Background & Aims
Chronic pain, affecting 20% globally, poses economic and health challenges1. Fibromyalgia (FM), as a severe chronic pain condition, remains to be poorly understood2,3. Emerging evidence underscores the pivotal role of the immune system in chronic pain development4–7. Studies have reported cytokine profile abnormalities and immune system dysregulation in peripheral blood of FM patients, pointing towards a potential link between immune dysfunction and FM pathogenesis7,8. It has previously been demonstrated that neutrophils are important in the development of chronic widespread pain9; however, the specific involvement of neutrophils, crucial immune effectors often overlooked in chronic pain contexts, remains inadequately explored. In this study, we conducted a comprehensive whole transcriptomics analysis of peripheral neutrophils from FM patients and healthy controls (HC), focusing on their responses to inflammatory stimuli as an objective indicator of immune system responsiveness.
Methods
STAR was used for mapping the RNA-seq data to the reference genome10. Differential gene expression, pathway, and transcription factor analyses were done using DESeq211 and gProfiler12 R packages. Neutrophils were isolated from whole blood using MACSxpress® Whole Blood Neutrophil Isolation Kit as per manufacturer instructions. 1 hour of 10 ng/mL LPS was used as stimulant to objectively quantify neutrophil responsiveness. Long-term outcomes were studied using Patient’s Global Impression of Change (PGIC) questionnaire13. Using PGIC we categorized patients into Improver and Persister groups based on symptom improvement.
Results
Comparing the neutrophils of FM and HC revealed an active inflammatory state in FM neutrophils. Despite this baseline inflammation, an impaired ability to respond efficiently to LPS stimulation was observed in FM neutrophils compared to HC. Moreover, analysis of Improver and Persister FM groups has uncovered a higher immune response in Persisters at baseline as well as a distinct response to LPS stimulation, with Improvers exhibiting a bigger response compared to Persisters, although both groups show a reduced response compared to HC. Notably, the two groups did not have significant difference in their symptoms and disease severity based on questionnaire responses. Transcription factor and pathway analysis identified NF-kB as a key regulator of both baseline differences and LPS responsiveness. Inhibition of NF-kB activity pathway was observed in both FM cases and Persisters. FM subgroups were also validated in animal models.
Conclusions
In conclusion, our investigation of the peripheral blood neutrophils has uncovered a distinct inflammatory state in FM patients. FM neutrophils exhibited an inefficiency in responding to LPS, implicating a potential cell signaling defect, leading to chronic low-grade chronic inflammation. NF-kB transcription factor inhibition emerged as a crucial contributor to this state. Furthermore, Persister FM subgroup showed increased levels of immune response at the baseline but blunted LPS responsiveness emphasizing the relevance of immune dynamics in chronic pain progression or improvement.
References
1.Gaskin, D. J. & Richard, P. The economic costs of pain in the United States. J Pain 13, 715–724 (2012).
2.Clauw, D. J. Fibromyalgia: a clinical review. JAMA 311, 1547–1555 (2014).
3.Bair, M. J. & Krebs, E. E. Fibromyalgia. Ann Intern Med 172, ITC33–ITC48 (2020).
4.Kavelaars, A. & Heijnen, C. J. Immune regulation of pain: Friend and foe. Sci Transl Med 13, (2021).
5.Ren, K. & Dubner, R. Interactions between the immune and nervous systems in pain. Nat Med 16, 1267–1276 (2010).
6.Bethea, J. R., Fischer, R. & Fischer, R. Role of Peripheral Immune Cells for Development and Recovery of Chronic Pain. 12, 1–7 (2021).
7.Marchand, F., Perretti, M. & Mcmahon, S. B. FO C U S O N PA I N ROLE OF THE IMMUNE SYSTEM IN CHRONIC PAIN. 6, 521–532 (2005).
8.O’Mahony, L. F., Srivastava, A., Mehta, P. & Ciurtin, C. Is fibromyalgia associated with a unique cytokine profile? A systematic review and meta-analysis. Rheumatology (Oxford) 60, 2602–2614 (2021).
9.Caxaria, S. et al. Neutrophils infiltrate sensory ganglia and mediate chronic widespread pain in fibromyalgia. Proc Natl Acad Sci U S A 120, (2023).
10.Dobin, A. et al. STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
11.Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 1–21 (2014).
12.Peterson, H., Kolberg, L., Kuzmin, I., Arak, T. & Adler, P. g?: Profiler?: a web server for functional enrichment analysis and conversions of gene lists ( 2019 update ). 47, 191–198 (2019).
13.Hurst, H. & Bolton, J. Assessing the clinical significance of change scores recorded on subjective outcome measures. J Manipulative Physiol Ther 27, 26–35 (2004).
Presenting Author
Sahel Jahangiri Esfahani
Poster Authors
Sahel Jahangiri Esfahani
PhD
McGill University
Lead Author
Topics
- Genetics