indeed, we found participants that would go on to develop cLBP differed in global DNA methylation status, candidate gene expression, and somatosensory function at the time of pain onset compared to aLBP participants. Identifying factors predictive of the transition to cLBP, can shed light on novel precision pain medicine interventions targeting specific pathological processes early on in order to prevent the transition all together.

Low back pain is one of the most common pain conditions, affecting nearly 1 in 10 people worldwide (1). Because not all individuals will transition from aLBP to cLBP, it is imperative to determine factors with predictive potential for transitioning to cLBP in order to identify those at the highest risk and develop precision pain medicine interventions. The present study was designed to examine whether differences in epigenetic modifications (global DNA methylation and/or H4 histone acetylation status), candidate gene expression, and/or somatosensory functioning (QST) could distinguish participants whose pain would resolve quickly (aLBP) from those who would eventually transition to cLBP.

Participants who would later develop cLBP exhibited decreased global DNA methylation status compared to participants whose pain resolved quickly (aLBP) and healthy controls, indicating a potential role for hypomethylation in subsequent candidate gene expression changes contributing to pain chronicity. We further found that a single candidate gene, IL2, was negatively correlated with global DNA methylation at both pain onset and 6 months later, a time when participants had transitioned to cLBP. In addition, multiple measures of somatosensory function were also associated with global DNA methylation which may reflect the contribution of epigenetic modifications to fundamental sensory processing changes seen in cLBP. While the present analyses do not test a hypothesized mechanism underlying the transition from aLBP to cLBP, our findings shed light on potential novel hypotheses for subsequent testing in other larger cohorts.

Histone acetylation occurs on the n-terminal of the histone tail and makes chromatin more accessible for transcription factors to bind (23), which usually leads to an increase in transcription (24, 25). Altered histone acetylation status has been associated with both inflammatory and neuropathic pain (26) in preclinical models. Interestingly, inhibitors of histone acetyltransferases, which add acetyl groups, and histone deacetylases, which remove the acetyl groups have been shown to relieve pain (35, 36) albeit through different mechanisms (26). We found no differences in H4 acetylation status between aLBP and cLBP at the time of pain onset although when both groups were combined, they were found to have elevated H4 acetylation compared to healthy controls. This pattern suggests that H4 acetylation may contribute to or result from the presence of acute pain but does not appear to discriminate those at increased risk for transition to chronic pain. As a result, we prioritized the examination of potential relationships between global DNA methylation, candidate gene expression, somatosensory function (QST) and pain phenotype (chronicity).

cLBP participants had significantly lower global DNA methylation compared to aLBP and healthy controls, in line with other studies that report differences in DNA methylation patterns in individuals with chronic pain (14–16, 18, 37), including cLBP (19). For example, Aroke et al., found that individuals with non-specific cLBP had 159 differentially methylated regions compared to healthy controls, the majority of which were located in CpG islands and promoter regions (19) for genes involved in immune signaling, endochondral ossification, and G-protein coupled transmissions. DNA methylation has also been implicated in the etiology of fibromyalgia (FM) (14, 18). Ciampi de Andrade et al., found 1,610 differentially methylated DNA positions in FM patients, the vast majority (69%) of which were hypomethylated (18). Genes associated with these CpG sites included those involved in metabolic pathways, the mitogen-activated protein kinase signaling pathway, and regulatory pathways of the actin cytoskeleton, among others. Conversely, Menzies et al., found 69 differentially methylated sites in a cohort of FM patients with most being hypermethylated compared to healthy controls (14) and an overrepresentation of genes involved in neuron differentiation and nervous system development. In a cohort of patients with limb amputations, the subset that developed complex regional pain syndrome (CRPS) had 48 differentially methylated CpG sites compared to individuals that developed non-CRPS neuropathic pain (37) with all but 7 of these sites found to be hypomethylated. Finally, a study assessing whole genome DNA methylation found significant differences in DNA methylation profiles in individuals with early vs. advanced intervertebral disc degeneration (38). Four loci were hypomethylated while 216 were hypermethylated in the advanced disc degeneration stage but none of these alterations in methylation were assessed for their association with pain, per se.

Hypomethylation is most often associated with increased gene expression (13), so we identified a single gene from our quantified 84 pain-related candidate genes, IL2, whose expression was significantly negatively correlated with DNA methylation level at both the time of pain onset and after transition to chronic pain. Eight other candidate genes were significantly positively correlated with global DNA methylation status at the time of pain onset: BDNF, CX3CR1, GCH1, P2RX4, P2RX7, P2RY1, PTGES3, and TNF. Expression levels of each of these candidate genes have previously been associated with various chronic pain conditions in humans and preclinical models (39–46), but these positively correlated candidate genes are unlikely to reflect the impact the hypomethylation noted in the cLBP group. As a result, IL2 was identified as the highest priority candidate gene.

cLBP participants exhibited nearly a 5-fold increase in IL2 expression relative to aLBP participants at pain onset. The IL2 gene encodes a cytokine that has been shown in preclinical studies to have both algesic (47, 48) and analgesic properties (49, 50). This conflict in the literature may depend on the pain model, location of IL2 gene or IL2 protein manipulation, as well as the pain-like behavior measured but points to the role of IL2 as a potent pain modulator. For example, studies examining thermal sensitivity after chronic constriction injury found that intrathecal administration of human IL2 gene transfection resulted in analgesia (49, 50), whereas others examining mechanical hypersensitivity following an intraplantar (47) or intraarticular (48) injection of IL2 protein found it to be algesic. These data suggest that an increased level of peripheral IL2, in particular, may contribute to the transition from aLBP to cLBP. This result suggests that expression of IL2 is elevated early in individuals with pain and is maintained in those individuals that eventually transition to a cLBP state.

IL2 has been shown to exert both anti- or pro-inflammatory (51) influence, but our data support the pro-inflammatory interpretation as IL2 remained increased in the cLBP group over time. Prior reports indicate patients with painful neuropathy exhibit 2-fold higher IL2 mRNA expression in circulation compared to healthy controls and 2-fold higher IL2 mRNA and IL2 protein compared to patients with painless neuropathy (52). Similarly, IL2 expression is higher in the blood of individuals with CRPS compared to healthy controls (53). We hypothesize that cells circulating in the blood, such as immune cells, produce IL2 due to decreased methylation of the IL2 gene. Taken together with prior reports, our data point to an algesic role for IL2 through its role as a proinflammatory mediator that fails to resolve in individuals with cLBP potentially resulting in peripheral nociceptor sensitization and persistent enhanced pain perception. Further work is needed to test this, and other, specific mechanisms in the transition to chronic pain.

P2RX4 is a gene that encodes the P2RX4 protein, which belongs to the family of receptors for ATP. In our participants, the expression level of P2RX4 went from a positive correlation with global DNA methylation at pain onset to a negative correlation at the 6 month time point, which suggests that increased P2RX4 signaling may play a separate role in the initiation and maintenance cLBP and that only the long term effects are under the influence of DNA methylation. This is in agreement with other studies that implicate P2RX4 in the chronification of neuropathic pain (54), potentially through astrocyte or other glial cell activation (55, 56). Interestingly, P2RX4 and IL2 appear to be linked as inhibition of P2RX4 has been shown to inhibit IL2 transcription (57) and ATP is required for IL2 transcription in immune cells (58). This represents one mechanism implicating both P2RX4 and IL2 in early balance of inflammatory responding and risk for pain chronicity.

In agreement with our previous reports (6, 27, 28) that included a larger cohort of these participants, cLBP participants displayed higher BPI subscale scores compared to aLBP participants at pain onset. cLBP participants also displayed altered somatosensory function across multiple domains measured at the site of pain compared to aLBP participants. Moreover, for the cLBP participants, multiple somatosensory function (QST) measures were correlated over time between pain onset and the 6 month follow up visit, indicating that increased sensitivity at the time of recruitment corresponded to a persistent increase in sensitivity over time. Only dynamic mechanical allodynia (ALL) taken at the control site which was found to increase over time for those in the cLBP group, suggesting that the mechanisms underlying ALL are engaged early on and escalate over time. Several QST measurements were negatively correlated with global DNA methylation as were all of the BPI subscales, with the exception of BPI relief. When we examined the relationship of IL2 expression with the QST measures, CPT at the site of pain was the only variable significantly associated with IL2 expression, indicating that increased cold sensitivity may be modulated by IL2 expression. Cold pain sensitivity has been shown elsewhere to be a predictive factor in the development of long-term pain (59) and others have reported a positive correlation between IL2 plasma concentrations and increasing pain intensity in chronic pain patients (60). While we prioritized potential associations with IL2, other pain-related candidate genes could also influence somatosensory function and/or chronic pain development.

Further evaluation of the associations of QST measures and BPI with methylation and IL2 expression revealed that methylation status, not IL2 expression level, was a major predictor of QST outcomes as adding IL2 to the model did not account for any additional variance in the regression analysis, suggesting the methylation effects and IL2 expression reflect a common process. Bruniquel and Schwartz describe a close relationship between Il2 expression and DNA methylation status in preclinical mouse models (61). They found both in vitro and in vivo evidence indicating that naïve T lymphocytes are highly methylated in the promoter-enhancer region of Il2, and therefore do not express Il2. Conversely, when T cells are activated, many of the CpG sites in the Il2 promoter-enhancer region are demethylated and Il2 is expressed. Clearly, active demethylation of the IL2 promoter region plays a key role in its expression, however the mechanism behind this remains unclear (62).

Our data align with previous studies demonstrating that individuals that will develop cLBP display significant differences in somatosensory function at pain onset compared to individuals whose pain will resolve. Further, our data suggest that global DNA methylation status is an epigenetic predictor of individuals that eventually transition from aLBP to cLBP, while histone acetylation is more accurately linked to an active pain state than to the risk for chronic pain transition. Finally, IL2 significantly negatively correlated with DNA methylation level and was significantly higher in our cLBP participants even at the onset of the low back pain episode. This research is significant because while many interventions for the treatment of cLBP exist, few are universally successful in reducing pain and increasing quality of life (63) once pain is established. This type of profiling has already been recognized as a way to identify subgroups of neuropathic pain patients and to personalize their pain management program (64). Improving prediction of those who will transition to cLBP will allow us to intervene before the transition to the more treatment-resistant chronic pain condition.

The current study has several caveats including the relatively small sample size and the use of global epigenetic modification data with specific candidate gene expression. While genotyping studies require substantial sample sizes, the use of candidate gene expression and quantifiable epigenetic measures are more amenable to analysis in smaller study cohorts. That being said, we did have a significantly greater percentage of Black individuals in our cLBP group, but we did not control for race in our analyses due to the small sample size and limited variance. However, previous studies have shown that Black individuals experience more frequent and severe chronic-pain compared to non-Hispanic whites (65), including back pain (66). Interestingly, this has been suggested to be due, in part, to epigenetic changes associated with increased adversity, stress, and racial discrimination (67), our findings support this theory but do not test it directly. Future studies in larger cohorts are warranted to tease apart the interrelationships between race, epigenetic status, gene expression, and chronic pain. An additional limitation is that we measured a subset of global epigenetic changes in our participants (i.e., global H4 acetylation and global DNA methylation). There are other histone modifications and gene specific epigenetic modifications that are not assessed with these methods. We acknowledge that more specific measurements could be taken, such as acetylation at specific lysine sites or methylation of specific gene promoters, including those for IL2, should be addressed in future work.

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by University of Connecticut Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.

OCE: data collection, data analyses, and manuscript preparation. NG, BK, NM, MP, and KMBC: data collection and data analyses. AS, EEY, and KMB: study design, supervision of data collection, and manuscript preparation. All authors contributed to the article and approved the submitted version.

This work was supported by R03NS096454-02, R21NS104789, Rita Allen Foundation Award in Pain (to KMB), R01 NR013932 (to AS), and NINR T32 NR013456 (mPIs) (to KMBC).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.