|Ahead of print publication
Serum interleukin-6, interleukin-8, and interleukin-1 receptor antagonist levels in South Indian fibromyalgia patients and its correlation with disease severity
Sandeep Surendran1, CB Mithun1, Vishnu S Chandran2, Suma Balan1, Arun Tiwari1
1 Department of Rheumatology and Clinical Immunology, Amrita Institute of Medical Sciences, Kochi, Kerala, India
2 Department of Rheumatology, Believers Church Medical College Hospital, Thiruvalla, Kerala, India
|Date of Submission||01-Oct-2020|
|Date of Acceptance||13-Aug-2021|
|Date of Web Publication||26-Nov-2021|
Department of Rheumatology and Clinical Immunology, Amrita Institute of Medical Sciences, Ponekkara, Kochi, Kerala
Source of Support: None, Conflict of Interest: None
Background: Fibromyalgia is a chronic pain syndrome characterized by widespread diffuse pain and multiple tender points along with sleep disturbances, fatigue, and psychological distress. Cytokines have been proposed to play an important role in the pathogenesis of fibromyalgia. Different studies, mainly done in the Western population, have shown dysregulated cytokines in fibromyalgia patients. This study was aimed to analyze the serum cytokine patterns of interleukin-1 receptor antagonist (IL-1Ra), interleukin-8 (IL-8), and interleukin-6 (IL-6) in Indian females with fibromyalgia and its correlation with the disease severity.
Materials and Methods: This single-center cross-sectional study compared the serum cytokines levels (IL-6, IL-8, and IL-1Ra) of 21 females fulfilling 2016 modification of the 2010/11 American College of Rheumatology fibromyalgia criteria and without co-existent major depressive or inflammatory diseases; with 20 age- and sex-matched controls. The outcome measures used to measure disease activity were visual analog scale pain, fibromyalgia impact questionnaire-revised, brief pain inventory pain scores, and SF-36 health survey. Statistical tests used were the Kolmogorov–Smirnov test of normality, Mann–Whitney's test, Chi-square tests, Spearman rank order, and Benjamini–Hochberg correlation.
Results: Serum IL1-Ra levels were significantly lower in fibromyalgia patients, median 226.90 pg/ml (interquartile range [IQR] 476.95) than the healthy controls, median 778.80 pg/ml (IQR 721) (P < 0.05). However, there was no statistical difference in the levels of IL-8 and IL-6 between patients and the control group. The analysis for the correlation between cytokine and outcome measures failed to show any statistically significant correlation.
Conclusion: Fibromyalgia patients in our study had lower serum IL-1Ra level, whereas IL-8 and IL-6 levels were normal. When compared with western studies, these contrasting results suggest a heterogeneous cytokine profile of fibromyalgia patients in different ethnic groups.
Keywords: Cytokines, fibromyalgia, interleukin-1 receptor antagonist, interleukin-6, interleukin-8
|How to cite this URL:|
Surendran S, Mithun C B, Chandran VS, Balan S, Tiwari A. Serum interleukin-6, interleukin-8, and interleukin-1 receptor antagonist levels in South Indian fibromyalgia patients and its correlation with disease severity. Indian J Rheumatol [Epub ahead of print] [cited 2022 Jan 16]. Available from: https://www.indianjrheumatol.com/preprintarticle.asp?id=331252
| Introduction|| |
Fibromyalgia is a chronic pain syndrome that is characterized by widespread diffuse pain and multiple tender points. It is often associated with insomnia or sleep disturbances, fatigue, and significant psychological distress besides the widespread generalized pain.
There is no single pathological pathway or process that can explain the development of fibromyalgia in a patient, but the dominant role of abnormal pain processing in the pathophysiology of the disease is well-known. Studies with functional magnetic resonance imaging scans have suggested that fibromyalgia patients may have a lowered pressure-pain threshold. Furthermore, the studies have shown that there is an increased response to peripheral stimulation via amplification of signaling in the central nervous system. This phenomenon is known as “central sensitization.” However, it is not just the central processing of pain in the brain that is affected; the studies have shown that there are abnormal ascending pain pathways and peripheral large nerves in fibromyalgia patients., In addition, newer research shows the existence of small fiber neuropathy in fibromyalgia., The exact process of this diffuse mal-activation of the neural networks is not known yet, but various studies have shown abnormal elevation of cytokines levels in these patients. Certain pro-inflammatory cytokines (interleukin-1 β, interleukin-6 [IL-6], and tumor necrosis factor-α [TNF-α]) have been shown to contribute directly to central and peripheral neuropathic pain in the other pain syndromes., It is likely that these cytokines also play a role in the neurobiology of fibromyalgia.
The etiopathogenic role of cytokines was first described by Wallace et al., and this has been further studied in Western populations; with many studies showing higher circulatory levels of IL-6 and interleukin-8 (IL-8) in fibromyalgia patients.,,, However, the pattern of cytokine elevations is not consistent across the world, with certain studies showing differing findings., A systematic review of these studies summarized that fibromyalgia patients had higher levels of interleukin-1 receptor antagonist (IL-1Ra), IL-6, and IL-8. Given the genetic heterogeneity of Indians when compared to the Western cohorts, it is not known whether these cytokines are elevated in Indian patients with fibromyalgia. There has been only a single study published from India regarding the cytokine pattern in fibromyalgia. In this study, Ghizal et al. showed higher circulatory levels of TNF-α in fibromyalgia patients than controls. However, IL-6 levels were not significantly different between the groups. This result sharply contrasts with the Western data. To the best of our knowledge, there have been no other studies published on the cytokine profile in fibromyalgia in the Indian population.
In our study, we aimed to analyze the serum cytokine pattern of IL-1Ra, IL-8, and IL-6 in Indian females with fibromyalgia and its correlation with the disease severity.
| Materials and Methods|| |
Study population and design
This was a cross-sectional study conducted at the Rheumatology clinic of a tertiary care center in South India. Date of approval is needed from authors ethical clearance was obtained from the Institutional Review Board of Amrita Institute of Medical Sciences-2018-309. Female subjects between the ages of 18 and 60 years who were newly diagnosed with fibromyalgia using the 2016 revised version of the 2010/2011 American College of Rheumatology Criteria were included in the study. All the patients had symptoms of fibromyalgia for at least 3 months. Patients who were suffering from other chronic rheumatological conditions, any acute or chronic infections; who had other chronic pain syndromes; who had a major depressive disorder or were already on treatment with serotonin and norepinephrine reuptake inhibitors and tricyclic antidepressants, were excluded from the study. We also excluded patients on steroids or any other anti-inflammatory medications which could alter cytokine profile this study patients were then compared with healthy controls. The controls were selected based on one-to-one propensity-based age and gender matching, asymptomatic healthy people visiting for routine health check-ups at our center. A written informed consent was obtained from all the study participants.
Twenty-one female fibromyalgia patients and 20controls were selected by convenience sampling and studied as per the above-mentioned protocol.
Outcome measures and other clinical data
The disease activity measures measured at the baseline included visual analog scale (VAS) Pain, fibromyalgia impact questionnaire-revised (FIQR) Score, and brief pain inventory (BPI) pain score (total and average), and SF-36 health survey (including physical and mental component scales). The demographic data were also collected at the baseline.
Serum cytokines analysis
Samples for cytokine analysis were also drawn at the baseline. For this, 5 ml of blood was drawn in a plain vacutainer tube and allowed clotting for 30 min in a serum separator tube, then centrifuged at 1000 rpm for 10 min to remove the serum layer. The serum samples were then stored at a temperature <-20°C, until the time of analysis. Serum samples were thawed for the cytokine testing (IL-6, IL-8, and Il-1Ra) which were done using enzyme-linked immunosorbent assay (ELISA) kits by Krishgen Biosystems. Serum levels were calculated using standard curves generated with specific standards; according to the manufacturer's instructions. The lower limit of detection of the ELISA kits for IL-1Ra, IL-6, and IL-8 was 40 pg/ml, 3.13 pg/ml, and 15.63 pg/ml, respectively.
The distribution of data was assessed by the Kolmogorov–Smirnov test of normality. In the case of normal distribution of data, results were presented as mean ± standard deviation and nonparametric data were presented, and median and interquartile range (IQR). Comparisons between IL8 and IL1Ra levels between investigated groups were tested for significance using the Mann–Whitney U-test. Comparisons for IL6 levels were done using the Chi-square test. Correlations between variables within the group were analyzed using Spearman's rank-order and Benjamini–Hochberg correlation. A P < 0.05 was considered statistically significant. Data analysis was done using Excel for data entry and SPSS v20.0 (IBM Corporation, USA) for analysis.
| Results|| |
Twenty-one female fibromyalgia patients and 20 age- and gender-matched controls were enrolled in the study. [Table 1] shows the demographic data, baseline clinical characteristics, and serum cytokine levels (IL-1Ra, IL-6, and IL-18) of fibromyalgia patients and controls. Serum IL1-Ra levels were significantly lower in fibromyalgia patients median 226.90 pg/ml (IQR 476.95) than the healthy controls median 778.80 pg/ml (IQR 721) (P < 0.05). This is graphically represented in [Figure 1]. Serum IL-6 levels were beyond the lower limit of detection of the ELISA kits.
|Figure 1: Serum interleukin 1Ra levels in fibromyalgia patients versus controls|
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The serum cytokine levels of IL-1Ra and IL-8 were analyzed for correlation with outcome measures (VAS Pain, BPI pain and function, FIQR scores, SF-36 health survey), and the results are shown in [Table 2]. A moderate positive correlation was seen between IL-8 levels and VAS pain score (r2 = 0.44 P = 0.05), but it was not found to be significant with Benjamini–Hochberg correction (P = 0.006). Although the IL-8 levels showed a trend for a positive correlation with BPI average pain (r2 = 0.42, P = 0.06), it was also not statistically significant. None of the other outcome measures also showed any significant correlation with the serum cytokine levels.
|Table 2: Baseline outcome measures of fibromyalgia arm and their correlation with serum cytokines (interleukin-1Ra and interleukin-8)|
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| Discussion|| |
In accordance with other studies, our study too demonstrated a statistically significant difference in serum cytokine IL1-Ra levels between fibromyalgia patients and controls, thereby suggesting a role of cytokines in the pathogenesis of fibromyalgia. However, in our study, IL-1Ra levels were found to be significantly lower in fibromyalgia patients as compared to healthy controls, which was contrary to our expectations based on a systematic review of previous studies done on cytokine profile in fibromyalgia patients. Furthermore, there was no statistically significant difference in levels of IL-6 and IL-8 between fibromyalgia patients and healthy controls. The cytokine profile in our fibromyalgia patients appears to be different from the Western population. A comparative summary of studies done for cytokines in fibromyalgia is summarized in [Table 3].
Our study results are in contrast to the data published by Wallace et al. and Iannuccelli et al. where higher levels of IL-1Ra were reported in fibromyalgia patients., On comparing the results of our cytokine analysis with these two studies where the IL-1Ra levels were reported as elevated, it could be noted that these studies also reported higher IL-6 levels, unlike our study. It is already known that IL-1Ra production is linked to the IL-6 levels. As our patients had very low IL-6 levels (majority undetectable), this could be one reason why IL-1Ra levels were low in fibromyalgia patients. Another explanation for the low IL-1Ra levels may be the IL-1Ra variable number of tandem repeats polymorphism which has been previously reported in the South Indian population by Panneer et al. in a study done in lupus patients. Of the two common patterns seen in South Indians, two allele repeats were reported to be associated with lower IL-1Ra levels. These polymorphisms may be higher in fibromyalgia patients also. Further studies of this are warranted. These polymorphisms could have a role in the severity of the disease and the refractory nature of the disease; similar to what is reported in lupus.
Baseline outcome measures failed to show any correlation with IL-8 and IL-1Ra levels, although there was a moderate correlation between VAS and IL-8 by Spearman rank order, it was not significant with Benjamin Hochberg correction. In addition, in our study, serum IL-6 levels were undetectable (<3.13 pg/ml) in the majority of the patients. These data are different from the Western studies, which showed elevated IL-6 levels in fibromyalgia patients. This heterogeneity in serum IL-6 levels in fibromyalgia patients is also reflected in the published studies on IL-6 levels in another related disorder-depression. While meta-analysis of the Western data in patients with depression showed elevated IL-6 levels, the Indian studies (Jeenger et al., Jangpangi et al.) showed contrasting results with low IL-6 levels., A possible hypothesis that can be made from these findings is that it is likely the pathways of neural mal-activation are different in the Indian population.
The translational importance of cytokines assessment in fibromyalgia cases could be with respect to treatment protocols. Cytokine-targeted therapies are being considered for similar spectrum illnesses like depression and chronic fatigue syndrome. O'Brien et al. have discussed in a review article summarizing the role of cytokines in acute stress, chronic stress, and major depression in animal and human studies from 1980 to 2003. They have also proposed cytokine-targeted therapy for depression. This is further supported by a meta-analysis by Kappelmann et al. for antidepressant activity of anti-cytokine treatment found that adalimumab, etanercept, infliximab, and tocilizumab all showed statistically significant improvements in depressive symptoms in a variety of diseases. Anakinra (Anti IL-1) therapy has also been tried in chronic fatigue syndrome in the Netherlands by Roerink et al., though this study didn't show any benefit, given the cytokine heterogenicity suggested by our study, the possibility of the role of cytokine targeted therapy could perhaps be considered.
Our study is, however, not without limitations. We were neither able to assess the correlation of serum IL-6 with the outcome measures nor able to do an absolute mean comparison between the groups as the majority of our patients had undetectable IL-6 levels. In addition, it was a single-center study from South India, and India is a country of ethnic diversity, a multicenter study involving centers from other parts of India may give a clearer picture of the cytokine pattern in Indian fibromyalgia patients. SF-36 questionnaire, which was developed as a self-reported questionnaire, was verbally asked by the authors to the patients in regional language in our study, which could cause a bias.
Like other studies from the West, the present study also suggests a key role for cytokines in the pathogenesis of fibromyalgia. The association of serum cytokine levels as a guide for pharmacotherapy in fibromyalgia patients (pre and post levels) is another area that could be studied in further studies. Given the widespread prevalence of fibromyalgia, understanding the pathways that lead to the small fiber mal-activation and their heterogeneity in cohorts across the world will help understanding an often misunderstood disease better.
| Conclusion|| |
In our study, IL-1Ra levels were found to be significantly low in fibromyalgia patients. However, our study showed a different cytokine profile in Indian fibromyalgia patients when compared to the western population. Further studies on the role of other cytokines and whether they correlate with other domains of fibromyalgia, like sleep and cognitive disturbances, would help in understanding the clinical role of the cytokines in this disease better. The role of cytokine targeted therapy for fibromyalgia remains unknown hence, understanding the cytokine heterogenicity in the Indian cohort of fibromyalgia perhaps would be the first step before trying cytokine targeted therapy.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]