Plasma Pro‐ and Anti‐Inflammatory Cytokines in an Observational Chronic Low Back Pain Cohort

ABSTRACT

Background

Chronic low back pain (cLBP) is a multifactorial condition that can have various contributing factors, including biological, biomechanical, and behavioral. Recent evidence suggests that systemic inflammation may contribute to cLBP, impacting pain sensitivity and individuals' functional status. Circulatory pro‐ and anti‐inflammatory cytokines are widely used to determine individuals' systemic inflammatory status. The University of Pittsburgh Mechanistic Research Center, part of the National Institutes of Health's (NIH) Helping to End Addiction Long‐term Initiative, conducted a prospective, observational study to identify phenotypes in a large cohort of individuals with cLBP. The present work reports the quantification of key circulatory cytokines in this cLBP cohort.

Methods

A total of 1007 individuals with cLBP were enrolled. Plasma samples were available from 936 participants, and concentrations of pro‐inflammatory (IL‐6, IFN‐γ, TNF, IL‐15, Leptin) and anti‐inflammatory (IL‐1ra, IL‐10) cytokines were measured via immunoassays. Pain and functional status were assessed using validated self‐reported numeric pain ratings scale and the Oswestry Disability Index. Descriptive statistics of analyzed cytokines were reported across the overall population, stratified by age (< 60 and ≥ 60 years old) and sex, and by pain levels as mild (0–5), moderate (6, 7), and severe (8–10), and ODI, categorized as minimal disability (0%–20%), moderate (21%–40%), and severe disability (> 40%).

Results

The values of circulating cytokines assessed in this study aligned with those reported in the literature for other painful inflammatory conditions and, in most cases, exceeded those documented for healthy populations. IL‐6, IL‐1ra, and Leptin demonstrated higher concentrations with higher pain and disability severity. TNF showed higher concentration in participants with higher disability severity. Concentration levels of IFN‐γ, IL‐15, and IL‐10 exhibited no differences across pain or ODI categories. Notably, TNF levels were higher in older adults (≥ 60 years), whereas Leptin levels were higher in females than in males.

Conclusion

This study provides a snapshot of key circulating cytokines in a large cLBP cohort, revealing differences in pro‐ and anti‐inflammatory cytokines across pain and disability for the overall population and in sex and age subgroups. Additional longitudinal and mechanistic studies are required to clarify how cytokines could serve as diagnostic, prognostic, or phenotyping markers, ultimately informing targeted, inflammation‐focused therapies that may reshape current treatment approaches.

The University of Pittsburgh Mechanistic Research Center, part of the National Institutes of Health's (NIH) Helping to End Addiction Long‐term Initiative, conducted a prospective, observational study to identify phenotypes in a large cohort of individuals with cLBP. The present work reports the quantification of key circulatory cytokines in this cLBP cohort.

1. Introduction

The experience of back pain profoundly influences people on multiple dimensions, from cellular processes [1, 2, 3, 4] to the broader spectrum of systemic dysfunctions, impacting physical and mental health [5, 6, 7, 8, 9, 10, 11, 12] and social relationships [13, 14, 15, 16]. Systemic inflammation has been increasingly studied as a distinct contributor among the different factors associated with cLBP [12, 17, 18, 19]. Broadly, systemic inflammation adversely affects health if it becomes chronic [20, 21, 22, 23]. A pro‐inflammatory state has been implicated in a myriad of health complications and diseases, such as cardiovascular conditions [24, 25], Type 2 diabetes [26, 27, 28], autoimmune diseases [29, 30, 31], cancer [32, 33, 34], neurological disorders [35, 36, 37, 38, 39, 40], and notably, chronic pain syndromes [41, 42, 43, 44, 45]. In persons experiencing pain, systemic inflammation may act as either a precursor or a consequence, depending on the situation and specific contributing factors [46].

In the context of cLBP, systemic inflammation can contribute to pain through tissue‐specific direct mechanisms, negatively impacting lumbar spine structures such as muscles, tendons, joints, intervertebral discs, and fascia. Studies have observed a correlation between inflammatory markers, such as C‐reactive protein, and increased spinal stiffness and disc degeneration in older adults with back pain, linking systemic inflammation with structural changes in the spine [47]. Systemic inflammation has also been shown to contribute to pain through non‐tissue‐specific or indirect mechanisms [48, 49]. For example, systemic inflammation has been shown to up‐regulate pain responses by modulating the neuroimmune status of the central nervous system and altering the functionality of specific portions of the brain or spinal cord that are linked to pain control [50, 51].

Cytokines, vital in cell communication during the immune response, play a pivotal role in inflammation by facilitating or suppressing inflammatory responses (Table 1) [125]. Interleukins, such as IL‐6, IL‐15, interferons (IFN)‐α and ‐γ, and tumor necrosis factor‐α (TNF) are primarily proinflammatory cytokines contributing to musculoskeletal (MSK) conditions such as osteoarthritis, rheumatoid arthritis, osteoporosis, and inflammatory myopathies [81, 86, 90, 120, 126, 127]. These cytokines promote inflammation, joint damage, and pain by sensitizing pain pathways and nociceptors. For example, TNF and IL‐6 have been associated with generating and sustaining inflammatory pain through modulation of nociceptive nerve cells [128, 129]. Also, TNF and IFN‐γ contribute to neuropathic pain through glial activation [130]. IFN‐α, elevated in autoimmune MSK diseases, contributes to both inflammation and pain via immune and nociceptive pathways [80, 82, 83]. IL‐1β drives joint inflammation and cartilage breakdown while also sensitizing neurons to increase pain perception [102, 103]. Other serum biomarkers, such as adipokines, may contribute indirectly to inflammatory processes that worsen disc health, as found in a study exploring adiponectin and leptin levels in cLBP patients [131]. Leptin influences MSK health by affecting bone and muscle processes, with elevated levels linked to increased pain sensitivity in inflammatory and neuropathic pain conditions [132, 133, 134].

TABLE 1.

Selected cytokines and their inflammatory roles.

Cytokines		Summary
Proinflammatory	IL‐6 [52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65]	IL‐6 is a proinflammatory cytokine involved in immune regulation, inflammation, and bone metabolism. It is implicated in musculoskeletal conditions such as RA, OA, ankylosing spondylitis, and spinal degeneration. IL‐6 can exacerbate inflammation, joint damage, and pain by promoting the production of other proinflammatory cytokines and increasing immune cell recruitment to affected tissues. It also contributes to pain by sensitizing peripheral and central pain pathways, leading to hyperalgesia and chronic pain states
	IFN‐α [66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78]	IFN‐α is an immunomodulatory cytokine involved in antiviral defense and autoimmune inflammation. It contributes to RA, SLE, and dermatomyositis by enhancing immune activation and correlates with disease severity. Clinically, IFN‐α is linked to pain symptoms such as myalgia, arthralgia, and neuropathic pain via peripheral and central nociceptive mechanisms, though it may also inhibit spinal nociception, reflecting a context‐dependent role in pain
	IFN‐γ [79, 80, 81, 82, 83, 84]	IFN‐γ is a key cytokine in immune regulation, playing roles in autoimmune diseases such as RA, osteoporosis, and inflammatory myopathies. It contributes to inflammation, bone loss, and muscle damage. IFN‐γ is linked to both neuropathic and inflammatory pain through its effects on glial cell activation, proinflammatory signaling, and sensitization of nociceptors, enhancing pain perception and contributing to chronic pain conditions
	TNF [85, 86, 87, 88, 89, 90, 91, 92, 93]	TNF is a proinflammatory cytokine that regulates immune responses and is implicated in musculoskeletal conditions like RA, OA, ankylosing spondylitis, osteoporosis, and tendonitis. It contributes to inflammation, tissue damage, and pain by directly sensitizing nociceptors, promoting the release of other proinflammatory cytokines, and increasing the activation of pain pathways. This makes TNF a key mediator of both inflammatory and neuropathic pain
	IL‐15 [94, 95]	IL‐15 has a role in musculoskeletal conditions by promoting muscle hypertrophy, maintaining muscle mass, and influencing bone metabolism. It also contributes to inflammation in autoimmune conditions such as RA. IL‐15 is linked to pain modulation, especially in joint‐related pain, by enhancing the inflammatory response and increasing nociceptive signaling and pain sensitivity
	IL‐1β [96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106]	IL‐1β is a proinflammatory cytokine involved in musculoskeletal conditions such as RA, OA, tendinitis, and intervertebral disc degeneration. It promotes joint inflammation, cartilage degradation, and bone resorption by inducing other inflammatory mediators and matrix‐degrading enzymes. IL‐1β also contributes to pain by sensitizing sensory neurons and enhancing the release of pain‐promoting substances like prostaglandins and bradykinin
	Leptin [107, 108, 109, 110, 111, 112, 113]	Leptin is a hormone implicated in musculoskeletal conditions, particularly in bone remodeling and inflammation. Elevated leptin levels are linked to joint inflammation and pain in conditions like osteoarthritis. Leptin can modulate pain by enhancing the sensitivity of nociceptors and promoting inflammatory signaling, contributing to increased pain perception and chronic pain development
Anti‐inflammatory	IL‐10 [114, 115, 116, 117, 118, 119, 120, 121, 122]	IL‐10 is an anti‐inflammatory cytokine that helps regulate immune responses by suppressing proinflammatory cytokines. It is involved in managing musculoskeletal conditions like RA, OA, and SLE, with potential roles in reducing inflammation and promoting healing. IL‐10's pain‐relieving effects are primarily linked to its ability to inhibit glial activation and decrease neuroinflammation, thereby reducing peripheral and central sensitization and relieving chronic pain
	IL‐1ra [123, 124]	IL‐1ra is an anti‐inflammatory cytokine that inhibits the action of IL‐1β, helping to reduce inflammation and pain in musculoskeletal conditions such as RA, OA, and Systemic Lupus Erythematosus (SLE). IL‐1ra is produced by the body to counteract the effects of IL‐1β, providing a regulatory mechanism that prevents excessive inflammatory responses. It plays a key role in maintaining balance in inflammatory processes by competitively binding to IL‐1 receptors, thereby blocking the proinflammatory effects of IL‐1β. IL‐1ra also helps regulate pain by preventing IL‐1β‐mediated sensitization of pain receptors, thereby reducing hyperalgesia and allodynia. Studies have shown that levels of IL‐1ra may be elevated in response to injury or inflammation, indicating its role as part of the body's natural anti‐inflammatory defense system

Anti‐inflammatory cytokines, such as IL‐1Ra and IL‐10, help modulate inflammation and may reduce pain in MSK conditions by blocking receptors, reducing proinflammatory mediators, and promoting tissue repair. IL‐1Ra and IL‐10 have also shown promise as targets in treating pain in animal models, though their clinical translation remains challenging [135, 136]. The cytokines selected for this study were chosen based on their well‐documented involvement in both musculoskeletal inflammation and pain pathways, as supported by prior clinical and translational literature. Their inclusion, although not exhaustive, provides a representative view of the dynamic interplay between pro‐ and anti‐inflammatory processes in cLBP. The current study aimed to measure the circulating concentrations of relevant selected inflammatory biomarkers in a large cLBP population and describes the cytokine levels based on age, sex, pain, and disability level.

2. Methods

Data presented herein was collected at the time of enrollment in the University of Pittsburgh's Low Back Pain: Biological, Biomechanical, Behavioral Phenotypes (LB³P) Mechanistic Research Center, a member of the National Institutes of Health's Back Pain Consortium (BACPAC) Research Program—which is part of the Helping to End Addiction Long‐term (HEAL) Initiative. The objective of LB³P is to perform in‐depth phenotyping of patients with cLBP, for which it enrolled 1007 people with cLBP and collected comprehensive demographic, behavioral, biomechanical, and biological data, including biosampling, providing a unique dataset to examine cytokines [137].

2.1. Participants

The study enrolled English‐speaking adults who experience chronic low back pain defined as “back pain (in the space between the lower posterior margin of the rib cage and the horizontal gluteal fold) that persisted at least three months and resulted in pain on at least half the days in the past six months” [138]. Participants were excluded if there they (1) were not identified in the University of Pittsburgh Medical Center (UPMC) Electronic Health Record system, (2) were participating in a masked intervention study for LBP, and/or (3) had a medical condition that would place the participant at increased risk or preclude them from complying with study procedures. Participants were enrolled by referral from clinicians, research registries, and community announcements between June 2020 and March 2024. The in‐person enrollment visit took place at the University of Pittsburgh Department of Physical Therapy—Clinical and Translational Research Center. Participants were compensated for their participation.

3. Cytokines

Circulating cytokine concentrations are commonly assessed to evaluate systemic inflammation [139, 140, 141]. This study selected seven pro‐inflammatory and two anti‐inflammatory cytokines (Table 1).

3.1. Blood Collection and Plasma Preparation

Blood was collected from participants using standard sterile phlebotomy techniques in 4 or 10 mL EDTA lavender plasma tubes, which were immediately kept on ice and centrifuged within 60 min post‐collection. Centrifugation was performed at 2000 g for 20 min at 4°C, and the plasma fraction was separated from the blood components within 20 min post‐centrifugation and inspected for signs of hemolysis. Lysed samples were aliquoted separately and excluded from the analysis. Non‐lysed plasma was transferred to a Falcon tube without disturbing the buffy coat or red blood cells, homogenized by gently mixing the sample, then aliquoted into cryovials and immediately frozen in a −80°C freezer within 1 h of processing and stored upright in a −80°C freezer to ensure long‐term integrity.

3.2. Plasma Analysis Methods

Cytokines quantification for IL‐1ra, IL‐1β, IL‐6, IL‐10, IL‐15, IFN‐α, IFN‐γ, and TNF was performed using ELLA kits (SPCKC‐PS‐000323 and ST01E‐PS‐003426; ProteinSimple, San Jose, CA) on plasma samples processed according to the manufacturer's specifications. Briefly, plasma samples and the cartridge, diluent, and wash buffer were allowed to reach room temperature before use. The dilution factor (MRD) specified in the Simple Plex Cartridge Kit was a 1:2 dilution, using 75 μL of plasma aliquoted into a microcentrifuge tube and centrifuged at 8000–10000 g at 4°C for 10 min to remove particulates. A total of 50 μL of supernatant was carefully collected, avoiding disturbance of the pellet, and mixed thoroughly with 50 μL of pre‐mixed well‐diluent. Subsequently, 50 μL of the diluted sample was pipetted into the specified cartridge inlet, ensuring no air bubbles were introduced. Finally, 1 mL of Wash Buffer A was added to all buffer inlets. All reagents were used as supplied by the manufacturer. Limits of quantitation and detection were cytokine‐specific and provided by the manufacturer. The limits were applied to the standard curves automatically generated by the machine for each cytokine.

Leptin was assayed using ELISA kits and reagents (Human Leptin Immunoassay DLP00, R&D Systems, INC., Minneapolis, MN, USA) according to the manufacturer's specifications. Plasma samples were prepared by adding 10 μL of the sample to 990 μL of Calibrator Diluent RD5P (diluted 1:5), followed by the addition of 100 μL of Assay Diluent RD1‐19 to each well. Then, 100 μL of standard, control, or sample was added per well and incubated for 2 h at room temperature. Wells were aspirated and washed four times using 400 μL of Wash Buffer. After washing, 200 μL of Human Leptin Conjugate was added to each well, the plate was covered, and incubated for 1 h at room temperature, followed by repeated washing. Subsequently, 200 μL of Substrate Solution was added, and the plate was incubated for 30 min at room temperature, protected from light. Finally, 50 μL of Stop Solution was added, and the optical density was measured at 450 nm within 30 min, with correction at 540 nm to account for optical imperfections.

4. Data Analysis

Cytokine descriptive statistics (mean, SD; median, IQR) were reported across the entire population and stratified by sex (male/female), age (< 60 and ≥ 60 years old), and pain and Oswestry Disability Index (ODI) severity. Age 60 was selected as the cutoff based on prior evidence showing that individuals over 60 have higher cLBP prevalence, distinct psychosocial factors, and more complex pain experiences [142]. Specifically, low back pain was categorized as mild (0–5), moderate (6, 7), and severe (8–10) according to published research [143]. ODI was categorized as minimal disability (0%–20%), moderate (21%–40%), and severe disability (> 40%), the latter representing the combined severe, crippling, and bed‐bound categories previously reported in the literature [144]. Data was visually explored for normal distribution. All data procedures were performed with JMP 18.1 Pro, JMP Statistical Discovery LLC, USA.

5. Results

5.1. Demographics

Of the 1007 participants in the study, 59 did not have blood drawn due to failed phlebotomy attempts, five did not have plasma available due to a limited amount of blood drawn, and nine had lysed samples—resulting in a total of 934 plasma samples available for analysis. One participant reported sex at birth as intersex and was therefore not included in the male/female comparison. For those 934 individuals, the mean age was 59.0 years (SD = 16.4), with 395 (42%) below the age of 60 years, 539 (58%) with age equal to or above 60 years, and 391 (42%) being males. The mean BMI was 31.3 kg/m² (SD = 7.5). The racial composition included 721 White (77%), 157 Black (17%), 19 Asian (2%), and 37 (4%) identified as other races. 97% of participants 90% (n = 836) were not Hispanic or Latino, with the remaining 3% (n = 24) reporting being Hispanic or Latino. The mean ODI was 30.7% (SD = 15.2), 275 with minimal/mild, 437 moderate (29%) in the mild, 438 (47%) in the moderate, and 222 (24%) in the severe disability groups. Mean low back pain was 5.4 (SD = 2.1), 479 with mild LBP (51%) in the mild, 293 (31%) in the moderate, and 162 (17%) in the severe pain group.

5.2. Cytokines Concentration

IFN‐α levels were below the manufacturer's provided quantitation limit (1.46 pg/mL) for over 70% of the samples and, therefore, the levels of IFN‐α were not reported along with the other cytokines (Table 2). IL‐1β levels were below the manufacturer's provided quantitation limit (0.064 pg/mL) for over 97% of the samples after performing the analysis in the first 250 participants' samples. Therefore, the analysis of IL‐1β was not completed for the remaining samples, and its levels were not reported along with the other cytokines.

TABLE 2.

Cytokine concentration.

	Analyzed cytokine	Non‐quantifiable	Quantified	Mean (SD)	Median	IQR
		N (%)	N (%)
Pro‐inflammatory	IL‐6 IFN‐γ TNF IL‐15 Leptin	4 (0.4%) 89 (9.5%) 2 (0.2%) 14 (1.5%) 17 (1.8%)	930 (99.6%) 845 (90.5%) 932 (99.8%) 920 (98.5%) 917 (98.2%)	5.56 (19.27) 1.45 (4.88) 13.49 (66.35) 3.19 (3.55) 27.0 (27.11)	3.24 0.80 9.46 2.57 18.17	3.23 0.72 4.02 1.12 25.61
Anti‐Inflammatory	IL‐10 IL‐1ra	48 (5.1%) 5 (0.5%)	886 (94.9%) 931 (99.5%)	3.35 (11.06) 399.04 (449.74)	2.28 282.00	1.14 240.50

Note: All measurement units are pg/mL, except for Leptin, which is reported as ng/mL. All cytokines were tested for n = 934.

Abbreviations: IQR = interquartile range; SD = standard deviation.

IL‐6, IFN‐γ, IL‐10, and IL‐15 showed consistent values across age and sex groups (Table 3). TNF had a noticeable difference between those under age 60 years (8.88 pg/mL) and those age 60 years or older (16.88 pg/mL), while similar values were observed between sexes. Leptin showed similar values across age groups, whereas it showed larger values for females (34.81 ng/mL) than males (16.14 ng/mL). Finally, IL‐1ra levels appear higher in participants under 60 years (428.81 pg/mL) compared to those 60 or older (377.02 pg/mL), while men showed lower IL‐1ra concentrations than women.

TABLE 3.

Cytokine concentrations categorized by age (< 60 or ≥ 60) and sex.

	Analyzed cytokines	Age < 60	Age ≥ 60	Male	Female
		Mean (SD)	Mean (SD)	Mean (SD)	Mean (SD)
Pro‐inflammatory	IL‐6 IFN‐γ TNF IL‐15 Leptin	6.02 (28.53)	5.22 (6.91)	5.81 (13.25)	5.39 (22.64)
		1.11 (1.59)	1.69 (6.20)	1.43 (3.85)	1.47 (5.51)
		8.88 (3.44)	16.88 (87.24)	14.27 (56.73)	12.93 (72.50)
		3.22 (2.84)	3.18 (4.0)	3.29 (4.69)	3.13 (2.43)
		29.72 (29.43)	25.05 (25.12)	16.14 (18.59)	34.89 (29.51)
Anti‐Inflammatory	IL‐10 IL‐1ra	2.70 (1.96)	3.83 (14.45)	3.57 (11.23)	3.20 (10.97)
		430.47 (564.34)	377.46 (341.19)	323.11 (254.50)	454.90 (542.54)

Note: All measurement units are pg/mL, except for Leptin, which is reported as ng/mL.

The concentration of cytokines based on pain and disability severity showed higher concentration of IL‐6, IL‐1ra, and Leptin with higher pain severity and ODI severity (Figures 1 and 2). TNF demonstrated higher concentrations in higher severity groups for ODI only. IL‐10, IL‐15, and IFN‐γ did not show differences in cytokine levels across severity groups of pain and ODI.

FIGURE 1.

Distribution of Pro‐Inflammatory cytokines across Pain and ODI categories. Each panel shows box‐and‐whisker plots of cytokine levels (TNF, IL‐15, Leptin, IFN‐γ) across three categories of pain (in blue; mild, moderate, severe) and three categories of disability (in red; mild, moderate, severe, per the Oswestry Disability Index [ODI]). The box boundaries represent the 25th and 75th percentiles, the horizontal line within each box is the median, and the whiskers extend to the minimum and maximum values (excluding outliers). Numeric annotations (e.g., mean, median, min, max) are displayed above each group.

FIGURE 2.

Distribution of Anti‐Inflammatory cytokines across Pain and ODI categories. Each panel shows box‐and‐whisker plots of cytokine levels (IL‐10, IL‐6, IL‐1ra) across three categories of pain (in blue; mild, moderate, severe) and three categories of disability (in red; mild, moderate, severe, per the Oswestry Disability Index [ODI]). The box boundaries represent the 25th and 75th percentiles, the horizontal line within each box is the median, and the whiskers extend to the minimum and maximum values (excluding outliers). Numeric annotations (e.g., mean, median, min, max) are displayed above each group.

6. Discussion

This study presents one of the most extensive characterizations to date of circulating cytokines in a large and representative cohort of individuals with chronic low back pain (cLBP), offering a valuable set of reference values for future research in this area. Our findings demonstrate that several pro‐ and anti‐inflammatory mediators (e.g., IL‐6, TNF, and IL‐1ra) not only align with previously reported ranges in healthy populations and smaller cLBP samples but also exhibit considerable variability, revealing potentially distinct subgroups with notably elevated cytokine levels. Taken together, these data serve as both a broad reference benchmark and highlight the need for more nuanced investigations of inflammatory mechanisms in cLBP, setting the stage for potential targeted interventions based on cytokine profiles.

For healthy adults, mean IL‐6 levels typically range from 1 to 5 pg/mL [145, 146, 147] and literature mean values for cLBP are generally between 2 and 5 pg/mL, aligning with the values in healthy adults [19]. In our cLBP cohort, while mean IL‐6 levels were on the high end of these values, they were characterized by high variability in the measurement of this marker, suggesting that a subset of these individuals may exhibit heightened IL‐6 levels. A similar observation could be made for IFN‐γ, another cytokine crucial to immune regulation. IFN‐γ typically ranges from 0.1 to 2 pg/mL in healthy individuals [148], though much broader ranges have been reported by other authors [149, 150]. In the current cLBP population, the IFN‐γ mean value for the overall population is smaller than mean values from the literature, but its SD is larger, highlighting again a greater range and potential specific subgroups with higher values. The levels of IL‐6 and IFN‐γ across age and sex suggest that these demographic factors may not meaningfully influence cytokine levels in cLBP.

IL‐10 and IL‐15 also showed similar concentrations across both sex and age, with values comparable to healthy and cLBP populations reported in the literature. In healthy adults, IL‐10 concentrations generally range from 1.0 to 3.5 pg/mL [122, 151, 152], and in other cLBP cohorts ranged between 2 and 4 pg/mL, thus aligning with the findings from our cohort. Similarly, IL‐15 has normative levels reported in the literature for both healthy and chronic low back pain (cLBP) populations, ranging from 2 to 4 pg/mL, and the same range was observed in this study's population [153, 154, 155].

TNF typically ranges from 1.0 to 8 pg/mL in healthy populations and between 10 and 15 pg/mL in other cLBP populations reported in the literature [148, 156]. Mean TNF levels in the current cLBP population are 13.47 pg/mL, thus simultaneously exceeding the normative values of healthy adults and aligning with other cLBP values reported in the literature. When looking at mean values across sexes, no major differences in TNF levels could be observed between males and females, suggesting that sex may not be meaningful in describing TNF levels in the included population. On the contrary, it is noted that the age group of individuals below 60 years. had mean levels of 8.88 pg/mL TNF, while individuals above 60 years. had mean levels of 16.86 pg/mL. This notable difference suggests the relevance of aging in TNF levels in people with cLBP. In fact, evidence from the literature notes that systemic inflammatory load often increases with aging. This condition has been described in the literature as inflammaging and may help account for the difference between the two age groups in the current cLBP population [157, 158]. Interestingly, though, TNF was the only cytokine that showed a noticeable difference across age groups.

IL‐6 has also been associated with inflammaging, but its concentration does not differ between the two age groups examined in our cohort [159]. Thus, inflammaging may differentially impact TNF levels in this cLBP population compared to other cytokines. Meanwhile, the other cytokines explored in the study may be associated with cLBP through different mechanisms that do not involve age, as supported by the fact that no large differences across age groups were identified for most other cytokines.

Leptin is influenced by body fat, sex, and metabolic health, with normative levels in healthy individuals ranging from 4 to 20 ng/mL [107, 160, 161]. In other cLBP populations, leptin levels vary widely, with mean levels between 16 and 22 ng/mL [162]. In the current cLBP population, leptin levels were comparable across age and slightly elevated compared to normative values of healthy individuals and other cLBP populations in the literature, a finding that could be at least partly explained by BMI levels higher in our population compared to the literature used for comparison [163]. More importantly, females exhibited more than twice the circulating leptin concentration of males. Although sex‐based differences in leptin levels have been widely documented, the magnitude of this difference in our study population exceeds previously reported values [164]. These findings suggest that additional leptin‐related mechanisms may contribute to chronic low back pain in females, potentially helping to explain the higher prevalence of cLBP among women reported in the literature.

Normative IL‐1ra mean levels in healthy individuals range between 150 and 300 pg/mL [165, 166]. In cLBP populations from the literature, means fall between 300 and 400 pg/mL [167], which aligns with the range observed in our overall cLBP population. This finding supports the broader observation of elevated circulating cytokines among individuals with cLBP. However, IL‐1ra is unique among anti‐inflammatory mediators due to its inclination to rise in chronic inflammatory states—a pattern not commonly seen with other anti‐inflammatory cytokines such as IL‐10 [166].

When examining differences by age and sex, we found higher IL‐1ra concentrations in participants under 60 and females. This intriguing result warrants replication and further study to clarify its implications. It may indicate more robust anti‐inflammatory mechanisms in younger adults, consistent with the “inflammaging” concept, in which proinflammatory activity increases with age. Meanwhile, the higher IL‐1ra levels in females could signal a compensatory response to proinflammatory tendencies characteristic of this sex [168]. This potential explanation, though, is challenged by the fact that we could not observe overall stronger inflammation in females compared to males for the observed cytokines in our population. These observations leave unanswered questions about the mechanisms driving increased IL‐1ra and whether its elevation leads to meaningful clinical outcomes. Another difficulty in interpreting the observed levels of IL‐1ra comes from the low circulating IL‐1β levels, largely below the detection limit in this cohort, despite IL‐1ra levels being elevated. IL‐1ra, a natural inhibitor of IL‐1 signaling, may reflect a systemic response to inflammation or tissue degeneration. However, the single timepoint assayed in the current study does not allow for inference regarding potential compensatory expression. The elevated IL‐1ra in the presence of low circulating IL‐1 suggests possible dysregulation in inflammatory pathways, warranting further investigation into the role of IL‐1ra as a marker or modulator of chronic systemic inflammation.

Several notable patterns emerged when examining mean cytokine levels across different tiers of pain severity and ODI scores. IL‐6, IL‐1ra, and Leptin showed larger concentrations with more pain and ODI severity, highlighting a potential role for systemic inflammation in cLBP. TNF exhibited more concentration with higher ODI but not with pain, implying a more marginal connection with the clinical presentation of cLBP. Meanwhile, IL‐10, IL‐15, and IFN‐γ demonstrated no distinct variation across the severity of pain and disability. These cross‐sectional findings provide a detailed snapshot of circulating cytokine levels that could contribute to—or reflect—the pathophysiological processes underlying cLBP.

This study makes several important and novel contributions to the literature on chronic low back pain (cLBP). Most notably, it is the largest investigation to date assessing circulating cytokines in a well‐characterized cLBP cohort, with over 930 participants providing plasma samples. The demographic diversity of the cohort, spanning age, sex, BMI, and race, enhances the generalizability of findings and allows for robust subgroup analyses that are rarely feasible in smaller studies. The breadth of the cytokine panel further distinguishes this work; alongside well‐established pro‐inflammatory markers such as IL‐6 and TNF, the study includes less commonly evaluated cytokines such as IL‐15 and Leptin, as well as two key anti‐inflammatory mediators, IL‐10 and IL‐1ra. Leptin, in particular, adds a unique metabolic dimension to the inflammatory profile and reveals interesting sex differences, suggesting potential relevance to sex‐specific mechanisms in chronic pain. Furthermore, the stratified analyses across age, sex, pain intensity, and disability severity provide new insights into how cytokine levels may reflect clinically meaningful subgroups within the cLBP population. For instance, TNF levels were notably higher in older adults, consistent with the concept of “inflammaging.” In contrast, IL‐1ra levels were elevated in younger individuals and females, suggesting age‐ and sex‐dependent regulatory patterns. Together, these findings support the potential utility of inflammatory profiling for identifying biologically distinct phenotypes in cLBP, and they lay the groundwork for additional analyses within this well characterized cohort as well as future precision medicine approaches that target specific immune pathways as a means to improve diagnosis, prognosis, and treatment response.

Overall, these findings indicate that select cytokines—particularly IL‐6, IL‐1ra, and leptin—tend to exceed reference ranges from the literature and vary along with pain and disability severity in cLBP. This pattern suggests a specific set of elevated cytokines underlying cLBP's unique clinical presentation, which may 1 day aid in more precise diagnosis and treatment strategies. Additionally, stratifying cytokine trends by age and sex underscores the heterogeneity within the cLBP population, highlighting the need for further sub‐classification to better address the diverse factors contributing to chronic low back pain.

7. Limitations

This analysis aims to provide a summary and overview of the characteristics of the cLBP sample. Descriptive statistics serve as an essential initial step in highlighting key features relevant to the broader objectives of the project. Subsequent research will involve inferential analyses, including the exploration of group differences and relationships, as well as comparisons to control populations. The study's cross‐sectional design provides only a snapshot of individuals with cLBP and prevents discerning whether inflammatory changes precede or follow the onset or exacerbation of cLBP, thereby precluding any inference of causality. Moreover, this design does not capture day‐to‐day variability in cytokine levels, which can fluctuate due to various factors. Longitudinal studies are needed to elucidate temporal relationships through repeated cytokine levels and clinical outcome measurements. Although cytokine selection was informed by the existing literature, the human body produces thousands of proteins; thus, using a broader panel of cytokines could improve insights into the role of inflammatory patterns in cLBP. These limitations underscore the need to build upon our current dataset by incorporating additional biomarkers, extending longitudinal follow‐up within the same cohort, and expanding the analysis to explore the associations of cytokines and outcome measures.

8. Conclusion

This study sought to elucidate the inflammatory landscape in cLBP by examining multiple pro‐ and anti‐inflammatory cytokines. Despite the limited number of cytokines analyzed, the findings highlight a unique pattern of inflammatory cytokines in cLBP, where differences in cytokines such as IL‐6, TNF, and Leptin were observed when examining the severity of pain and disability levels in this population.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1: Supporting Information.

Enrico V. T., Anderst W., Bell K. M., et al., “Plasma Pro‐ and Anti‐Inflammatory Cytokines in an Observational Chronic Low Back Pain Cohort,” JOR Spine 8, no. 3 (2025): e70095, 10.1002/jsp2.70095.